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NORMAL GENOMIC

• DeSalle LM, Pagano M
• Regulation of the G1 to S transition by the ubiquitin pathway.
• FEBS Lett. 2001; 490: 179-89
• Display abstract

• This year the most prestigious prize in medical sciences, the Lasker Award, has been presented to the three scientists who discovered the ubiquitin pathway: Aaron Ciechanover, Avram Hershko, and Alexander Varshavsky [Nature Med. 6 (2000) 1073-1081]. During a time when the scientific community was focused on understanding how proteins were synthesized, they intently pursued the novel idea that cells were programmed to selectively destroy proteins. Their work led to the identification of an elaborate system of protein degradation targeting a myriad of cellular substrates. A small protein called ubiquitin is at the center of this process. Although the ubiquitin pathway was first described in the early 1980s, it has only more recently advanced to the forefront of basic research as a significant regulatory network within the cell. The field continues to grow as new ubiquitination enzymes and novel functions of this system are identified. Scientists are focused on elucidating the mechanisms by which cells deploy the ubiquitin pathway to control levels of selected proteins, such as cell cycle regulatory proteins, transcription factors and signaling molecules. Accelerated or decelerated rates of degradation of particular substrates participate in the genesis of many human diseases. Thus, understanding the mechanisms that confer specificity to the ubiquitin system will allow the development of novel therapeutic approaches to target aberrations in this pathway underlying tumorigenesis and other human pathologies.

• Coffino P
• Antizyme, a mediator of ubiquitin-independent proteasomal degradation.
• Biochimie. 2001; 83: 319-23
• Display abstract

• Ornithine decarboxylase (ODC) is among the small set of proteasome substrates that is not ubiquitinated. It is instead degraded in conjunction with the protein antizyme (AZ). ODC and AZ are participants in a regulatory circuit that restricts pools of polyamines, the downstream products of ODC enzymatic activity. Functional studies using directed mutagenesis have identified regions of ODC and AZ required for the process of ODC degradation. Within ODC, there is a region that is required for AZ binding which lies on the surface of an alpha-beta barrel forming one domain of the ODC monomer. A carboxy-terminal ODC domain is needed for both AZ-dependent and AZ-independent degradation. Within AZ, the carboxy-terminal half molecule is sufficient for binding to ODC, but an additional domain found within the AZ amino terminus must be present for stimulation of ODC degradation by the proteasome. Recently, the AZs have been found to consist of an ancient gene family. Within vertebrate species, multiple isoforms are found, with distinct functions that remain to be sorted out. Although AZ homologs have been found in some yeast species, homology searches have failed to identify an AZ homolog in Saccharomyces cerevisiae. Nevertheless, the close parallel between polyamine-induced ODC degradation in S. cerevisiae and in animal cells suggests that this organism will also be found to harbor an AZ-like protein.

• Ingvardsen C, Veierskov B
• Ubiquitin- and proteasome-dependent proteolysis in plants.
• Physiol Plant. 2001; 112: 451-459
• Display abstract

• In recent years it has become obvious that protein degradation is an important catabolic process during development in plants and animals. One very conserved degradative system is the ubiquitin- and proteasome-dependent proteolytic pathway, which is found in all eukaryotes from yeast to mammals and plants. The pathway consists of two parts, one in which chains of ubiquitin are conjugated to substrate proteins, and one in which these substrate proteins are either degraded by the 26S proteasome or are de-ubiquitinated. The ubiquitin- and proteasome-dependent pathway degrades a wide range of proteins in the nucleus and cytoplasm. It is highly specific, but controls a large number of cellular events due to the diversity in the conjugating enzymes. This pathway is important for removal of abnormal/damaged proteins that have had their recognition sites exposed as well as for control of specific transcription factors and cell cycle regulators. In plants, ubiquitin- and proteasome-dependent proteolysis is known to be involved in regulation of the cell cycle and transcription factors as well as endoplasmic reticulum-associated protein degradation, stress response and developmental processes, such as xylogenesis and senescence.

• Attaix D, Combaret L, Pouch MN, Taillandier D
• Regulation of proteolysis.
• Curr Opin Clin Nutr Metab Care. 2001; 4: 45-9
• Display abstract

• The mechanisms of proteolysis remain to be fully defined. This review focuses on recent advances in our understanding of the ubiquitin-proteasome-dependent pathway, which is involved in the control of many major biological functions. The ubiquitinylation/deubiquitinylation system is a complex machinery responsible for the specific tagging and proof-reading of substrates degraded by the 26S proteasome, as well as having other functions. The formation of a polyubiquitin degradation signal is required for proteasome-dependent proteolysis. Several families of enzymes, which may comprise hundreds of members to achieve high selectivity, control this process. The substrates tagged by ubiquitin are then recognized by the 26S proteasome and degraded into peptides. In addition, the 26S proteasome also recognizes and degrades some non-ubiquitinylated proteins. In fact, there are multiple ubiquitin- or proteasome-dependent pathways. These systems presumably degrade specific classes of substrates and single proteins by alternative mechanisms and could be interconnected. They may also interfere or cooperate with other proteolytic pathways.

• Layfield R, Alban A, Mayer RJ, Lowe J
• The ubiquitin protein catabolic disorders.
• Neuropathol Appl Neurobiol. 2001; 27: 171-9
• Display abstract

• The ubiquitin-proteasome system of intracellular proteolysis is essential for cell viability. We propose the concept that neurodegenerative diseases such as Alzheimer's and Parkinson's, as well as other conditions including some types of cancer, collectively represent a raft of 'ubiquitin protein catabolic disorders' in which altered function of the ubiquitin-proteasome system can cause or directly contribute to disease pathogenesis. Genetic abnormalities within the ubiquitin pathway, either in ubiquitin-ligase (E3) enzymes or in deubiquitinating enzymes, cause disease because of problems associated with substrate recognition or supply of free ubiquitin, respectively. In some cases, mutations in protein substrates of the ubiquitin-proteasome system may directly contribute to disease progression because of inefficient substrate recognition. Mutations in transcripts for the ubiquitin protein itself (as a result of 'molecular misreading') also affect ubiquitin-dependent proteolysis with catastrophic consequences. This has been shown in Alzheimer's disease and could apply to other age-associated neurodegenerative conditions. Within the nervous system, accumulation of unwanted proteins as a result of defective ubiquitin-dependent proteolysis may contribute to aggregation events, which underlie the pathogenesis of several major human neurodegenerative diseases.

• Lee C, Schwartz MP, Prakash S, Iwakura M, Matouschek A
• ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal.
• Mol Cell. 2001; 7: 627-37
• Display abstract

• Protein unfolding is a key step in several cellular processes, including protein translocation across some membranes and protein degradation by ATP-dependent proteases. ClpAP protease and the proteasome can actively unfold proteins in a process that hydrolyzes ATP. Here we show that these proteases seem to catalyze unfolding by processively unraveling their substrates from the attachment point of the degradation signal. As a consequence, the ability of a protein to be degraded depends on its structure as well as its stability. In multidomain proteins, independently stable domains are unfolded sequentially. We show that these results can explain the limited degradation by the proteasome that occurs in the processing of the precursor of the transcription factor NF-kappaB.

• Wang C, Xi J, Begley TP, Nicholson LK
• Solution structure of ThiS and implications for the evolutionary roots of ubiquitin.
• Nat Struct Biol. 2001; 8: 47-51
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• ThiS is a sulfur carrier protein that plays a central role in thiamin biosynthesis in Escherichia coli. Here we report the solution NMR structure of ThiS, the first for this class of sulfur carrier proteins. Although ThiS shares only 14% sequence identity with ubiquitin, it possesses the ubiquitin fold. This structural homology, combined with established functional similarities involving sulfur chemistry, demonstrates that the eukaryotic ubiquitin and the prokaryotic ThiS evolved from a common ancestor. This illustrates how structure determination is essential in establishing evolutionary links between proteins in which structure and function have been conserved through eons of evolution despite loss of sequence identity. The ThiS structure reveals both hydrophobic and electrostatic surface features that are likely determinants for interactions with binding partners. Comparison with surface features of ubiquitin and ubiquitin homologs SUMO-1, RUB-1 and NEDD8 suggest how Nature has utilized this single fold to incorporate similar chemistry into a broad array of highly specific biological processes.

• Hicke L
• Protein regulation by monoubiquitin.
• Nat Rev Mol Cell Biol. 2001; 2: 195-201
• Display abstract

• Multi-ubiquitin chains at least four subunits long are required for efficient recognition and degradation of ubiquitylated proteins by the proteasome, but other functions of ubiquitin have been discovered that do not involve the proteasome. Some proteins are modified by a single ubiquitin or short ubiquitin chains. Instead of sending proteins to their death through the proteasome, monoubiquitylation regulates processes that range from membrane transport to transcriptional regulation.

• Sommer T, Jarosch E, Lenk U
• Compartment-specific functions of the ubiquitin-proteasome pathway.
• Rev Physiol Biochem Pharmacol. 2001; 142: 97-160

• Sloper-Mould KE, Jemc JC, Pickart CM, Hicke L
• Distinct functional surface regions on ubiquitin.
• J Biol Chem. 2001; 276: 30483-9
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• The characterized functions of the highly conserved polypeptide ubiquitin are to target proteins for proteasome degradation or endocytosis. The formation of a polyubiquitin chain of at least four units is required for efficient proteasome binding. By contrast, monoubiquitin serves as a signal for the endocytosis of plasma membrane proteins. We have defined surface residues that are important for ubiquitin's vital functions in Saccharomyces cerevisiae. Surprisingly, alanine scanning mutagenesis showed that only 16 of ubiquitin's 63 surface residues are essential for vegetative growth in yeast. Most of the essential residues localize to two hydrophobic clusters that participate in proteasome recognition and/or endocytosis. The others reside in or near the tail region, which is important for conjugation and deubiquitination. We also demonstrate that the essential residues comprise two distinct functional surfaces: residues surrounding Phe(4) are required for endocytosis, whereas residues surrounding Ile(44) are required for both endocytosis and proteasome degradation.

• Hofmann K, Falquet L
• A ubiquitin-interacting motif conserved in components of the proteasomal and lysosomal protein degradation systems.
• Trends Biochem Sci. 2001; 26: 347-50
• Display abstract

• Ubiquitination generally serves as a signal for targeting cytoplasmic and nuclear proteins to the proteasome for subsequent degradation. Recently, evidence has accumulated indicating that ubiquitination also plays an important role in targeting integral membrane proteins for degradation by the lytic vacuole or the lysosome. This article describes a conserved protein motif, based on a sequence of the proteasomal component Rpn10/S5a, that is known to recognize ubiquitin. The presence of this motif in Eps15, Epsin and HRS, proteins involved in ligand-activated receptor endocytosis and degradation, suggest a more general role in ubiquitin recognition.

• DiAntonio A, Haghighi AP, Portman SL, Lee JD, Amaranto AM, Goodman CS
• Ubiquitination-dependent mechanisms regulate synaptic growth and function.
• Nature. 2001; 412: 449-52
• Display abstract

• The covalent attachment of ubiquitin to cellular proteins is a powerful mechanism for controlling protein activity and localization. Ubiquitination is a reversible modification promoted by ubiquitin ligases and antagonized by deubiquitinating proteases. Ubiquitin-dependent mechanisms regulate many important processes including cell-cycle progression, apoptosis and transcriptional regulation. Here we show that ubiquitin-dependent mechanisms regulate synaptic development at the Drosophila neuromuscular junction (NMJ). Neuronal overexpression of the deubiquitinating protease fat facets leads to a profound disruption of synaptic growth control; there is a large increase in the number of synaptic boutons, an elaboration of the synaptic branching pattern, and a disruption of synaptic function. Antagonizing the ubiquitination pathway in neurons by expression of the yeast deubiquitinating protease UBP2 (ref. 5) also produces synaptic overgrowth and dysfunction. Genetic interactions between fat facets and highwire, a negative regulator of synaptic growth that has structural homology to a family of ubiquitin ligases, suggest that synaptic development may be controlled by the balance between positive and negative regulators of ubiquitination.

• Muller S, Hoege C, Pyrowolakis G, Jentsch S
• SUMO, ubiquitin's mysterious cousin.
• Nat Rev Mol Cell Biol. 2001; 2: 202-10
• Display abstract

• Covalent modification of cellular proteins by the ubiquitin-like modifier SUMO regulates various cellular processes, such as nuclear transport, signal transduction, stress response and cell-cycle progression. But, in contrast to ubiquitylation, sumoylation does not tag proteins for degradation, but seems to enhance their stability or modulate their subcellular compartmentalization.

• Xiao W, Jang J
• F-box proteins in Arabidopsis.
• Trends Plant Sci. 2000; 5: 454-7

• Callis J, Vierstra RD
• Protein degradation in signaling.
• Curr Opin Plant Biol. 2000; 3: 381-6
• Display abstract

• Recent studies have linked proteolysis by the ubiquitin/proteasome pathway to a variety of signaling pathways in higher plants. These links were uncovered by characterization of mutants altered in signaling or by targeted disruption of components of the proteolytic pathway. Significant advances have recently revealed connections between proteolysis and hormone responses, light perception, environmental adaptation, and floral development.

• Ciechanover A, Orian A, Schwartz AL
• Ubiquitin-mediated proteolysis: biological regulation via destruction.
• Bioessays. 2000; 22: 442-51
• Display abstract

• The ubiquitin proteolytic system plays an important role in a broad array of basic cellular processes. Among these are regulation of cell cycle, modulation of the immune and inflammatory responses, control of signal transduction pathways, development and differentiation. These complex processes are controlled via specific degradation of a single or a subset of proteins. Degradation of a protein by the ubiquitin system involves two successive steps, conjugation of multiple moieties of ubiquitin and degradation of the tagged protein by the 26S proteasome. An important question concerns the identity of the mechanisms that underlie the high degree of specificity of the system. Substrate recognition is governed by a large family ubiquitin ligases that recognize the substrates, bind them and catalyze/facilitate their interaction with ubiquitin.

• Pickart CM
• Ubiquitin in chains.
• Trends Biochem Sci. 2000; 25: 544-8
• Display abstract

• The ubiquitin-proteasome system fulfills an essential function in eukaryotes by controlling the levels of crucial intracellular regulatory proteins. In this system, a specific type of polyubiquitin chain acts as the proximal signal for targeting substrates to 26S proteasomes for degradation. Recent results have revealed important determinants of polyubiquitin-chain recognition by proteasomes, helping to explain the biological rationale behind this novel signaling mechanism.

• Nonaka M
• Origin and evolution of the complement system.
• Curr Top Microbiol Immunol. 2000; 248: 37-50

• Amerik AY, Nowak J, Swaminathan S, Hochstrasser M
• The Doa4 deubiquitinating enzyme is functionally linked to the vacuolar protein-sorting and endocytic pathways.
• Mol Biol Cell. 2000; 11: 3365-80
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• The Saccharomyces cerevisiae DOA4 gene encodes a deubiquitinating enzyme that is required for rapid degradation of ubiquitin-proteasome pathway substrates. Both genetic and biochemical data suggest that Doa4 acts in this pathway by facilitating ubiquitin recycling from ubiquitinated intermediates targeted to the proteasome. Here we describe the isolation of 12 spontaneous extragenic suppressors of the doa4-1 mutation; these involve seven different genes, six of which were cloned. Surprisingly, all of the cloned DID (Doa4-independent degradation) genes encode components of the vacuolar protein-sorting (Vps) pathway. In particular, all are class E Vps factors, which function in the maturation of a late endosome/prevacuolar compartment into multivesicular bodies that then fuse with the vacuole. Four of the six Did proteins are structurally related, suggesting an overlap in function. In wild-type and several vps strains, Doa4-green fluorescent protein displays a cytoplasmic/nuclear distribution. However, in cells lacking the Vps4/Did6 ATPase, a large fraction of Doa4-green fluorescent protein, like several other Vps factors, concentrates at the late endosome-like class E compartment adjacent to the vacuole. These results suggest an unanticipated connection between protein deubiquitination and endomembrane protein trafficking in which Doa4 acts at the late endosome/prevacuolar compartment to recover ubiquitin from ubiquitinated membrane proteins en route to the vacuole.

• Sheaff RJ, Singer JD, Swanger J, Smitherman M, Roberts JM, Clurman BE
• Proteasomal turnover of p21Cip1 does not require p21Cip1 ubiquitination.
• Mol Cell. 2000; 5: 403-10
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• The Cdk inhibitor p21Cip1 is an unstable protein. Pharmacologic inhibition of the proteasome increases the half-life of p21 from less than 30 min to more than 2 hr and results in the accumulation of p21-ubiquitin conjugates. To determine whether ubiquitination was required for proteasomal degradation of p21, we constructed mutant versions of p21 that were not ubiquitinated in vivo. Remarkably, these mutants remained unstable and increased in abundance upon proteasome inhibition, indicating that direct ubiquitination of p21 is not necessary for its turnover by the proteasome. The frequently observed correlation between protein ubiquitination and proteasomal degradation is insufficient to conclude that ubiquitination is a prerequisite for degradation.

• Mossessova E, Lima CD
• Ulp1-SUMO crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast.
• Mol Cell. 2000; 5: 865-76
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• Modification of cellular proteins by the ubiquitin-like protein SUMO is essential for nuclear processes and cell cycle progression in yeast. The Ulp1 protease catalyzes two essential functions in the SUMO pathway: (1) processing of full-length SUMO to its mature form and (2) deconjugation of SUMO from targeted proteins. Selective reduction of the proteolytic reaction produced a covalent thiohemiacetal transition state complex between a Ulp1 C-terminal fragment and its cellular substrate Smt3, the yeast SUMO homolog. The Ulp1-Smt3 crystal structure and functional testing of elements within the conserved interface elucidate determinants of SUMO recognition, processing, and deconjugation. Genetic analysis guided by the structure further reveals a regulatory element N-terminal to the proteolytic domain that is required for cell growth in yeast.

• Jentsch S, Pyrowolakis G
• Ubiquitin and its kin: how close are the family ties?
• Trends Cell Biol. 2000; 10: 335-42
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• Modification of proteins by the covalent attachment of ubiquitin is known to target them for degradation by proteasomes. Several proteins have been discovered recently that are related to ubiquitin or function similarly. Some of these proteins act as modifiers; others bear ubiquitin-like domains embedded in their polypeptide chain but do not form conjugates with cellular proteins. Ubiquitin-like proteins mediate an impressive range of cellular functions, including cell-cycle progression, DNA repair and apoptosis. Recent discoveries endorse the view that, in many cases, the function of the relatives of ubiquitin is linked to the ubiquitin pathway.

• Yeh ET, Gong L, Kamitani T
• Ubiquitin-like proteins: new wines in new bottles.
• Gene. 2000; 248: 1-14
• Display abstract

• Ubiquitin is a small polypeptide that covalently modifies other cellular proteins and targets them to the proteasome for degradation. In recent years, ubiquitin-dependent proteolysis has been demonstrated to play a critical role in the regulation of many cellular processes, such as cell cycle progression, cell signaling, and immune recognition. The recent discovery of three new ubiquitin-like proteins, NEDD8, Sentrin/SUMO, and Apg12, has further broadened the horizon of this type of post-translational protein modification. This review will focus on the biology and biochemistry of the Sentrin/SUMO and NEDD8 modification pathways, which are clearly distinct from the ubiquitination pathway and have unique biological functions.

• Gereben B, Goncalves C, Harney JW, Larsen PR, Bianco AC
• Selective proteolysis of human type 2 deiodinase: a novel ubiquitin-proteasomal mediated mechanism for regulation of hormone activation.
• Mol Endocrinol. 2000; 14: 1697-708
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• We investigated the mechanism by which T4 regulates its activation to T3 by the type 2 iodothyronine deiodinase (D2). D2 is a short- lived (t1/2 50 min), 31-kDa endoplasmic reticulum (ER) integral membrane selenoenzyme that generates intracellular T3. Inhibition of the ubiquitin (Ub) activating enzyme, E1, or MG132, a proteasome blocker, inhibits both the basal and substrate-induced acceleration of D2 degradation. Using a catalytically active transiently expressed FLAG-tagged-NH2-D2, we found rapid synthesis of high molecular mass (100-300 kDa) Ub-D2 conjugates that are catalytically inactive. Ub-D2 increases when cells are exposed to D2 substrate or MG132 and disappears rapidly after E1 inactivation. Fusion of FLAG epitope to the COOH terminus of D2 prolongs its half-life approximately 2.5-fold and increases the levels of active and, especially, Ub-D2. This indicates that COOH-terminal modification interferes with proteasomal uptake of Ub-D2 that can then be deubiquitinated. Interestingly, the type 1 deiodinase, a related selenoenzyme that also converts T4 to T3 but with a half-life of >12 h, is inactivated but not ubiquitinated or degraded after exposure to substrate. Thus, ubiquitination of the ER-resident enzyme D2 constitutes a specific posttranslational mechanism for T4 regulation of its own activation in the central nervous system and pituitary tissues in which D2-catalyzed T4 to T3 conversion is the major source of intracellular T3.

