SMART MODE:

NORMAL GENOMIC

• Marmorstein LY et al.
• A human BRCA2 complex containing a structural DNA binding component influences cell cycle progression.
• Cell. 2001; 104: 247-57
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• Germline mutations of the human BRCA2 gene confer susceptibility to breast cancer. Although the function of the BRCA2 protein remains to be determined, murine cells homozygous for BRCA2 inactivation display chromosomal aberrations. We have isolated a 2 MDa BRCA2-containing complex and identified a structural DNA binding component, designated as BRCA2-Associated Factor 35 (BRAF35). BRAF35 contains a nonspecific DNA binding HMG domain and a kinesin-like coiled coil domain. Similar to BRCA2, BRAF35 mRNA expression levels in mouse embryos are highest in proliferating tissues with high mitotic index. Strikingly, nuclear staining revealed a close association of BRAF35/BRCA2 complex with condensed chromatin coincident with histone H3 phosphorylation. Importantly, antibody microinjection experiments suggest a role for BRCA2/BRAF35 complex in modulation of cell cycle progression.

• Ptak C, Gwozd C, Huzil JT, Gwozd TJ, Garen G, Ellison MJ
• Creation of a pluripotent ubiquitin-conjugating enzyme.
• Mol Cell Biol. 2001; 21: 6537-48
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• We describe the creation of a pluripotent ubiquitin-conjugating enzyme (E2) generated through a single amino acid substitution within the catalytic domain of RAD6 (UBC2). This RAD6 derivative carries out the stress-related function of UBC4 and the cell cycle function of CDC34 while maintaining its own DNA repair function. Furthermore, it carries out CDC34's function in the absence of the CDC34 carboxy-terminal extension. By using sequence and structural comparisons, the residues that define the unique functions of these three E2s were found on the E2 catalytic face partitioned to either side by a conserved divide. One of these patches corresponds to a binding site for both HECT and RING domain proteins, suggesting that a single substitution in the catalytic domain of RAD6 confers upon it the ability to interact with multiple ubiquitin protein ligases (E3s). Other amino acid substitutions made within the catalytic domain of RAD6 either caused loss of its DNA repair function or modified its ability to carry out multiple E2 functions. These observations suggest that while HECT and RING domain binding may generally be localized to a specific patch on the E2 surface, other regions of the functional E2 face also play a role in specificity. Finally, these data also indicate that RAD6 uses a different functional region than either UBC4 or CDC34, allowing it to acquire the functions of these E2s while maintaining its own. The pluripotent RAD6 derivative, coupled with sequence, structural, and phylogenetic data, suggests that E2s have diverged from a common multifunctional progenitor.

• Kakhniashvili DG, Chaudhary T, Zimmer WE, Bencsath FA, Jardine I, Goodman SR
• Erythrocyte spectrin is an E2 ubiquitin conjugating enzyme.
• Biochemistry. 2001; 40: 11630-42
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• The involvement of red blood cell spectrin in the ubiquitination process was studied. Spectrin was found to form two ubiquitin-associated derivatives, a DTT-sensitive ubiquitin adduct and a DTT-insensitive conjugate, characteristic intermediate and final products of the ubiquitination reaction cascade. In addition to spectrin and ubiquitin, ubiquitin-activating enzyme (E1) and ATP were necessary and sufficient to form both the spectrin-ubiquitin adduct and conjugate. No exogenous ubiquitin-conjugating (E2) or ligase (E3) activities were required, suggesting that erythrocyte spectrin is an E2 ubiquitin-conjugating enzyme able to target itself. Both ubiquitin adduct and conjugate were linked to the alpha subunit of spectrin, suggesting that the ubiquitin-conjugating (UBC) domain and its target regions reside on the same subunit.

• Niwa J, Ishigaki S, Doyu M, Suzuki T, Tanaka K, Sobue G
• A novel centrosomal ring-finger protein, dorfin, mediates ubiquitin ligase activity.
• Biochem Biophys Res Commun. 2001; 281: 706-13
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• We cloned a novel gene, Dorfin (double ring-finger protein), from human spinal cord. The Dorfin mRNA transcript was 4.4 kb and expressed ubiquitously in many organs as well as in the central nervous system, including the spinal cord. Dorfin encoded 838 amino acid protein Dorfin, which contains two RING-finger motifs and an IBR (in between RING-fingers) motif at its N-terminus. Dorfin is a short-lived protein. Treatment with MG132, a potent proteasome inhibitor, resulted in the accumulation of ubiquitinated Dorfin and Dorfin-associated cellular proteins in cultured cells. Dorfin bound specifically with human ubiquitin-conjugating enzymes UbcH7 and UbcH8 through the RING-finger/IBR domain. Partial deletion of the RING-finger/IBR domain eliminated these interaction and ubiquitination activities. These results strongly suggest that Dorfin is a new member of RING-finger type ubiquitin ligase. Dorfin is localized in the centrosome and probably functions in the microtubule organizing centers. Copyright 2001 Academic Press.

• Moraes TF et al.
• Crystal structure of the human ubiquitin conjugating enzyme complex, hMms2-hUbc13.
• Nat Struct Biol. 2001; 8: 669-73
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• The ubiquitin conjugating enzyme complex Mms2-Ubc13 plays a key role in post-replicative DNA repair in yeast and the NF-kappaB signal transduction pathway in humans. This complex assembles novel polyubiquitin chains onto yet uncharacterized protein targets. Here we report the crystal structure of a complex between hMms2 (Uev1) and hUbc13 at 1.85 A resolution and a structure of free hMms2 at 1.9 A resolution. These structures reveal that the hMms2 monomer undergoes a localized conformational change upon interaction with hUbc13. The nature of the interface provides a physical basis for the preference of Mms2 for Ubc13 as a partner over a variety of other structurally similar ubiquitin-conjugating enzymes. The structure of the hMms2-hUbc13 complex provides the conceptual foundation for understanding the mechanism of Lys 63 multiubiquitin chain assembly and for its interactions with the RING finger proteins Rad5 and Traf6.

• Huang G, Shigesada K, Ito K, Wee HJ, Yokomizo T, Ito Y
• Dimerization with PEBP2beta protects RUNX1/AML1 from ubiquitin-proteasome-mediated degradation.
• EMBO J. 2001; 20: 723-33
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• The RUNX family genes are the mammalian homologs of the DROSOPHILA: genes runt and lozenge, and members of this family function as master regulators of definitive hematopoiesis and osteogenesis. The RUNX genes encode the alpha subunit of the transcription factor PEBP2/CBF. The beta subunit consists of the non-RUNX protein PEBP2beta. We found that RUNX1/AML1, which is essential for hematopoiesis, is continuously subjected to proteolytic degradation mediated by the ubiquitin-proteasome pathway. When PEBP2beta is present, however, the ubiquitylation of RUNX1 is abrogated and this causes a dramatic inhibition of RUNX1 proteolysis. Heterodimerization between PEBP2beta and RUNX1 thus appears to be an essential step in the generation of transcriptionally competent RUNX1. Consistent with this notion, RUNX1 was barely detected in PEBP2beta(-/-) mouse. CBF(PEBP2)beta- SMMHC, the chimeric protein associated with inv(16) acute myeloid leukemia, was found to protect RUNX1 from proteolytic degradation more efficiently than PEBP2beta. These results reveal a hitherto unknown and major role of PEBP2beta, namely that it regulates RUNX1 by controlling its turnover. This has allowed us to gain new insights into the mechanism of leukemogenesis by CBFbeta-SMMHC.

• Goodman SR et al.
• Erythrocyte Spectrin Is An E2 Ubiquitin Conjugating Enzyme.
• Cell Mol Biol Lett. 2001; 6: 205-205

• Lee SJ, Choi JY, Sung YM, Park H, Rhim H, Kang S
• E3 ligase activity of RING finger proteins that interact with Hip-2, a human ubiquitin-conjugating enzyme.
• FEBS Lett. 2001; 503: 61-4
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• To identify proteins that interact with Huntingtin-interacting protein-2 (Hip-2), a ubiquitin-conjugating enzyme, a yeast two-hybrid screen system was used to isolate five positive clones. Sequence analyses showed that, with one exception, all Hip-2-interacting proteins contained the RING finger motifs. The interaction of Hip-2 with RNF2, one of the clones, was further confirmed through in vitro and in vivo experiments. Mutations in the RING domain of RNF2 prevented the clone from binding to Hip-2, an indication that the RING domain is the binding determinant. RNF2 showed a ubiquitin ligase (E3) activity in the presence of Hip-2, suggesting that a subset of RING finger proteins may have roles as E3s.

• Ardley HC, Tan NG, Rose SA, Markham AF, Robinson PA
• Features of the parkin/ariadne-like ubiquitin ligase, HHARI, that regulate its interaction with the ubiquitin-conjugating enzyme, Ubch7.
• J Biol Chem. 2001; 276: 19640-7
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• We recently reported the identification of a RING finger-containing protein, HHARI (human homologue of Drosophila ariadne), which binds to the human ubiquitin-conjugating enzyme UbcH7 in vitro. We now demonstrate that HHARI interacts and co-localizes with UbcH7 in mammalian cells, particularly in the perinuclear region. We have further defined a minimal interaction region of HHARI comprising residues 186-254, identified individual amino acid residues essential for the interaction, and determined that the distance between the RING1 finger and IBR (in between RING fingers) domains is critical to maintaining binding. We have also established that the RING1 finger of HHARI cannot be substituted for by the highly homologous RING finger domains of either of the ubiquitin-protein ligase components c-CBL or Parkin, despite their similarity in structure and their independent capabilities to bind UbcH7. Furthermore, mutation of the RING1 finger domain of HHARI from a RING-HC to a RING-H2 type abolishes interaction with UbcH7. These studies demonstrate that very subtle changes to the domains that regulate recognition between highly conserved components of the ubiquitin pathway can dramatically affect their ability to interact.

• Kulikova DA, Mertsalov IB, Ninkina NN, Simonova OB, Bukhman VL, Korochkin LI
• Genomic organization of the mouse ubi-d4/requiem gene.
• Dokl Biol Sci. 2000; 370: 84-7

• Lester D, Farquharson C, Russell G, Houston B
• Identification of a family of noncanonical ubiquitin-conjugating enzymes structurally related to yeast UBC6.
• Biochem Biophys Res Commun. 2000; 269: 474-80
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• Ubiquitin-conjugating enzymes (UBCs) selectively target proteins for proteasomal degradation by the covalent attachment of ubiquitin moieties. Yeast UBC6 is unusual in having an active site distinct from all other UBCs and in possessing a transmembrane domain that anchors it to the cytoplasmic surface of the endoplasmic reticulum. During a differential display analysis on chick growth plate chondrocytes we isolated a cDNA encoding a noncanonical ubiquitin-conjugating enzyme (NCUBE1) structurally similar to yeast UBC6. Chick NCUBE1 transcripts were detected in all tissues examined and decreased threefold during chondrocyte terminal differentiation. Database searches identified other related proteins; the human and mouse orthologues of NCUBE1, a second human homologue of yeast UBC6 (NCUBE2), and related proteins from S. pombe, C. elegans, and P. mariana. Together with yeast UBC6 these proteins constitute a distinct family of UBCs sharing a conserved noncanonical active site sequence and a C-terminal transmembrane domain. By analogy with yeast UBC6 they are likely to be localised to the endoplasmic reticulum where they may be involved in targeting retrotranslocated, ER-associated proteins for proteasomal degradation.

• Freiberg G, Mesecar AD, Huang H, Hong JY, Liebman SW
• Characterization of novel rad6/ubc2 ubiquitin-conjugating enzyme mutants in yeast.
• Curr Genet. 2000; 37: 221-33
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• Null mutations in the RAD6/UBC2 gene encoding an E2 ubiquitin-conjugating enzyme cause deficiencies in DNA repair, N-end-rule protein degradation, sporulation and telomeric silencing, and alter the preferred integration positions for Ty1 retrotransposons. Here we selected for mutants of RAD6 that cause a release of telomeric silencing. Some alleles retained nearly wild-type ability for sporulation, DNA repair and the degradation of proteins. Alteration in Ty1 integration-site bias accompanied some of these alleles. The possibility that some mutations specifically affect binding of an unknown protein that works with Rad6 in its silencing role, but is not required for DNA repair or N-end-rule activity, is discussed in terms of the Rad6 crystal structure.

• Richards DE, Peng J, Harberd NP
• Plant GRAS and metazoan STATs: one family?
• Bioessays. 2000; 22: 573-7
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• GRAS is a recently discovered family of plant-specific proteins that play important regulatory roles in diverse aspects of plant development. Several of the motifs present in the GRAS proteins suggest that they function as transcription factors, although homology-searching programs have revealed no significant similarity to any non-plant proteins. Here we propose that the GRAS proteins are related to the Signal Transducers and Activators of Transcription (STAT) family of proteins. STATs are known in many non-plant species, and act as intracellular intermediaries between extracellular ligands and the transcription and activation of genes. Our hypothesis is that the GRAS proteins perform this function in plants, with mechanisms similar to those of the animal STATs. If true, this hypothesis has important implications for the evolution of phosphotyrosine based signal transduction systems in eukaryotic organisms. BioEssays 22:573-577, 2000.

• Molkentin JD
• The zinc finger-containing transcription factors GATA-4, -5, and -6. Ubiquitously expressed regulators of tissue-specific gene expression.
• J Biol Chem. 2000; 275: 38949-52

• van Dongen MJ, Cederberg A, Carlsson P, Enerback S, Wikstrom M
• Solution structure and dynamics of the DNA-binding domain of the adipocyte-transcription factor FREAC-11.
• J Mol Biol. 2000; 296: 351-9
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• Transcription factors of the forkhead type share a highly conserved DNA-binding domain of about 100 amino acid residues. FREAC-11, expressed in adipocytes, belongs to this class. Here, we report on NMR studies that established the three-dimensional structure of the FREAC-11, DNA-binding domain. Although apparent similarities to the structures of other members within the forkhead family are observed, the structure also reveals some remarkable differences. Along with the complementary dynamics, the data provide insight into the fundamentals of sequence specificity within a highly conserved motif.