• Swanson R, Hochstrasser M
• A viable ubiquitin-activating enzyme mutant for evaluating ubiquitin system function in Saccharomyces cerevisiae.
• FEBS Lett. 2000; 477: 193-8
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• Ligation of proteins to ubiquitin requires activation of ubiquitin by E1, the ubiquitin-activating enzyme. Mutant alleles of E1 in mammalian cells have been crucial for dissecting the contribution of the ubiquitin system to cell function. Comparable mutants have been unavailable for Saccharomyces cerevisiae. Here we describe the isolation and characterization of a hypomorphic allele of S. cerevisiae E1. Protein modification by ubiquitin is strongly impaired in the mutant, inhibiting degradation of ubiquitin-proteasome pathway substrates as well as ubiquitin-dependent but proteasome-independent degradation of membrane receptors. This allele will be a useful tool for evaluating the ubiquitin-dependence of cellular processes in yeast, even those in which the proteasome is not involved.

• Kleijnen MF et al.
• The hPLIC proteins may provide a link between the ubiquitination machinery and the proteasome.
• Mol Cell. 2000; 6: 409-19
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• Although there is a binding site on the proteasome for the polyubiquitin chains attached to degradation substrates by the ubiquitination machinery, it is currently unclear whether in vivo the activities of the ubiquitination machinery and the proteasome are coupled. Here we show that two human homologs of the yeast ubiquitin-like Dsk2 protein, hPLIC-1 and hPLIC-2, physically associate with both proteasomes and ubiquitin ligases in large complexes. Overexpression of hPLIC proteins interferes with the in vivo degradation of two unrelated ubiquitin-dependent proteasome substrates, p53 and IkappaBalpha, but not a ubiquitin-independent substrate. Our findings raise the possibility that the hPLIC proteins, and possibly related ubiquitin-like family members, may functionally link the ubiquitination machinery to the proteasome to affect in vivo protein degradation.

• Mayer RJ
• The meteoric rise of regulated intracellular proteolysis.
• Nat Rev Mol Cell Biol. 2000; 1: 145-8
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• It is often the case in biology that research into breaking things down lags behind research into synthesizing them, and this is certainly true for intracellular proteolysis. Now that we recognize that intracellular proteolysis, triggered by attaching multiple copies of a small protein called ubiquitin to target proteins, is fundamental to life, it is hard to believe that 20 years ago this field was little more than a backwater of biochemistry studied by a handful of laboratories. Among the few were Avram Hershko, Aaron Ciechanover and Alexander Varshavsky, who were recently awarded the Albert Lasker award for basic medical research for discovering the importance of protein degradation in cellular physiology. This Timeline traces how they and their collaborators triggered the rapid movement of ubiquitin-mediated proteolysis to centre stage.

• Hochstrasser M
• Evolution and function of ubiquitin-like protein-conjugation systems.
• Nat Cell Biol. 2000; 2: 1537-1537
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• Ubiquitin functions by covalently modifying other proteins. In the past few years, a surprising number of other proteins have been identified that, despite often being only slightly similar to ubiquitin, can also be attached to proteins. Newly discovered parallels between the activation of ubiquitin and the biosynthesis of certain enzyme cofactors now hint at the possible evolutionary origins of the ubiquitin system.

• Wilkinson KD
• Ubiquitination and deubiquitination: targeting of proteins for degradation by the proteasome.
• Semin Cell Dev Biol. 2000; 11: 141-8
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• The post-translational modification of proteins by covalent attachment of ubiquitin targets these proteins for degradation by the proteasome. An astounding number of proteins are involved in ubiquitination and deubiquitination of proteins. The pathways are combinatorial, and selectivity of proteolysis will depend strongly on the exact combination of ubiquitinating and deubiquitinating enzymes present at any time. In addition to temporal control, it is likely that these modifications are also regulated spatially. In this review, we discuss the regulation of ubiquitination by enzymes of this pathway and highlight some of the outstanding problems in understanding this regulation.

• Varshavsky A, Turner G, Du F, Xie Y
• Felix Hoppe-Seyler Lecture 2000. The ubiquitin system and the N-end rule pathway.
• Biol Chem. 2000; 381: 779-89
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• Eukaryotes contain a highly conserved multienzyme system which covalently links a small protein, ubiquitin, to a variety of intracellular proteins that bear degradation signals recognized by this system. The resulting ubiquitin-protein conjugates are degraded by the 26S proteasome, an ATP-dependent protease. Pathways that involve ubiquitin play major roles in a huge variety of processes, including cell differentiation, cell cycle, and responses to stress. In this article we briefly review the design of the ubiquitin system, and describe two recent advances, the finding that ubiquitin ligases interact with specific components of the 26S proteasome, and the demonstration that peptides accelerate their uptake into cells by activating the N-end rule pathway, one of several proteolytic pathways of the ubiquitin system.

• Nakayama K, Nakayama K
• [Ubiquitin-dependent proteolysis by SCF complex]
• Seikagaku. 2000; 72: 113-7

• Luders J, Demand J, Hohfeld J
• The ubiquitin-related BAG-1 provides a link between the molecular chaperones Hsc70/Hsp70 and the proteasome.
• J Biol Chem. 2000; 275: 4613-7
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• The BAG-1 protein modulates the chaperone activity of Hsc70 and Hsp70 in the mammalian cytosol and nucleus. Remarkably, BAG-1 possesses a ubiquitin-like domain at its amino terminus, suggesting a link to the ubiquitin/proteasome system. Here we show that BAG-1 is indeed associated with the 26 S proteasome in HeLa cells. Binding of the chaperone cofactor to the proteolytic complex is regulated by ATP hydrolysis and is not mediated by Hsc70 and Hsp70. The presented findings reveal a role of BAG-1 as a physical link between the Hsc70/Hsp70 chaperone system and the proteasome. In fact, targeting of BAG-1 to the proteasome promotes an association of the chaperones with the proteolytic complex in vitro and in vivo. A regulatory function of the chaperone cofactor at the interface between protein folding and protein degradation is thus indicated.

• Kornitzer D, Ciechanover A
• Modes of regulation of ubiquitin-mediated protein degradation.
• J Cell Physiol. 2000; 182: 1-11
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• The ubiquitin-proteasome pathway is responsible for the major portion of specific cellular protein degradation. Ubiquitin-mediated degradation is involved in physiological regulation of many cellular processes, including cell cycle progression, differentiation, and signal transduction. Here, we review the basic mechanisms of the ubiquitin system and the various ways in which ubiquitin-mediated degradation can be modulated by physiological signals.

• Hochstrasser M
• Biochemistry. All in the ubiquitin family.
• Science. 2000; 289: 563-4

• Minty A, Dumont X, Kaghad M, Caput D
• Covalent modification of p73alpha by SUMO-1. Two-hybrid screening with p73 identifies novel SUMO-1-interacting proteins and a SUMO-1 interaction motif.
• J Biol Chem. 2000; 275: 36316-23
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• Two-hybrid screening in yeast with p73alpha isolated SUMO-1 (small ubiquitin-like modifier 1), the enzyme responsible for its conjugation, Ubc-9, and a number of novel SUMO-1-interacting proteins, including thymine DNA glycosylase, PM-Scl75, PIASx, PKY, and CHD3/ZFH. A subset of these proteins contain a common motif, hhXSXS/Taaa, where h is a hydrophobic amino acid and a is an acidic amino acid, that is shown to interact with SUMO-1 in the two-hybrid system. We show here that p73alpha, but not p73beta, can be covalently modified by SUMO-1. The major SUMO-1-modified residue in p73alpha is the C-terminal lysine (Lys(627)). The sequence surrounding this lysine conforms to a consensus SUMO-1 modification site b(X)XXhKXE, where b is a basic amino acid. SUMO-1-modified p73 is more rapidly degraded by the proteasome than unmodified p73, although SUMO-1 modification is not required for p73 degradation. SUMO-1 modification does not affect the transcriptional activity of p73alpha on an RGC-luciferase reporter gene in SK-N-AS cells. Instead, SUMO-1 modification may alter the subcellular localization of p73, because SUMO-1-modified p73 is preferentially found in detergent-insoluble fractions. Alternatively, it may modulate the interaction of p73 with other proteins that are substrates for SUMO-1 modification or which interact with SUMO-1, such as those identified here.

• Melchior F
• SUMO--nonclassical ubiquitin.
• Annu Rev Cell Dev Biol. 2000; 16: 591-626
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• SUMO (small ubiquitin-related modifier) is the best-characterized member of a growing family of ubiquitin-related proteins. It resembles ubiquitin in its structure, its ability to be ligated to other proteins, as well as in the mechanism of ligation. However, in contrast to ubiquitination-often the first step on a one-way road to protein degradation-SUMOlation does not seem to mark proteins for degradation. In fact, SUMO may even function as an antagonist of ubiquitin in the degradation of selected proteins. While most SUMO targets are still at large, available data provide compelling evidence for a role of SUMO in the regulation of protein-protein interactions and/or subcellular localization.

• Swaminathan S, Amerik AY, Hochstrasser M
• The Doa4 deubiquitinating enzyme is required for ubiquitin homeostasis in yeast.
• Mol Biol Cell. 1999; 10: 2583-94
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• Attachment of ubiquitin to cellular proteins frequently targets them to the 26S proteasome for degradation. In addition, ubiquitination of cell surface proteins stimulates their endocytosis and eventual degradation in the vacuole or lysosome. In the yeast Saccharomyces cerevisiae, ubiquitin is a long-lived protein, so it must be efficiently recycled from the proteolytic intermediates to which it becomes linked. We identified previously a yeast deubiquitinating enzyme, Doa4, that plays a central role in ubiquitin-dependent proteolysis by the proteasome. Biochemical and genetic data suggest that Doa4 action is closely linked to that of the proteasome. Here we provide evidence that Doa4 is required for recycling ubiquitin from ubiquitinated substrates targeted to the proteasome and, surprisingly, to the vacuole as well. In the doa4Delta mutant, ubiquitin is strongly depleted under certain conditions, most notably as cells approach stationary phase. Ubiquitin depletion precedes a striking loss of cell viability in stationary phase doa4Delta cells. This loss of viability and several other defects of doa4Delta cells are rescued by provision of additional ubiquitin. Ubiquitin becomes depleted in the mutant because it is degraded much more rapidly than in wild-type cells. Aberrant ubiquitin degradation can be partially suppressed by mutation of the proteasome or by inactivation of vacuolar proteolysis or endocytosis. We propose that Doa4 helps recycle ubiquitin from both proteasome-bound ubiquitinated intermediates and membrane proteins destined for destruction in the vacuole.

• Kim E et al.
• Interaction between RGS7 and polycystin.
• Proc Natl Acad Sci U S A. 1999; 96: 6371-6
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• Regulators of G protein signaling (RGS) proteins accelerate the intrinsic GTPase activity of certain Galpha subunits and thereby modulate a number of G protein-dependent signaling cascades. Currently, little is known about the regulation of RGS proteins themselves. We identified a short-lived RGS protein, RGS7, that is rapidly degraded through the proteasome pathway. The degradation of RGS7 is inhibited by interaction with a C-terminal domain of polycystin, the protein encoded by PKD1, a gene involved in autosomal-dominant polycystic kidney disease. Furthermore, membranous expression of C-terminal polycystin relocalized RGS7. Our results indicate that rapid degradation and interaction with integral membrane proteins are potential means of regulating RGS proteins.

• Vierstra RD, Callis J
• Polypeptide tags, ubiquitous modifiers for plant protein regulation.
• Plant Mol Biol. 1999; 41: 435-42
• Display abstract

• Evidence has emerged over the past few years that plants, like animals and fungi, employ a variety of polypeptides as tags to reversibly or irreversibly affect the function, structure, location, and/or turnover of numerous intracellular proteins. In plants, known polypeptide tags include ubiquitin, SUMO, RUB, and APG12, with the possibility of others. These modifiers are typically added post-translationally using individual sets of conjugase pathways that attach the polypeptides via an isopeptide bond to epsilon-lysyl amino group(s) in the targets. Often the tags can be removed subsequently by unique proteases that specifically cleave only the isopeptide bond. Examples also exist where the tag is added during translation upon fusion of the coding sequence of the tag with that of the target. Based on the number and diversity of targets, ubiquitin is the most influential modifier which mainly serves as a reusable signal for selective protein degradation by the 26S proteasome. In contrast, SUMO, RUB and APG12 become attached to a more limited number of targets and appear to have specialized functions, including roles in nuclear pore assembly/function, cell-cycle regulation, and lysosomal/vacuole trafficking, respectively. Based on their widespread occurrence in plants and their pervasive role in various biological processes, polypeptide tags likely play a prominent role in plant cell regulation.

• Hochstrasser M et al.
• The Saccharomyces cerevisiae ubiquitin-proteasome system.
• Philos Trans R Soc Lond B Biol Sci. 1999; 354: 1513-22
• Display abstract

• Our studies of the yeast ubiquitin-proteasome pathway have uncovered a number of general principles that govern substrate selectivity and proteolysis in this complex system. Much of the work has focused on the destruction of a yeast transcription factor, MAT alpha 2. The alpha 2 protein is polyubiquitinated and rapidly degraded in alpha-haploid cells. One pathway of proteolytic targeting, which depends on two distinct endoplasmic reticulum-localized ubiquitin-conjugating enzymes, recognizes the hydrophobic face of an amphipathic helix in alpha 2. Interestingly, degradation of alpha 2 is blocked in a/alpha-diploid cells by heterodimer formation between the alpha 2 and a1 homeodomain proteins. The data suggest that degradation signals may overlap protein-protein interaction surfaces, allowing a straightforward steric mechanism for regulated degradation. Analysis of alpha 2 degradation led to the identification of both 20S and 26S proteasome subunits, and several key features of proteasome assembly and active-site formation were subsequently uncovered. Finally, it has become clear that protein (poly) ubiquitination is highly dynamic in vivo, and our studies of yeast de-ubiquitinating enzymes illustrate how such enzymes can facilitate the proteolysis of diverse substrates.

• Gabdoulline RR, Wade RC, Walther D
• MolSurfer: two-dimensional maps for navigating three-dimensional structures of proteins.
• Trends Biochem Sci. 1999; 24: 285-7

• Laney JD, Hochstrasser M
• Substrate targeting in the ubiquitin system.
• Cell. 1999; 97: 427-30

• Smith TF
• The art of matchmaking: sequence alignment methods and their structural implications.
• Structure Fold Des. 1999; 7: 712-712

• Chung CH, Baek SH
• Deubiquitinating enzymes: their diversity and emerging roles.
• Biochem Biophys Res Commun. 1999; 266: 633-40
• Display abstract

• A growing number of important regulatory proteins within cells are modified by conjugation of ubiquitin, a well-conserved 76-amino-acid polypeptide. The ubiquitinated proteins are targeted to proteasome for degradation or alternative metabolic fates, such as triggering of plasma membrane endocytosis and trafficking to vacuoles or lysosomes. Deubiquitination, reversal of this modification, is being recognized as an important regulatory step. Deubiquitinating enzymes are cysteine proteases that specifically cleave off ubiquitin from ubiquitin-conjugated protein substrates as well as from its precursor proteins. Genome sequencing projects have identified more than 90 deubiquitinating enzymes, making them the largest family of enzymes in the ubiquitin system. This review will concentrate on recent important findings as well as new insights into the diversity and emerging roles of deubiquitinating enzymes in the ubiquitin-dependent pathway.

• Bounpheng MA, Dimas JJ, Dodds SG, Christy BA
• Degradation of Id proteins by the ubiquitin-proteasome pathway.
• FASEB J. 1999; 13: 2257-64
• Display abstract

• Id proteins act as negative regulators of bHLH transcription factors by forming transcriptionally inactive protein complexes. The proposed function of these proteins includes promotion of cell growth and cell cycle progression, induction of apoptosis, and inhibition of cellular differentiation. We investigated the role of the ubiquitin-mediated proteolytic pathway in the degradation of the Id3 protein. We found Id3 to be a short-lived protein and estimated the half-life to be approximately 20 min in 293 cells. Using specific inhibitors of the 26S proteasome and mutant fibroblast cells with a temperature-sensitive defect in the essential E1 ubiquitin-activating enzyme, we show that Id3 and the related Id1 and Id2 proteins are degraded through the ubiquitin-proteasome pathway. We found the Id4 protein to be much less sensitive to inhibitors of the 26S proteasome, but its degradation was dependent on the E1 enzyme. In addition, we observed that coexpression of the bHLH protein E47 with Id3 significantly reduced the rate of degradation of Id3, suggesting that Id3 is less susceptible to degradation by the 26S proteasome when complexed to a bHLH protein. -Bounpheng, M. A., Dimas, J. J., Dodds, S. G., Christy, B. A. Degradation of Id proteins by the ubiquitin-proteasome pathway.

• Yamao F
• Ubiquitin system: selectivity and timing of protein destruction.
• J Biochem (Tokyo). 1999; 125: 223-9
• Display abstract

• A growing number of cellular functions have been shown to be regulated through protein degradation. The selective degradation of many short-lived proteins in eukaryotic cells is mediated by the ubiquitin system, by which proteins covalently ligated to ubiquitin are targeted for degradation. The selectivity of the destruction is ensured by the substrate specificity in the ubiquitination steps composed of a series of enzymatic reactions. Ubiquitin-ligase (E3), in conjunction with ubiquitin-conjugating enzyme (E2), has been implicated as playing an essential role in the substrate recognition. The substantial character, however, of the ligase was not clear until several recent studies demonstrated ligases that exert key roles in irreversible steps of the cell-cycle control. In this review, attention is focused on the molecular basis of target recognition of ubiquitination, particularly as exemplified in the ubiquitin-ligases in the cell-cycle control mechanisms.

• Wilkinson KD
• Ubiquitin-dependent signaling: the role of ubiquitination in the response of cells to their environment.
• J Nutr. 1999; 129: 1933-6
• Display abstract

• The response of a cell to its external environment requires rapid and significant alteration of protein amount, localization and/or function. This regulation involves a complex combination of processes that control synthesis, localization and degradation. All of these processes must be properly regulated and are often interrelated. Intracellular proteolysis is largely accomplished by the ubiquitin-dependent system and has been shown to be required for growth control, cell cycle regulation, receptor function, development and the stress response. Substrates subject to regulated degradation by this system include cyclins and cyclin-dependent kinase inhibitors, tumor suppressors, transcription factors and cell surface receptors. In addition, proteins that are damaged by oxidation or that are improperly folded or localized are substrates whose degradation by this system often leads to antigen presentation on the surface of the cell in the context of Class I major histocompatibility complex molecules. A very large body of work in the last fifteen years has shown that degradation by this system requires the covalent attachment of a small protein called ubiquitin and that this modification serves to direct target proteins for degradation by a 26S proteolytic particle, the proteasome. Thus, the attachment of the ubiquitin domain is of vital importance in regulating normal growth and differentiation, as well as in defending against cellular damage caused by xenobiotics, environmental insults, infection and mutation. This review focuses on the role of ubiquitination in the cellular signaling pathways that deal with these external influences.

• Mimnaugh EG, Bonvini P, Neckers L
• The measurement of ubiquitin and ubiquitinated proteins.
• Electrophoresis. 1999; 20: 418-28
• Display abstract

• Ubiquitination of key cellular proteins involved in signal transduction, gene transcription and cell-cycle regulation usually condemns those proteins to proteasomal or lysosomal degradation. Additionally, cycles of reversible ubiquitination regulate the function of certain proteins in a manner analogous to phosphorylation. In this short review we describe the current methodology for measuring ubiquitin and ubiquitination, provide examples which illustrate how various techniques have been used to study protein ubiquination, alert the readers of pitfalls to avoid, and offer guidelines to investigators newly interested in this novel post-translational protein modification.

• Okuma T, Honda R, Ichikawa G, Tsumagari N, Yasuda H
• In vitro SUMO-1 modification requires two enzymatic steps, E1 and E2.
• Biochem Biophys Res Commun. 1999; 254: 693-8
• Display abstract

• The SUMO-1 has been identified as a protein that is highly similar to ubiquitin and shown to conjugate to RanGAP1, PML, Sp200 and I kappa B alpha. The conjugation steps are thought to be similar to those of ubiquitination; and human Ubc9, which is homologous to the E2 enzyme for the ubiquitin conjugation step, was identified and shown to be necessary for the conjugation of SUMO-1 to its target protein. Other essential enzymes involved in this modification, however, remain to be clarified. Here we cloned human Sua1 (SUMO-1 activating enzyme) and hUba2, which are human homologs of yeast Saccharomyces cerevisiae Aos1 and Uba2, respectively. The recombinant proteins, Sua1p and hUba2p, formed a complex. In this complex, hUba2 bound SUMO-1 and this complex had the activity of the SUMO-1 activating enzyme. Furthermore, in an in vitro system, RanGAP1 was modified by SUMO-1 in the presence of Sua1p/Uba2p and hUbc9p, showing that the modification of SUMO-1 could be catalyzed by two enzyme steps, although ubiquitination usually requires three enzyme steps.