• Bell KM, Western PS, Sinclair AH
• SOX8 expression during chick embryogenesis.
• Mech Dev. 2000; 94: 257-60
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• We have isolated the SOX8 gene from the chicken embryo. This gene shows a high degree of sequence homology to SOX9 and SOX10. Detailed analysis of SOX8 expression by whole-mount in situ shows a dynamic and restricted expression pattern during chick development. SOX8 is expressed in the somitic derivative, the dermomyotome, the developing heart, pancreas, enteric neurone system, limb and the neural tube. This is the first detailed expression analysis of SOX8 in any species

• Monzingo AF, Breksa A, Ernst S, Appling DR, Robertus JD
• The X-ray structure of the NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Saccharomyces cerevisiae.
• Protein Sci. 2000; 9: 1374-81
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• Eucaryotes possess one or more NADP-dependent methylene-THF dehydrogenases as part of multifunctional enzymes. In addition, yeast expresses an unusual monofunctional NAD-dependent enzyme, yMTD. We report X-ray structures for the apoenzyme and its complex with NAD+ at 2.8 and 3.0 A resolution, respectively. The protein fold resembles that seen for the human and Escherichia coli dehydrogenase/cyclohydrolase bifunctional enzymes. The enzyme has two prominent domains, with the active site cleft between them. yMTD has a noncanonical NAD-binding domain that has two inserted strands compared with the NADP-binding domains of the bifunctional enzymes. This insert precludes yMTD from dimerizing in the same way as the bifunctional enzymes. yMTD functions as a dimer, but the mode of dimerization is novel. It does not appear that the difference in dimerization accounts for the difference in cofactor specificity or for the loss of cyclohydrolase activity. These functional differences are probably accounted for by minor differences within the tertiary structure of the active site of the monomeric protein.

• Perdomo J, Holmes M, Chong B, Crossley M
• Eos and pegasus, two members of the Ikaros family of proteins with distinct DNA binding activities.
• J Biol Chem. 2000; 275: 38347-54
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• Members of the Ikaros family of transcription factors, Ikaros, Aiolos, and Helios, are expressed in lymphocytes and have been implicated in controlling lymphoid development. These proteins contain two characteristic clusters of zinc fingers, an N-terminal domain important for DNA recognition, and a C-terminal domain that mediates homo- and heterotypic associations between family members. The conservation of these domains is such that all three proteins recognize related DNA sequences, and all are capable of dimerizing with other family members. Here we describe two additional Ikaros family proteins, Eos and Pegasus. Eos is most highly related to Helios and shares its DNA binding and protein association properties. Pegasus is related to other Ikaros proteins in its C-terminal dimerization domain but contains a divergent N-terminal zinc finger domain. Pegasus self-associates and binds to other family members but recognizes distinct DNA-binding sites. Eos and Pegasus repress the expression of reporter genes containing their recognition elements. Our results suggest that these proteins may associate with previously described Ikaros family proteins in lymphoid cells and play additional roles in other tissues.

• Feng GH, Lih CJ, Cohen SN
• TSG101 protein steady-state level is regulated posttranslationally by an evolutionarily conserved COOH-terminal sequence.
• Cancer Res. 2000; 60: 1736-41
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• Antisense inactivation of the tsg101 tumor susceptibility gene in murine NIH3T3 fibroblasts leads to neoplastic transformation and tumorigenesis, which are reversed by restoration of tsg101 activity. tsg101 deficiency is associated with a series of mitosis-related abnormalities, whereas overexpression of TSG101 can also result in neoplastic transformation and the perturbation of cell cycling. Together, these observations imply that TSG101 production outside of a narrow range can lead to abnormal cell growth. We report here that the TSG101 protein is maintained at an almost constant steady-state level in cultured murine and human cells and that this occurs through a posttranslational process involving TSG101 protein degradation. Sustained overproduction of TSG101 from chromosomally inserted adventitious constructs resulted in compensatory down-regulation of endogenous TSG101 and replacement of the native protein by the adventitious one. Using deletion mutants of TSG101, we mapped the region responsible for autoregulation of the TSG101 steady-state level to an evolutionarily conserved sequence, here termed the "steadiness box," located near TSG101's COOH-terminal end. Our results suggest a model in which the biological effects of TSG101 are modulated either by self-promoted proteolysis or participation with other cellular protein(s) in a proteolytic feedback-control loop.

• Leverson JD, Joazeiro CA, Page AM, Huang Hk, Hieter P, Hunter T
• The APC11 RING-H2 finger mediates E2-dependent ubiquitination.
• Mol Biol Cell. 2000; 11: 2315-25
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• Polyubiquitination marks proteins for degradation by the 26S proteasome and is carried out by a cascade of enzymes that includes ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). The anaphase-promoting complex or cyclosome (APC/C) comprises a multisubunit ubiquitin ligase that mediates mitotic progression. Here, we provide evidence that the Saccharomyces cerevisiae RING-H2 finger protein Apc11 defines the minimal ubiquitin ligase activity of the APC. We found that the integrity of the Apc11p RING-H2 finger was essential for budding yeast cell viability, Using purified, recombinant proteins we showed that Apc11p interacted directly with the Ubc4 ubiquitin conjugating enzyme (E2). Furthermore, purified Apc11p was capable of mediating E1- and E2-dependent ubiquitination of protein substrates, including Clb2p, in vitro. The ability of Apc11p to act as an E3 was dependent on the integrity of the RING-H2 finger, but did not require the presence of the cullin-like APC subunit Apc2p. We suggest that Apc11p is responsible for recruiting E2s to the APC and for mediating the subsequent transfer of ubiquitin to APC substrates in vivo.

• Warren AJ, Bravo J, Williams RL, Rabbitts TH
• Structural basis for the heterodimeric interaction between the acute leukaemia-associated transcription factors AML1 and CBFbeta.
• EMBO J. 2000; 19: 3004-15
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• Mutations in the genes encoding the interacting proteins AML1 and CBFbeta are the most common genetic abnormalities in acute leukaemia, and congenital mutations in the related AML3 gene are associated with disorders of osteogenesis. Furthermore, the interaction of AML1 with CBFbeta is essential for haematopoiesis. We report the 2.6 A resolution crystal structure of the complex between the AML1 Runt domain and CBFbeta, which represents a paradigm for the mode of interaction of this highly conserved family of transcription factors. The structure demonstrates that point mutations associated with cleidocranial dysplasia map to the conserved heterodimer interface, suggesting a role for CBFbeta in osteogenesis, and reveals a potential protein interaction platform composed of conserved negatively charged residues on the surface of CBFbeta.

• Aramburu J, Rao A, Klee CB
• Calcineurin: from structure to function.
• Curr Top Cell Regul. 2000; 36: 237-95

• Jiang J, Horowitz DS, Xu RM
• Crystal structure of the functional domain of the splicing factor Prp18.
• Proc Natl Acad Sci U S A. 2000; 97: 3022-7
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• The splicing factor Prp18 is required for the second step of pre-mRNA splicing. We have isolated and determined the crystal structure of a large fragment of the Saccharomyces cerevisiae Prp18 that lacks the N-terminal 79 amino acids. This fragment, called Prp18Delta79, is fully active in yeast splicing in vitro and includes the sequences of Prp18 that have been evolutionarily conserved. The core structure of Prp18Delta79 is compact and globular, consisting of five alpha-helices that adopt a novel fold that we have designated the five-helix X-bundle. The structure suggests that one face of Prp18 interacts with the splicing factor Slu7, whereas the more evolutionarily conserved amino acids in Prp18 form the opposite face. The most highly conserved region of Prp18, a nearly invariant stretch of 19 aa, forms part of a loop between two alpha-helices and may interact with the U5 small nuclear ribonucleoprotein particles. The structure is consistent with a model in which Prp18 forms a bridge between Slu7 and the U5 small nuclear ribonucleoprotein particles.

• Palm GJ, Billy E, Filipowicz W, Wlodawer A
• Crystal structure of RNA 3'-terminal phosphate cyclase, a ubiquitous enzyme with unusual topology.
• Structure Fold Des. 2000; 8: 13-23
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• BACKGROUND: RNA cyclases are a family of RNA-modifying enzymes that are conserved in eucarya, bacteria and archaea. They catalyze the ATP-dependent conversion of the 3'-phosphate to the 2',3'-cyclic phosphodiester at the end of RNA, in a reaction involving formation of the covalent AMP-cyclase intermediate. These enzymes might be responsible for production of the cyclic phosphate RNA ends that are known to be required by many RNA ligases in both prokaryotes and eukaryotes. RESULTS: The high-resolution structure of the Escherichia coli RNA 3'-terminal phosphate cyclase was determined using multiwavelength anomalous diffraction. Two orthorhombic crystal forms of E. coli cyclase (space group P2(1)2(1)2(1) and P2(1)2(1)2) were used to solve and refine the structure to 2.1 A resolution (R factor 20.4%; R(free) 27.6%). Each molecule of RNA cyclase consists of two domains. The larger domain contains three repeats of a folding unit comprising two parallel alpha helices and a four-stranded beta sheet; this fold was previously identified in translation initiation factor 3 (IF3). The large domain is similar to one of the two domains of 5-enolpyruvylshikimate-3-phosphate synthase and UDP-N-acetylglucosamine enolpyruvyl transferase. The smaller domain uses a similar secondary structure element with different topology, observed in many other proteins such as thioredoxin. CONCLUSIONS: The fold of RNA cyclase consists of known elements connected in a new and unique manner. Although the active site of this enzyme could not be unambiguously assigned, it can be mapped to a region surrounding His309, an adenylate acceptor, in which a number of amino acids are highly conserved in the enzyme from different sources. The structure of E. coli cyclase will be useful for interpretation of structural and mechanistic features of this and other related enzymes.

• Shigesada K
• [Molecular mechanisms of PEBP2/CBF as a master regulator of tissue-specific gene expression]
• Tanpakushitsu Kakusan Koso. 2000; 45: 55-62

• Lorick KL, Jensen JP, Fang S, Ong AM, Hatakeyama S, Weissman AM
• RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination.
• Proc Natl Acad Sci U S A. 1999; 96: 11364-9
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• A RING finger-containing protein (AO7) that binds ubiquitin-conjugating enzymes (E2s) and is a substrate for E2-dependent ubiquitination was identified. Mutations of cation-coordinating residues within AO7's RING finger abolished ubiquitination, as did chelation of zinc. Several otherwise-unrelated RING finger proteins, including BRCA1, Siah-1, TRC8, NF-X1, kf-1, and Praja1, were assessed for their ability to facilitate E2-dependent ubiquitination. In all cases, ubiquitination was observed. The RING fingers were implicated directly in this activity through mutations of metal-coordinating residues or chelation of zinc. These findings suggest that a large number of RING finger-containing proteins, with otherwise diverse structures and functions, may play previously unappreciated roles in modulating protein levels via ubiquitination.

• Ulmasov T, Hagen G, Guilfoyle TJ
• Dimerization and DNA binding of auxin response factors.
• Plant J. 1999; 19: 309-19
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• Auxin response factors (ARFs) are transcription factors that bind with specificity to TGTCTC auxin response elements (AuxREs) found in promoters of primary/early auxin response genes. ARFs are encoded by a multi-gene family, consisting of more than 10 genes. Ten ARFs have been analyzed by Northern analysis and were found to be expressed in all major plant organs and suspension culture cells of Arabidopsis. The predicted amino acid sequences indicate that the 10 ARFs contain a novel amino-terminal DNA binding domain and a carboxyl-terminal dimerization domain, with the exception of ARF3 which lacks this dimerization domain. All ARFs tested bind with specificity to the TGTCTC AuxRE, but there are subtle variations in the sequence requirements at positions 5 (T) and 6 (C) of the AuxRE. While the amino-terminal domain of about 350 amino acids is sufficient for binding ARF1 to TGTCTC AuxREs, this domain is not sufficient for the binding of some other ARFs to palindromic AuxREs. Our results suggest that ARFs must form dimers on palindromic TGTCTC AuxREs to bind stably, and this dimerization may be facilitated by conserved motifs found in ARF carboxyl-terminal domains. Dimerization in at least some cases may dictate which ARF(s) are targeted to AuxREs.

• Rosinski JA, Atchley WR
• Molecular evolution of helix-turn-helix proteins.
• J Mol Evol. 1999; 49: 301-9
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• The helix-turn-helix domain-containing family of transcriptional regulators is of ancient origin and has been incorporated into numerous disparate biological processes. As a consequence, the forces shaping its early evolution have been difficult to reconstruct. Herein, we analyze this large and diverse family with a combination of traditional phylogenetic techniques and newer sequence analysis tools to determine whether the helix-turn-helix family arose from a single common ancestor. Our analyses of the DNA-binding domain show that amino acid chemistry is conserved at many sites in the first helix and the turn. The high level of divergence combined with the short length of the domain hinders robust reconstruction of the entire phylogeny, but some level of deep node inference is possible. All analyses point to a predominantly monophyletic origin for the helix-turn-helix domain. The consequences of such an origin for a diverse group of proteins, and guidelines for the identification of future members of the HTH family are discussed.

• Perkins A
• Erythroid Kruppel like factor: from fishing expedition to gourmet meal.
• Int J Biochem Cell Biol. 1999; 31: 1175-92
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• Erythroid Kruppel like factor (EKLF) is the founding member of a family of transcription factors which are defined by the presence of three C-terminal C2H2-type zinc fingers. Since its discovery 6 years ago, the study of EKLF has been intense. In this review I will revisit the discovery of EKLF, and highlight recent advances in our understanding of how it interacts with other proteins to regulate erythroid gene transcription. The current knowledge of the biological role/s of EKLF in erythroid cell differentiation and globin gene switching are summarized.

• Miura T, Klaus W, Gsell B, Miyamoto C, Senn H
• Characterization of the binding interface between ubiquitin and class I human ubiquitin-conjugating enzyme 2b by multidimensional heteronuclear NMR spectroscopy in solution.
• J Mol Biol. 1999; 290: 213-28
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• Ubiquitin-conjugating enzymes (Ubc) are involved in ubiquitination of proteins in the protein degradation pathway of eukaryotic cells. Ubc transfers the ubiquitin (Ub) molecules to target proteins by forming a thioester bond between their active site cysteine residue and the C-terminal glycine residue of ubiquitin. Here, we report on the NMR assignment and secondary structure of class I human ubiquitin-conjugating enzyme 2b (HsUbc2b). Chemical shift perturbation studies allowed us to map the contact area and binding interface between ubiquitin and HsUbc2b by1H-15N HSQC NMR spectroscopy. The serine mutant of the active site Cys88 of HsUbc2b was employed to obtain a relatively stable covalent ubiquitin complex of HsUbc2b(C88S). Changes in chemical shifts of amide protons and nitrogen atoms induced by the formation of the covalent complex were measured by preparing two segmentally labeled complexes with either ubiquitin or HsUbc2b(C88S)15N-labeled. In ubiquitin, the interaction is primarily sensed by the C-terminal segment Val70 - Gly76, and residues Lys48 and Gln49. The surface area on ubiquitin, as defined by these residues, overlaps partially with the presumed binding site with ubiquitin-activating enzyme (E1). In HsUbc2b, most of the affected residues cluster in the vicinity of the active site, namely, around the active site Cys88 itself, the second alpha-helix, and the flexible loop which connects helices alpha2 and alpha3 and which is adjacent to the active site. An additional site on HsUbc2b for a weak interaction with ubiquitin could be detected in a titration study where the two proteins were not covalently linked. This site is located on the backside of HsUbc2b opposite to the active site and is part of the beta-sheet. The covalent and non-covalent interaction sites are clearly separated on the HsUbc2b surface, while no such clear-cut segregation of the interaction area was observed on ubiquitin.

• Huang L et al.
• Structure of an E6AP-UbcH7 complex: insights into ubiquitination by the E2-E3 enzyme cascade.
• Science. 1999; 286: 1321-6
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• The E6AP ubiquitin-protein ligase (E3) mediates the human papillomavirus-induced degradation of the p53 tumor suppressor in cervical cancer and is mutated in Angelman syndrome, a neurological disorder. The crystal structure of the catalytic hect domain of E6AP reveals a bilobal structure with a broad catalytic cleft at the junction of the two lobes. The cleft consists of conserved residues whose mutation interferes with ubiquitin-thioester bond formation and is the site of Angelman syndrome mutations. The crystal structure of the E6AP hect domain bound to the UbcH7 ubiquitin-conjugating enzyme (E2) reveals the determinants of E2-E3 specificity and provides insights into the transfer of ubiquitin from the E2 to the E3.

• Turner J, Crossley M
• Mammalian Kruppel-like transcription factors: more than just a pretty finger.
• Trends Biochem Sci. 1999; 24: 236-40
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• The transcription factor SP1 contains three Kruppel-like zinc fingers. Recently, several related proteins, including erythroid, lung and gut-enriched Kruppel-like factors, have been identified. Together with SP1, these proteins form a sizeable family of transcription factors that share homology in their zinc-finger domains but differ elsewhere. Analysis of these differences is illuminating specific mechanisms by which transcription is regulated.

• Fujii Y, Shimizu T, Kusumoto M, Kyogoku Y, Taniguchi T, Hakoshima T
• Crystal structure of an IRF-DNA complex reveals novel DNA recognition and cooperative binding to a tandem repeat of core sequences.
• EMBO J. 1999; 18: 5028-41
• Display abstract

• There has been growing interest in the role of the IRF (interferon regulatory factor) family of transcription factors in the regulation of immune responses, cytokine signaling, and oncogenesis. These members are characterized by their well-conserved DNA binding domains at the N-terminal regions. Here we report the 2.2 A resolution crystal structure of the DNA binding domain of one such family member, IRF-2, bound to DNA. The structure reveals its recognition sequence, AANNGAAA (here, recognized bases are underlined and in bold, and N indicates any base), and its cooperative binding to a tandem repeat of the GAAA core sequence induced by DNA structure distortions. These facts explain well the diverse binding properties of the IRF family members, which bind to both single and tandemly repeated sequences. Furthermore, we also identified the 'helix-hairpin-strand motif' at the C terminus of the recognition helix as a metal binding site that is commonly found in certain classes of DNA-interactive proteins. Our results provide new insights into the structure and function of this family of transcription factors.

• Jiang F, Basavappa R
• Crystal structure of the cyclin-specific ubiquitin-conjugating enzyme from clam, E2-C, at 2.0 A resolution.
• Biochemistry. 1999; 38: 6471-8
• Display abstract

• The destruction of the cyclin B protein is necessary for the cell to exit from mitosis. The destruction of cyclin B occurs via the ubiquitin/proteasome system and involves a specific ubiquitin-conjugating enzyme (Ubc) that donates ubiquitin to cyclin B. Here we present the crystal structure of the cyclin-specific Ubc from clam, E2-C, determined at 2.0 A resolution. The E2-C enzyme contains an N-terminal extension in addition to the Ubc core domain. The N-terminal extension is disordered, perhaps reflecting a need for flexibility as it interacts with various partners in the ubiquitination system. The overall structure of the E2-C core domain is quite similar to those in previously determined Ubc proteins. The interaction between particular pairs of E2-C proteins in the crystal has some of the hallmarks of a functional dimer, though solution studies suggest that the E2-C protein exists as a monomer. Comparison of the E2-C structure with that of the other available Ubc structures indicates conserved surface residues that may interact with common components of the ubiquitination system. Such comparison also reveals a remarkable spine of conserved hydrophobic residues in the center of the protein that may drive the protein to fold and stabilize the protein once folded. Comparison of residues conserved only among E2-C and its homologues indicates surface areas that may be involved in mitotic-specific ubiquitination.

• Cairns BR, Schlichter A, Erdjument-Bromage H, Tempst P, Kornberg RD, Winston F
• Two functionally distinct forms of the RSC nucleosome-remodeling complex, containing essential AT hook, BAH, and bromodomains.
• Mol Cell. 1999; 4: 715-23
• Display abstract

• RSC is an essential 15 protein nucleosome-remodeling complex from S. cerevisiae. We have identified two closely related RSC members, Rsc1 and Rsc2. Biochemical analysis revealed Rsc1 and Rsc2 in distinct complexes, defining two forms of RSC. Genetic analysis has shown that Rsc1 and Rsc2 possess shared and unique functions. Rsc1 and Rsc2 each contain two bromodomains, a bromo-adjacent homology (BAH) domain, and an AT hook. One of the bromodomains, the BAH domain, and the AT hook are each essential for Rsc1 and Rsc2 functions, although they are not required for assembly into RSC complexes. Therefore, these domains are required for RSC function. Additional genetic analysis provides further evidence that RSC function is related to transcriptional control.

• Linden H, Ballario P, Arpaia G, Macino G
• Seeing the light: news in Neurospora blue light signal transduction.
• Adv Genet. 1999; 41: 35-54

• Simon H
• T-box genes and the formation of vertebrate forelimb- and hindlimb specific pattern.
• Cell Tissue Res. 1999; 296: 57-66
• Display abstract

• Limb patterning is thought to be a multistep process involving specification of the limb fields, establishment of defined signaling centers that globally inform cells of their position, interpretation of positional signals, and regulated growth and differentiation of the limb structures. Great progress has been made over the past few years in identifying the molecular players that control limb outgrowth and patterning, in particular, how the limb axes are specified. However, the molecular mechanism for determination of the morphological and functional differences between forelimbs and hindlimbs has remained elusive. The recent identification of a series of limb-specific transcription factors has now provided excellent candidates for such upstream regulators of limb identity, and has allowed new insights into the regulatory network of making a hand or a foot.

• Hofmann RM, Pickart CM
• Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair.
• Cell. 1999; 96: 645-53
• Display abstract

• Ubiquitin-conjugating enzyme variant (UEV) proteins resemble ubiquitin-conjugating enzymes (E2s) but lack the defining E2 active-site residue. The MMS2-encoded UEV protein has been genetically implicated in error-free postreplicative DNA repair in Saccharomyces cerevisiae. We show that Mms2p forms a specific heteromeric complex with the UBC13-encoded E2 and is required for the Ubc13p-dependent assembly of polyubiquitin chains linked through lysine 63. A ubc13 yeast strain is UV sensitive, and single, double, and triple mutants of the UBC13, MMS2, and ubiquitin (ubiK63R) genes display a comparable phenotype. These findings support a model in which an Mms2p/Ubc13p complex assembles novel polyubiquitin chains for signaling in DNA repair, and they suggest that UEV proteins may act to increase diversity and selectivity in ubiquitin conjugation.

• Hester G, Stark W, Moser M, Kallen J, Markovic-Housley Z, Jansonius JN
• Crystal structure of phosphoserine aminotransferase from Escherichia coli at 2.3 A resolution: comparison of the unligated enzyme and a complex with alpha-methyl-l-glutamate.
• J Mol Biol. 1999; 286: 829-50
• Display abstract

• Phosphoserine aminotransferase (PSAT; EC 2.6.1.52), a member of subgroup IV of the aminotransferases, catalyses the conversion of 3-phosphohydroxypyruvate to l-phosphoserine. The crystal structure of PSAT from Escherichia coli has been solved in space group P212121 using MIRAS phases in combination with density modification and was refined to an R-factor of 17.5% (Rfree=20.1 %) at 2.3 A resolution. In addition, the structure of PSAT in complex with alpha-methyl-l-glutamate (AMG) has been refined to an R-factor of 18.5% (Rfree=25.1%) at 2.8 A resolution. Each subunit (361 residues) of the PSAT homodimer is composed of a large pyridoxal-5'-phosphate binding domain (residues 16-268), consisting of a seven-stranded mainly parallel beta-sheet, two additional beta-strands and seven alpha-helices, and a small C-terminal domain, which incorporates a five-stranded beta-sheet and two alpha-helices. A three-dimensional structural comparison to four other vitamin B6-dependent enzymes reveals that three alpha-helices of the large domain, as well as an N-terminal domain (subgroup II) or subdomain (subgroup I) are absent in PSAT. Its only 15 N-terminal residues form a single beta-strand, which participates in the beta-sheet of the C-terminal domain. The cofactor is bound through an aldimine linkage to Lys198 in the active site. In the PSAT-AMG complex Ser9 and Arg335 bind the AMG alpha-carboxylate group while His41, Arg42 and His328 are involved in binding the AMG side-chain. Arg77 binds the AMG side-chain indirectly through a solvent molecule and is expected to position itself during catalysis between the PLP phosphate group and the substrate side-chain. Comparison of the active sites of PSAT and aspartate aminotransferase suggests a similar catalytic mechanism, except for the transaldimination step, since in PSAT the Schiff base is protonated. Correlation of the PSAT crystal structure to a published profile sequence analysis of all subgroup IV members allows active site modelling of nifs and the proposal of a likely molecular reaction mechanism.

• Zheng N, Fraenkel E, Pabo CO, Pavletich NP
• Structural basis of DNA recognition by the heterodimeric cell cycle transcription factor E2F-DP.
• Genes Dev. 1999; 13: 666-74
• Display abstract

• The E2F and DP protein families form heterodimeric transcription factors that play a central role in the expression of cell cycle-regulated genes. The crystal structure of an E2F4-DP2-DNA complex shows that the DNA-binding domains of the E2F and DP proteins both have a fold related to the winged-helix DNA-binding motif. Recognition of the central c/gGCGCg/c sequence of the consensus DNA-binding site is symmetric, and amino acids that contact these bases are conserved among all known E2F and DP proteins. The asymmetry in the extended binding site TTTc/gGCGCc/g is associated with an amino-terminal extension of E2F4, in which an arginine binds in the minor groove near the TTT stretch. This arginine is invariant among E2Fs but not present in DPs. E2F4 and DP2 interact through an extensive protein-protein interface, and structural features of this interface suggest it contributes to the preference for heterodimers over homodimers in DNA binding.

• Atchley WR, Terhalle W, Dress A
• Positional dependence, cliques, and predictive motifs in the bHLH protein domain.
• J Mol Evol. 1999; 48: 501-16
• Display abstract

• Quantitative analyses were carried out on a large number of proteins that contain the highly conserved basic helix-loop-helix domain. Measures derived from information theory were used to examine the extent of conservation at amino acid sites within the bHLH domain as well as the extent of mutual information among sites within the domain. Using the Boltzmann entropy measure, we described the extent of amino acid conservation throughout the bHLH domain. We used position association (pa) statistics that reflect the joint probability of occurrence of events to estimate the "mutual information content" among distinct amino acid sites. Further, we used pa statistics to estimate the extent of association in amino acid composition at each site in the domain and between amino acid composition and variables reflecting clade and group membership, loop length, and the presence of a leucine zipper. The pa values were also used to describe groups of amino acid sites called "cliques" that were highly associated with each other. Finally, a predictive motif was constructed that accurately identifies bHLH domain-containing proteins that belong to Groups A and B.

• Xie Y, Varshavsky A
• The E2-E3 interaction in the N-end rule pathway: the RING-H2 finger of E3 is required for the synthesis of multiubiquitin chain.
• EMBO J. 1999; 18: 6832-44
• Display abstract

• We dissected physical and functional interactions between the ubiquitin-conjugating (E2) enzyme Ubc2p and Ubr1p, the E3 component of the N-end rule pathway in Saccharomyces cerevisiae. The binding of the 20 kDa Ubc2p by the 225 kDa Ubr1p is shown to be mediated largely by the basic residue-rich (BRR) region of Ubr1p. However, mutations of the BRR domain that strongly decrease the interaction between Ubr1p and Ubc2p do not prevent the degradation of N-end rule substrates. In contrast, this degradation is completely dependent on the RING-H2 finger of Ubr1p adjacent to the BRR domain. Specifically, the first cysteine of RING-H2 is required for the ubiquitylation activity of the Ubr1p-Ubc2p complex, although this cysteine plays no detectable role in either the binding of N-end rule substrates by Ubr1p or the physical affinity between Ubr1p and Ubc2p. These results defined the topography of the Ubc2p-Ubr1p interaction and revealed the essential function of the RING-H2 finger, a domain that is present in many otherwise dissimilar E3 proteins of the ubiquitin system.

• Toyoda T, Suzuki K, Sekiguchi T, Reed LJ, Takenaka A
• Crystal structure of eucaryotic E3, lipoamide dehydrogenase from yeast.
• J Biochem (Tokyo). 1998; 123: 668-74
• Display abstract

• The crystal structure of eucaryotic lipoamide dehydrogenase from yeast has been determined by an X-ray analysis at 2.7 (partially at 2.4) A resolution. The enzyme has two identical subunits related by a pseudo twofold symmetry. The tertiary structure is similar to those of other procaryotic enzymes. The active site, consisting of FAD, Cys44, and Cys49 from one subunit and His457' from the other subunit, is highly conserved. This enzyme is directly bound to the core protein E2 of the 2-oxoglutarate dehydrogenase complex, whereas it is bound to the pyruvate dehydrogenase complex through a protein X. The calculated electrostatic potential suggests two characteristic regions for binding with these two proteins.

• Jeffery CJ, Barry T, Doonan S, Petsko GA, Ringe D
• Crystal structure of Saccharomyces cerevisiae cytosolic aspartate aminotransferase.
• Protein Sci. 1998; 7: 1380-7
• Display abstract

• The crystal structure of Saccharomyces cerevisiae cytoplasmic aspartate aminotransferase (EC 2.6.1.1) has been determined to 2.05 A resolution in the presence of the cofactor pyridoxal-5'-phosphate and the competitive inhibitor maleate. The structure was solved by the method of molecular replacement. The final value of the crystallographic R-factor after refinement was 23.1% with good geometry of the final model. The yeast cytoplasmic enzyme is a homodimer with two identical active sites containing residues from each subunit. It is found in the "closed" conformation with a bound maleate inhibitor in each active site. It shares the same three-dimensional fold and active site residues as the aspartate aminotransferases from Escherichia coli, chicken cytoplasm, and chicken mitochondria, although it shares less than 50% sequence identity with any of them. The availability of four similar enzyme structures from distant regions of the evolutionary tree provides a measure of tolerated changes that can arise during millions of years of evolution.