• Deshaies RJ
• SCF and Cullin/Ring H2-based ubiquitin ligases.
• Annu Rev Cell Dev Biol. 1999; 15: 435-67
• Display abstract

• Protein degradation is deployed to modulate the steady-state abundance of proteins and to switch cellular regulatory circuits from one state to another by abrupt elimination of control proteins. In eukaryotes, the bulk of the protein degradation that occurs in the cytoplasm and nucleus is carried out by the 26S proteasome. In turn, most proteins are thought to be targeted to the 26S proteasome by covalent attachment of a multiubiquitin chain. Ubiquitination of proteins requires a multienzyme system. A key component of ubiquitination pathways, the ubiquitin ligase, controls both the specificity and timing of substrate ubiquitination. This review is focused on a conserved ubiquitin ligase complex known as SCF that plays a key role in marking a variety of regulatory proteins for destruction by the 26S proteasome.

• Wyndham AM, Baker RT, Chelvanayagam G
• The Ubp6 family of deubiquitinating enzymes contains a ubiquitin-like domain: SUb.
• Protein Sci. 1999; 8: 1268-75
• Display abstract

• A sequence motif that is Similar to Ubiquitin (SUb) has been identified in the Saccharomyces cerevisiae ubiquitin-specific protease Ubp6. SUb is conserved in all known Ubp6 homologues from a spectrum of eukaryotic species and is also present in a group of hypothetical proteins of unknown function (Unk1-3) present in sequence databases. An N-terminal deletion mutant of Ubp6 that lacks SUb is still capable of cleaving alpha-linked ubiquitin fusions, suggesting that SUb forms a separate domain to the catalytic core of Ubp6 and demonstrating that it is not required for in vitro cleavage activity. A homology model of the 78 N-terminal amino acids of human Ubp6, based on the known fold of ubiquitin, is presented. In human Ubp6, SUb shares only 20% sequence identity with ubiquitin. Even weaker similarity occurs between S. cerevisiae SUb and ubiquitin. The homology model supports a ubiquitin-like fold for SUb and suggests that two conserved Lys residues, corresponding to Lys48 and Lys63 of ubiquitin, are functionally important.

• Tanaka K
• [The ubiquitin-ligating system: from the discovery to the present]
• Tanpakushitsu Kakusan Koso. 1999; 44: 737-43

• Papa FR, Amerik AY, Hochstrasser M
• Interaction of the Doa4 deubiquitinating enzyme with the yeast 26S proteasome.
• Mol Biol Cell. 1999; 10: 741-56
• Display abstract

• e Saccharomyces cerevisiae Doa4 deubiquitinating enzyme is required for the rapid degradation of protein substrates of the ubiquitin-proteasome pathway. Previous work suggested that Doa4 functions late in the pathway, possibly by deubiquitinating (poly)-ubiquitin-substrate intermediates associated with the 26S proteasome. We now provide evidence for physical and functional interaction between Doa4 and the proteasome. Genetic interaction is indicated by the mutual enhancement of defects associated with a deletion of DOA4 or a proteasome mutation when the two mutations are combined. Physical association of Doa4 and the proteasome was investigated with a new yeast 26S proteasome purification procedure, by which we find that a sizeable fraction of Doa4 copurifies with the protease. Another yeast deubiquitinating enzyme, Ubp5, which is related in sequence to Doa4 but cannot substitute for it even when overproduced, does not associate with the proteasome. DOA4-UBP5 chimeras were made by a novel PCR/yeast recombination method and used to identify an N-terminal 310-residue domain of Doa4 that, when appended to the catalytic domain of Ubp5, conferred Doa4 function, consistent with Ubp enzymes having a modular architecture. Unlike Ubp5, a functional Doa4-Ubp5 chimera associates with the proteasome, suggesting that proteasome binding is important for Doa4 function. Together, these data support a model in which Doa4 promotes proteolysis through removal of ubiquitin from proteolytic intermediates on the proteasome before or after initiation of substrate breakdown.

• Bonifacino JS, Weissman AM
• Ubiquitin and the control of protein fate in the secretory and endocytic pathways.
• Annu Rev Cell Dev Biol. 1998; 14: 19-57
• Display abstract

• The modification of proteins by chains of ubiquitin has long been known to mediate targeting of cytosolic and nuclear proteins for degradation by proteasomes. In this article, we discuss recent developments that reveal the involvement of ubiquitin in the degradation of proteins retained within the endoplasmic reticulum (ER) and in the internalization of plasma membrane proteins. Both luminal and transmembrane proteins retained in the ER are now known to be retrotranslocated into the cytosol in a process that involves ER chaperones and components of the protein import machinery. Once exposed to the cytosolic milieu, retro-translocated proteins are degraded by the proteasome, in most cases following polyubiquitination. There is growing evidence that both the ubiquitin-conjugating machinery and proteasomes may be associated with the cytosolic face of the ER membrane and that they could be functionally coupled to the process of retrotranslocation. The ubiquitination of plasma membrane proteins, on the other hand, mediates internalization of the proteins, which in most cases is followed by lysosomal/vacuolar degradation. There is, however, a well-documented case of a plasma membrane protein (the c-Met receptor) for which ubiquitination results in proteasomal degradation. These recent findings imply that ubiquitin plays more diverse roles in the regulation of the fate of cellular proteins than originally anticipated.

• Tanaka K, Suzuki T, Chiba T
• The ligation systems for ubiquitin and ubiquitin-like proteins.
• Mol Cells. 1998; 8: 503-12
• Display abstract

• Ubiquitin (Ub) is a highly conserved small protein present universally in eukaryotic cells, which is covalently attached to substrate proteins by a cascade system, consisting of activating (E1), conjugating (E2), and/or ligating (E3) enzymes. The modification of cellular proteins with Ub targets them for degradation by a large multisubunit protease, called the 26S proteasome. The unexpected existence of many genes encoding E2 and E3 reveals that a number of distinct Ub-ligating pathways operate for selective proteolysis in cells, implying its involvement in divergent biologically important processes. Currently, it becomes clear that a set of novel molecules with a structural similarity to Ub, called Ub-like proteins (Ubls), is present in various eukaryotic cells. They are divided into two subclasses: type-1 Ubls with small sizes, such as SUMO1 and NEDD8, that are ligated to target proteins in a fashion similar, but not identical, to the ubiquitination pathway, and another type-2 Ubls that contain Ub-like structure in a variety of different classes of large proteins having apparently distinct functions, such as Rad23, Elongin B, and Parkin. Ub and type-1 Ubls are central players consisting of a new type of post-translational protein-modifying system, although the significance of type-2 Ubl remains obscure.

• Hershko A, Ciechanover A
• The ubiquitin system.
• Annu Rev Biochem. 1998; 67: 425-79
• Display abstract

• The selective degradation of many short-lived proteins in eukaryotic cells is carried out by the ubiquitin system. In this pathway, proteins are targeted for degradation by covalent ligation to ubiquitin, a highly conserved small protein. Ubiquitin-mediated degradation of regulatory proteins plays important roles in the control of numerous processes, including cell-cycle progression, signal transduction, transcriptional regulation, receptor down-regulation, and endocytosis. The ubiquitin system has been implicated in the immune response, development, and programmed cell death. Abnormalities in ubiquitin-mediated processes have been shown to cause pathological conditions, including malignant transformation. In this review we discuss recent information on functions and mechanisms of the ubiquitin system. Since the selectivity of protein degradation is determined mainly at the stage of ligation to ubiquitin, special attention is focused on what we know, and would like to know, about the mode of action of ubiquitin-protein ligation systems and about signals in proteins recognized by these systems.

• Fomenkova NP, Nevskaia NA, Nikulin AD, Nikonov SV
• [Structural aspects of protein thermostability]
• Mol Biol (Mosk). 1998; 32: 301-8

• Mayer RJ, Landon M, Layfield R
• Ubiquitin superfolds: intrinsic and attachable regulators of cellular activities?
• Fold Des. 1998; 3: 979-979
• Display abstract

• Ubiquitinylation, the post-translational covalent conjugation of ubiquitin to other proteins, mediates diverse cellular processes in addition to the proteasome-catalysed degradation signalled by multiple ubiquitinylation. Ubiquitin superfolds have also been found in other proteins. The amino acid sequences of these superfolds are unrelated to ubiquitin, but they have an almost identical three-dimensional shape to that of ubiquitin. Additionally, a number of 'ubiquitin-like' proteins, some of which can be conjugated to other proteins, may also contain the ubiquitin superfold. Intrinsic and attachable ubiquitin superfolds can act as powerful ligands and probably have important roles in protein-protein interactions in the cell.

• Liakopoulos D, Doenges G, Matuschewski K, Jentsch S
• A novel protein modification pathway related to the ubiquitin system.
• EMBO J. 1998; 17: 2208-14
• Display abstract

• Ubiquitin conjugation is known to target protein substrates primarily to degradation by the proteasome or via the endocytic route. Here we describe a novel protein modification pathway in yeast which mediates the conjugation of RUB1, a ubiquitin-like protein displaying 53% amino acid identity to ubiquitin. We show that RUB1 conjugation requires at least three proteins in vivo. ULA1 and UBA3 are related to the N- and C-terminal domains of the E1 ubiquitin-activating enzyme, respectively, and together fulfil E1-like functions for RUB1 activation. RUB1 conjugation also requires UBC12, a protein related to E2 ubiquitin-conjugating enzymes, which functions analogously to E2 enzymes in RUB1-protein conjugate formation. Conjugation of RUB1 is not essential for normal cell growth and appears to be selective for a small set of substrates. Remarkably, CDC53/cullin, a common subunit of the multifunctional SCF ubiquitin ligase, was found to be a major substrate for RUB1 conjugation. This suggests that the RUB1 conjugation pathway is functionally affiliated to the ubiquitin-proteasome system and may play a regulatory role.

• Hochstrasser M
• There's the rub: a novel ubiquitin-like modification linked to cell cycle regulation.
• Genes Dev. 1998; 12: 901-7

• Johnson PR, Swanson R, Rakhilina L, Hochstrasser M
• Degradation signal masking by heterodimerization of MATalpha2 and MATa1 blocks their mutual destruction by the ubiquitin-proteasome pathway.
• Cell. 1998; 94: 217-27
• Display abstract

• Proteolysis by the ubiquitin-proteasome pathway is often regulated, but the mechanisms underlying such regulation remain ill-defined. In Saccharomyces cerevisiae, cell type is controlled by the MAT transcription factors. The alpha2 repressor is a known ubiquitin pathway substrate in alpha haploid cells. We show that a1 is rapidly degraded in a haploids. In a/alpha diploids, alpha2 and a1 are stabilized by heterodimerization. Association depends on N-terminal coiled-coil interactions between a1 and alpha2. Residues in alpha2 important for these interactions overlap a critical determinant of an alpha2 degradation signal, which we delimit by extensive mutagenesis. Our data provide a detailed description of a natural ubiquitin-dependent degradation signal and point to a molecular mechanism for regulated turnover in which proteolytic signals are differentially masked in alternative multiprotein complexes.

• Ciechanover A
• The ubiquitin-proteasome pathway: on protein death and cell life.
• EMBO J. 1998; 17: 7151-60

• Lustig AJ
• Mechanisms of silencing in Saccharomyces cerevisiae.
• Curr Opin Genet Dev. 1998; 8: 233-9
• Display abstract

• In the yeast Saccharomyces cerevisiae, heterochromatin-like regions are formed at the silent mating type loci and at telomeres. The past year of investigations has led to a clearer understanding of the nature of nucleation and spreading of heterochromatin, as well as uncovering a fascinating link between silencing, the nucleolus and aging.

• Bahrami AR, Gray JE
• Conservation of proteasome structure and activity between plants and other eukaryotes.
• Biochem Soc Trans. 1998; 26: 395-395

• Whitby FG, Xia G, Pickart CM, Hill CP
• Crystal structure of the human ubiquitin-like protein NEDD8 and interactions with ubiquitin pathway enzymes.
• J Biol Chem. 1998; 273: 34983-91
• Display abstract

• The NEDD8/Rub1 class of ubiquitin-like proteins has been implicated in progression of the cell cycle from G1 into S phase. These molecules undergo a metabolism that parallels that of ubiquitin and involves specific interactions with many different proteins. We report here the crystal structure of recombinant human NEDD8 refined at 1.6-A resolution to an R factor of 21.9%. As expected from the high sequence similarity (57% identical), the NEDD8 structure closely resembles that reported previously for ubiquitin. We also show that recombinant human NEDD8 protein is activated, albeit inefficiently, by the ubiquitin-activating (E1) enzyme and that NEDD8 can be transferred from E1 to the ubiquitin conjugating enzyme E2-25K. E2-25K adds NEDD8 to a polyubiquitin chain with an efficiency similar to that of ubiquitin. A chimeric tetramer composed of three ubiquitins and one histidine-tagged NEDD8 binds to the 26 S proteasome with an affinity similar to that of tetraubiquitin. Seven residues that differ from the corresponding residues in ubiquitin, but are conserved between NEDD8 orthologs, are candidates for mediating interactions with NEDD8-specific partners. One such residue, Ala-72 (Arg in ubiquitin), is shown to perform a key role in selecting against reaction with the ubiquitin E1 enzyme, thereby acting to prevent the inappropriate diversion of NEDD8 into ubiquitin-specific pathways.

• Bayer P et al.
• Structure determination of the small ubiquitin-related modifier SUMO-1.
• J Mol Biol. 1998; 280: 275-86
• Display abstract

• The recently discovered small ubiquitin-related modifier SUMO-1 belongs to the growing family of ubiquitin-related proteins involved in postranslational protein modification. Unlike ubiquitin, SUMO-1 does not appear to target proteins for degradation but seems to be involved in the modulation of protein-protein interactions. Independent studies demonstrate an essential function of SUMO-1 in the regulation of nucleo-cytoplasmic transport, and suggest a role in cell-cycle regulation and apoptosis. Here, we present the first three-dimensional structure of SUMO-1 solved by NMR. Although having only 18% amino acid sequence identity with ubiquitin, the overall structure closely resembles that of ubiquitin, featuring the betabetaalphabetabetaalphabeta fold of the ubiquitin protein family. In addition, the position of the two C-terminal Gly residues required for isopeptide bond formation is conserved between ubiquitin and SUMO-1. The most prominent feature of SUMO-1 is a long and highly flexible N terminus, which protrudes from the core of the protein and which is absent in ubiquitin. Furthermore, ubiquitin Lys48, required to generate ubiquitin polymers, is substituted in SUMO-1 by Gln69 at the same position, which provides an explanation of why SUMO-1 has not been observed to form polymers. Moreover, the hydrophobic core of SUMO-1 and ubiquitin is maintained by conserved hydrophobic residues, whereas the overall charge topology of SUMO-1 and ubiquitin differs significantly, suggesting specific modifying enzymes and target proteins for both proteins.

• Attaix D et al.
• Ubiquitin-proteasome-dependent proteolysis in skeletal muscle.
• Reprod Nutr Dev. 1998; 38: 153-65
• Display abstract

• The ubiquitin-proteasome proteolytic pathway has recently been reported to be of major importance in the breakdown of skeletal muscle proteins. The first step in this pathway is the covalent attachment of polyubiquitin chains to the targeted protein. Polyubiquitylated proteins are then recognized and degraded by the 26S proteasome complex. In this review, we critically analyse recent findings in the regulation of this pathway, both in animal models of muscle wasting and in some human diseases. The identification of regulatory steps of ubiquitin conjugation to protein substrates and/or of the proteolytic activities of the proteasome should lead to new concepts that can be used to manipulate muscle protein mass. Such concepts are essential for the development of anti-cachectic therapies for many clinical situations.

• Zhang Z et al.
• Identification of an activation region in the proteasome activator REGalpha.
• Proc Natl Acad Sci U S A. 1998; 95: 2807-11
• Display abstract

• Proteasomes can be markedly activated by associating with 19S regulatory complexes to form the 26S protease or by binding 11S protein complexes known as REG or PA28. Three REG subunits, alpha, beta, and gamma, have been expressed in Escherichia coli, and each recombinant protein can activate human proteasomes. Combining PCR mutagenesis with an in vitro activity assay, we have isolated and characterized 36 inactive, single-site mutants of recombinant REGalpha. Most are monomers that produce functional proteasome activators when mixed with REGbeta subunits. Five REGalpha mutants that remain inactive in the mixing assay contain amino acid substitutions clustered between Arg-141 and Gly-149. The crystal structure of the REGalpha heptamer shows that this region forms a loop at the base of each REGalpha subunit. One mutation in this loop (N146Y) yields a REGalpha heptamer that binds the proteasome as tightly as wild-type REGalpha but does not activate peptide hydrolysis. Corresponding amino acid substitutions in REGbeta (N135Y) and REGgamma (N151Y) produce inactive proteins that also bind the proteasome and inhibit proteasome activation by their normal counterparts. Our studies clearly demonstrate that REG binding to the proteasome can be separated from activation of the enzyme. Moreover, the dominant negative REGs identified here should prove valuable for elucidating the role(s) of these proteins in antigen presentation.

• Ciechanover A, Schwartz AL
• The ubiquitin-proteasome pathway: the complexity and myriad functions of proteins death.
• Proc Natl Acad Sci U S A. 1998; 95: 2727-30

• Pearce DA, Sherman F
• Differential ubiquitin-dependent degradation of the yeast apo-cytochrome c isozymes.
• J Biol Chem. 1997; 272: 31829-36
• Display abstract

• The yeast Saccharomyces cerevisiae contains two forms of cytochrome c, iso-1- and iso-2-cytochrome c, which are encoded by the nuclear genes CYC1 and CYC7, respectively. The cytochromes c are synthesized in the cytosol, imported into mitochondria, and subsequently modified by the covalent attachment of heme through the action of cytochrome c heme lyase, which is encoded by CYC3. Apo-iso-2-cytochrome c but not apo-iso-1-cytochrome c was observed in cyc3(-) mutants. Furthermore, pulse-chase experiments previously demonstrated that the lack of apo-iso-1-cytochrome c was due to its rapid degradation. We report herein that this degradation of apo-iso-1-cytochrome c is dependent on ubiquitination and on the action of the proteasome. Diminished degradation of apo-iso-1-cytochrome c was observed in pre2-2 and pre1-1 mutants having altered proteasome subunits; in ubc1, ubc4, and ubc5 strains lacking one or more of the ubiquitin-conjugating enzymes; and in strains blocked in multi-ubiquitination by overproduction of the abnormal ubiquitin-K48R ubiquitin. In addition, we have used epitope-tagged ubiquitin to demonstrate that apo-iso-1-cytochrome c but not apo-iso-2-cytochrome c is ubiquitinated. Furthermore, the degradation of apo-iso-1-cytochrome c was diminished when the N-terminal region was replaced with the N-terminal region of apo-iso-2-cytochrome c, indicating that this region may be the target for degradation. We suggest that ubiquitin-dependent degradation of apo-iso-1-cytochrome c is part of the regulatory process controlling the preferential expression of the iso-cytochromes c.

• Cruz M, Nandi D, Hendil KB, Monaco JJ
• Cloning and characterization of mouse Lmp3 cDNA, encoding a proteasome beta subunit.
• Gene. 1997; 190: 251-6
• Display abstract

• We isolated and sequenced a cDNA encoding mouse proteasome subunit LMP3 from a macrophage cDNA library. The gene encodes a 264-amino-acid protein with a calculated molecular mass of 29.11 kDa and an isoelectric point (pI) of 5.44. Comparison of the predicted protein sequence with that of the human and rat homologues, N3, revealed 11 and eight changes, respectively, in the cleaved NH2-terminal presequence of the precursor protein (pre-LMP3), and six and 10 changes, respectively, in the processed product. To corroborate the predicted molecular mass and pI, we analyzed LMP3 by immunoprecipitation with a mAb to human N3 that crossreacts with mouse LMP3. Precursor and processed forms of LMP3 were identified by 2D NEPHGE-PAGE, and their mobilities suggest the Lmp3 clone encodes the entire protein sequence.

• Lupas A et al.
• Eubacterial proteasomes.
• Mol Biol Rep. 1997; 24: 125-31
• Display abstract

• Proteasomes are large, multisubunit proteases with highly conserved structures. The 26S proteasome of eukaryotes is an ATP-dependent enzyme of about 2 MDa, which acts as the central protease of the ubiquitin-dependent pathway of protein degradation. The core of the 26S complex is formed by the 20S proteasome, an ATP-independent, barrel-shaped protease of about 700 kDa, which has also been detected in archaebacteria and, more recently, in eubacteria. Currently, the distribution of 20S proteasomes in eubacteria appears limited to the actinomycetes, while most other eubacteria contain a related complex of simpler structure.