• Yamamoto KR, Darimont BD, Wagner RL, Iniguez-Lluhi JA
• Building transcriptional regulatory complexes: signals and surfaces.
• Cold Spring Harb Symp Quant Biol. 1998; 63: 587-98

• Katsanis N, Fisher EM
• Identification, expression, and chromosomal localization of ubiquitin conjugating enzyme 7 (UBE2G2), a human homologue of the Saccharomyces cerevisiae ubc7 gene.
• Genomics. 1998; 51: 128-31
• Display abstract

• Protein degradation is an essential mechanism for the maintenance of cellular homeostasis, in which excess or aberrant proteins are eliminated from the cell. In eukaryotes, conjugation of target proteins to ubiquitin is an essential step in the proteasome-dependent degradation process and is mediated by a family of ubiquitin conjugating enzymes (UBC). Several of these have been identified in a variety of organisms. Here we report the identification of UBE2G2, a human homologue of the yeast Ubc7 gene. We describe a 2.9-kb cDNA sequence encoding a 165-amino-acid protein that shares significant similarity with other members of the UBC family. We have found UBE2G2 to be ubiquitously expressed, with high levels of expression seen in adult muscle, and have detected two transcripts of 2.9 and 7.0 kb in all tissues. In addition, we have mapped UBC7 to human chromosome 21q22.3 close to 21qtel.

• Teakle GR, Gilmartin PM
• Two forms of type IV zinc-finger motif and their kingdom-specific distribution between the flora, fauna and fungi.
• Trends Biochem Sci. 1998; 23: 100-2

• Oughtred R, Bedard N, Vrielink A, Wing SS
• Identification of amino acid residues in a class I ubiquitin-conjugating enzyme involved in determining specificity of conjugation of ubiquitin to proteins.
• J Biol Chem. 1998; 273: 18435-42
• Display abstract

• The ubiquitin pathway is a major system for selective proteolysis in eukaryotes. However, the mechanisms underlying substrate selectivity by the ubiquitin system remain unclear. We previously identified isoforms of a rat ubiquitin-conjugating enzyme (E2) homologous to the Saccharomyces cerevisiae class I E2 genes, UBC4/UBC5. Two isoforms, although 93% identical, show distinct features. UBC4-1 is expressed ubiquitously, whereas UBC4-testis is expressed in spermatids. Interestingly, although these isoforms interacted similarly with some ubiquitin-protein ligases (E3s) such as E6-AP and rat p100 and an E3 that conjugates ubiquitin to histone H2A, they also supported conjugation of ubiquitin to distinct subsets of testis proteins. UBC4-1 showed an 11-fold greater ability to support conjugation of ubiquitin to endogenous substrates present in a testis nuclear fraction. Site-directed mutagenesis of the UBC4-testis isoform was undertaken to identify regions of the molecule responsible for the observed difference in substrate specificity. Four residues (Gln-15, Ala-49, Ser-107, and Gln-125) scattered on surfaces away from the active site appeared necessary and sufficient for UBC4-1-like conjugation. These four residues identify a large surface of the E2 core domain that may represent an area of binding to E3s or substrates. These findings demonstrate that a limited number of amino acid substitutions in E2s can dictate conjugation of ubiquitin to different proteins and indicate a mechanism by which small E2 molecules can encode a wide range of substrate specificities.

• Kuhne C, Banks L
• E3-ubiquitin ligase/E6-AP links multicopy maintenance protein 7 to the ubiquitination pathway by a novel motif, the L2G box.
• J Biol Chem. 1998; 273: 34302-9
• Display abstract

• Ubiquitin ligases are generally assumed to play a major role in substrate recognition and thus provide specificity to a particular ubiquitin modification system. The multicopy maintenance protein (Mcm) 7 subunit of the replication licensing factor-M was identified as a substrate of the E3-ubiquitin ligase/E6-AP by its interaction with human papillomavirus-18E6. Mcm7 is ubiquitinated in vivo in both an E6-AP-dependent and -independent manner. E6-AP functions in these reactions independently of the viral oncogene E6. We show that recognition of Mcm7 by E6-AP is mediated by a homotypic interaction motif present in both proteins, called the L2G box. These findings served as the basis for the definition of substrate specificity for E6-AP. A small cluster of proteins whose function is intimately associated with the control of cell growth and/or proliferation contains the L2G box and is thereby implicated in an E6-AP and, by default, HPV-E6-dependent ubiquitination pathway.

• Thomson TM, Khalid H, Lozano JJ, Sancho E, Arino J
• Role of UEV-1A, a homologue of the tumor suppressor protein TSG101, in protection from DNA damage.
• FEBS Lett. 1998; 423: 49-52
• Display abstract

• The open reading frame YGL087c in the budding yeast Saccharomyces cerevisiae genome encodes a polypeptide highly similar to the human UEV (ubiquitin-conjugating E2 enzyme variant) proteins, which have been proposed to belong to a family of putative dominant negative ubiquitin regulators. Deletion of the YGL087c open reading frame yields viable cells which are sensitive to UV irradiation or methyl methanesulfonate, but not to hydroxyurea. This phenotype is reminiscent of that of rad mutants and suggests that the YGL087c-encoded protein functions in a process related to tolerance to DNA damage. We also show that the mutant phenotype is fully complemented by expression of the human UEV-1A cDNA and we propose that UEV-1 proteins could also have a role in protecting higher eukaryotic cells from DNA damaging agents.

• Aravind L, Koonin EV
• Eukaryotic transcription regulators derive from ancient enzymatic domains.
• Curr Biol. 1998; 8: 1113-1113

• Hori R, Carey M
• Transcription: TRF walks the walk but can it talk the talk?
• Curr Biol. 1998; 8: 1247-1247
• Display abstract

• The TBP-related factor TRF binds to a TATA box promoter sequence and mediates basal transcription in vitro. A central question is: does TRF function in vivo to transduce the effects of activators in a tissue-specific fashion?

• Koldamova RP, Lefterov IM, DiSabella MT, Lazo JS
• An evolutionarily conserved cysteine protease, human bleomycin hydrolase, binds to the human homologue of ubiquitin-conjugating enzyme 9.
• Mol Pharmacol. 1998; 54: 954-61
• Display abstract

• Bleomycin hydrolase (BH) is a highly conserved cysteine proteinase that deamidates and inactivates the anticancer drug bleomycin. Yeast BH self-assembles to form a homohexameric structure, which resembles a 20 S proteasome and may interact with other proteins. Therefore, we searched for potential human BH (hBH) partners using the yeast two-hybrid system with a HeLa cDNA library and identified the full-length human homologue of yeast ubiquitin-conjugating enzyme 9 (UBC9). Cotransformation assays using hBH deletion mutants revealed that the carboxyl terminus of hBH (amino acids 356-455), which contains two of the three essential catalytic amino acids, was not critical for protein binding in the yeast two-hybrid environment. In vitro translated human UBC9 was precipitated by glutathione S-transferase-hBH fusion protein but not by glutathione S-transferase. Efficient in vitro binding occurred in the absence of the first 24 amino acids of UBC9 and the catalytic Cys93 of UBC9. We confirmed that hBH and UBC9 interacted in vivo by affinity copurification of proteins overexpressed in mammalian cells. Using immunocytochemical analysis, hBH was colocalized with UBC9. Coexpression of hBH and UBC9 in mammalian cells did not markedly alter the bleomycin-hydrolyzing activity of hBH or apparent small ubiquitin-related modifier 1 addition. This is the first reported heteromeric interaction with hBH, and it suggests a role for hBH in intracellular protein processing and degradation.

• Nakatsu T, Kato H, Oda J
• Crystal structure of asparagine synthetase reveals a close evolutionary relationship to class II aminoacyl-tRNA synthetase.
• Nat Struct Biol. 1998; 5: 15-9
• Display abstract

• The crystal structure of E. coli asparagine synthetase has been determined by X-ray diffraction analysis at 2.5 A resolution. The overall structure of the enzyme is remarkably similar to that of the catalytic domain of yeast aspartyl-tRNA synthetase despite low sequence similarity. These enzymes have a common reaction mechanism that implies the formation of an aminoacyl-adenylate intermediate. The active site architecture and most of the catalytic residues are also conserved in both enzymes. These proteins have probably evolved from a common ancestor even though their sequence similarities are small. The functional and structural similarities of both enzymes suggest that new enzymatic activities would generally follow the recruitment of a protein catalyzing a similar chemical reaction.

• Osaka F et al.
• A new NEDD8-ligating system for cullin-4A.
• Genes Dev. 1998; 12: 2263-8
• Display abstract

• NEDD8 is a ubiquitin (Ub)-like protein. Here we report a novel ubiquitinylation-related pathway for modification by NEDD8. NEDD8 was activated by an E1 (Ub-activating enzyme)-like complex, consisting of APP-BP1 and hUba3 with high respective homologies to the amino- and carboxy-terminal regions of E1 and then linked to hUbc12 (a human homolog of yeast Ub-conjugating enzyme Ubc12p). The major target protein modified by NEDD8 was found to be Hs-cullin-4A (Cul-4A), a member of the family of human cullin/Cdc53 proteins functioning as an essential component of a multifunctional Ub-protein ligase E3 complex that has a critical role in Ub-mediated proteolysis.

• Worthylake DK, Prakash S, Prakash L, Hill CP
• Crystal structure of the Saccharomyces cerevisiae ubiquitin-conjugating enzyme Rad6 at 2.6 A resolution.
• J Biol Chem. 1998; 273: 6271-6
• Display abstract

• The Saccharomyces cerevisiae ubiquitin-conjugating enzyme (UBC) Rad6 is required for several functions, including the repair of UV damaged DNA, damage-induced mutagenesis, sporulation, and the degradation of cellular proteins that possess destabilizing N-terminal residues. Rad6 mediates its role in N-end rule-dependent protein degradation via interaction with the ubiquitin-protein ligase Ubr1 and in DNA repair via interactions with the DNA binding protein Rad18. We report here the crystal structure of Rad6 refined at 2.6 A resolution to an R factor of 21.3%. The protein adopts an alpha/beta fold that is very similar to other UBC structures. An apparent difference at the functionally important first helix, however, has prompted a reassessment of previously reported structures. The active site cysteine lies in a cleft formed by a coil region that includes the 310 helix and a loop that is in different conformations for the three molecules in the asymmetric unit. Residues important for Rad6 interaction with Ubr1 and Rad18 are on the opposite side of the structure from the active site, indicating that this part of the UBC surface participates in protein-protein interactions that define Rad6 substrate specificity.

• Cavarelli J, Delagoutte B, Eriani G, Gangloff J, Moras D
• L-arginine recognition by yeast arginyl-tRNA synthetase.
• EMBO J. 1998; 17: 5438-48
• Display abstract

• The crystal structure of arginyl-tRNA synthetase (ArgRS) from Saccharomyces cerevisiae, a class I aminoacyl-tRNA synthetase (aaRS), with L-arginine bound to the active site has been solved at 2.75 A resolution and refined to a crystallographic R-factor of 19.7%. ArgRS is composed predominantly of alpha-helices and can be divided into five domains, including the class I-specific active site. The N-terminal domain shows striking similarity to some completely unrelated proteins and defines a module which should participate in specific tRNA recognition. The C-terminal domain, which is the putative anticodon-binding module, displays an all-alpha-helix fold highly similar to that of Escherichia coli methionyl-tRNA synthetase. While ArgRS requires tRNAArg for the first step of the aminoacylation reaction, the results show that its presence is not a prerequisite for L-arginine binding. All H-bond-forming capability of L-arginine is used by the protein for the specific recognition. The guanidinium group forms two salt bridge interactions with two acidic residues, and one H-bond with a tyrosine residue; these three residues are strictly conserved in all ArgRS sequences. This tyrosine is also conserved in other class I aaRS active sites but plays several functional roles. The ArgRS structure allows the definition of a new framework for sequence alignments and subclass definition in class I aaRSs.

• Choo Y
• Recognition of DNA methylation by zinc fingers.
• Nat Struct Biol. 1998; 5: 264-5

• Loiseau L, Pasteau S, Brun G
• Molecular cloning and expression pattern of the DP members of the chicken E2F transcription factor.
• Gene Expr. 1997; 6: 259-73
• Display abstract

• The DP proteins are components of the E2F transcription factor. They form heterodimers with the E2F proteins and these complexes bind efficiently to E2F response elements in promoters of genes that are involved in cell cycle regulation. The properties of the DP proteins are less documented than those of their E2F counterpart and the present work was aimed at characterizing avian DP genes (named chDP) and their products. Here we describe the cloning of the chicken homologues of the mammalian DP-1 and DP-2 proteins. This work also suggests that DP-2 isoforms have an additional 60 amino acid extension at the N-terminus compared to its human counterpart. Gel-shift assays and coimmunoprecipitation show that both DP-1 and DP-2 dimerize to chE2F-1 and activate transcription efficiently, as demonstrated by transient expression assays. However, contrary to the expression patterns exhibited by E2F-1 during the cell cycle or during neuroretina development, DP member's expression appears more invariant, suggesting that E2F activity is limited by the availability of the E2F proteins.

• Haldeman MT, Xia G, Kasperek EM, Pickart CM
• Structure and function of ubiquitin conjugating enzyme E2-25K: the tail is a core-dependent activity element.
• Biochemistry. 1997; 36: 10526-37
• Display abstract

• Individual members of the conserved family of ubiquitin conjugating enzymes (E2s) mediate the ubiquitination and turnover of specific substrates of the ubiquitin-dependent degradation pathway. E2 proteins have a highly conserved core domain of approximately 150 amino acids which contains the active-site Cys. Certain E2s have unique terminal extensions, which are thought to contribute to selective E2 function by interacting either with substrates or with trans-acting factors such as ubiquitin-protein ligases (E3s). We used the mammalian ubiquitin conjugating enzyme E2-25K in a biochemical test of this hypothesis. The properties of two truncated derivatives show that the 47-residue tail of E2-25K is necessary for three of the enzyme's characteristic properties: high activity in the synthesis of unanchored K48-linked polyubiquitin chains; resistance of the active-site Cys residue to alkylation; and an unusual discrimination against noncognate (nonmammalian) ubiquitin activating (E1) enzymes. However, the tail is not sufficient to generate these properties, as shown by the characteristics of a chimeric enzyme in which the tail of E2-25K was fused to the core domain of yeast UBC4. These and other results indicate that the specific biochemical function of the tail is strongly dependent upon unique features of the E2-25K core domain. Thus, divergent regions within the conserved core domains of E2 proteins may be highly significant for function. Expression of truncated E2-25K as a glutathione S-transferase (GST) fusion protein resulted in the apparent recovery of E2-25K-specific properties, including activity in chain synthesis. However, the catalytic mechanism utilized by the truncated fusion protein proved to be distinct from the mechanism utilized by the wild-type enzyme. The unexpected properties of the fusion protein were due to GST-induced dimerization. These results indicate the potential for self-association to modulate the polyubiquitin chain synthesis activities of E2 proteins, and indicate that caution should be applied in interpreting the activities of GST fusion proteins.

• Schreiber-Agus N et al.
• A biochemical and biological analysis of Myc superfamily interactions.
• Curr Top Microbiol Immunol. 1997; 224: 159-68

• Sun B, Jeyaseelan K, Chung MC, Tan TW, Chock PB, Teo TS
• Cloning, characterization and expression of a cDNA clone encoding rabbit ubiquitin-conjugating enzyme, E2(32k).
• Biochim Biophys Acta. 1997; 1351: 231-8
• Display abstract

• A cDNA clone encoding rabbit E2(32k) was obtained by library screening and PCR. The cDNA contains an open reading frame coding for 238 amino acids which shows an overall identity of 81% to human CDC34, the cell cycle-related ubiquitin-conjugating enzyme. A 50% homology to yeast CDC34 within the conserved core domain was also observed. Northern blot analysis indicated that three transcripts existed in all six rabbit tissues examined but their expression levels varied over a wide range. The putative cDNA coding region was highly expressed in Escherichia coli as a his-tagged protein which was purified to homogeneity. The ability of this expressed protein to form a thiolester bond with ubiquitin showed that it was functionally active. The ability of this protein to catalyze the conjugation of ubiquitin to histone H2A and H2B was also examined.

• Desterro JM, Thomson J, Hay RT
• Ubch9 conjugates SUMO but not ubiquitin.
• FEBS Lett. 1997; 417: 297-300
• Display abstract

• Ubiquitin conjugating enzymes participate in the thioester cascade that leads to protein ubiquitination. Although Ubc9 is homologous to E2 ubiquitin conjugating enzymes we have shown that it is unable to form a thioester with ubiquitin, but can form a thioester with the small ubiquitin-like protein SUMO. Thus Ubc9 is a SUMO conjugating enzyme rather than a ubiquitin conjugating enzyme. Transacetylation of Ubc9 by SUMO is not mediated by the E1 ubiquitin activating enzyme, but by a distinct enzymatic activity. SUMO conjugation to target proteins is mediated by a different, but parallel pathway to ubiquitination.

• Gong L, Kamitani T, Fujise K, Caskey LS, Yeh ET
• Preferential interaction of sentrin with a ubiquitin-conjugating enzyme, Ubc9.
• J Biol Chem. 1997; 272: 28198-201
• Display abstract

• Sentrin is a ubiquitin-like molecule that has been shown to interact with the death domains of Fas and tumor necrosis factor receptor 1 (TNFR1), PML, Rad51, Rad52, and RanGAP1. We have reported previously that sentrin can be conjugated to other proteins in a manner analogous to protein ubiquitination (Kamitani, T., Nguyen, H. P., and Yeh, E. T. H. (1997) J. Biol. Chem. 272, 14001-14004). Furthermore, the conserved C-terminal Gly-Gly residues are required for sentrinization to occur. To identify enzymes which play a role in sentrinization, the yeast two-hybrid system was used to screen a human placenta cDNA library using sentrin as bait. A strong positive interacting clone was found to contain a cDNA insert encoding the ubiquitin-conjugating enzyme, Ubc9. The interaction between sentrin and Ubc9 required the ubiquitin domain and the C-terminal Gly-Gly residues of sentrin. This interaction appears to be specific because sentrin could only interact weakly with UbcH5B, but could not interact with HHR6B, UbcH6 nor E2-EPF. In vitro translated sentrin could be precipitated by a GST-Ubc9 fusion protein, but not by glutathione S-transferase. A beta-mercaptoethanol-sensitive Ubc9-sentrin conjugate could also be identified in the in vitro binding assay. Substitution of the conserved cysteine residue of Ubc9 by serine abolished the formation of the Ubc9-sentrin conjugate. Taken together, Ubc9 is a strong candidate to be the key conjugating enzyme in the sentrinization pathway.

• Johnston SC, Larsen CN, Cook WJ, Wilkinson KD, Hill CP
• Crystal structure of a deubiquitinating enzyme (human UCH-L3) at 1.8 A resolution.
• EMBO J. 1997; 16: 3787-96
• Display abstract

• Ubiquitin C-terminal hydrolases catalyze the removal of adducts from the C-terminus of ubiquitin. We have determined the crystal structure of the recombinant human Ubiquitin C-terminal Hydrolase (UCH-L3) by X-ray crystallography at 1.8 A resolution. The structure is comprised of a central antiparallel beta-sheet flanked on both sides by alpha-helices. The beta-sheet and one of the helices resemble the well-known papain-like cysteine proteases, with the greatest similarity to cathepsin B. This similarity includes the UCH-L3 active site catalytic triad of Cys95, His169 and Asp184, and the oxyanion hole residue Gln89. Papain and UCH-L3 differ, however, in strand and helix connectivity, which in the UCH-L3 structure includes a disordered 20 residue loop (residues 147-166) that is positioned over the active site and may function in the definition of substrate specificity. Based upon analogy with inhibitor complexes of the papain-like enzymes, we propose a model describing the binding of ubiquitin to UCH-L3. The UCH-L3 active site cleft appears to be masked in the unliganded structure by two different segments of the enzyme (residues 9-12 and 90-94), thus implying a conformational change upon substrate binding and suggesting a mechanism to limit non-specific hydrolysis.

• Schlake T, Schorpp M, Nehls M, Boehm T
• The nude gene encodes a sequence-specific DNA binding protein with homologs in organisms that lack an anticipatory immune system.
• Proc Natl Acad Sci U S A. 1997; 94: 3842-7
• Display abstract

• In the mouse, the product of the nude locus, Whn, is required for the keratinization of the hair shaft and the differentiation of epithelial progenitor cells in the thymus. A bacterially expressed peptide representing the presumptive DNA binding domain of the mouse whn gene in vitro specifically binds to a 11-bp consensus sequence containing the invariant tetranucleotide 5'-ACGC. In transient transfection assays, such binding sites stimulated reporter gene expression about 30- to 40-fold, when positioned upstream of a minimal promotor. Whn homologs from humans, bony fish (Danio rerio), cartilaginous fish (Scyliorhinus caniculus), agnathans (Lampetra planeri), and cephalochordates (Branchiostoma lanceolatum) share at least 80% of amino acids in the DNA binding domain. In agreement with this remarkable structural conservation, the DNA binding domains from zebrafish, which possesses a thymus but no hair, and amphioxus, which possesses neither thymus nor hair, recognize the same target sequence as the mouse DNA binding domain in vitro and in vivo. The genomes of vertebrates and cephalochordates contain only a single whn-like gene, suggesting that the primordial whn gene was not subject to gene-duplication events. Although the role of whn in cephalochordates and agnathans is unknown, its requirement in the development of the thymus gland and the differentiation of skin appendages in the mouse suggests that changes in the transcriptional control regions of whn genes accompanied their functional reassignments during evolution.

• Loveys DA, Streiff MB, Schaefer TS, Kato GJ
• The mUBC9 murine ubiquitin conjugating enzyme interacts with the E2A transcription factors.
• Gene. 1997; 201: 169-77
• Display abstract

• The ubiquitin-mediated degradation of cellular proteins requires the sequential activity of E1, E2 and, in some cases, E3 enzymes. Using the yeast two-hybrid system, we have cloned 1.0- and 2.5-kb cDNAs encoding the identical murine E2, or ubiquitin conjugating enzyme by virtue of its interaction with the E2A transcription factor. This cDNA encodes the 158-amino-acid protein, mUBC9, which has considerable sequence homology to UBC9 from Saccharomyces cerevisiae and HUS5 from Schizosaccharomyces pombe and is identical to the human UBC9 protein. HUS5 is essential for DNA damage repair, whereas UBC9 is necessary for G2/M progression. The human protein has been shown to correct the UBC9 defect in yeast. Antisera raised against bacterially expressed mUBC9 fusion protein recognize a murine cellular protein of approximately 18 kDa, corresponding to the predicted mobility. Unlike E2A, the mUBC9 protein level is not regulated by serum growth factors. The activity of the apparent homologues UBC9 and HUS5 suggests that mUBC9 may be involved in the degradation of key nuclear proteins that regulate cell cycle progression.

• Peters H, Schuster G, Neubuser A, Richter T, Hofler H, Balling R
• Isolation of the Pax9 cDNA from adult human esophagus.
• Mamm Genome. 1997; 8: 62-4

• de Launoit Y et al.
• Structure-function relationships of the PEA3 group of Ets-related transcription factors.
• Biochem Mol Med. 1997; 61: 127-35
• Display abstract

• The PEA3 group of transcription factors belongs to the Ets family and is composed of PEA3, ERM, and ER81, which are more than 95% identical within the DNA-binding domain--the ETS domain--and which demonstrate 50% aa identity overall. We present here a review of the current knowledge of these transcription factors, which possess functional domains responsible for DNA-binding, DNA-binding inhibition, and transactivation. Recent data suggest that these factors are targets for signaling cascades, such as the Ras-dependent ones, and thus may contribute first to the nuclear response to cell stimulation and second to Ras-induced cell transformation. The expression of the PEA3 group members in numerous developing murine organs, and, especially, in epithelial-mesenchymal interaction events, suggests a key role in murine organogenesis. Moreover, their expression in certain breast cancer cells suggests a possible involvement of these genes in the appearance, progression, and invasion of malignant cells.

• Tong H, Hateboer G, Perrakis A, Bernards R, Sixma TK
• Crystal structure of murine/human Ubc9 provides insight into the variability of the ubiquitin-conjugating system.
• J Biol Chem. 1997; 272: 21381-7
• Display abstract

• Murine/human ubiquitin-conjugating enzyme Ubc9 is a functional homolog of Saccharomyces cerevisiae Ubc9 that is essential for the viability of yeast cells with a specific role in the G2-M transition of the cell cycle. The structure of recombinant mammalian Ubc9 has been determined from two crystal forms at 2.0 A resolution. Like Arabidopsis thaliana Ubc1 and S. cerevisiae Ubc4, murine/human Ubc9 was crystallized as a monomer, suggesting that previously reported hetero- and homo-interactions among Ubcs may be relatively weak or indirect. Compared with the known crystal structures of Ubc1 and Ubc4, which regulate different cellular processes, Ubc9 has a 5-residue insertion that forms a very exposed tight beta-hairpin and a 2-residue insertion that forms a bulge in a loop close to the active site. Mammalian Ubc9 also possesses a distinct electrostatic potential distribution that may provide possible clues to its remarkable ability to interact with other proteins. The 2-residue insertion and other sequence and structural heterogeneity observed at the catalytic site suggest that different Ubcs may utilize catalytic mechanisms of varying efficiency and substrate specificity.

• Bailly V, Prakash S, Prakash L
• Domains required for dimerization of yeast Rad6 ubiquitin-conjugating enzyme and Rad18 DNA binding protein.
• Mol Cell Biol. 1997; 17: 4536-43
• Display abstract

• The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme required for postreplicational repair of UV-damaged DNA and for damage-induced mutagenesis. In addition, Rad6 functions in the N end rule pathway of protein degradation. Rad6 mediates its DNA repair role via its association with Rad18, whose DNA binding activity may target the Rad6-Rad18 complex to damaged sites in DNA. In its role in N end-dependent protein degradation, Rad6 interacts with the UBR1-encoded ubiquitin protein ligase (E3) enzyme. Previous studies have indicated the involvement of N-terminal and C-terminal regions of Rad6 in interactions with Ubr1. Here, we identify the regions of Rad6 and Rad18 that are involved in the dimerization of these two proteins. We show that a region of 40 amino acids towards the C terminus of Rad18 (residues 371 to 410) is sufficient for interaction with Rad6. This region of Rad18 contains a number of nonpolar residues that have been conserved in helix-loop-helix motifs of other proteins. Our studies indicate the requirement for residues 141 to 149 at the C terminus, and suggest the involvement of residues 10 to 22 at the N terminus of Rad6, in the interaction with Rad18. Each of these regions of Rad6 is indicated to form an amphipathic helix.

• Gibbons RJ et al.
• Mutations in transcriptional regulator ATRX establish the functional significance of a PHD-like domain.
• Nat Genet. 1997; 17: 146-8

• Tashiro K, Pando MP, Kanegae Y, Wamsley PM, Inoue S, Verma IM
• Direct involvement of the ubiquitin-conjugating enzyme Ubc9/Hus5 in the degradation of IkappaBalpha.
• Proc Natl Acad Sci U S A. 1997; 94: 7862-7
• Display abstract

• The NF-kappaB/Rel proteins are sequestered in the cytoplasm in association with IkappaBalpha. In response to external signals, IkappaBalpha is phosphorylated, multi-ubiquitinated, and degraded by proteasomes, thereby releasing NF-kappaB/Rel proteins to migrate to the nucleus. We have cloned a mouse ubiquitin-conjugating enzyme (mE2), which associates with IkappaBalpha. mE2 is homologous to the yeast Ubc9/Hus5 ubiquitin-conjugating enzyme. A transdominant-negative mutant of mE2 had no effect on phosphorylation of IkappaBalpha, but delayed its degradation. Correspondingly, tumor necrosis factor-alpha-inducible NF-kappaB activity was diminished. We propose that mE2 is directly involved in the ubiquitin conjugation of IkappaBalpha, a pivotal step in its degradation pathway.

• Bradley RL, Reddy KJ
• Cloning, sequencing, and regulation of the global nitrogen regulator gene ntcA in the unicellular diazotrophic cyanobacterium Cyanothece sp. strain BH68K.
• J Bacteriol. 1997; 179: 4407-10
• Display abstract

• In cyanobacteria, ammonium represses expression of proteins involved in nitrogen fixation and assimilation. The global nitrogen regulator gene ntcA encodes a DNA-binding protein, NtcA, that is a transcriptional activator of genes subject to nitrogen control. We report the cloning and sequencing of the ntcA gene from a nitrogen-fixing unicellular cyanobacterium, Cyanothece sp. strain BH68K. The gene comprises 678 nucleotides, and the deduced NtcA protein contains 226 amino acids with a predicted molecular weight of 25,026. In addition, ntcA mRNA levels were measured in cells grown under different nitrogen regimes. Under nitrogen-fixing conditions, ntcA transcripts were weakly expressed. Furthermore, ntcA expression was diminished or inversely proportional to nifHDK expression. Conversely, ntcA expression increased in nitrate-grown cells, and a concentration-dependent increase was seen in ammonium-grown cells up to 1 mM NH4Cl. These results indicate that ntcA is involved more in nitrogen assimilation than in nitrogen fixation and also imply that the rhythmic expression of ntcA and nifHDK transcription may be under the control of a circadian clock.