• Gilchrist CA, Gray DA, Baker RT
• A ubiquitin-specific protease that efficiently cleaves the ubiquitin-proline bond.
• J Biol Chem. 1997; 272: 32280-5
• Display abstract

• Ubiquitin is a small eukaryotic protein that is synthesized naturally as one of several fusion proteins, which are processed by ubiquitin-specific proteases to release free ubiquitin. The expression of heterologous proteins as fusions to ubiquitin in either prokaryotic or eukaryotic hosts often dramatically enhances their yield, and allows the exposure of any amino acid following cleavage of ubiquitin. The single exception is when proline is the amino acid immediately following ubiquitin; the ubiquitin-proline bond is poorly cleaved by presently studied ubiquitin-specific proteases. We show that the mouse ubiquitin-specific protease Unp, and its human homolog Unph, can efficiently cleave the ubiquitin-proline bond in ubiquitin fusion proteins of different sizes. N-terminal sequencing of the cleavage products reveals that cleavage occurs precisely at the ubiquitin-proline junction. The biological significance of this cleavage activity is unclear, as ubiquitin-proline fusions do not occur naturally. However, it may indicate a different catalytic mechanism for these ubiquitin-specific proteases and/or that they can cleave ubiquitin-like proteins. Unp and Unph thus represent versatile ubiquitin-specific proteases for cleaving ubiquitin-fusion proteins in biotechnology and basic research, regardless of both the amino acid immediately following ubiquitin, and the size of the fusion partner.

• Tanaka K, Tanahashi N, Tsurumi C, Yokota KY, Shimbara N
• Proteasomes and antigen processing.
• Adv Immunol. 1997; 64: 1-38

• Wilkinson KD
• Regulation of ubiquitin-dependent processes by deubiquitinating enzymes.
• FASEB J. 1997; 11: 1245-56
• Display abstract

• An astounding number of important regulatory and structural proteins are subject to modification by the attachment of ubiquitin or ubiquitin-like proteins. This modification acts as a targeting signal, delivering the modified protein to different locations in the cell and modifying its activity, macromolecular interactions, or half-life. Deubiquitination, or the removal of this modification, is being recognized as an important regulatory strategy. This reaction is catalyzed by processing proteases known as deubiquitinating enzymes (DUBs). More than 60 DUBs are already known, although little is known about their biological roles. This review concentrates on recent findings and new insights into this fascinating class of enzymes.

• Kamitani T, Nguyen HP, Yeh ET
• Preferential modification of nuclear proteins by a novel ubiquitin-like molecule.
• J Biol Chem. 1997; 272: 14001-4
• Display abstract

• Sentrin is a novel ubiquitin-like protein that protects cells against both anti-Fas and tumor necrosis factor-induced cell death. Antiserum recognizing the N terminus of sentrin revealed the presence of a 18-kDa sentrin monomer, a 90-kDa band (p90), and multiple high molecular mass bands. Because sentrin possesses the conserved Gly-Gly residues near the C terminus, it is likely that these additional bands represent conjugation of sentrin to other proteins in a manner that is similar to the ubiquitination pathway. Transient expression of hemagglutinin epitope-tagged sentrin mutants in COS cells demonstrated that the sentrin C terminus is cleaved, which allows it to be conjugated to other proteins via the conserved C-terminal Gly residue. Immunocytochemical staining and cell fractionation analysis demonstrated that sentrin monomer is localized predominantly to the cytosol. However, p90 and the majority of sentrinized proteins appeared to be localized to the nucleus. When the conserved Gly-Gly residues of sentrin were changed to Gly-Ala, only sentrin monomer and p90 but not the high molecular mass bands were observed. Thus, p90 generation appears to be required for the formation of high molecular mass bands in the nucleus. Taken together, sentrinization represents a novel pathway for nuclear protein modification, which is distinct from ubiquitination.

• Kho CJ, Huggins GS, Endege WO, Hsieh CM, Lee ME, Haber E
• Degradation of E2A proteins through a ubiquitin-conjugating enzyme, UbcE2A.
• J Biol Chem. 1997; 272: 3845-51
• Display abstract

• The helix-loop-helix E2A proteins (E12 and E47) govern cellular growth and differentiation. To identify binding partners that regulate the function of these ubiquitous transcription factors, we screened for proteins that interacted with the C terminus of E12 by the yeast interaction trap. UbcE2A, a rat enzyme that is highly homologous to and functionally complements the yeast ubiquitin-conjugating enzyme UBC9, was identified and cloned. UbcE2A appears to be an E2A-selective ubiquitin-conjugating enzyme because it interacts specifically with a 54-amino acid region in E47-(477-530) distinct from the helix-loop-helix domain. In contrast, most of the UbcE2A protein is required for interaction with an E2A protein. The E2A proteins appear to be degraded by the ubiquitin-proteasome pathway because the E12 half-life of 60 min is extended by the proteasome inhibitor MG132, and E12 is multi-ubiquitinated in vivo. Finally, antisense UbcE2A reduces E12 degradation. By participating in the degradation of the E2A proteins, UbcE2A may regulate cell growth and differentiation.

• Mitch WE, Price SR
• Protein degradation by proteasomes: molecular mechanisms of muscle catabolism.
• Nephrol Dial Transplant. 1997; 12: 13-5

• Haracska L, Udvardy A
• Mapping the ubiquitin-binding domains in the p54 regulatory complex subunit of the Drosophila 26S protease.
• FEBS Lett. 1997; 412: 331-6
• Display abstract

• Short-lived intracellular proteins, after being marked by multiubiquitination, are degraded by the 26S protease. This large ATP-dependent protease is composed of two multiprotein complexes: the regulatory complex and the 20S proteosome. The selective recognition of ubiquitinated proteins is ensured by the regulatory complex. Using an overlay assay a single 54-kDa multiubiquitin-chain-binding subunit was detected in the regulatory complex of the Drosophila 26S protease. Overlay assay with the recombinant p54 subunit confirmed its ubiquitin-binding property. The recombinant protein showed pronounced preference for higher ubiquitin multimers, in agreement with the known preference of the 26S protease for multiubiquitinated proteins as substrates. To map the ubiquitin-binding domain of the p54 subunit different segments of the recombinant protein were expressed in E. coli and tested by the overlay assay. The p54 subunit carries two independent ubiquitin-binding domains. The central domain carries two highly conserved sequence blocks: the FGVDP sequence (at position 207), which is 100% conserved from yeast till human, and the DPELALALRVSMEE sequence (at position 214), which is 100% conserved in higher eukaryotes with two amino acid changes in yeast. In the C-terminal ubiquitin-binding domain the GVDP sequence motif is repeated and 100% conserved in higher eukaryotes. This domain, however, due to the shorter size of the yeast multiubiquitin-binding subunit, is present only in higher eukaryotes.

• Rolfe M, Chiu MI, Pagano M
• The ubiquitin-mediated proteolytic pathway as a therapeutic area.
• J Mol Med. 1997; 75: 5-17
• Display abstract

• Ubiquitin-mediated proteolysis is involved in the turnover of many short-lived regulatory proteins. This pathway leads to the covalent attachment of one or more multiubiquitin chains to target substrates which are then degraded by the 26S multicatalytic proteasome complex. Multiple classes of regulatory enzymes have been identified that mediate either ubiquitin conjugation or ubiquitin deconjugation from target substrates. Timed destruction of cellular regulators by the ubiquitin-proteasome pathway plays a critical role in ensuring normal cellular processes. This review provides multiple examples of key growth regulatory proteins whose levels are regulated by ubiquitin-mediated proteolysis. Pharmacological intervention which alters the half-lives of these cellular proteins may have wide therapeutic potential. Specifically, prevention of p53 ubiquitination (and subsequent degradation) in human papilloma virus positive tumors, and perhaps all tumors retaining wild-type p53 but lacking the retinoblastoma gene function, should lead to programmed cell death. Specific inhibitors of p27 and cyclin B ubiquitination are predicted to be potent antiproliferative agents. Inhibitors of IkappaB ubiquitination should prevent NFkappaB activation and may have utility in a variety of autoimmune and inflammatory conditions. Finally, we present a case for deubiquitination enzymes as novel, potential drug targets.

• Crinelli R, Bianchi M, Serafini G, Magnani M
• Structural determinants that make hexokinase susceptible to ubiquitin- and ATP-dependent proteolysis.
• Biochem Soc Trans. 1997; 25: 69-69

• Kawahara H
• [Sequence motifs in proteasome subunits and their possible functions]
• Tanpakushitsu Kakusan Koso. 1997; 42: 2154-64

• Tobiasz A, Zoladek T
• [The ubiquitin conjugation system and its role in the Saccharomyces cerevisiae yeasts]
• Postepy Biochem. 1997; 43: 91-7

• Kamitani T, Kito K, Nguyen HP, Yeh ET
• Characterization of NEDD8, a developmentally down-regulated ubiquitin-like protein.
• J Biol Chem. 1997; 272: 28557-62
• Display abstract

• NEDD8 is a novel 81 amino acid polypeptide which is 60% identical and 80% homologous to ubiquitin. Northern blot analysis showed that the NEDD8 message was developmentally down-regulated. In adult tissues, NEDD8 expression was mostly restricted to the heart and skeletal muscle. Antiserum specific for NEDD8 detected a 6-kDa monomer in SK-N-SH, BJAB, and HL60 cell lysates. A 14-kDa band was also detected in BJAB, HL60, and SK-MEL28 but not in SK-N-SH and K562 cell lysates. An approximately 90-kDa band was detected in all cell lines tested. Thus, NEDD8 is likely to be conjugated to other proteins in a manner analogous to ubiquitination. However, the conjugation pattern of NEDD8 is entirely different from that of ubiquitin in all cell lines tested. To study NEDD8 conjugation in more detail, hemagglutinin-epitope-tagged NEDD8 was expressed in COS cells. Western blot analysis revealed an NEDD8 monomer and a series of higher molecular weight NEDD8-conjugated proteins or NEDD8 multimers. Immunocytochemical analysis showed that NEDD8 expression was highly enriched in the nucleus and was much weaker in the cytosol. In contrast, ubiquitin expression was detectable equally well in the nucleus and cytosol. Mutational analysis showed that the C terminus of NEDD8 was efficiently cleaved and that Gly-76 was required for conjugation of NEDD8 to other proteins. Taken together, NEDD8 provides another substrate for covalent protein modification and may play a unique role during development.

• Ciechanover A, Hargrove JL, Gross-Mesilaty S
• Ubiquitin-mediated degradation of tyrosine aminotransferase (TAT) in vitro and in vivo.
• Mol Biol Rep. 1997; 24: 27-33
• Display abstract

• Degradation of a protein via the ubiquitin proteolytic pathway involves two successive steps. Covalent attachment of ubiquitin to the target protein and degradation of the tagged substrate by the 26S proteasome. Most native cellular proteins that are targeted by the ubiquitin system are short-lived transcriptional activators and growth and cell cycle regulators, as well as unstable membrane proteins. In the present study we demonstrate the involvement of the system in the degradation of tyrosine aminotransferase (TAT), a key enzyme in intermediary metabolism. In vitro, we have shown that the native enzyme is conjugated and degraded in a system that requires ATP and ubiquitin. Degradation was monitored by following the decrease of catalytic activity as well as disappearance of the protein molecule. The enzyme could be protected from degradation by association with its specific cofactor, pyridoxal phosphate (PLP). In vivo, we prepared cell extracts from livers of animals in which TAT was induced by starvation and corticosteroid administration. The dramatic increase in the level of the enzyme was accompanied by a concomitant increase in the level of specific TAT-ubiquitin adducts.

• Lupas A, Baumeister W, Hofmann K
• A repetitive sequence in subunits of the 26S proteasome and 20S cyclosome (anaphase-promoting complex).
• Trends Biochem Sci. 1997; 22: 195-6

• Smith SE, Koegl M, Jentsch S
• Role of the ubiquitin/proteasome system in regulated protein degradation in Saccharomyces cerevisiae.
• Biol Chem. 1996; 377: 437-46
• Display abstract

• Selective degradation of proteins in eukaryotes is mediated primarily by the ubiquitin system in conjunction with the 26S proteasome. The yeast Saccharomyces cerevisiae has proved a powerful model system to study protein degradation in vivo. Biochemical and genetic studies complemented by the sequence analysis of the entire yeast genome have identified more than 70 genes presumed to function in the ubiquitin/proteasome system. Moreover, a number of physiological substrates of the ubiquitin system have been identified in yeast which are key regulatory proteins involved in the control of the cell cycle and transcription. In this review we will describe the enzymes effecting ubiquitin-protein conjugation and degradation. In addition we will discuss several targets of this system and describe the cellular functions mediated by this pathway.

• Baboshina OV, Haas AL
• Novel multiubiquitin chain linkages catalyzed by the conjugating enzymes E2EPF and RAD6 are recognized by 26 S proteasome subunit 5.
• J Biol Chem. 1996; 271: 2823-31
• Display abstract

• Targeting of substrates for degradation by the ATP, ubiquitin-dependent pathway requires formation of multiubiquitin chains in which the 8.6-kDa polypeptide is linked by isopeptide bonds between carboxyl termini and Lys-48 residues of successive monomers. Binding of Lys-48-linked chains by subunit 5 of the 26 S proteasome regulatory complex commits the attached target protein to degradation with concomitant release of free ubiquitin monomers following disassembly of the chains. Point mutants of ubiquitin (Lys-->Arg) were used to map the linkage specificity for ubiquitin-conjugating enzymes previously demonstrated to form novel multiubiquitin chains not attached through Lys-48. Recombinant human E2EPF catalyzed multiubiquitin chain formation exclusively through Lys-11 of ubiquitin while recombinant yeast RAD6 formed chains linked only through Lys-6. Multiubiquitin chains linked through Lys-6, Lys-11, or Lys-48 each bound to subunit 5 of partially purified human 26 S proteasome with comparable affinities. Since chains bearing different linkages are expected to pack into distinct structures, competition between Lys-11 and Lys-48 chains for binding to subunit 5 demonstrates that the latter possesses determinants for recognizing alternatively linked chains and precludes the existence of subunit 5 isoforms recognizing distinct structures. In addition, competition studies provided an estimate of Kd < or = 18 nM for the intrinsic binding of Lys-48-linked chains of linkage number n > 4. This result suggests that the principal mechanistic advantage of multiubiquitin chain formation is to enhance the affinity of the associated substrate for the 26 S complex relative to that of unconjugated target protein. Complementation studies with E1/E2-depleted rabbit reticulocyte extract demonstrated RAD6 supported isopeptide ligase-dependent degradation only through Lys-48-linked chains, while E2EPF retained the ability to target a model radiolabeled substrate through Lys-11-linked chains. Therefore, the linkage specificity exhibited by these E2 isozymes depends on their catalytic context with respect to isopeptide ligase.

• Haas AL, Katzung DJ, Reback PM, Guarino LA
• Functional characterization of the ubiquitin variant encoded by the baculovirus Autographa californica.
• Biochemistry. 1996; 35: 5385-94
• Display abstract

• The marked evolutionary conservation of ubiquitin is assumed to arise from constraints imposed by folding, stability, and interaction of the polypeptide with various components of the ATP, ubiquitin-dependent degradative pathway. The present studies characterize the most divergent (75% identity) of the species-specific ubiquitin isoforms encoded as a late gene product of the baculovirus Autographa californica [Guarino, L. A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 409-413]. Viral ubiquitin supports 40% of the rate of ATP-dependent degradation exhibited by eukaryotic ubiquitin. Inhibition of proteolysis correlated with a lower steady-state concentration of ubiquitin-conjugated degradative intermediates. Rate studies revealed that viral ubiquitin exerts its effect at the step of isopeptide ligase-catalyzed (E3) ubiquitin conjugation since viral and eukaryotic polypeptides are identical in their abilities to support ATP-coupled activation by E1 and transthiolation to E2 carrier proteins. Other studies demonstrated viral ubiquitin severely attenuated the rate of K48-linked multiubiquitin chain formation in E3-independent conjugation catalyzed by recombination yeast CDC34 or rabbit reticulocyte E232K but not chain elongation of alternate linkages formed by yeast RAD6 or human E2EPF. The latter observations suggest nonconserved positions on viral ubiquitin constitute recognition signals for K48-linked chain formation. Sequence comparison of species-specific ubiquitin isoforms indicates that nonconserved positions localized to a defined region on the polypeptide surface distinct from the basic face required for E1 binding. These results suggest this novel ubiquitin isoform may function in baculoviral replication to block destruction of a short-lived protein(s) by the host degradative pathway, targeted through either E2-catalyzed K48-linked multibiquitin chain formation or general E3-mediated conjugation.

• Shaeffer JR, Cohen RE
• Differential effects of ubiquitin aldehyde on ubiquitin and ATP-dependent protein degradation.
• Biochemistry. 1996; 35: 10886-93
• Display abstract

• ATP-dependent proteolysis of 125I-labeled human alpha-globin, bovine alpha-lactalbumin, bovine serum albumin, or chicken lysozyme was assessed in a rabbit reticulocyte extract supplemented with ATP, excess ubiquitin, and variable amounts of ubiquitin aldehyde (Ubal), an inhibitor of many ubiquitin-protein isopeptidases. Low concentrations (0.8 microM) of Ubal increased the ATP-dependent degradation of 125I-alpha-globin by approximately 30% after 2 h at 37 degrees C, had little effect on 125I-lysozyme turnover, and decreased 125I-alpha-lactalbumin or 125I-albumin degradation by approximately 20%. The ATP-dependent degradation of all substrates was inhibited by high concentrations (> 3 microM) of Ubal throughout the incubation (15 min to 2 h); after 2 h, this inhibition ranged from 15% for 125I-alpha-globin to approximately 85% for 125I-alpha-lactalbumin and 125I-albumin. Levels of ubiquitin-125I-protein conjugates were increased significantly with Ubal; with > or = 8.0 microM Ubal, high molecular mass multiubiquitinated conjugates were particularly evident for 125I-alpha-globin and 125I-alpha-lactalbumin. These mixtures also accumulated ubiquitin conjugates with sizes expected for di- through pentaubiquitin oligomers. The results are consistent with the following proposed events: The ATP-dependent degradation of 125I-alpha-lactalbumin or 125I-albumin is probably mediated almost exclusively through polyubiquitinated intermediates. High Ubal concentrations inhibit an isopeptidase(s) which normally disassembles "unanchored" polyubiquitin chains that remain after substrate degradation by the 26S proteasome; these chains accumulate to inhibit further conjugate degradation. Much of the ATP-dependent degradation of 125I-alpha-globin and, to a lesser degree, 125I-lysozyme may occur through alternative structures where ubiquitin monomers or short oligomers are ligated to one or more substrate lysines. For 125I-alpha-globin, even low concentrations of Ubal effectively inhibit deubiquitination of these conjugates to enhance alpha-globin degradation.

• Chun KT, Mathias N, Goebl MG
• Ubiquitin-dependent proteolysis and cell cycle control in yeast.
• Prog Cell Cycle Res. 1996; 2: 115-27
• Display abstract

• Genetic and biochemical data indicate that ubiquitin-mediated proteolysis is involved in the regulated turnover of proteins required for controlling cell cycle progression. In general, mutations in some genes that encode proteins involved in the ubiquitin pathway cause cell cycle defects and affect the turnover of cell cycle regulatory proteins. Furthermore, some cell cycle regulatory proteins are short-lived, ubiquitinated, and degraded by the ubiquitin pathway. This review will examine how the ubiquitin pathway plays a role in regulating progression from the G1 to the S phase of the cell cycle, as well as the G2 to M phase transition.

• Aristarkhov A, Eytan E, Moghe A, Admon A, Hershko A, Ruderman JV
• E2-C, a cyclin-selective ubiquitin carrier protein required for the destruction of mitotic cyclins.
• Proc Natl Acad Sci U S A. 1996; 93: 4294-9
• Display abstract

• Ubiquitin-dependent proteolysis of the mitotic cyclins A and B is required for the completion of mitosis and entry into the next cell cycle. This process is catalyzed by the cyclosome, an approximately 22S particle that contains a cyclin-selective ubiquitin ligase activity, E3-C, that requires a cyclin-selective ubiquitin carrier protein (UBC) E2-C. Here we report the purification and cloning of E2-C from clam oocytes. The deduced amino acid sequence of E2-C indicates that it is a new UBC family member. Bacterially expressed recombinant E2-C is active in in vitro cyclin ubiquitination assays, where it exhibits the same substrate specificities seen with native E2-C. These results demonstrate that E2-C is not a homolog of UBC4 or UBC9, proteins previously suggested to be involved in cyclin ubiquitination, but is a new UBC family member with unique properties.

• King RW, Glotzer M, Kirschner MW
• Mutagenic analysis of the destruction signal of mitotic cyclins and structural characterization of ubiquitinated intermediates.
• Mol Biol Cell. 1996; 7: 1343-57
• Display abstract

• Mitotic cyclins are abruptly degraded at the end of mitosis by a cell-cycle-regulated ubiquitin-dependent proteolytic system. To understand how cyclin is recognized for ubiquitin conjugation, we have performed a mutagenic analysis of the destruction signal of mitotic cyclins. We demonstrate that an N-terminal cyclin B segment as short as 27 residues, containing the 9-amino-acid destruction box, is sufficient to destabilize a heterologous protein in mitotic Xenopus extracts. Each of the three highly conserved residues of the cyclin B destruction box is essential for ubiquitination and subsequent degradation. Although an intact destruction box is essential for the degradation of both A- and B-type cyclins, we find that the Xenopus cyclin A1 destruction box cannot functionally substitute for its B-type counterpart, because it does not contain the highly conserved asparagine necessary for cyclin B proteolysis. Physical analysis of ubiquitinated cyclin B intermediates demonstrates that multiple lysine residues function as ubiquitin acceptor sites, and mutagenic studies indicate that no single lysine residue is essential for cyclin B degradation. This study defines the key residues of the destruction box that target cyclin for ubiquitination and suggests there are important differences in the way in which A- and B-type cyclins are recognized by the cyclin ubiquitination machinery.

• Galan JM, Moreau V, Andre B, Volland C, Haguenauer-Tsapis R
• Ubiquitination mediated by the Npi1p/Rsp5p ubiquitin-protein ligase is required for endocytosis of the yeast uracil permease.
• J Biol Chem. 1996; 271: 10946-52
• Display abstract

• Uracil uptake by Saccharomyces cerevisiae is mediated by the FUR4-encoded uracil permease. This permease undergoes endocytosis and subsequent degradation in cells subjected to adverse conditions. The data presented here show that uracil permease also undergoes basal turnover under normal growth conditions. Both basal and induced turnover depend on the essential Npi1p/Rsp5p ubiquitin-protein ligase. Epitope-tagged ubiquitin variants have been used to show that uracil permease is ubiquitinated in vivo. The ubiquitin-permease conjugates that are readily demonstrated in wild type cells were barely detectable in npi1 mutant cells, indicating that uracil permease may be a physiological substrate of the Npi1p ubiquitin ligase. The lack of ubiquitination of the permease in npi1 cells resulted in an increase in active, i.e. plasma membrane-located, permease, suggesting that there is a direct relationship between ubiquitination and removal of the permease from the plasma membrane. The accumulation of ubiquitin-permease conjugates in thermosensitive act1 mutant cells, deficient in the internalization step of endocytosis is consistent with this idea. On the other hand, the degradation of uracil permease does not require a functional proteasome since the permease was not stabilized in either pre1 pre2 or cim3 and cim5 mutant cells that have impaired catalytic (pre) or regulatory (cim) proteasome subunits. In contrast, both basal and stress-stimulated turnover rates were greatly reduced in pep4 mutant cells having defective vacuolar protease activities. We therefore propose that ubiquitination of uracil permease acts as a signal for endocytosis of the protein that is subsequently degraded in the vacuole.

• Tsurumi C, Shimizu Y, Tanaka K
• [Degradation mechanism of cell cycle factors by the proteasome]
• Nippon Rinsho. 1996; 54: 861-9
• Display abstract

• Cell cycle progression is mainly controlled by the hetero-dimeric protein kinase complex named SPF (S-phase promoting factor) and MPF (M-phase promoting factor), consisting of CDKs and the regulator cyclins, which are involved in G1/S and G2/M transitions, respectively. Moreover, SPF is modulated by not only various oncoproteins positively, but also tumor suppresive gene products negatively. These regulator proteins are extremely unstable in cells, oscillating during cell cycle, and cell cycle stage-dependent destruction of specific factors is required for cell cycle progression, but molecular mechanism of their destabilization remains to be clarified. The ubiquitin-proteasome system is responsible for selective- and ATP-dependent degradation of various types of short-lived proteins in the cytoplasm and the nucleus. In this article, we review briefly the proteolytic pathway mediated by ubiquitin and the proteasome, and the degradation mechanism of major cell cycle protein factors, such as Mos, p53, cyclin B, Fos/Jun and NFkappaB/IkappaB.

• van Nocker S, Deveraux Q, Rechsteiner M, Vierstra RD
• Arabidopsis MBP1 gene encodes a conserved ubiquitin recognition component of the 26S proteasome.
• Proc Natl Acad Sci U S A. 1996; 93: 856-60
• Display abstract

• Multiubiquitin chain attachment is a key step leading to the selective degradation of abnormal polypeptides and many important regulatory proteins by the eukaryotic 26S proteasome. However, the mechanism by which the 26S complex recognizes this posttranslational modification is unknown. Using synthetic multiubiquitin chains to probe an expression library for interacting proteins, we have isolated an Arabidopsis cDNA, designated MBP1, that encodes a 41-kDa acidic protein exhibiting high affinity for chains, especially those containing four or more ubiquitins. Based on similar physical and immunological properties, multiubiquitin binding affinities, and peptide sequence, MBP1 is homologous to subunit 5a of the human 26S proteasome. Structurally related proteins also exist in yeast, Caenorhabditis, and other plant species. Given their binding properties, association with the 26S proteasome, and widespread distribution, MBP1, S5a, and related proteins likely function as essential ubiquitin recognition components of the 26S proteasome.

• Hoffman L, Rechsteiner M
• Regulatory features of multicatalytic and 26S proteases.
• Curr Top Cell Regul. 1996; 34: 1-32
• Display abstract

• It should be clear from the foregoing accounts that our understanding of MCP and 26S regulation is still rudimentary. Moreover, we have only recently identified about a dozen natural substrates of these two proteases. Those outside the field may view the situation with some dismay. Those who study the MCP and 26S enzymes are provided with rich opportunities to address fundamental questions of protein catabolism and metabolic regulation.

• Liu Z, Haas AL, Diaz LA, Conrad CA, Giudice GJ
• Characterization of a novel keratinocyte ubiquitin carrier protein.
• J Biol Chem. 1996; 271: 2817-22
• Display abstract

• A novel member of the ubiquitin carrier protein family, designated E2EPF, has been cloned by our laboratory and expressed in a bacterial system in an active form. Ubiquitin carrier proteins, or E2s, catalyze one step in a multistep process that leads to the covalent conjugation of ubiquitin to substrate proteins. In this paper, we show that recombinant E2EPF catalyzes auto/multiubiquitination, the conjugation of multiple ubiquitin molecules to itself. Multiubiquitination has been shown previously to be required for targeting of a substrate protein for rapid degradation. Using a rabbit reticulocyte lysate system, E2EPF was shown to support the degradation of a model substrate in an ATP- and ubiquitin-dependent fashion. In contrast to a previous study which showed that selective protein degradation in one system is dependent upon multiubiquitination via the lysine 48 residue of ubiquitin, multiubiquitination, and proteolytic targeting by E2EPF was shown here to be independent of the lysine 48 multiubiquitin linkage. This functional characterization of E2EPF revealed a combination of features that distinguishes this enzyme from all previously characterized members of the ubiquitin carrier protein family. These results also suggest several possible autoregulatory models for E2EPF involving auto- and multiubiquitination.

• Vierstra RD
• Proteolysis in plants: mechanisms and functions.
• Plant Mol Biol. 1996; 32: 275-302
• Display abstract

• Proteolysis is essential for many aspects of plant physiology and development. It is responsible for cellular housekeeping and the stress response by removing abnormal/misfolded proteins, for supplying amino acids needed to make new proteins, for assisting in the maturation of zymogens and peptide hormones by limited cleavages, for controlling metabolism, homeosis, and development by reducing the abundance of key enzymes and regulatory proteins, and for the programmed cell death of specific plant organs or cells. It also has potential biotechnological ramifications in attempts to improve crop plants by modifying protein levels. Accumulating evidence indicates that protein degradation in plants is a complex process involving a multitude of proteolytic pathways with each cellular compartment likely to have one or more. Many of these have homologous pathways in bacteria and animals. Examples include the chloroplast ClpAP protease, vacuolar cathepsins, the KEX2-like proteases of the secretory system, and the ubiquitin/26S proteasome system in the nucleus and cytoplasm. The ubiquitin-dependent pathway requires that proteins targeted for degradation become conjugated with chains of multiple ubiquitins; these chains then serve as recognition signals for selective degradation by the 26S proteasome, a 1.5 MDa multisubunit protease complex. The ubiquitin pathway is particularly important for developmental regulation by selectively removing various cell-cycle effectors, transcription factors, and cell receptors such as phytochrome A. From insights into this and other proteolytic pathways, the use of phosphorylation/dephosphorylation and/or the addition of amino acid tags to selectively mark proteins for degradation have become recurring themes.

• Schmidtke G et al.
• Analysis of mammalian 20S proteasome biogenesis: the maturation of beta-subunits is an ordered two-step mechanism involving autocatalysis.
• EMBO J. 1996; 15: 6887-98
• Display abstract

• Maturation of eukaryotic 20S proteasomes involves the processing of beta-subunits by limited proteolysis. To study the processing mechanism we analysed different point mutations of the beta-subunit LMP2 in transfected human T2 cells. Here we show that the presence of the intact Gly-1Thr1 consensus motif and Lys33 are essential for correct processing. Mutation of Thr1, the active site residue in mature subunits, or of Lys33, results in complete inhibition of processing at the consensus site. In addition, proprotein processing in vitro of wild-type LMP2, incorporated in immature 16S precursor complexes, can be blocked by a proteasome-specific inhibitor. While the processing of inhibitor-treated wild-type proprotein was completely prevented, the site-directed mutagenesis of LMP2 results in processing intermediates carrying an extension of 8-10 residues preceding Thr1, suggesting an additional cleavage event within the prosequence. Furthermore, exchange of mammalian prosequences interferes with processing efficiency and suggests subunit specificity. Based on our data we propose a model for self-activation of proteasomal beta-subunits in which residue Thr1 serves as nucleophile and Lys33 as proton donor/acceptor. We provide evidence that subunit processing of mammalian beta-subunits proceeds via a novel ordered two-step mechanism involving autocatalysis.

• Kalderon D
• Protein degradation: de-ubiquitinate to decide your fate.
• Curr Biol. 1996; 6: 662-5
• Display abstract

• The ubiquitination/de-ubiquitination system that controls the degradation of many cellular proteins can be regulated at several of its distinct steps; one recently discovered control is important in Drosophila eye development.

• Isozaki U, Mitch WE, England BK, Price SR
• Protein degradation and increased mRNAs encoding proteins of the ubiquitin-proteasome proteolytic pathway in BC3H1 myocytes require an interaction between glucocorticoids and acidification.
• Proc Natl Acad Sci U S A. 1996; 93: 1967-71
• Display abstract

• In rats and humans, metabolic acidosis stimulates protein degradation and glucocorticoids have been implicated in this response. To evaluate the importance of glucocorticoids in stimulating proteolysis, we measured protein degradation in BC3H1 myocytes cultured in 12% serum. Acidification accelerated protein degradation but dexamethasone did not augment this response. To reduce the influence of glucocorticoids and other hormones and cytokines in 12% serum that could mediate proteolysis, we studied BC3H1 myocytes maintained in only 1% serum. Acidification of the medium or addition of dexamethasone at pH 7.4 did not significantly increase protein degradation, while acidification plus dexamethasone accelerated proteolysis. The steroid receptor antagonist RU 486 prevented this proteolytic response. Acidification of the medium with 1% serum did increase the mRNAs for ubiquitin and the C2 proteasome subunit, but when dexamethasone was added the mRNAs were increased significantly more. The steroid-receptor antagonist RU 486 suppressed this response to the addition of dexamethasone but the mRNAs remained at the levels measured in cells at pH 7.1 alone. Thus, acidification alone can increase the mRNAs of the ubiquitin-proteasome proteolytic pathway, but both acidosis and glucocorticoids are required to stimulate protein degradation. Since these changes occur without adding cytokines or other hormones, we conclude that the proteolytic response to acidification requires glucocorticoids.

• Matuschewski K, Hauser HP, Treier M, Jentsch S
• Identification of a novel family of ubiquitin-conjugating enzymes with distinct amino-terminal extensions.
• J Biol Chem. 1996; 271: 2789-94
• Display abstract

• The ubiquitin/proteasome system is the main eukaryotic nonlysosomal protein degradation system. Substrate selectivity of this pathway is thought to be mediated in part by members of a large family of ubiquitin-conjugating (E2) enzymes, which catalyze the covalent attachment of ubiquitin to proteolytic substrates. E2 enzymes have a conserved approximately 150-residue so-called UBC domain, which harbors the cysteine residue required for enzyme-ubiquitin thioester formation. Some E2 enzymes possess additional carboxyl-terminal extensions that are involved in substrate specificity and intracellular localization of the enzyme. Here we describe a novel family of E2 enzymes from higher eukaryotes (Drosophila, mouse, and man) that have amino-terminal extensions but lack carboxyl-terminal extensions. We have identified four different variants of these enzymes that have virtually identical UBC domains (94% identity) but differ in their amino-terminal extensions. In yeast, these enzymes can partially complement mutants deficient in the UBC4 E2 enzyme. This indicates that members of this novel E2 family may operate in UBC4-related proteolytic pathways.

• Obin MS, Jahngen-Hodge J, Nowell T, Taylor A
• Ubiquitinylation and ubiquitin-dependent proteolysis in vertebrate photoreceptors (rod outer segments). Evidence for ubiquitinylation of Gt and rhodopsin.
• J Biol Chem. 1996; 271: 14473-84
• Display abstract

• In corroboration of the hypothesized regulation of phototransduction proteins by the ubiquitin-dependent pathway, we identified free ubiquitin (8 kDa) and ubiquitin-protein conjugates (50 to >200 kDa; pI 5.3-6.8 by two-dimensional electrophoresis) in bovine rod outer segments (ROS). A 38-kDa ubiquitinylated protein and transducin (Gt) were eluted together from light-adapted ROS membranes with GTP. When ROS were dark-adapted, this 38-kDa ubiquitinylated species and Gt were readily solubilized in buffer lacking GTP. These data are consistent with ubiquitinylation of Gt and corroborate previous cell-free experiments identifying Gt as a substrate for ubiquitin-dependent proteolysis (Obin, M. S., Nowell, T., and Taylor, A. (1994) Biochem. Biophys. Res. Commun. 200, 1169-1176). Evidence for ubiquitinylation of rhodopsin (36 kDa), the (photo)receptor coupled to Gt, included (i) the presence in ROS membranes "stripped" of peripheral membrane proteins of numerous ubiquitin-protein conjugates, including two whose masses (44 and 50 kDa) are consistent with mono- and diubiquitinylated rhodopsin; (ii) catalysis by permeabilized ROS of 125I-labeled ubiquitin-protein conjugates whose masses (42, 50, and 58 kDa) suggest a "ladder" of mono-, di-, and triubiquitinylated rhodopsin; (iii) parallel mobility shifts on SDS-polyacrylamide gels of rhodopsin and these 125I-labeled ubiquitin-protein conjugates; and (iv) generation of enhanced levels of 125I-labeled ubiquitin-protein conjugates when stripped, detergent-solubilized ROS membranes (95% rhodopsin) were incubated with reticulocyte lysate. A functional ubiquitin-dependent pathway in ROS is demonstrated by the presence of (i) the ubiquitin-activating enzyme (E1); (ii) four ubiquitin carrier proteins (E214K, E220K, E225K, and E235K) and pronounced activity of E214K, an enzyme required for "N-end rule" proteolysis; (iii) ATP-dependent 26 S proteasome activity that rapidly degrades high mass 125I-labeled ubiquitin-ROS protein conjugates; and (iv) distinct ubiquitin C-terminal isopeptidase/hydrolase activities, including potent ubiquitin-aldehyde-insensitive activity directed at high mass ubiquitinylated moieties. Considered together, the data support a novel role for the ubiquitin-dependent pathway in the regulation of mammalian phototransduction protein levels and/or activities and provide the first identification of a non-calpain proteolytic system in photoreceptors.

• Narasimhan J, Potter JL, Haas AL
• Conjugation of the 15-kDa interferon-induced ubiquitin homolog is distinct from that of ubiquitin.
• J Biol Chem. 1996; 271: 324-30
• Display abstract

• The biological effect of type 1 interferons is proposed to arise in part from the conjugation of ubiquitin cross-reactive protein (UCRP), the ISG15 gene product, to intracellular target proteins in a process analogous to that of its sequence homolog ubiquitin, a highly conserved 8.6-kDa polypeptide whose ligation marks proteins for degradation via the 26 S proteasome. Inclusion of CoCl2 during the purification of recombinant UCRP blocks the proteolytic inactivation of the polypeptide occurring by cleavage of the carboxyl-terminal glycine dipeptide required for activation and subsequent ligation. Intact UCRP supports a low rate of ubiquitin-activating enzyme (E1)-dependent ATP:PPi exchange but fails to form a stoichiometric E1-UCRP thiol ester or undergo transfer to ubiquitin carrier protein (E2). The binding affinity of E1 for UCRP is significantly diminished relative to that of ubiquitin. These results suggest that UCRP conjugation proceeds through an enzyme pathway distinct from that of ubiquitin, at least with respect to the step of activation. This was confirmed for an in vitro conjugation assay in which 125I-UCRP could be ligated in an ATP-dependent reaction to proteins present within an A549 human lung carcinoma cell extract and could be competitively inhibited by excess unlabeled UCRP but not ubiquitin. Other results demonstrate that 125I-UCRP conjugation is significantly increased in cell extracts after 24 h of incubation in the presence of interferon-beta, consistent with the late induction of UCRP conjugating activity. Thus, interferon-responsive cells contain a pathway for UCRP ligation that is parallel but distinct from that of ubiquitin.

• Takada K, Hibi N, Tsukada Y, Shibasaki T, Ohkawa K
• Ability of ubiquitin radioimmunoassay to discriminate between monoubiquitin and multi-ubiquitin chains.
• Biochim Biophys Acta. 1996; 1290: 282-8
• Display abstract

• Free ubiquitin (mainly monoubiquitin) and multi-ubiquitin chains coexist in eukaryote cells and serve distinct cellular roles. However, any immunoassay systems established previously have not been proved to be applicable for measuring the former without cross-reactive responses with the latter. For this purpose, we developed a radioimmunoassay specific to monoubiquitin by employing antiserum US-1 against ubiquitin. In this assay, ubiquitin-protein conjugates, prepared by a reticulocyte lysate fraction II and fractionated on Moro Q and Superdex 200 columns, exhibited practically no cross-reactivity. The cross-reactivity of fractionated ubiquitin-lysozyme conjugates was also analyzed as a function of their multi-ubiquitin chain size. As a result, the larger the conjugates were found to be, the weaker were the cross-reactive responses they showed, and the multi-ubiquitin chains (n > approx. 20) were substantially unreactive in the radioimmunoassay. By using the radioimmunoassay, heat-shock-induced decrease in the level of cellular free (mono)ubiquitin was detected. In addition, the standard preparation of multi-ubiquitin chains was not cross-reactive in all other five radioimmunoassays employing distinct antibodies to ubiquitin (four antisera and a monoclonal antibody). These data suggest that radioimmunoassays employing ubiquitin antibodies raised by the general methods can discriminate between monoubiquitin and multi-ubiquitin chains and quantitate cellular free ubiquitin.

• Bohley P
• Surface hydrophobicity and intracellular degradation of proteins.
• Biol Chem. 1996; 377: 425-35
• Display abstract

• Cellular proteins turn over with rates varying more than 1000 fold and all organelles are involved in this process of renewal. Although the surface of protein molecules bears many peptide bonds, which could be potential cleavage sites for proteases, only a small fraction of all cellular proteins is subject to very rapid turnover, with half-lives of less than one hour in mammalian cells. Many of these proteins play key roles in basic regulatory mechanisms. One of the features that make proteins short-lived is surface hydrophobicity which is increased in nascent polypeptide chains before association with chaperones, in oligomeric proteins before the association of the monomers, and in many enzymes in absence of their substrates. Cellular proteases tend to act most rapidly on peptide bonds involving (or near to) apolar amino acids. A striking correlation has been found between the half-lives of cellular proteins and their surface hydrophobicity. Clusters of hydrophobic residues appear to be necessary for ubiquitination. Apolar amino acid residues that are hidden can be exposed after oxidation of proteins or after binding of ubiquitin molecules. Additionally, tetraubiquitin exposes many hydrophobic residues which are essential for targeting of the ubiquitinated substrate proteins to a 50 kDa-subunit of the 26S-protease.

• Hershko A
• Mechanisms and regulation of ubiquitin-mediated cyclin degradation.
• Adv Exp Med Biol. 1996; 389: 221-7

• Muller S, Schwartz LM
• Ubiquitin in homeostasis, development and disease.
• Bioessays. 1995; 17: 677-84
• Display abstract

• Ubiquitin is the most phylogenetically conserved protein known. This 8,500 Da polypeptide can be covalently attached to cellular proteins as a posttranslational modification. In most cases, the addition of multiple ubiquitin adducts to a protein targets it for rapid degradation by a multisubunit protease known as the 26S proteasome. While the ubiquitin/26S proteasome pathway is responsible for the degradation of the bulk of cellular proteins during homeostasis, it may also be responsible for the rapid loss of protein during the programmed death of certain cells, such as skeletal muscle during insect metamorphosis. In addition, alterations in the expression and regulation of ubiquitin may play significant roles in pathological disorders. For example, dramatic increases in ubiquitin and ubiquitin-protein conjugates are observed in a wide variety of neurodegenerative disorders, including Alzheimer's disease. Patients suffering from the autoimmune disease systemic lupus erythematosus generate antibodies reacting with ubiquitin and ubiquitinated histones. At present, it is not known whether these changes in ubiquitin expression and regulation initiate pathological changes in these diseases or if they are altered as a consequence of these disorders.