• Verma-Kurvari S, Johnson JE
• Identification of an achaete-scute homolog, Fash1, from Fugu rubripes.
• Gene. 1997; 200: 145-8
• Display abstract

• Proteins of the achaete-scute family of transcription factors play important roles in neurogenesis in both invertebrates and vertebrates. Here, we report the cloning and characterization of a Japanese pufferfish, Fugu rubripes achaete-scute homolog 1, Fash1. Sequence alignment of the predicted amino acid sequence of Fash1 with other vertebrate homologs of the achaete scute homolog 1 subclass shows that the carboxyl 2/3 of the protein, including the basic helix-loop-helix, a putative nuclear localization signal and several consensus phosphorylation sites, is highly conserved. Strikingly, the similarity in this region between eight vertebrate species is close to 90%.

• Wingender E
• [Classification of eukaryotic transcription factors]
• Mol Biol (Mosk). 1997; 31: 584-600

• Stenson C, McNair A, Byrnes L, Gannon F
• Cloning of an Atlantic salmon transcription factor.
• Biochem Soc Trans. 1996; 24: 106-106

• Bienstock RJ, Darden T, Wiseman R, Pedersen L, Barrett JC
• Molecular modeling of the amino-terminal zinc ring domain of BRCA1.
• Cancer Res. 1996; 56: 2539-45
• Display abstract

• The equine herpes virus zinc ring domain nuclear magnetic resonance structure was used for homology-based modeling of the amino-terminal zinc ring domain of the BRCA1 breast and ovarian cancer susceptibility gene. The zinc ring domain of BRCA1 is of particular interest because it is the location of significant and frequently occurring missense (Cys(61)Gly, Cys(64)Gly, and Cys(64)Tyr) and frameshift (185delAG) mutations observed in several high-risk kindreds. The BRCA1 zinc ring domain possesses 54% sequence similarity with the equine herpes virus zinc ring domain. The model structure undergoes little conformational variance after 140 ps of solvated molecular dynamics. This model proposes BRCA1 zinc ring domain residues that may play a role in DNA binding and/or protein-protein interactions. These predictions provide a point of departure for the design of mutants to probe BRCA1 zinc ring domain functionality.

• Berleth ES, Pickart CM
• Mechanism of ubiquitin conjugating enzyme E2-230K: catalysis involving a thiol relay?
• Biochemistry. 1996; 35: 1664-71
• Display abstract

• Covalent conjugation of ubiquitin to intracellular proteins is a signal for degradation by the 26S protease. Conjugation is usually accomplished by the sequential action of activating (E1), conjugating (E2), and ligase (E3) enzymes. Each of these enzymes forms a covalent thiol ester with ubiquitin as part of its catalytic cycle. In most cases, the apparent role of the ubiquitin conjugating enzyme (E2) is to transfer ubiquitin from the E1 active site to the E3 active site. Ubiquitin is then delivered from E3 to the substrate lysine residue. An unusually large, reticulocyte-specific enzyme, known as E2-230K, is unique among the large family of E2 enzymes is being susceptible to inhibition by inorganic arsenite [Klemperer et al. (1989) Biochemistry 28, 6035-6041]. We show that phenylarsenoxides potently inhibit E2-230K, apparently by binding to vicinal Cys residues of the enzyme: bound aminophenylarsenoxide partially protects the enzyme against inactivation by N-ethylmalemide (NEM), and prior enzyme inactivation with NEM blocks enzyme binding to immobilized phenylarsenoxide. Studies on the mechanistic basis of inhibition showed that a concentration of (aminophenyl)arsenoxide that produced complete inhibition of steady-state turnover had no effect on the turnover of the preformed E2-ubiquitin adduct. However, when the enzyme was preincubated with this concentration of inhibitor prior to initiation of adduct formation, the level of E2-associated ubiquitin was reduced by 60%. These results are consistent with a model in which two Cys residues of the enzyme sequentially form thiol esters with ubiquitin and the second of these Cys residues is bound to arsenic in the enzyme-inhibitor complex. In this model, E2-230K functions as an E2-E3 hybrid.

• Wang ZY et al.
• Molecular cloning of the cDNA and chromosome localization of the gene for human ubiquitin-conjugating enzyme 9.
• J Biol Chem. 1996; 271: 24811-6
• Display abstract

• We report a novel human gene whose product specifically associates with the negative regulatory domain of the Wilms' tumor gene product (WT1) in a yeast two-hybrid screen and with WT1 in immunoprecipitation and glutathione S-transferase (GST) capture assays. The gene encodes a 17-kDa protein that has 56% amino acid sequence identity with yeast ubiquitin-conjugating enzyme (yUBC) 9, a protein required for cell cycle progression in yeast, and significant identity with other subfamilies of ubiquitin-conjugating enzymes. The human gene fully complements yeast that have a temperature-sensitive yUBC9 gene mutation to fully restore normal growth, indicating that we have cloned a functionally conserved human (h) homolog of yUBC9. Transcripts of hUBC9 of 4.4 kilobases (kb), 2.8 kb, and 1.3 kb were found in all human tissues tested. A single copy of the hUBC9 gene was found and localized to human chromosome 16p13.3. We conclude that hUBC9 retains striking structural and functional conservation with yUBC9 and suggest a possible link of the ubiquitin/proteosome proteolytic pathway and the WT1 transcriptional repressor system.

• Dor Y, Raboy B, Kulka RG
• Role of the conserved carboxy-terminal alpha-helix of Rad6p in ubiquitination and DNA repair.
• Mol Microbiol. 1996; 21: 1197-206
• Display abstract

• RAD6 in the yeast Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme essential for DNA repair as well as for a number of other biological processes. It is believed that the functions of Rad6p require the ubiquitination of target proteins, but its substrates as well as other interacting proteins are largely unknown. Rad6p homologues of higher eukaryotes have a number of amino acid residues in the C-terminal alpha-helix, which are conserved from yeast to man but are absent from most other yeast ubiquitin-conjugating enzymes (Ubcs). This specific conservation suggests that the C-terminal alpha-helix is important for the unique activities of the Rad6p family of Ubcs. We have investigated the effects of mutating this highly conserved region on the ubiquitination of model substrates in vitro and on error-free DNA repair in vivo. C-terminal point and deletion mutants of Rad6p differentially affected its in vitro activity on various substrates, raising the possibility that Rad6p interacts with its substrates in vivo by similar mechanisms. The distal part of the C-terminal alpha-helix is also essential for error-free DNA repair in vivo. Overexpression of Rad18p, a single-stranded DNA-binding protein that also interacts with Rad6p, alleviates the DNA repair defects of the C-terminal alpha-helix mutants to different degrees. This indicates that the C-terminal alpha-helix of Rad6p mediates its interaction with Rad18p, an essential step in DNA repair. Models of Rad6p action propose that its ubiquitination function is followed by proteolysis of unknown ubiquitinated targets. Mutants affecting several functions of the 26S proteasome retain wild-type capacity for error-free DNA repair. This raises the possibility that ubiquitination by Rad6p in DNA repair does not target proteins for proteasomal degradation.

• Hsiao CD, Sun YJ, Rose J, Wang BC
• The crystal structure of glutamine-binding protein from Escherichia coli.
• J Mol Biol. 1996; 262: 225-42
• Display abstract

• The crystal structure of the glutamine-binding protein (GlnBP) from Escherichia coli in a ligand-free "open" conformational state has been determined by isomorphous replacement methods and refined to an R-value of 21.4% at 2.3 A resolution. There are two molecules in the asymmetric unit, related by pseudo 4-fold screw symmetry. The refined model consists of 3587 non-hydrogen atoms from 440 residues (two monomers), and 159 water molecules. The structure has root-mean-square deviations of 0.013 A from "ideal" bond lengths and 1.5 degrees from "ideal" bond angles. The GlnBP molecule has overall dimensions of approximately 60 A x 40 A x 35 A and is made up of two domains (termed large and small), which exhibit a similar supersecondary structure, linked by two antiparallel beta-strands. The small domain contains three alpha-helices and four parallel and one antiparallel beta-strands. The large domain is similar to the small domain but contains two additional alpha-helices and three more short antiparallel beta-strands. A comparison of the secondary structural motifs of GlnBP with those of other periplasmic binding proteins is discussed. A model of the "closed form" GlnBP-Gln complex has been proposed based on the crystal structures of the histidine-binding protein-His complex and "open form" GlnBP. This model has been successfully used as a search model in the crystal structure determination of the "closed form" GlnBP-Gln complex by molecular replacement methods. The model agrees remarkably well with the crystal structure of the Gln-GlnBP complex with root-mean-square deviation of 1.29 A. Our study shows that, at least in our case, it is possible to predict one conformational state of a periplasmic binding protein from another conformational state of the protein. The glutamine-binding pockets of the model and the crystal structure are compared and the modeling technique is described.

• Kovalenko OV et al.
• Mammalian ubiquitin-conjugating enzyme Ubc9 interacts with Rad51 recombination protein and localizes in synaptonemal complexes.
• Proc Natl Acad Sci U S A. 1996; 93: 2958-63
• Display abstract

• Hsubc9, a human gene encoding a ubiquitin-conjugating enzyme, has been cloned. The 18-kDa HsUbc9 protein is homologous to the ubiquitin-conjugating enzymes Hus5 of Schizosaccharomyces pombe and Ubc9 of Saccharomyces cerevisiae. The Hsubc9 gene complements a ubc9 mutation of S. cerevisiae. It has been mapped to chromosome 16p13.3 and is expressed in many human tissues, with the highest levels in testis and thymus. According to the Ga14 two-hybrid system analysis, HsUbc9 protein interacts with human recombination protein Rad51. A mouse homolog, Mmubc9, encodes an amino acid sequence that is identical to the human protein. In mouse spermatocytes, MmUbc9 protein, like Rad51 protein, localizes in synaptonemal complexes, which suggests that Ubc9 protein plays a regulatory role in meiosis.

• Yamazaki RK, Chau V
• Bacterial expression of the Saccharomyces cerevisiae ubiquitin-conjugating enzyme Ubc7.
• Protein Expr Purif. 1996; 7: 122-7
• Display abstract

• The coding sequence for the yeast ubiquitin-conjugating enzyme Ubc7 was obtained by PCR from Saccharomyces cerevisiae genomic DNA. This sequence was placed in a plasmid containing the lambdaPL promoter and was used for temperature-regulated expression in Escherichia coli. The expressed 18-kDa protein was isolated in the inclusion body fraction from bacterial lysates, in contrast to the soluble nature of other yeast ubiquitin-conjugating enzymes expressed in E. coli. Selective solubilization of the protein using 5 M urea followed by dialysis, MonoQ FPLC, and Superdex-75 FPLC yielded electrophoretically pure Ubc7 protein. The purified protein was enzymatically active as determined by formation of enzyme-linked thiolester with ubiquitin. The ability of Ubc7 protein to regain enzymatic activity after urea denaturation appears to be attributable to the stable core alpha/beta folded structure common to the ubiquitin-conjugating enzymes whose structures have been determined to date.

• Kalchman MA et al.
• Huntingtin is ubiquitinated and interacts with a specific ubiquitin-conjugating enzyme.
• J Biol Chem. 1996; 271: 19385-94
• Display abstract

• Using the yeast two-hybrid system, we have identified a human ubiquitin-conjugating enzyme (hE2-25K) as a protein that interacts with the gene product for Huntington disease (HD) (Huntingtin). This protein has complete amino acid identity with the bovine E2-25K protein and has striking similarity to the UBC-1, -4 and -5 enzymes of Saccharomyces cerevisiae. This protein is highly expressed in brain and a slightly larger protein recognized by an anti-E2-25K polyclonal antibody is selectively expressed in brain regions affected in HD. The huntingtin-E2-25K interaction is not obviously modulated by CAG length. We also demonstrate that huntingtin is ubiquitinated. These findings have implications for the regulated catabolism of the gene product for HD.

• Yasugi T, Howley PM
• Identification of the structural and functional human homolog of the yeast ubiquitin conjugating enzyme UBC9.
• Nucleic Acids Res. 1996; 24: 2005-10
• Display abstract

• Ubiquitin conjugating enzymes (UBCs) are a family of proteins directly involved in ubiquitination of proteins. Ubiquitination is known to be involved in control of a variety of cellular processes, including cell proliferation, through the targeting of key regulatory proteins for degradation. The ubc9 gene of the yeast Saccharomyces cerevisiae (Scubc9) is an essential gene which is required for cell cycle progression and is involved in the degradation of S phase and M phase cyclins. We have identified a human homolog of Scubc9 (termed hubc9) using the two hybrid screen for proteins that interact with the human papillomavirus type 16 E1 replication protein. The hubc9 encoded protein shares a very high degree of amino acid sequence similarity with ScUBC9 and with the homologous hus5+ gene product of Schizosaccharomyces pombe. Genetic complementation experiments in a S.cerevisiae ubc9ts mutant reveal that hUBC9 can substitute for the function of ScUBC9 required for cell cycle progression.

• Kaiser P, Mandl S, Schweiger M, Schneider R
• Characterization of functionally independent domains in the human ubiquitin conjugating enzyme UbcH2.
• FEBS Lett. 1995; 377: 193-6
• Display abstract

• UbcH2 encodes a human ubiquitin conjugating enzyme (E2) able to conjugate ubiquitin to histone H2A in an E3 independent manner in vitro, which indicates that UbcH2 directly interacts with its substrates. To identify parts of the enzyme that are capable of binding H2A, we expressed several deletion mutants of UbcH2 in E. coli and tested the ability of the affinity purified mutant proteins to ubiquitinate H2A in the presence of bacterial expressed E1 and ubiquitin. With this in vitro assay we identified a C-terminal part of UbcH2 to be important for the interaction with H2A. Transfer of this C-terminal domain to another human E2, which is unable to catalyze ubiquitination of histones, leads to a fully active hybrid human ubiquitin conjugating enzyme capable of H2A ubiquitination. These results demonstrate that UbcH2 consists of two functionally independent domains. A N-terminal core domain with ubiquitin conjugating activity, and a C-terminal domain which interacts with substrate proteins.