• Shean BS, Mykles DL
• Polyubiquitin in crustacean striated muscle: increased expression and conjugation during molt-induced claw muscle atrophy.
• Biochim Biophys Acta. 1995; 1264: 312-22
• Display abstract

• The claw muscles of decapod crustaceans undergo a molt-induced atrophy to facilitate withdrawal of the claws at ecdysis. Polyubiquitin expression, as well as the levels of ubiquitin conjugates, a ubiquitin-conjugating enzyme involved in the ATP/ubiquitin-dependent proteolytic pathway (crustacean E2(16 kDa) homolog of Drosophila UbcD1), and proteasome, were examined to determine the role of ATP/ubiquitin-dependent proteolysis in the enhanced degradation of myofibrillar proteins during muscle atrophy. A partial-length clone (1.7 kb) of polyubiquitin was isolated from a lobster muscle cDNA library; the 5' end lacked the 5' untranslated region (UTR) and the beginning of the first ubiquitin monomer, while the 3' end contained the terminal ubiquitin monomer and 3' UTR. The deduced amino acid sequence was 100% identical with that from Manduca, Drosophila, and human. In land crab claw muscle, the polyubiquitin mRNA (2.7 kb) increased about 5-fold and ubiquitin-protein conjugates (> 200 kDa) increased about 8-fold during atrophy. In contrast, the level of a ubiquitin-conjugating enzyme (E2(16 kDa)) remained unchanged. The proteasome, which constitutes the catalytic core of the ATP/ubiquitin-dependent proteinase complex, increased about 2-fold during proecdysis, reaching its highest level immediately before ecdysis. These results suggest that the ATP/ubiquitin-dependent proteolytic pathway contributes to the changes in protein metabolism that occur during molt-induced muscle atrophy.

• Hochstrasser M
• Ubiquitin, proteasomes, and the regulation of intracellular protein degradation.
• Curr Opin Cell Biol. 1995; 7: 215-23
• Display abstract

• Rapid degradation of specific proteins by ubiquitin/proteaseome-dependent pathways is a component of many cellular regulatory mechanisms. Recent work has shown that protein ubiquitination and deubiquitination are both mediated by large families of enzymes and that proteolysis can be modulated by alterations of the proteasome itself. The complexity of the ubiquitin system is reflected in the broad range of processes it regulates; these include key steps in cell cycle progression, processing of foreign proteins for presentation by class I MHC molecules, and the control of cell proliferation.

• Scheffner M, Nuber U, Huibregtse JM
• Protein ubiquitination involving an E1-E2-E3 enzyme ubiquitin thioester cascade.
• Nature. 1995; 373: 81-3
• Display abstract

• Ubiquitination of proteins involves the concerted action of the E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzymes and E3 ubiquitin-protein ligases. It has been proposed that E3s function as 'docking proteins', specifically binding substrate proteins and specific E2s, and that ubiquitin is then transferred directly from E2s to substrates. We show here that formation of a ubiquitin thioester on E6-AP, an E3 involved in the human papillomavirus E6-induced ubiquitination of p53 (refs 6-10), is an intermediate step in E6-AP-dependent ubiquitination. The order of ubiquitin transfer is from E1 to E2, from E2 to E6-AP, and finally from E6-AP to a substrate. This cascade of ubiquitin thioester complexes suggests that E3s have a defined enzymatic activity and do not function simply as docking proteins. The cysteine residue of E6-AP responsible for ubiquitin thioester formation was mapped to a region that is highly conserved among several proteins of unknown function, suggesting that these proteins share the ability to form thioesters with ubiquitin.

• Jennissen HP
• Ubiquitin and the enigma of intracellular protein degradation.
• Eur J Biochem. 1995; 231: 1-30
• Display abstract

• Contrary to widespread belief, the regulation and mechanism of degradation for the mass of intracellular proteins (i.e. differential, selective protein turnover) in vertebrate tissues is still a major biological enigma. There is no evidence for the conclusion that ubiquitin plays any role in these processes. The primary function of the ubiquitin-dependent protein degradation pathway appears to lie in the removal of abnormal, misfolded, denatured or foreign proteins in some eukaryotic cells. ATP/ubiquitin-dependent proteolysis probably also plays a role in the degradation of some so-called 'short-lived' proteins. Evidence obtained from the covalent modification of such natural substrates as calmodulin, histones (H2A, H2B) and some cell membrane receptors with ubiquitin indicates that the reversible interconversion of proteins with ubiquitin followed by concomitant functional changes may be of prime importance.

• Falquet L, Paquet N, Frutiger S, Hughes GJ, Hoang-Van K, Jaton JC
• A human de-ubiquitinating enzyme with both isopeptidase and peptidase activities in vitro.
• FEBS Lett. 1995; 359: 73-7
• Display abstract

• Some enzymatic and physicochemical properties of a human ubiquitin-specific isopeptidase are reported. The enzyme was purified to homogeneity from red blood cells and its specificity towards polymeric ubiquitin substrates suggests a de-ubiquitinating activity capable of cleaving 'head-to-tail' polyUb chains as well as isoamide 'branched' Ub dimers. KM values show a 10 fold preference for the cleavage of branched Ub dimers over head-to-tail Ub dimers. The enzymatic activity can be strongly inhibited by various peptides containing either of the cleavage site sequences found in Ub polymers, but not by unrelated peptides. The enzyme is monomeric under reducing conditions and exhibits a globular shape with an average diameter of 9 nm, an S20,w value of 5.2 S and a molar mass of 110 kDa +/- 10%. Because the enzyme cleaves both peptide-linked and isopeptide-linked Ub moieties from substrates, we propose to name it de-ubiquitinase rather than isopeptidase.

• Dohmen RJ et al.
• An essential yeast gene encoding a homolog of ubiquitin-activating enzyme.
• J Biol Chem. 1995; 270: 18099-109
• Display abstract

• Ubiquitin (Ub) activation by the Ub-activating (E1) enzyme is the initial and essential step common to all of the known processes that involve post-translational conjugation of Ub to itself or other proteins. The "activated" Ub, linked via a thioester bond to a specific cysteine residue in one of several Ub-conjugating (E2) enzymes, which catalyze the formation of isopeptide bonds between the C-terminal glycine of Ub and lysine residues of acceptor proteins. In the yeast Saccharomyces cerevisiae, a 114-kDa E1 enzyme is encoded by an essential gene termed UBA1 (McGrath, J.P., Jentsch, S., and Varshavsky, A. (1991) EMBO J. 10, 227-236). We describe the isolation and analysis of another essential gene, termed UBA2, that encodes a 71-kDa protein with extensive sequence similarities to both the UBA1-encoded yeast E1 and E1 enzymes of other organisms. The regions of similarities between Uba1p and Uba2p encompass a putative ATP-binding site as well as a sequence that is highly conserved between the known E1 enzymes and contains the active-site cysteine of E1. This cysteine is shown to be required for an essential function of Uba2p, suggesting that Uba2p-catalyzed reactions involved a transient thioester bond between Uba2p and either Ub or another protein. Uba2p is located largely in the nucleus. The putative nuclear localization signal of Uba2p is near its C terminus. The Uba1p (E1 enzyme) and Uba2p cannot complement each others essential functions even if their subcellular localization is altered by mutagenesis. Uba2p appears to interact with itself and several other S. cerevisiae proteins with apparent molecular masses of 52, 63, 87, and 120 kDa. Uba2p is multiubiquitinated in vivo, suggesting that at least a fraction of Uba2p is metabolically unstable. Uba2p is likely to be a component of the Ub system that functions as either an E2 or E1/E2 enzyme.

• Stein RL, Chen Z, Melandri F
• Kinetic studies of isopeptidase T: modulation of peptidase activity by ubiquitin.
• Biochemistry. 1995; 34: 12616-23
• Display abstract

• We have investigated the specificity of isopeptidase T toward peptide-AMC substrates based on the C-termini of ubiquitin. The substrates investigated were Z-Gly-Gly-AMC, Z-Arg-Gly-Gly-AMC, Z-Leu-Arg-Gly-Gly-AMC, and Z-Arg-Leu-Arg-Gly-Gly-AMC and were hydrolyzed by isopeptidase T with kc/Km values of < 0.1, 1, 18, and 95 M-1 s-1, respectively. In the course of these experiments, we observed that the hydrolytic activity of isopeptidase T toward these substrates is modulated by ubiquitin in a biphasic fashion. While submicromolar concentrations of ubiquitin activate isopeptidase T, higher concentrations are inhibitory. In the activation phase, the extent of stimulation of kc/Km varies with substrate and is 8-, 50-, and 70-fold for Z-Arg-Gly-Gly-AMC, Z-Leu-Arg-Gly-Gly-AMC, and Z-Arg-Leu-Arg-Gly-Gly-AMC, respectively. Kd for ubiquitin in this phase is, of course, independent of substrate and equals 0.10 +/- 0.03 microM. At higher concentrations, ubiquitin is inhibitory and titrates kc/Km with an average Ki value of 3.0 +/- 1.3 microM for all three substrates. To explain these observations, we propose a structural model for isopeptidase T that involves two binding sites for ubiquitin. We propose that the two sites are adjacent to one another and are the extended active site that binds two ubiquitin moieties of a polyubiquitin chain for isopeptide bond hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

• Hochstrasser M et al.
• The DOA pathway: studies on the functions and mechanisms of ubiquitin-dependent protein degradation in the yeast Saccharomyces cerevisiae.
• Cold Spring Harb Symp Quant Biol. 1995; 60: 503-13

• Goldberg AL, Stein R, Adams J
• New insights into proteasome function: from archaebacteria to drug development.
• Chem Biol. 1995; 2: 503-8
• Display abstract

• The proteasome is not simply a 'garbage disposal unit' but also has functions in the control of the cell cycle and immune responses. The structure of an archaebacterial proteasome has recently been determined to high resolution, and provides insight into the unusual mechanism of proteolytic cleavage by the proteasome.

• Wunsch AM, Haas AL
• Ubiquitin-protein conjugates selectively distribute during early chicken embryogenesis.
• Dev Dyn. 1995; 204: 118-32
• Display abstract

• The major mechanism for proteolysis in eucaryotes involves an ATP-dependent pathway for which the covalent attachment of ubiquitin targets proteins for degradation. The involvement of ubiquitin conjugation in early embryonic vertebrate development was investigated by examining the amounts and localization of ubiquitin conjugates at different stages of development in the chicken using an affinity-purified antibody specific for conjugated ubiquitin. Solid phase immunochemical assays measuring whole embryo pools of free and conjugated ubiquitin demonstrated a progressive increase in conjugate pools to stage 18, followed by a decline to stage 24. In contrast, levels of free polypeptide showed a dramatic increase after stage 5, indicating a change in the dynamics of the two pools during development. Immunohistochemistry revealed that the distribution of ubiquitin adducts between stages 3 and 22 was pronounced in regions undergoing extensive cellular remodeling. Ubiquitin conjugates were detected in the primitive streak where cells ingress during gastrulation. The presence of these degradative intermediates in both neuroectodermal cells of the neural folds and subsequent neural crest cells migrating from the dorsum of the neural tube is consistent with an involvement in key morphogenetic events. The localization of ubiquitin conjugates at other selected tissue interfaces including limb bud ectoderm/mesoderm, and cardiac atrioventricular myocardium/endothelium suggests an active role for ubiquitin-mediated protein modification in similar developmental interactions. Conjugates were distributed first between somites, then in myotomes with a pattern spatially identical that of the ubiquitin conjugating enzyme, E214K, the major cognate isozyme for isopeptide ligase (E3)-dependent degradation. The potential involvement of ubiquitin conjugation at sites of epithelial-mesenchymal associations was further analyzed in culture using atrioventricular canal (AV) endothelium. Immunoreactivity was abundant in cells immediately prior to and during their transformation into mesenchyme. Collectively, the specific temporal and spatial changes in ubiquitin conjugates during early vertebrate development suggest a regulatory role for this degradative pathway in the cellular remodeling accompanying embryonic growth and differentiation.

• Oh CE, McMahon R, Benzer S, Tanouye MA
• bendless, a Drosophila gene affecting neuronal connectivity, encodes a ubiquitin-conjugating enzyme homolog.
• J Neurosci. 1994; 14: 3166-79
• Display abstract

• The Drosophila bendless (ben) gene was originally isolated as a mutation affecting the escape jump response. This behavioral defect was ascribed to a single lesion affecting the connectivity between the giant fiber and the tergotrochanter motor neuron. A closer examination of the ben phenotype suggests that ben activity is broader and affects a variety of other neurons including photoreceptor cells and their axons. Mosaic analysis indicates that the focus of ben activity is presynaptic. We have cloned the ben gene through a chromosomal walk and show that it is homologous to a class of ubiquitin-conjugating enzymes. The major role of ubiquitination in the protein degradative pathway suggests that ben regulates neural developmental processes such as growth cone guidance by targeting specific proteins for degradation.

• Thompson HS, Scordilis SP
• Ubiquitin changes in human biceps muscle following exercise-induced damage.
• Biochem Biophys Res Commun. 1994; 204: 1193-8
• Display abstract

• Changes in ubiquitin levels were characterized in human biceps muscle following high-force eccentric exercise. Volunteers performed damaging eccentric-isokinetic actions of the biceps muscle with the non-dominant arm. Protein extracts of biopsy tissue samples taken two days post-exercise were run on SDS polyacrylamide gels, analyzed densitometrically and revealed a 64% higher level of a protein band at 12 kD. New monoclonal immunoblotting techniques identified the band as free ubiquitin. On these blots, free ubiquitin increased in the exercised sample by 55% over the control, and ubiquitin conjugates of varying molecular weights follow a similar pattern. The changes seen in both free and conjugated ubiquitin suggest that their increases are involved in the response to exercise-induced muscle damage.

• del Monte U, Costa RC, Bianchi C
• Effect of aging on ubiquitin-mediated proteolysis.
• Exp Gerontol. 1994; 29: 101-2

• Ciechanover A
• The ubiquitin-mediated proteolytic pathway: mechanisms of action and cellular physiology.
• Biol Chem Hoppe Seyler. 1994; 375: 565-81
• Display abstract

• Ubiquitin modification of many protein targets within cells plays important roles in a variety of biological processes. Among these are regulation of gene expression, regulation of cell cycle and division, involvement in the cellular stress response, modification of cell surface receptors, DNA repair, import of proteins into mitochondria, uptake of precursors of neurotransmitters into synaptosomes, biogenesis of peroxisomes, assembly of ribosomes, and programmed cell death. The mechanisms that underlie these complex processes are poorly understood. The best studied modification occurs in the ubiquitin-mediated proteolytic pathway. Recent experimental evidence indicates that the ubiquitin system is involved in the degradation of mitotic cyclins, oncoproteins and tumor suppressors, in the removal of abnormal and otherwise damaged proteins, and in processing of antigens restricted to class I MHC molecules. Degradation of a protein via the ubiquitin system involves two discrete steps. Initially, multiple ubiquitin molecules are covalently linked in an ATP-dependent mode to the protein substrate. The targeted protein is then degraded by a specific, energy-dependent and high molecular mass protease complex into peptides and free amino acids, and free and reutilizable ubiquitin is released. In addition, stable mono-ubiquitin adducts are also found in the cell, for example, those involving nucleosomal histones. Despite the considerable progress that has been made in elucidating the mode of action and roles of the ubiquitin system, many problems remain unsolved. For example, little is known on the signals that target proteins for degradation. While a few proteins are targeted for degradation following recognition of their N-terminal amino acid residue, the vast majority of cellular proteins are targeted by other signals. The identity of the native cellular substrates of the system is another important, yet unresolved problem: only a few proteins have been recognized so far as substrates of the system in vivo. The scope of this review is to discuss the mechanisms involved in ubiquitin activation, selection of substrates for conjugation, and degradation of ubiquitin-conjugated proteins in the cell-free system. In addition, we shall summarize what is currently known of the physiological roles of ubiquitin-mediated proteolysis in vivo.

• Deveraux Q, Ustrell V, Pickart C, Rechsteiner M
• A 26 S protease subunit that binds ubiquitin conjugates.
• J Biol Chem. 1994; 269: 7059-61
• Display abstract

• Ubiquitin-mediated proteolysis provides an important mechanism for regulating a variety of cellular processes. Ubiquitin-conjugated proteins are degraded by a 26 S protease that contains more than 30 different subunits. Of these, a single 50-kDa polypeptide, subunit 5, specifically binds ubiquitin-lysozyme conjugates. Binding is inhibited by short polymeric chains of ubiquitin but not by ubiquitin monomers or by lysozyme. In addition, subunit 5 binds free ubiquitin chains with efficient association requiring at least four ubiquitins. Thus, proteins conjugated to polymers of ubiquitin may be selected for degradation by a single subunit of the 26 S protease complex.

• Magnani M, Crinelli R, Antonelli A, Casabianca A, Serafini G
• The soluble but not mitochondrially bound hexokinase is a substrate for the ATP- and ubiquitin-dependent proteolytic system.
• Biochim Biophys Acta. 1994; 1206: 180-90
• Display abstract

• Intracellular protein degradation is highly selective, however, the mechanism(s) underlying this selectivity are not fully understood. We have previously shown that purified rabbit hexokinase type I, an enzyme present in mammalian brain both in soluble and mitochondrial bound form, is conjugate to ubiquitin and then degraded by a rabbit reticulocyte fraction II. In the present study we report that the mitochondrial bound hexokinase is stable for several hours in the same proteolytic system both in the presence or absence of ATP. E1, E2 and E3, the enzymes of the ubiquitin conjugating system, are able to incorporate 125I- or biotin-labelled ubiquitin in an ATP-dependent manner in soluble hexokinase as well as in a number of mitochondrial proteins. Furthermore, the mitochondria by themselves have a pronounced ATP-dependent ability to conjugate 125I-ubiquitin. However, Western blotting experiments, using a specific antibody against hexokinase, or against ubiquitin, showed that the mitochondrial bound enzyme is neither ubiquitinated nor degraded. This result has been confirmed by purification of bound hexokinase from the brain mitochondrial fraction or following the incubation of intact mitochondria with ATP, 125I-ubiquitin and E1, E2 and E3. Thus, mitochondrial bound hexokinase is not recognized by the ubiquitin conjugating system while the soluble enzyme is conjugate to ubiquitin and then degraded. Furthermore, the soluble hexokinase from rabbit brain was isolated by immunoaffinity chromatography and shown to be recognized by an anti-ubiquitin antibody. These results suggest that the intracellular distribution of protein is an important feature of a protein which determines its susceptibility to ubiquitin-dependent degradation.

• Huet JC, Salle-Tourne M, Pernollet JC
• Amino acid sequence and toxicity of the alpha elicitin secreted with ubiquitin by Phytophthora infestans.
• Mol Plant Microbe Interact. 1994; 7: 302-4
• Display abstract

• A single alpha elicitin, phytotoxin secreted by Phytophthora infestans, responsible for systemic HR-like necroses in tobacco, was purified from the culture filtrate. Its sequence was compared to other alpha elicitins in correlation with toxicity. In the filtrate, we also found ubiquitin, whose biological function remains unclear.

• Xiao W, Fontanie T, Tang M
• UBP5 encodes a putative yeast ubiquitin-specific protease that is related to the human Tre-2 oncogene product.
• Yeast. 1994; 10: 1497-502
• Display abstract

• A gene from chromosome V of the yeast Saccharomyces cerevisiae has been cloned and sequenced. The deduced amino acid sequence encoded by this gene is similar to several ubiquitin-specific proteases from yeast, especially at the highly conserved domain. It is thus named UBP5. UBP5 is also closely related to the human Tre-2 and the mouse Unp oncogene products. This study adds a new member to the ubiquitin protease family and suggests that alteration of ubiquitin protease activity may result in cancer in mammals. However, disruption of the UBP5 gene in a haploid strain did not result in a noticeable phenotypic alteration.

• Sutrave P, Shafer BK, Strathern JN, Hughes SH
• Isolation, identification and characterization of the FUN12 gene of Saccharomyces cerevisiae.
• Gene. 1994; 146: 209-13
• Display abstract

• We have cloned and characterized the FUN12 gene which is found on chromosome 1 of Saccharomyces cerevisiae. The complete nucleotide (nt) sequence of the cDNA and the genomic clones shows that FUN12 is expressed as a 3.7-kb message and should encode a 97 kDa-protein. Immunoprecipitations using antipeptide antibodies showed that the cells contain a Fun12p of this size. The databases contain no nt sequences that are homologous to FUN12 and no protein homologous to Fun12p. Gene disruption experiments showed that FUN12 is an essential gene.

• Ciechanover A
• The ubiquitin-mediated proteolytic pathway.
• Brain Pathol. 1993; 3: 67-75
• Display abstract

• Ubiquitin modification of a variety of protein targets within the cell plays important roles in many cellular processes. Among these are regulation of gene expression, regulation of cell cycle and division, involvement in the cellular stress response, modification of cell surface receptors, DNA repair, and biogenesis of mitochondria and ribosomes. The best studied modification occurs in the ubiquitin-dependent proteolytic pathway. Degradation of a protein by the ubiquitin system involves two discrete steps. Initially, multiple ubiquitin molecules are covalently linked in an ATP-dependent mode to the protein substrate. The protein moiety of the conjugate is then degraded by a specific protease into free amino acids with the release of free and reutilizable ubiquitin. This process also requires energy. In addition, stable mono-ubiquitin adducts are also found intracellularly, for example, those involving nucleosomal histones. Despite the considerable progress that has been made in elucidating the mode of action and roles of the ubiquitin system, many problems remain unsolved. For example, very little is known about the cellular substrates of the system and the signals that target them for conjugation and degradation. The scope of this review is to summarize briefly what is currently known on the role of the ubiquitin system in protein turnover, and to discuss in detail the mechanisms involved in selection of substrates for conjugation and in degradation of ubiquitin-conjugated proteins.

• Kambouris NG, Burke DJ, Creutz CE
• Cloning and genetic analysis of the gene encoding a new protein kinase in Saccharomyces cerevisiae.
• Yeast. 1993; 9: 141-50
• Display abstract

• We have isolated a single gene from the yeast Saccharomyces cerevisiae encoding a potential 800 amino acid polypeptide of calculated M(r) 90,098 Da. This protein consists of an N-terminal region that shares significant homology with the catalytic domains of several serine- and threonine-specific protein kinases, as well as a large, unique, C-terminal domain of unknown function. Haploid disruption mutants are viable and do not exhibit any readily observable growth defects under varying conditions of temperature, nutrients or osmotic strength. Due to the apparent structural similarity between this kinase and the protein products of the KIN1 and KIN2 genes, we have chosen to name this new gene KIN3.

• Ishida N et al.
• Mos is degraded by the 26S proteasome in a ubiquitin-dependent fashion.
• FEBS Lett. 1993; 324: 345-8
• Display abstract

• Mos, the c-mos proto-oncogene product, is a key regulator of cell cycle progression. Recently, rapid turnover of Mos in an early stage of meiotic maturation of Xenopus oocytes was found to be mediated by the ubiquitin pathway, but the protease responsible for its breakdown was not identified. In the present study, we found that 35S-labeled Mos synthesized in an in vitro transcription/translation system was degraded ATP- and time-dependently by the 26S proteasome, but not by the 20S proteasome, in the presence of a ubiquitin-ligation system. The 26S proteasome did not degrade a mutant Mos in which Ser3 was replaced by Asp3 that is metabolically stable in oocytes, indicating a similarity in the proteolytic events in vivo to those observed in vitro in the present work. This is the first demonstration that the proteasome catalyzes the ATP-dependent degradation of a naturally occurring, short-lived oncoprotein by the ubiquitin pathway. This finding suggests that the proteasome may regulate the intracellular stability of various oncoproteins.

• Sullivan ML, Vierstra RD
• Formation of a stable adduct between ubiquitin and the Arabidopsis ubiquitin-conjugating enzyme, AtUBC1+.
• J Biol Chem. 1993; 268: 8777-80
• Display abstract

• Ubiquitin conjugating enzymes (E2s) are an integral part of a multienzyme pathway that ligates ubiquitin to intracellular target proteins. This ligation has been implicated in a number of fundamental processes including protein degradation, cell cycle progression, DNA repair, and organelle biogenesis. To function, E2s form a labile thiol-ester intermediate between a specific cysteine within the E2 and the carboxyl terminus of ubiquitin; this high energy intermediate then serves as the donor for ubiquitin ligation. To aid in the characterization of E2s, we have created a stable ubiquitin-E2 intermediate using a mutant form of the 16-kDa E2 encoded by the Arabidopsis thaliana AtUBC1 gene in which the active-site cysteine at residue 88 was replaced with serine. The mutant protein synthesized in Escherichia coli formed an adduct with ubiquitin in vitro, but in this case the E2 and ubiquitin were linked via a more stable ester bond. The ester-linked ubiquitin could not be transferred subsequently to substrate proteins in an E3 alpha-dependent conjugation reaction. The ester adduct was sufficiently stable to survive purification by anion exchange high performance liquid chromatography. As a result, this adduct may prove useful for the structural analysis of ubiquitin-E2 intermediates and in the study of E2s interacting with other ubiquitin pathway enzymes.

• Tanaka K, Tamura T
• [Proteolytic bio-system]
• Tanpakushitsu Kakusan Koso. 1993; 38: 1075-88

• Lassalle F, Lassegues M, Roch P
• Serological evidence and amino acid sequence of ubiquitin-like protein isolated from coelomic fluid and cells of the earthworm Eisenia fetida andrei.
• Comp Biochem Physiol B. 1993; 104: 623-8
• Display abstract

• 1. A small protein of M(r) 10 kDa has been isolated by reverse-phase chromatography of the basic proteins contained in the coelomic fluid and cell lysate of the earthworm Eisenia fetida andrei. 2. The protein crossreacted in dot-blot with an anti-bovine ubiquitin antiserum. 3. Its N-terminal primary structure was determined by automatic Edman degradation on 26 consecutive amino acids and showed 69% (based on the 26 amino acids) or 82% (based on the first 19 consecutive amino acids) identity with many ubiquitins and similar charge and hydrophobicity profiles and secondary structure conformation.

• Papa FR, Hochstrasser M
• The yeast DOA4 gene encodes a deubiquitinating enzyme related to a product of the human tre-2 oncogene.
• Nature. 1993; 366: 313-9
• Display abstract

• Modification of specific intracellular proteins by ubiquitin targets them for degradation. We describe a yeast enzyme, Doa4, that is integral to the degradation of ubiquitinated proteins and is required in diverse physiological processes. Doa4 appears to function late in the proteolytic pathway by cleaving ubiquitin from substrate remnants still bound to protease. The human tre-2 oncogene encodes a deubiquitinating enzyme similar to Doa4, indicating a role for the ubiquitin system in mammalian growth control.

• Bednar B, Mandys V
• [Inclusions of cytoplasmic fibrillar degradation and ubiquitin]
• Cesk Patol. 1993; 29: 3-5
• Display abstract

• Cell degradation of various origin often starts with an ubiquitinization of damaged proteins and with later production of roughly fibrillar electron-microscopical inclusions. There is a strong immunohistological positivity of ubiquitin in the early intracellular phase. It tends toward an apoptotic lysis either ending with extracellular immunohistologically inert refuse of amyloid type or representing various inter-steps of the degradation process. The ubiquitinization is the most common in neurons and paraneurons. A general importance of ubiquitinization is proved by ubiquitin fibrillar inclusions which were observed in growing old tissue cultures as an expression of programmed cell death. Immunodetection of increased ubiquitin is labour-saving as compared with some classical methods detecting similar lesions particularly in neuropathology.

• Takizawa N, Takada K, Ohkawa K
• Inhibitory effect of nonenzymatic glycation on ubiquitination and ubiquitin-mediated degradation of lysozyme.
• Biochem Biophys Res Commun. 1993; 192: 700-6
• Display abstract

• We examined ubiquitination and ubiquitin-mediated degradation of glycated protein by rabbit reticulocyte lysate fraction II (ubiquitin-free preparation). Non-glycated lysozyme and three glycated lysozyme preparations with different glucose binding ratios were used as substrates. Glycation sites of the lysozyme were mostly the epsilon-NH2 group of lysine residues, since modification at the alpha-NH2 group of the amino terminal was not detectable. Ubiquitin was conjugated with three glycated lysozyme preparations, which contained 1.4, 2.8 and 4.5 mol glucose per mol, by fraction II supplemented with hemin. The extent of formed conjugates was reduced 81, 72 and 56% of those of ubiquitin-non-glycated lysozyme conjugates, respectively. Additionally, ubiquitin-mediated degradation of the resultant conjugates was reduced and their respective rates were 97, 56 and 19% of that of the non-glycated lysozyme. These results indicated that both ubiquitin conjugation and ubiquitin-mediated degradation of the lysozyme were inhibited by nonenzymatic glucose binding to the lysozyme.

• Yamao F
• [Cell cycle regulation by ubiquitin system]
• Tanpakushitsu Kakusan Koso. 1993; 38: 1527-30

• Hill CP, Johnston NL, Cohen RE
• Crystal structure of a ubiquitin-dependent degradation substrate: a three-disulfide form of lysozyme.
• Proc Natl Acad Sci U S A. 1993; 90: 4136-40
• Display abstract

• Covalent attachment of ubiquitin marks substrates for proteolysis, but features that identify ubiquitination targets such as chicken egg white lysozyme are poorly understood. Recognition of lysozyme first requires reduction of Cys-6 Cys-127, one of its four native disulfide bonds, and Cys-6,Cys-127-carboxymethylated (6,127-rcm) lysozyme can mimic this three-disulfide intermediate. The 6,127-rcm form of lysozyme is known to retain a substantially native-like conformation in solution, and we demonstrate that it is this folded structure that is recognized for ubiquitination. Because native lysozyme is not a substrate, differences between the native and three-disulfide structures must include features responsible for selective ubiquitination. The 1.9-A resolution crystal structure of 6,127-rcm-lysozyme, reported here, affords a view of this ubiquitin-dependent degradation substrate. Two conformers of 6,127-rcm-lysozyme were obtained in the crystal. These differ uniquely from crystal forms of native lysozyme by displacement of the C-terminal residues. The structures suggest that localized unfolding at the C terminus of three-disulfide lysozyme allows the complex of E3 alpha (ubiquitin-protein ligase) and E2 (ubiquitin-carrier protein) to bind to a surface that includes Lys-1 and the putative ubiquitination site Lys-13. From this we infer that the N-terminal and internal substrate recognition sites on the E3 alpha.E2 complex are separated by approximately 20 A.

• Hochstrasser M
• Ubiquitin and intracellular protein degradation.
• Curr Opin Cell Biol. 1992; 4: 1024-31
• Display abstract

• In eukaryotes, the ubiquitin-dependent protoelytic pathway is one of the major routes by which intracellular proteins are selectively destroyed. Recent work has shown that conjugation of ubiquitin to substrate proteins is mediated by a remarkably diverse array of enzymes. Proteolytic targeting may also be regulated at steps between ubiquitination of the substrate and its degradation to peptides by the multisubunit 26S protease. The complexity of the ubiquitin system suggests a central role for protein turnover in eukaryotic cell regulation.

• Wajih N, Siddiqi AR, Kaiser R, Persson B, Zaidi ZH, Jornvall H
• Structural characterization of rabbit brain ubiquitin.
• Protein Seq Data Anal. 1992; 5: 31-2
• Display abstract

• Ubiquitin has been isolated and purified from rabbit brain using gel permeation and reverse-phase high-performance liquid chromatography. The 76-residue protein exhibits one difference towards a murine form, is identical to other characterized vertebrate ubiquitins, and confirms an extensive conservation of the ubiquitin structure. No positional microheterogeneities were detectable between two sub-forms.

• Wiebel FF, Kunau WH
• The Pas2 protein essential for peroxisome biogenesis is related to ubiquitin-conjugating enzymes.
• Nature. 1992; 359: 73-6
• Display abstract

• In the yeast Saccharomyces cerevisiae, PAS genes are essential for the biogenesis and proliferation of peroxisomes. Recently, the first two genes, PAS1 (ref. 3) and PAS3 (ref. 4), have been characterized. Here we report the cloning and sequencing of the PAS2 gene. It encodes a new member of the ubiquitin-conjugating (UBC) protein family and is the first member associated with peroxisomes. The proposed function of the Pas2 protein as a UBC enzyme (UBC10) is supported by the fact that site-directed mutagenesis of a strictly conserved and functionally essential cysteine residue of UBC proteins leads to mutant Pas2 proteins unable to complement pas2 mutant strains. Ubiquitination of proteins is known to play an important part in DNA repair, sporulation, cell cycle control and degradation of abnormal proteins. We provide evidence for a crucial role of the ubiquitin-conjugation pathway in organelle formation.

• Schwartz AL, Ciechanover A
• Ubiquitin-mediated protein modification and degradation.
• Am J Respir Cell Mol Biol. 1992; 7: 463-8
• Display abstract

• Ubiquitin is a small, 8 kD protein found in all eukaryotic cells. It is involved in a wide variety of regulatory roles within the cell, including gene expression, ribosome biosynthesis, receptor expression, and the stress response. The best understood of these is that of ubiquitin-mediated proteolysis, in which ubiquitin is covalently attached to specific protein target substrates that are then recognized and degraded by a high molecular weight protease.

• Jentsch S
• The ubiquitin-conjugation system.
• Annu Rev Genet. 1992; 26: 179-207

• Hershko A, Ciechanover A
• The ubiquitin system for protein degradation.
• Annu Rev Biochem. 1992; 61: 761-807

• von Kampen J, Wettern M
• [Ubiquitin-dependent degradation and modification of proteins]
• Naturwissenschaften. 1992; 79: 163-70
• Display abstract

• A large part of cellular proteins is in a dynamic state of turnover. Protein breakdown is responsible for essential cellular functions like modulation of key enzyme levels or removal of abnormal proteins. A major pathway for this selective proteolysis is mediated by the ubiquitin system, in which proteins are committed to degradation by their ligation to ubiquitin, a highly conserved 76 amino acid polypeptide. Recent evidence indicates that ubiquitination serves other functions besides marking proteins for destruction. As originally described for histones, the activities of several cellular proteins are reversibly regulated by ubiquitination and a successive de-ubiquitination step mediated by the activity of one or more isopeptidases.

• Sommer T, Seufert W
• Genetic analysis of ubiquitin-dependent protein degradation.
• Experientia. 1992; 48: 172-8
• Display abstract

• Selective degradation of cellular proteins serves to eliminate abnormal proteins and to mediate the turnover of certain short-lived proteins, many of which have regulatory functions. In eukaryotes a major pathway for selective protein degradation is ATP-dependent and is mediated by the ubiquitin system. This pathway involves substrate recognition by components of a ubiquitin-protein ligase system, covalent attachment of ubiquitin moieties to proteolytic substrates, and subsequent degradation of these conjugates by a multicatalytic protease complex. Recent genetic evidence suggests that the remarkable selectivity of this process is largely controlled at the level of substrate recognition by the ubiquitin ligase system. In Saccharomyces cerevisiae, ubiquitin-conjugating enzymes UBC1, UBC4 and UBC5 have been identified as key components of this highly conserved degradation pathway. Genetic analysis indicates that ubiquitin-dependent proteolysis is essential for cell viability and that UBC4 and UBC5 enzymes are essential components of the eukaryotic stress response.

• Mayer RJ, Doherty FJ
• Ubiquitin.
• Essays Biochem. 1992; 27: 37-48

• Silver ET, Gwozd TJ, Ptak C, Goebl M, Ellison MJ
• A chimeric ubiquitin conjugating enzyme that combines the cell cycle properties of CDC34 (UBC3) and the DNA repair properties of RAD6 (UBC2): implications for the structure, function and evolution of the E2s.
• EMBO J. 1992; 11: 3091-8
• Display abstract

• The CDC34 (UBC3) protein from Saccharomyces cerevisiae has a 125 residue tail that contains a polyacidic region flanked on either side by sequences of mixed composition. We show that although a catalytic domain is essential for CDC34 activity, a major cell cycle determinant of this enzyme is found within a 74 residue segment of the tail that does not include the polyacidic stretch or downstream sequences. Transposition of the CDC34 tail onto the catalytic domain of a functionally unrelated E2 such as RAD6 (UBC2) results in a chimeric E2 that combines RAD6 and CDC34 activities within the same polypeptide. In addition to the tail, the cell cycle function exhibited by the chimera and CDC34 is probably dependent on a conserved region of the catalytic domain that is shared by both RAD6 and CDC34. Despite this similarity, the CDC34 catalytic domain cannot substitute for the DNA repair and growth functions of the RAD6 catalytic domain, indicating that although these domains are structurally related, sufficient differences exist to maintain their functional individuality. Expression of the CDC34 catalytic domain and tail as separate polypeptides are capable of only partial function; thus, while the tail displays autonomous structural characteristics, there is considerable advantage gained when both domains coexist within the same polypeptide. The ability of these and other derivatives to restore partial function to a cdc34 temperature-sensitive mutant but not to a disruption mutant suggests that interaction between two CDC34 polypeptides is a requirement of CDC34 activity. Based on this idea we propose a model that accounts for the initiating steps leading to multi-ubiquitin chain synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

• Ciechanover A
• The ubiquitin-mediated system for intracellular protein degradation.
• J Basic Clin Physiol Pharmacol. 1991; 2: 141-59
• Display abstract

• Degradation of proteins by the ubiquitin system involves several discrete steps. Initially, multiple molecules of ubiquitin are covalently conjugated to the target substrate in an energy-requiring reaction. The protein thus marked is degraded by a specific ATP-dependent protease, and free and reutilizable ubiquitin is released. In this review we discuss the mechanisms involved in ubiquitin activation, selection of substrates for conjugation, and subsequent degradation of ubiquitin-conjugated proteins in the cell-free system. In addition, we summarize briefly what is currently known of the physiological roles of the ubiquitin system in vivo.

• Rechsteiner M
• Natural substrates of the ubiquitin proteolytic pathway.
• Cell. 1991; 66: 615-8

• Mayer RJ, Arnold J, Laszlo L, Landon M, Lowe J
• Ubiquitin in health and disease.
• Biochim Biophys Acta. 1991; 1089: 141-57
• Display abstract

• Studies in recent years have shown that ubiquitin has increasingly important functions in eukaryotic cells; roles which were previously not suspected in healthy and diseased cells. The interplay between molecular pathological and molecular cell biological findings has indicated that ubiquitin may be pivotal in the cell stress response in chronic degenerative and viral diseases. Furthermore, the studies have led to the notion that ubiquitination may not only serve as a signal for nonlysosomal protein degradation but may be a unifying covalent protein modification for the major intracellular protein catabolic systems; these can act to identify proteins for cytosolic proteinases or direct intact and fragmented proteins into the lysosome system for breakdown to amino acids. This unifying role could explain why ubiquitin is restricted to eukaryotic cells, which possess extensive endomembrane systems in addition to a nuclear envelope. Protein ubiquitination is a feature of most filamentous inclusions and certain other intracellular conglomerates that are found in some degenerative and viral diseases. The detection of ubiquitin-protein conjugates is not of great diagnostic importance in these diseases. Protein ubiquitination is not only essential for the normal physiological turnover of proteins but appears to have been adapted as part of an intracellular surveillance system that can be activated by altered, damaged, or foreign proteins and organelles. The purpose of this system is to isolate and eliminate these noxious structures from the cell: as a cytoprotective mechanism this appears to have evolved in the cell akin perhaps to an 'intracellular immune system'. Other heat shock proteins such as hsp 70 may be involved in this process. It is apparent that ubiquitin has a role in embryonic development. Protein ubiquitination is presumably involved in the reorganisation of cytoplasm that accompanies cell differentiation. Ubiquitin is also necessary for the gross intracellular degradative processes which are consequent upon programmed cell death. Cell elimination is of key importance for a number of developmental morphogenetic changes. An understanding of the molecular details of these processes will no doubt provide further insights into the wide ranging roles of ubiquitin in the life process. As it says in the book 'Ubiquitin'; there is no doubt that ubiquitin is a 'lucky' protein. It is lucky in many ways: lucky for scientific progress, lucky for biomedical scientists and lucky for life! If you have not already done so, why don't you get lucky and look for a role for ubiquitin in your experimental system. As Avram Hershko has said "there is plenty to go round"!

• Hershko A
• The ubiquitin pathway of protein degradation and proteolysis of ubiquitin-protein conjugates.
• Biochem Soc Trans. 1991; 19: 726-9

• Ishibashi Y, Takada K, Joh K, Ohkawa K, Aoki T, Matsuda M
• Ubiquitin immunoreactivity in human malignant tumours.
• Br J Cancer. 1991; 63: 320-2

• Chen Z, Pickart CM
• A 25-kilodalton ubiquitin carrier protein (E2) catalyzes multi-ubiquitin chain synthesis via lysine 48 of ubiquitin.
• J Biol Chem. 1990; 265: 21835-42
• Display abstract

• Target protein multi-ubiquitination involving lysine 48 of ubiquitin (Ub) is known to occur during protein degradation in the ATP- and Ub-dependent proteolytic pathway (Chau, V., Tobias, J. W., Bachmair, A., Marriott, D., Ecker, D. J., Gonda, D. K., and Varshavsky, A. (1989) Science 243, 1576-1583). However, little is known about the enzymatic mechanism of multi-ubiquitination. We show that a purified Ub carrier protein, E2(25)K, catalyzes multi-Ub chain synthesis from purified Ub. Incubation of E2(25)K with Ub activating enzyme (E1), MgATP, and radiolabeled Ub (Mr = 8500) resulted in time dependent appearance of a "ladder" of radiolabeled Ub conjugates with molecular masses of 8.5n kDa, where n = 1, 2, 3, 4... (up to at least n = 10). The kinetics of this conjugative process were consistent with Ub2 acting as a steady-state intermediate. The putative Ub2 product of E2(25)K catalysis was purified and cleaved with a partially purified isopeptidase preparation. The sole cleavage product (Mr = 8500) had a tryptic digest identical to that of authentic Ub, confirming that the original conjugate was Ub2. Tryptic digestion of intact Ub2 gave products consistent with the existence of an isopeptide linkage between the COOH terminus of one Ub and Lys-48 of the other; this structure was confirmed by sequence analysis of the unique Ub2 tryptic fragment. Tryptic digestion of higher order Ubn adducts (n greater than or equal to 4) yielded fragments identical to those of Ub2, indicating that E2(25)K ligates successive Ub molecules primarily or exclusively via Lys-48. Although several other E2s supported synthesis of an apparent Ub2 adduct of undetermined linkage, only E2(25)K was capable of synthesizing multi-Ub chains from isolated Ub. Quantitative analysis of single turnovers showed that transfer from E2(25)K-Ub to Ub and Ub2 occurred with kappa 2 = 488 and 1170 M-1 min-1, respectively, at pH 7.3 and 37 degrees C. These results show that increasing the number of Ub molecules in a chain increases susceptibility to further ubiquitination by E2(25)K. Ub2 was a good substrate for activation by E1 and was readily transferred to E2(25)K. The labile E2(25)K-Ub2 adduct was catalytically active, and exhibited preference for Ub2 (versus Ub) as acceptor. These results suggest that E2(25)K may function as a multi-ubiquitinating enzyme in the Ub-dependent proteolytic pathway.