• Jensen JP, Bates PW, Yang M, Vierstra RD, Weissman AM
• Identification of a family of closely related human ubiquitin conjugating enzymes.
• J Biol Chem. 1995; 270: 30408-14
• Display abstract

• Two very closely related human E2 ubiquitin conjugating enzymes, UbfH5B and UbcH5C, have been identified. These enzymes are products of distinct genes and are 88-89% identical in amino acid sequence to the recently described human E2, UbcH5 (now designated UbcH5A), UbcH5A-C are homologous to a family of five ubiquitin conjugating enzymes from Arabidopsis thaliana, AtUBC8-12. They are also closely related to Saccharomyces cerevisiae ScUBC4 and ScUBC5, which are involved in the stress response, and play a central role in the targeting of short-lived regulatory proteins for degradation. mRNAs encoding UbcH5A-C were co-expressed in all cell lines and tissues evaluated, with UbcH5C transcripts generally expressed at the highest levels. Analysis of Southern blots suggests that there are likely to be other related members of this family. Both UbcH5B and UbcH5C form thiol ester adducts with ubiquitin, and have activities similar to UbcH5A and AtUBC8 in the conjugation of ubiquitin to target proteins in the presence of the human ubiquitin protein ligase E6-AP. These results establish the existence of a highly conserved, and widely expressed, family of human ubiquitin conjugating enzymes.

• Rolfe M et al.
• Reconstitution of p53-ubiquitinylation reactions from purified components: the role of human ubiquitin-conjugating enzyme UBC4 and E6-associated protein (E6AP).
• Proc Natl Acad Sci U S A. 1995; 92: 3264-8
• Display abstract

• The E6 protein of the high-risk human papillomaviruses inactivates the tumor suppressor protein p53 by stimulating its ubiquitinylation and subsequent degradation. Ubiquitinylation is a multistep process involving a ubiquitin-activating enzyme, one of many distinct ubiquitin-conjugating enzymes, and in certain cases, a ubiquitin ligase. In human papillomavirus-infected cells, E6 and the E6-associated protein are thought to act as a ubiquitin-protein ligase in the ubiquitinylation of p53. Here we describe the cloning of a human ubiquitin-conjugating enzyme that specifically ubiquitinylates E6-associated protein. Furthermore, we define the biochemical pathway of p53 ubiquitinylation and demonstrate that in vivo inhibition of various components in the pathway leads to an inhibition of E6-stimulated p53 degradation.

• Gwozd CS, Arnason TG, Cook WJ, Chau V, Ellison MJ
• The yeast UBC4 ubiquitin conjugating enzyme monoubiquitinates itself in vivo: evidence for an E2-E2 homointeraction.
• Biochemistry. 1995; 34: 6296-302
• Display abstract

• Here we report that the stress-related conjugating enzyme UBC4 from Saccharomyces cerevisiae is monoubiquintinated in vivo. The UBC4-ubiquitin conjugate was detected by the coexpression in yeast of epitope-tagged ubiquitin in combination with either untagged or epitope-tagged versions of UBC4. Under these conditions the UBC4 conjugate proved to be the most abundant conjugate detected. Using chemical mapping and site-directed mutation, the site of ubiquitination was localized to a single lysine (K144) near the carboxy terminus of UBC4. A second lysine within UBC4 (K64) was also identified whose mutation resulted in the loss of ubiquitination at K144. The mutation of either K64 or K144 had no obvious effect on the known in vivo functions associated with UBC4. In another experiment, a nonfunctional UBC4 derivative with a mutation at the active site was also found to be monoubiquitinated in a manner that depended on the expression of active UBC4. This result indicated that ubiquitin was transferred in an intermolecular reaction from one UBC4 monomer to another. Cross-linking analysis demonstrated that UBC4 monomers directly and specifically interact with one another in vitro. Both the in vivo and in vitro observations reported here, in combination with previous findings, support the view that interactions between ubiquitin conjugating enzymes represent a general phenomenon.

• Ptak C, Prendergast JA, Hodgins R, Kay CM, Chau V, Ellison MJ
• Functional and physical characterization of the cell cycle ubiquitin-conjugating enzyme CDC34 (UBC3). Identification of a functional determinant within the tail that facilitates CDC34 self-association.
• J Biol Chem. 1994; 269: 26539-45
• Display abstract

• Like several other ubiquitin-conjugating enzymes, the yeast cell cycle enzyme CDC34 (UBC3) has a carboxyl-terminal extension or tail. These tails appear to carry out unique functions that can vary from one ubiquitin-conjugating enzyme to the next. Using biophysical techniques we have determined that the tail of CDC34 constitutes a highly structured and extended domain. Although the tail of CDC34 is the largest tail identified to date (125 residues), we have found that only 39 residues lying adjacent to the catalytic domain are necessary and sufficient for full cell cycle function and that this region fulfills a novel function that may be common to the tails of other ubiquitin-conjugating enzymes. Cross-linking studies demonstrate that this region facilitates a physical interaction between CDC34 monomers in vitro. Furthermore, phenotypic analysis of various CDC34 derivatives expressed in different cdc34 mutant strains indicates that this region facilitates the same interaction in vivo. Based on these findings, it appears that the cell cycle function of CDC34 is dependent upon the ability of CDC34 monomers to interact with one another and that this interaction is mediated by a small region of the CDC34 tail. The similarity of this region with sequences contained within the tails of the UBC1 and UBC6 enzymes suggests that these tails may function in a similar manner.

• Mathieu M, Zeelen JP, Pauptit RA, Erdmann R, Kunau WH, Wierenga RK
• The 2.8 A crystal structure of peroxisomal 3-ketoacyl-CoA thiolase of Saccharomyces cerevisiae: a five-layered alpha beta alpha beta alpha structure constructed from two core domains of identical topology.
• Structure. 1994; 2: 797-808
• Display abstract

• BACKGROUND: The peroxisomal enzyme 3-ketoacyl-coenzyme A thiolase of the yeast Saccharomyces cerevisiae is a homodimer with 417 residues per subunit. It is synthesized in the cytosol and subsequently imported into the peroxisome where it catalyzes the last step of the beta-oxidation pathway. We have determined the structure of this thiolase in order to study the reaction mechanism, quaternary associations and intracellular targeting of thiolases generally, and to understand the structural basis of genetic disorders associated with human thiolases. RESULTS: Here we report the crystal structure of unliganded yeast thiolase refined at 2.8 A resolution. The enzyme comprises three domains; two compact core domains having the same fold and a loop domain. Each of the two core domains is folded into a mixed five-stranded beta-sheet covered on each side by helices and the two are assembled into a five-layered alpha beta alpha beta alpha structure. The central layer is formed by two helices, which point with their amino termini towards the active site. The loop domain, which is to some extent stabilized by interactions with the other subunit, runs over the surface of the two core domains, encircling the active site of its own subunit. CONCLUSIONS: The crystal structure of thiolase shows that the active site is a shallow pocket, shaped by highly conserved residues. Two conserved cysteines and a histidine at the floor of this pocket probably play key roles in the reaction mechanism. The two active sites are on the same face of the dimer, far from the amino and carboxyl termini of both subunits and the disordered amino-terminal import signal sequence.

• Raboy B, Kulka RG
• Role of the C-terminus of Saccharomyces cerevisiae ubiquitin-conjugating enzyme (Rad6) in substrate and ubiquitin-protein-ligase (E3-R) interactions.
• Eur J Biochem. 1994; 221: 247-51
• Display abstract

• The product of the RAD6 (UBC2) gene of Saccharomyces cerevisiae is a ubiquitin-conjugating enzyme (Rad6) which is implicated in DNA repair, induced mutagenesis, retrotransposition, sporulation and the degradation of proteins with destabilizing N-terminal amino acid residues. Deletion of the 23-residue acidic C-terminus of Rad6 impairs sporulation and N-end rule protein degradation in vivo but does not affect other functions such as DNA repair and induced mutagenesis. We have investigated the role of the C-terminus of Rad6 in in vitro interactions with various substrates and with a putative ubiquitin-protein ligase, E3-R. The removal of the Rad6 C-terminus had significant different effects on enzyme activity for individual substrates. Although the 23-residue truncated Rad6-149 protein had markedly impaired activity for histone H2B and micrococcal nuclease, the activity for cytochrome c was the same as that of the intact Rad6 protein. Similarly, truncation of Rad6 had no effect on its activity for several poor substrates, namely, beta-casein, beta-lactoglobulin and oxidized RNase. E3-R stimulated the activities of both Rad6 and Rad6-149 for the latter three substrates to similar degrees. E3-R appears to act by enhancing the low intrinsic affinity of Rad6 and Rad6-149 for these substrates. Thus Rad6 can act in three different modes in vitro depending on the substrate, namely unassisted C-terminus-dependent, unassisted C-terminus-independent and E3-R-assisted C-terminus-independent modes. We also examined the results of removing the C-terminal acidic region of Cdc34 (Ubc3), a ubiquitin-conjugating enzyme closely related to Rad6. Truncation of Cdc34 like that of Rad6 had no effect on activity for beta-casein, beta-lactoglobulin or oxidized RNase in the presence or absence of E3-R.

• Kim TK, Roeder RG
• CTD-like sequences are important for transcriptional activation by the proline-rich activation domain of CTF1.
• Nucleic Acids Res. 1994; 22: 251-251

• Crane DI, Kalish JE, Gould SJ
• The Pichia pastoris PAS4 gene encodes a ubiquitin-conjugating enzyme required for peroxisome assembly.
• J Biol Chem. 1994; 269: 21835-44
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• We report here the cloning and initial characterization of PAS4, a gene required for peroxisome assembly in the yeast Pichia pastoris. The PAS4 gene encodes a 24-kDa protein (Pas4p) that is located on the cytoplasmic surface of peroxisomes and is induced during peroxisome proliferation. Analysis of the Pas4p sequence revealed a high degree of similarity to ubiquitin-conjugating enzymes, particularly in the region surrounding the putative active-site cysteine residue with which ubiquitin forms a thioester bond. As expected for a ubiquitin-conjugating enzyme, substitution of alanine or serine for the conserved active-site cysteine residue abolished PAS4 function. In addition, a small amount of a 32 kDa form of Pas4p (the predicted size of a Pas4p-ubiquitin conjugate) was detected both in vivo and in vitro. This species was eliminated by reducing agents and was not detected in the cysteine to alanine substitution mutant, suggesting that it is a Pas4p-ubiquitin conjugate. Using a yeast strain that overexpresses a Myc-ubiquitin fusion protein, we demonstrate directly that this conjugate contains ubiquitin. We conclude from these observations that PAS4 is a member of the ubiquitin-conjugating enzyme gene family and that one or more ubiquitination reactions are required for peroxisome assembly.

• Watkins JF, Sung P, Prakash S, Prakash L
• The extremely conserved amino terminus of RAD6 ubiquitin-conjugating enzyme is essential for amino-end rule-dependent protein degradation.
• Genes Dev. 1993; 7: 250-61
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• The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme that is required for DNA repair, damage-induced mutagenesis, and sporulation. In addition, RAD6 mediates the multiubiquitination and degradation of amino-end rule protein substrates. The structure and function of RAD6 have been remarkably conserved during eukaryotic evolution. Here, we examine the role of the extremely conserved amino terminus, which has remained almost invariant among RAD6 homologs from yeast to human. We show that RAD6 is concentrated in the nucleus and that the amino-terminal deletion mutation, rad6 delta 1-9, does not alter the location of the protein. The amino-terminal domain, however, is essential for the multiubiquitination and degradation of amino-end rule substrates. In the rad6 delta 1-9 mutant, beta-galactosidase proteins bearing destabilizing amino-terminal residues become long lived, and purified rad6 delta 1-9 protein is ineffective in ubiquitin-protein ligase (E3)-dependent protein degradation in the proteolytic system derived from rabbit reticulocytes. The amino terminus is required for physical interaction of RAD6 with the yeast UBR1-encoded E3 enzyme, as the rad6 delta 1-9 protein is defective in this respect. The rad6 delta 1-9 mutant is defective in sporulation, shows reduced efficiency of DNA repair, but is proficient in UV mutagenesis. E3-dependent protein degradation by RAD6 could be essential for sporulation and could affect the efficiency of DNA repair.

• Girod PA, Vierstra RD
• A major ubiquitin conjugation system in wheat germ extracts involves a 15-kDa ubiquitin-conjugating enzyme (E2) homologous to the yeast UBC4/UBC5 gene products.
• J Biol Chem. 1993; 268: 955-60
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• In eukaryotes, conjugation of ubiquitin to proteins serves as a committed step for intracellular protein degradation. Formation of ubiquitin-protein conjugates involves the transfer of ubiquitin-conjugating enzyme (E2)-bound ubiquitin to the target proteins with or without the assistance of ubiquitin-protein ligase (E3). We report the isolation and characterization of an E2 purified from wheat germ that accounts for the majority of ubiquitin conjugation activity observed in vitro. This E2 is basic, has an apparent molecular mass of 15 kDa, and forms oligomers that dissociate upon treatment with sulfhydryl reducing agents. E(2)15kDa will not work alone in vitro but requires an additional factor putatively identified as an E3 for substrate recognition. This E3 is distinct from E3 alpha previously described to be required for N-terminal recognition of target proteins. Partial amino acid sequence analysis of E(2)15kDa revealed a substantial identity (approximately 80% in two peptide regions) with yeast E2s encoded by UBC4/UBC5 genes. This homology was confirmed by immunodetection of a 16-kDa yeast protein corresponding to the molecular mass of the UBC4/UBC5 proteins with E(2)15kDa antisera. The products of yeast UBC4 and UBC5 genes along with that of UBC1 gene constitute a subfamily of functionally overlapping E2s that mediate the selective degradation of short-lived and abnormal proteins in vivo. Considering the high degree of functional and structural similarity of wheat E(2)15kDa with that of yeast UBC4/UBC5, it is likely that yeast UBC4/UBC5 and their homologs from other eukaryotes exhibit the same E3 dependence in performing their roles in protein degradation.