• Orlowski M
• The multicatalytic proteinase complex, a major extralysosomal proteolytic system.
• Biochemistry. 1990; 29: 10289-97

• Ciechanover A, Gonen H, Elias S, Mayer A
• Degradation of proteins by the ubiquitin-mediated proteolytic pathway.
• New Biol. 1990; 2: 227-34
• Display abstract

• Degradation of a protein by the ubiquitin system involves two distinct processes. In the first step, ubiquitin is covalently linked in an ATP-dependent mode to the protein substrate. The protein moiety of the conjugate is then degraded by a specific protease into free amino acids, resulting in the release of free and reutilizable ubiquitin. This process also requires energy. In this review we will briefly summarize our current knowledge of the role of the ubiquitin system in protein turnover and discuss in detail the mechanism involved in selection of substrates for conjugation and in degradation of ubiquitin-conjugated proteins.

• Seufert W, Jentsch S
• Ubiquitin-conjugating enzymes UBC4 and UBC5 mediate selective degradation of short-lived and abnormal proteins.
• EMBO J. 1990; 9: 543-50
• Display abstract

• Ubiquitin-conjugating enzymes catalyse the covalent attachment of ubiquitin to target proteins. Members of this enzyme family are involved in strikingly diverse cellular functions: UBC2 (RAD6) is central to DNA repair, UBC3 (CDC34) is involved in cell cycle control. We have cloned the genes for two novel ubiquitin-conjugating enzymes, UBC4 and UBC5, from the yeast Saccharomyces cerevisiae. These enzymes mediate selective degradation of short-lived and abnormal proteins. UBC4 and UBC5 are closely related in sequence and complementing in function. Expression of UBC4 and UBC5 genes is heat inducible. UBC4 and UBC5 enzymes generate high mol. wt ubiquitin-protein conjugates in vivo consistent with previous studies which suggested that attachment of multiple ubiquitin molecules to proteolytic substrates is required for their selective degradation. UBC4 and UBC5 enzymes comprise a major part of total ubiquitin-conjugation activity in stressed cells. Turnover of short-lived proteins and canavanyl-peptides but not of long-lived proteins is markedly reduced in ubc4ubc5 mutants. Loss of UBC4 and UBC5 activity impairs cell growth, leads to inviability at elevated temperatures or in the presence of an amino acid analog, and induces the stress response.

• Meyers G, Rumenapf T, Thiel HJ
• Ubiquitin in a togavirus.
• Nature. 1989; 341: 491-491

• Doherty FJ, Laszlo L, Lowe J, Lennox G, Landon M, Mayer RJ
• Ubiquitin-protein conjugates: clinical and experimental findings.
• Revis Biol Celular. 1989; 20: 255-73
• Display abstract

• Ubiquitin has been extensively studied as a protein which is a cofactor in extralysosomal protein degradation, particularly in reticulocyte lysates. Ubiquitin is also found conjugated to nuclear histones and surface receptors in somatic cells. Until recently the occurrence of stable cellular ubiquitin-protein conjugates in physiological and pathological states had not been considered and studied. Recently we have shown that ubiquitin-protein conjugate immunoreactivity is a clinical feature in several ostensibly unrelated chronic human degenerative diseases as well as in some viral diseases. The consistent observation is the occurrence of intracellular extralysosomal inclusions containing intermediate filaments and ubiquitin conjugates as determined by immunohistochemical methods. These diseases are therefore part of a family of intermediate filament-ubiquitin diseases. The involvement of intermediate filaments with ubiquitin, a protein of known significance in protein degradation, ties in with separate evidence for a close role between intermediate filaments and protein degradation. We have previously shown that intermediate filaments may be involved in protein sequestration for degradation in the lysosomal system. Clinical immunohistochemical observations suggest that elements of the lysosomal degradation system and the ubiquitin-dependent extralysosomal system are involved in the molecular pathogenesis of some diseases. To underpin these clinical observations, we have recently shown that ubiquitin-protein conjugates accumulate in lysosome-related multivesicular bodies in cells in which lysosomal degradation is impaired. This phenomenon may result from increased ubiquitin protein-conjugate formation in cells with a compromised lysosomal system followed by chance uptake into multivesicular bodies. Alternatively, ubiquitination may normally serve as a signal for protein uptake into the lysosomal system, ubiquitinated protein-conjugates may therefore accumulate in cells with a functionally impaired lysosomal system.

• Sullivan ML, Vierstra RD
• A ubiquitin carrier protein from wheat germ is structurally and functionally similar to the yeast DNA repair enzyme encoded by RAD6.
• Proc Natl Acad Sci U S A. 1989; 86: 9861-5
• Display abstract

• The RAD6 gene from the yeast Saccharomyces cerevisiae encodes a ubiquitin carrier protein (E2) required for a variety of cellular processes including DNA repair, induced mutagenesis, and sporulation. Here we identify an E2 from a higher plant, wheat, that is similar to RAD6 with respect to both structure and in vitro substrate specificity. The protein was purified from wheat germ by a combination of ubiquitin covalent affinity chromatography and anion-exchange HPLC and has an apparent molecular mass of 23 kDa [referred to as E2(23 kDa)]. E2(23 kDa) was capable of binding ubiquitin by means of a thiol ester linkage in an ATP-dependent and ubiquitin-activating enzyme-dependent reaction. In the presence of a variety of target proteins, E2(23 kDa), like the RAD6 gene product, formed covalent ubiquitin-protein conjugates in vitro only with histones in a ubiquitin protein ligase-independent reaction. E2(23 kDa) recognized both core and linker histones with an apparent order of preference of H2A greater than or equal to H1 greater than H2B greater than H3 greater than H4. This E2 protein was approximately 17-fold more effective at conjugating ubiquitin to histones than three other purified wheat germ E2 proteins tested. Mouse anti-E2(23 kDa) antibodies were used to isolate E2(23 kDa) DNA sequences from a wheat cDNA expression library. Antibody-positive clones were confirmed by amino acid identity of the sequence deduced from the cDNA to the peptide sequence of an E2(23 kDa) tryptic fragment. Protein expressed in Escherichia coli by the E2(23 kDa) cDNA was capable of both thiol ester adduct formation and conjugation of ubiquitin to histones. Analysis of the E2(23 kDa) cDNA shows that it encodes a protein with considerable amino acid sequence similarity to the yeast RAD6 gene product. Similarities exist at the amino terminus, the region surrounding the putative ubiquitin binding site, and at the carboxyl terminus, which is unusually acidic. Based on both the structural and enzymatic similarities to the RAD6 gene product, E2(23 kDa) may represent the first DNA repair enzyme identified in higher plants.

• Wang XM, Huang CH
• [Structure and function of ubiquitin]
• Sheng Li Ke Xue Jin Zhan. 1988; 19: 64-5

• Hershko A
• Ubiquitin-mediated protein degradation.
• J Biol Chem. 1988; 263: 15237-40

• McGuire MJ, Reckelhoff JF, Croall DE, DeMartino GN
• An enzyme related to the high molecular weight multicatalytic proteinase, macropain, participates in a ubiquitin-mediated, ATP-stimulated proteolytic pathway in soluble extracts of BHK 21/C13 fibroblasts.
• Biochim Biophys Acta. 1988; 967: 195-203
• Display abstract

• Soluble, cell-free extracts of BHK 21/C13 fibroblasts degraded a variety of exogenous proteins to acid-soluble peptides at pH 8.0. ATP stimulated the rates of proteolysis. Both the absolute rate of proteolysis and the magnitude of the ATP effect depended on the specific substrate. For example, casein was degraded approximately 10-fold faster than lysozyme, but lysozyme degradation was more highly stimulated by ATP than was casein degradation. Ubiquitin enhanced the ATP-stimulated proteolysis of each substrate in both postmicrosomal extracts and DEAE-cellulose fractionated extracts. In each extract, ubiquitin enhanced the ATP-stimulated degradation of lysozyme to a greater degree than that of casein. These results suggested that lysozyme was degraded by a pathway that was more dependent upon ubiquitin than was casein. Further evidence for this conclusion was obtained in studies using substrates whose amino groups were blocked by extensive methylation or carbamoylation. The high molecular weight proteinase, macropain, appears to be involved in the ATP-stimulated degradation of both substrates. Specific immunoprecipitation of macropain with polyclonal antibodies resulted in the inhibition of ATP-stimulated proteinase activity both in the absence and presence of ubiquitin. These results indicate that macropain plays a role in both ubiquitin-mediated and ubiquitin-independent ATP-stimulated proteolysis in BHK cell extracts.

• Cook J, Chock PB
• Ubiquitin: a review on a ubiquitous biofactor in eukaryotic cells.
• Biofactors. 1988; 1: 133-46

• Vierstra RD, Sullivan ML
• Hemin inhibits ubiquitin-dependent proteolysis in both a higher plant and yeast.
• Biochemistry. 1988; 27: 3290-5
• Display abstract

• In eukaryotes, a major route for ATP-dependent protein breakdown proceeds through covalent intermediates of target proteins destined for degradation and the highly conserved, 76 amino acid protein ubiquitin. In rabbit reticulocytes, it has been shown that hemin effectively inhibits this pathway by blocking the catabolism of ubiquitin-protein conjugates [KI = 25 microM (Haas, A. L., & Rose, I. A. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6845-6848)]. Here, we demonstrate that hemin is also an effective inhibitor of the ubiquitin-dependent proteolytic pathway in both a higher plant, oats (Avena sativa), and yeast (Saccharomyces cerevisiae). Hemin inhibits all stages of the pathway in vitro, including ATP-dependent formation of ubiquitin-protein conjugates, disassembly of conjugates by ubiquitin-protein lyase(s) (or isopeptidases), and degradation of ubiquitin-protein conjugates by ATP-dependent protease(s). Using ubiquitin-125I-lysozyme conjugates synthesized in vitro as substrates, we determined the specific effects of hemin on the rates of disassembly and degradation separately. The concentration of hemin required for half-maximal inhibition of both processes was identical in each species, approximately 60 microM in oats and approximately 50 microM in yeast. Similar inhibitory effects were observed when two hemin analogues, mesoheme or protoporphyrin IX, were employed. These results demonstrate that the effect of hemin on ubiquitin-dependent proteolysis is not restricted to erythroid cells and as a result hemin may be a useful tool in studies of this pathway in all eukaryotic cells. These results also question models where hemin serves as a specific negative modulator of proteolysis in erythroid cells.

• Ecker DJ et al.
• Gene synthesis, expression, structures, and functional activities of site-specific mutants of ubiquitin.
• J Biol Chem. 1987; 262: 14213-21
• Display abstract

• To study the structure and function of ubiquitin we have chemically synthesized a ubiquitin gene that encodes the amino acid sequence of animal ubiquitin, inserting a series of restriction enzyme sites that divide the gene into eight "mutagenesis modules." A series of site-specific mutations were constructed to selectively perturb various regions of the molecule. The mutant genes were expressed in a large quantity of Escherichia coli, and the modified proteins were purified. To determine the structural effects of the amino acid substitutions, the solution structure of ubiquitin was investigated by two-dimensional NMR and each of the mutant proteins were screened for structural perturbations. With one exception, virtually no changes were seen other than at the point of mutation. Functional studies of the mutant proteins with the ubiquitin-activating enzyme E1 and in the reticulocyte protein degradation assay were used to identify regions of the molecule important to ubiquitin's activity in intracellular proteolysis.

• Jentsch S, McGrath JP, Varshavsky A
• The yeast DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme.
• Nature. 1987; 329: 131-4
• Display abstract

• The RAD6 gene of the yeast Saccharomyces cerevisiae is required for a variety of cellular functions including DNA repair. The discovery that the RAD6 gene product can catalyse the covalent attachment of ubiquitin to other proteins suggests that the multiple functions of the RAD6 protein are mediated by its ubiquitin-conjugating activity.

• Baker RT, Board PG
• Nucleotide sequence of a human ubiquitin Ub B processed pseudogene.
• Nucleic Acids Res. 1987; 15: 4352-4352

• Ciechanover A
• Regulation of the ubiquitin-mediated proteolytic pathway: role of the substrate alpha-NH2 group and of transfer RNA.
• J Cell Biochem. 1987; 34: 81-100
• Display abstract

• Degradation of intracellular proteins via the ubiquitin pathway involves several steps. In the initial event, ubiquitin becomes covalently linked to the protein substrate in an ATP-requiring reaction. Following ubiquitin conjugation, the protein moiety of the adduct is selectively degraded with the release of free and reusable ubiquitin. Ubiquitin modification of a variety of protein targets in the cell plays a role in basic cellular functions. Modification of core nucleosomal histones is probably involved in regulation of gene expression at the level of chromatin structure. Ubiquitin attachment to cell surface proteins may play roles in processes of cell-cell interaction and adhesion, and conjugation of ubiquitin to other yet to be identified protein(s) could be involved in the progression of cells through the cell cycle. Despite the considerable progress that has been made in the elucidation of the mode of action and cellular roles of the ubiquitin pathway, many major problems remain unsolved. A problem of central importance is the specificity in the ubiquitin ligation system. Why are certain proteins conjugated and committed for degradation, whereas other proteins are not? A free alpha-NH2 group is an important feature of the protein structure recognized by the ubiquitin conjugation system, and tRNA is required for the conjugation of ubiquitin to selective proteolytic substrates and for their subsequent degradation. These findings can shed light on some of the features of a substrate that render it susceptible to ubiquitin-mediated degradation.

• Rechsteiner M
• Ubiquitin-mediated pathways for intracellular proteolysis.
• Annu Rev Cell Biol. 1987; 3: 1-30
• Display abstract

• Ubiquitination is one of several ways in which cells modify their proteins. As for phosphorylation or acetylation, there are distinct enzymes for adding and removing Ub from the surfaces of protein substrates. The dynamic equilibration of Ub with cellular proteins is also typical of most posttranslational modifications. Ubiquitination differs, however, in that the added group is large compared to acetate or phosphate. Its size must provide great potential for recognition by other cellular proteins. Ub may be the cell's reversible cross-linking reagent, covalently bound to protein substrates at one end and noncovalently associated with various Ub binding proteins at the other. It is likely that one ubiquitin binding protein is a component of the 26S ATP-dependent protease. The presence of Ub on histones and on the lymphocyte homing receptor suggests that ubiquitination does not serve exclusively to mark proteins for degradation. There are probably various ubiquitin binding proteins since Ub appears to be a multifunctional protein that affects chromatin structure, intracellular proteolysis, cellular interactions, and the stress response. This abundant protein may serve as an intracellular barometer whose distribution among several pools regulates a variety of processes.

• Cox MJ, Haas AL, Wilkinson KD
• Role of ubiquitin conformations in the specificity of protein degradation: iodinated derivatives with altered conformations and activities.
• Arch Biochem Biophys. 1986; 250: 400-9
• Display abstract

• Three iodinated derivatives of ubiquitin have been synthesized and these derivatives have been characterized in the ubiquitin-dependent protein degradation system. With chloramine-T as the oxidant, a derivative containing monoiodotyrosine is formed in the presence of 1 M KI and a derivative containing diiodotyrosine is produced in the presence of 1 mM KI. These derivatives exhibit phenolate ionizations at pH 9.2 and 7.9 with absorbance maxima at 305 and 314 nm, respectively. In addition to modification of the tyrosine residue, these conditions lead to the oxidation of the single methionine residue and iodination of the single histidine residue [M.J. Cox, R. Shapira, and K.D. Wilkinson (1986) Anal. Biochem. 154, 345-352]. Iodination of ubiquitin under these conditions renders the protein sensitive to hydrolysis by trypsin and results in an enhanced susceptibility to alcohol-induced helix formation. When the derivatives are tested in the ATP: pyrophosphate exchange reaction catalyzed by the ubiquitin adenylating enzyme, they are found to exhibit activity comparable to the native protein. When these derivatives are tested for the ability to act as a cofactor in the ubiquitin-dependent protein degradation system, they are both found to support a rate of protein degradation that is twice that of native ubiquitin. At high concentrations of derivatives, the rate of protein degradation is inhibited, while the steady state level of conjugates increases. Thus, the free derivatives inhibit the protease portion of the reaction, but are fully active in the activation and conjugation portions of the reaction. With iodine as the modification reagent, monoiodination of tyrosine is the predominant reaction. This derivative exhibits activity similar to native ubiquitin. Thus, it appears that modification of the histidine residue is responsible for the increased activity of the more highly iodinated derivatives. The enzymes of the system must recognize different portions of the ubiquitin structure, or different conformations of ubiquitin that are affected by the iodination of the histidine residue. These results suggest a conformational change of the ubiquitin molecule may be important in determining the rate and specificity of proteolysis.

• Hershko A, Ciechanover A
• The ubiquitin pathway for the degradation of intracellular proteins.
• Prog Nucleic Acid Res Mol Biol. 1986; 33: 19-56

• Ciechanover A, Finley D, Varshavsky A
• Mammalian cell cycle mutant defective in intracellular protein degradation and ubiquitin-protein conjugation.
• Prog Clin Biol Res. 1985; 180: 17-31
• Display abstract

• Ubiquitin, a 76 residue protein, occurs in eukaryotic cells either free or covalently joined via its carboxyl terminus to epsilon-amino groups of lysine residues in a wide variety of protein species. Previous work has shown that ubiquitin-protein conjugates are preferred substrates in vitro for a non-lysosomal ATP-dependent proteolytic pathway, suggesting that ubiquitin may function as a signal for attack by proteinases specific for ubiquitin-protein conjugates. One strategy to define the potential significance of the ubiquitin-dependent proteolytic pathway is to identify conditional mutants in the pathway. ts85 is a mouse derived cell-cycle mutant which has been shown to lose uH2A, a specific ubiquitin-histone H2A conjugate, at the nonpermissive temperature. We show that the loss of uH2A from ts85 cells is due to reduced ubiquitin-protein conjugation. We further show that the reduced conjugation is due to the specific thermolability of ubiquitin activating enzyme, E1, one of the three enzymic components of the ubiquitin-protein ligase system. We therefore proceeded to test whether the degradation of short-lived proteins is also temperature-sensitive in ts85 cells. Indeed, while more than 70% of the prelabeled abnormal (amino acid analog-containing) proteins or puromycyl peptides are degraded within 4 hours at the permissive temperature in the mutant (ts85), wild type (FM3A), and revertant (ts85R-MN3) cells, less than 15% of these proteins are degraded in ts85 cells at the nonpermissive temperature. In contrast, the rate of degradation of these proteins does not change significantly in either wild-type or revertant cells between permissive and nonpermissive temperatures. Degradation of normal short-lived proteins is also specifically temperature-sensitive in ts85 cells. Immunochemical analysis shows a strong and specific reduction in ubiquitin-protein conjugate levels in vivo at the nonpermissive temperature in ts85 cells. Taken together, our in vitro and in vivo findings with ts85 cells demonstrate that the degradation of the bulk of short-lived proteins in this higher eukaryotic cell is accomplished through a ubiquitin-mediated pathway.

• Hershko A, Heller H, Eytan E, Kaklij G, Rose IA
• Role of the alpha-amino group of protein in ubiquitin-mediated protein breakdown.
• Proc Natl Acad Sci U S A. 1984; 81: 7021-5
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• Previous studies suggest that the conjugation of ubiquitin to NH2 groups of proteins is required for protein breakdown. We now show that the selective modification of NH2-terminal alpha-NH2 groups of globin and lysozyme prevents their degradation by the ubiquitin proteolytic system from reticulocytes. The conjugation by ubiquitin of epsilon-NH2 groups of lysine residues, usually seen in multiples, was also inhibited in alpha-NH2-blocked proteins. Naturally occurring N alpha-acetylated proteins are not degraded by the ubiquitin system at a significant rate, while their nonacetylated counterparts from other species are good substrates. This suggests that one function of N alpha-acetylation of cellular proteins is to prevent their degradation by the ubiquitin system. alpha-NH2-blocked proteins can have their activity as substrates for degradation increased by incorporation of alpha-NH2 groups through the introduction of polyalanine side chains. Proteins in which most epsilon-NH2 groups are blocked but the alpha-NH2 group is free are degraded by the ubiquitin system, but at a reduced rate. It is therefore suggested that the exposure of a free NH2 terminus of proteins is required for degradation and probably initiates the formation of ubiquitin conjugates committed for degradation.

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