• Ko TP, Ng JD, McPherson A
• The three-dimensional structure of canavalin from jack bean (Canavalia ensiformis).
• Plant Physiol. 1993; 101: 729-44
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• The three-dimensional structure of the vicilin storage protein canavalin, from Canavalia ensiformis, has been determined in a hexagonal crystal by x-ray diffraction methods. The model has been refined at 2.6 A resolution to an R factor of 0.197 with acceptable geometry. Because of proteolysis, 58 of 419 amino acids of the canavalin polypeptide are not visible in the electron density map. The canavalin subunit is composed of two extremely similar structural domains that reflect the tandem duplication observed in the cDNA and in the amino acid sequence. Each domain consists of two elements, a compact, eight-stranded beta-barrel having the "Swiss roll" topology and an extended loop containing several short alpha-helices. The root mean square deviation between 84 pairs of corresponding C alpha atoms making up the strands of the two beta-barrels in a subunit is 0.78 A, and for 112 pairs of structurally equivalent C alpha atoms of the two domains the deviation is 1.37 A. The interface between domains arises from the apposition of broad hydrophobic surfaces formed by side chains originating from one side of the beta-barrels, supplemented by at least four salt bridges. The interfaces between subunits in the trimer are supplied by the extended loop elements. These interfaces are also composed primarily of hydrophobic residues supplemented by six salt bridges. The canavalin subunits have dimensions about 40 x 40 x 86 A, and the oligomer is a disk-shaped molecule about 88 A in diameter with a thickness of about 40 A. The distribution of domains lends a high degree of pseudo-32-point group symmetry to the molecule. There is a large channel of 18 A diameter, lined predominantly by hydrophilic and charged amino acids, running through the molecule along the 3-fold axis. The majority of residues conserved between domains and among vicilins occur at the interface between subunits but appear otherwise arbitrarily distributed within the subunit, although predominantly on its exterior.

• Andersen B, Schonemann MD, Flynn SE, Pearse RV 2nd, Singh H, Rosenfeld MG
• Skn-1a and Skn-1i: two functionally distinct Oct-2-related factors expressed in epidermis.
• Science. 1993; 260: 78-82
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• Two forms of a member of the POU domain family of transcriptional regulators, highly related to Oct-2, are selectively expressed in terminally differentiating epidermis and hair follicles. One form, referred to as Skn-1i, contains an amino-terminal domain that inhibits DNA binding and can inhibit transactivation by Oct-1. A second form, Skn-1a, contains an alternative amino terminus and serves to activate cytokeratin 10 (K10) gene expression. The pattern of expression of the Skn-1a/i gene products and the effect of the alternative products on the expression of other genes suggest that these factors serve regulatory functions with respect to epidermal development.

• Chen P, Johnson P, Sommer T, Jentsch S, Hochstrasser M
• Multiple ubiquitin-conjugating enzymes participate in the in vivo degradation of the yeast MAT alpha 2 repressor.
• Cell. 1993; 74: 357-69
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• Attachment of ubiquitin to proteins is catalyzed by a family of ubiquitin-conjugating (UBC) enzymes. Although these enzymes are essential for many cellular processes; their molecular functions remain unclear because no physiological target has been identified for any of them. Here we show that four UBC proteins (UBC4, UBC5, UBC6, and UBC7) target the yeast MAT alpha 2 transcriptional regulator for intracellular degradation by two distinct ubiquitination pathways. UBC6 and UBC7 define one of the pathways and can physically associate. The UBC6/UBC7-containing complex targets the Deg1 degradation signal of alpha 2, a conclusion underscored by the finding that UBC6 is encoded by DOA2, a gene previously implicated in Deg1-mediated degradation. These data reveal an unexpected overlap in substrate specificity among diverse UBC enzymes and suggest a combinatorial mechanism of substrate selection in which UBC enzymes partition into multiple ubiquitination complexes.

• Jungmann J, Reins HA, Schobert C, Jentsch S
• Resistance to cadmium mediated by ubiquitin-dependent proteolysis.
• Nature. 1993; 361: 369-71
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• Cadmium is a potent poison for living cells. In man, chronic exposure to low levels of cadmium results in damage to kidneys and has been linked to neoplastic disease and ageing, and acute exposure can cause damage to a variety of organs and tissues. Cadmium reacts with thiol groups and can substitute for zinc in certain proteins, but the reason for its toxicity in vivo remains uncertain. In eukaryotes, an important selective proteolysis pathway for the elimination of abnormal proteins that are generated under normal or stress conditions is ATP-dependent and mediated by the ubiquitin system. Substrates of this pathway are first recognized by ubiquitin-conjugating enzymes (or auxiliary factors) which covalently attach ubiquitin, a small and highly conserved protein, to specific internal lysine residues of proteolytic substrates. Ubiquitinated substrates are then degraded by the proteasome, a multisubunit protease complex. Here we show that expression of this ubiquitin-dependent proteolysis pathway in yeast is activated in response to cadmium exposure and that mutants deficient in specific ubiquitin-conjugating enzymes are hypersensitive to cadmium. Moreover, mutants in the proteasome are hypersensitive to cadmium, suggesting that cadmium resistance is mediated in part by degradation of abnormal proteins. This indicates that a major reason for cadmium toxicity may be cadmium-induced formation of abnormal proteins.

• Girod PA, Carpenter TB, van Nocker S, Sullivan ML, Vierstra RD
• Homologs of the essential ubiquitin conjugating enzymes UBC1, 4, and 5 in yeast are encoded by a multigene family in Arabidopsis thaliana.
• Plant J. 1993; 3: 545-52
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• The covalent attachment of the 76 amino acid protein ubiquitin is an important prerequisite for the degradation of many eukaryotic proteins. The specificity of this ligation is accomplished in part by a family of distinct ubiquitin conjugating enzymes (E2s) working in concert with specific ubiquitin-protein ligases (E3s). Three essential E2s in yeast encoded by ScUBC1, -4, and -5 comprise a functionally overlapping E2 subfamily that appears responsible for degrading most abnormal and short-lived proteins. A 15 kDa E2 protein homologous to this family has been identified previously in wheat germ, designated TaE2(15kDa) (Girod and Vierstra (1993) J. Biol. Chem. 268, 955-960). This E2 is responsible for much of the ubiquitin conjugating activity observed in wheat germ extracts and works together with a unique E3 (designated E3 gamma) for substrate recognition. In this paper, the cloning of five genes encoding E2(15kDa) from Arabidiopsis thaliana is described (designated AtUBC8-12). They encode 149 amino acid basic proteins 94-98% similar to each other and 88-92% similar to ScUBC4 at the amino acid sequence level. In contrast, AtUBC8-12 are only 55-65% similar to the Arabidopsis E2s encoded by AtUBC1, -4, and -7. The AtUBC8-12 proteins do not contain N- or C-terminal extensions and have the active site at residue Cys-86, based on their homology with other E2s. Analyses of genomic Southern blots are consistent with the existence of multiple members encoding this E2 subfamily. AtUBC8-12 are transcribed to yield about 800 nucleotide mRNAs that, unlike ScUBC4 and -5, are not strongly induced by heat shock.(ABSTRACT TRUNCATED AT 250 WORDS)

• Cook WJ, Jeffrey LC, Carson M, Chen Z, Pickart CM
• Structure of a diubiquitin conjugate and a model for interaction with ubiquitin conjugating enzyme (E2).
• J Biol Chem. 1992; 267: 16467-71
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• Covalent ligation of multiubiquitin chains targets eukaryotic proteins for degradation. In such multiubiquitin chains, successive ubiquitins are linked by an isopeptide bond involving the side chain of Lys48 and the carboxyl group of Gly76. The crystal structure of a diubiquitin conjugate has been determined and refined at 2.3-A resolution. The molecule has internal approximate 2-fold symmetry with multiple hydrophobic and hydrophilic contacts along the 2-fold axis. The structure of the diubiquitin conjugate suggests determinants for recognition of multiubiquitin chains. A model for the interaction of diubiquitin and a ubiquitin conjugating enzyme (E2) is proposed.

• Chun PW, Jou WS
• Molecular conformation of ubiquitinated structures and the implications for regulatory function.
• J Mol Graph. 1992; 10: 7-11
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• The molecular conformation of ubiquitinated structures and the validity of the N-end rule were examined by simulating the molecular mechanics to ascertain the global energy-minimized structure. We examined the chemical linkage involved in attaching the ubiquitin carboxyl terminus to the N-terminus of three different x-hexapeptides, where x is the amino group of the acceptor peptide--either valine, arginine or glutamic acid--(x-K linkage) and to the epsilon-amino group of lysine of the acceptor hexapeptide x-glu1-his2-lys3-gly4-lys5-val6 (K-K linkage) through the formation of an isopeptide bond. Changes in conformation and molecular stability of the multi-ubiquitinated structures were determined by energy-minimization procedures using the SYBYL program developed by Tripos Associates. In the x-K linkage, the ubiquitin molecule is stretched in the beta-pleated sheets and beta-turns while the alpha-helices expand, as the molecule continues to unfold linearly. In the K-K linkage, the ubiquitin molecules have turned into a u-shaped, semi-circular alignment, contracting into a compact, folded structure.

• Sullivan ML, Vierstra RD
• Cloning of a 16-kDa ubiquitin carrier protein from wheat and Arabidopsis thaliana. Identification of functional domains by in vitro mutagenesis.
• J Biol Chem. 1991; 266: 23878-85
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• Ubiquitin carrier proteins (E2s) are involved in the covalent attachment of ubiquitin to a variety of cellular target proteins in eukaryotes. Here, we report the cloning of genes from wheat and Arabidopsis thaliana that encode 16-kDa E2s and a domain analysis of E2s by in vitro mutagenesis. The genes for E216kDa, which we have designated wheat and At UBC1, encode proteins that are only 33% identical (58% similar) with a 23-kDa E2 from wheat (encoded by the gene now designated wheat UBC4), but are 63% identical (82% similar) with the E2 encoded by the Saccharomyces cerevisiae DNA repair gene, RAD6. Unlike the proteins encoded by RAD6 and wheat UBC4, the UBC1 gene products lack acidic C-terminal domains extending beyond the conserved core of the proteins and are incapable of efficient in vitro ligation of ubiquitin to histones. From enzymatic analysis of the UBC1 and UBC4 gene products mutagenized in vitro, we have identified several domains important for E2 function, including the active site cysteine and N-terminal and C-terminal domains. Cysteine residues 88 and 85 in the UBC1 and UBC4 gene products, respectively, are necessary for formation of the ubiquitin-E2 thiol ester intermediate. Whereas the UBC1 gene product does not require its additional cysteine residue at position 116 for thiol ester formation, alteration of cysteine 143 in the UBC4 gene product greatly diminishes this ability. The N terminus of UBC1 contains two domains that affect activity: a proximal region containing hydroxylated and uncharged residues whose removal increases the rate of thiol ester formation and a distal tract rich in basic residues. Deletion or substitution of these basic residues with neutral residues diminishes the rate of thiol ester formation. We have demonstrated also that C-terminal extensions can function to confer substrate specificity to E2s. When the acidic extension was deleted from UBC4, the protein was unable to efficiently conjugate ubiquitin to histones in vitro. Furthermore, fusion of the UBC4 acidic extension to the C terminus of UBC1 resulted in a chimeric protein capable of efficient histone conjugation, as did fusion of short tracts of alternating aspartate and glutamate residues. This result suggests that the target protein specificity of E2s can be altered by the addition of appropriate C-terminal extensions, thus providing a way to modify the selectivity of the ubiquitin system.

• Jentsch S, Seufert W, Hauser HP
• Genetic analysis of the ubiquitin system.
• Biochim Biophys Acta. 1991; 1089: 127-39

• Seufert W, Jentsch S
• Yeast ubiquitin-conjugating enzymes involved in selective protein degradation are essential for cell viability.
• Acta Biol Hung. 1991; 42: 27-37
• Display abstract

• Ubiquitin-mediated proteolysis is a major pathway for selective protein degradation in eukaryotic cells. This proteolysis pathway involves the processive covalent attachment of ubiquitin to proteolytic substrates and their subsequent degradation by a specific ATP-dependent protease complex. We have cloned the genes and characterized the function of ubiquitin-conjugating enzymes (UBCs) from the yeast Saccharomyces cerevisiae. UBC1, UBC4 and UBC5 enzymes were found to mediate selective degradation of short-lived and abnormal proteins. These enzymes have overlapping functions and constitute a UBC subfamily essential for growth. UBC1 is specifically required at early stages of growth after germination of spores. UBC4 and UBC5 enzymes generate high molecular weight ubiquitin-protein conjugates and comprise a major ubiquitin-conjugation activity in yeast cells. Moreover, these enzymes are central components of the cellular stress response.

• Babu YS, Bugg CE, Cook WJ
• Structure of calmodulin refined at 2.2 A resolution.
• J Mol Biol. 1988; 204: 191-204
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• The crystal structure of mammalian calmodulin has been refined at 2.2 A (1 A = 0.1 nm) resolution using a restrained least-squares method. The final crystallographic R-factor, based on 6685 reflections in the range 2.2 A less than or equal to d less than or equal to 5.0 A with intensities exceeding 2.5 sigma, is 0.175. Bond lengths and bond angles in the molecule have root-mean-square deviations from ideal values of 0.016 A and 1.7 degrees, respectively. The refined model includes residues 5 to 147, four Ca2+ and 69 water molecules per molecule of calmodulin. The electron density for residues 1 to 4 and 148 is poorly defined, and they are not included in the model. The molecule is shaped somewhat like a dumbbell, with an overall length of 65 A; the two lobes are connected by a seven-turn alpha-helix. Prominent secondary structural features include seven alpha-helices, four Ca2+-binding loops, and two short, double-stranded antiparallel beta-sheets between pairs of adjacent Ca2+-binding loops. The four Ca2+-binding domains in calmodulin have a typical EF hand conformation (helix-loop-helix) and are similar to those described in other Ca2+-binding proteins. The X-ray structure determination of calmodulin shows a large hydrophobic cleft in each half of the molecule. These hydrophobic regions probably represent the sites of interaction with many of the pharmacological agents known to bind to calmodulin.

• Vijay-Kumar S, Bugg CE, Cook WJ
• Structure of ubiquitin refined at 1.8 A resolution.
• J Mol Biol. 1987; 194: 531-44
• Display abstract

• The crystal structure of human erythrocytic ubiquitin has been refined at 1.8 A resolution using a restrained least-squares procedure. The crystallographic R-factor for the final model is 0.176. Bond lengths and bond angles in the molecule have root-mean-square deviations from ideal values of 0.016 A and 1.5 degrees, respectively. A total of 58 water molecules per molecule of ubiquitin are included in the final model. The last four residues in the molecule appear to have partial occupancy or large thermal motion. The overall structure of ubiquitin is extremely compact and tightly hydrogen-bonded; approximately 87% of the polypeptide chain is involved in hydrogen-bonded secondary structure. Prominent secondary structural features include three and one-half turns of alpha-helix, a short piece of 3(10)-helix, a mixed beta-sheet that contains five strands, and seven reverse turns. There is a marked hydrophobic core formed between the beta-sheet and alpha-helix. The molecule features a number of unusual secondary structural features, including a parallel G1 beta-bulge, two reverse Asx turns, and a symmetrical hydrogen-bonding region that involves the two helices and two of the reverse turns.

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