Faculty Opinions recommendation of Arabidopsis COP10 forms a complex with DDB1 and DET1 in vivo and enhances the activity of ubiquitin conjugating enzymes.

OTUB1 is a deubiquitinating enzyme that cleaves Lys-48–linked polyubiquitin chains and also regulates ubiquitin signaling through a unique, noncatalytic mechanism. OTUB1 binds to a subset of E2 ubiquitin-conjugating enzymes and inhibits their activity by trapping the E2∼ubiquitin thioester and preventing ubiquitin transfer. The same set of E2s stimulate the deubiquitinating activity of OTUB1 when the E2 is not charged with ubiquitin. Previous studies have shown that, in cells, OTUB1 binds to E2-conjugating enzymes of the UBE2D (UBCH5) and UBE2E families, as well as to UBE2N (UBC13). Cellular roles have been identified for the interaction of OTUB1 with UBE2N and members of the UBE2D family, but not for interactions with UBE2E E2 enzymes. We report here a novel role for OTUB1–E2 interactions in modulating E2 protein ubiquitination. We observe that Otub1−/− knockout mice exhibit late-stage embryonic lethality. We find that OTUB1 depletion dramatically destabilizes the E2-conjugating enzyme UBE2E1 (UBCH6) in both mouse and human OTUB1 knockout cell lines. Of note, this effect is independent of the catalytic activity of OTUB1, but depends on its ability to bind to UBE2E1. We show that OTUB1 suppresses UBE2E1 autoubiquitination in vitro and in cells, thereby preventing UBE2E1 from being targeted to the proteasome for degradation. Taken together, we provide evidence that OTUB1 rescues UBE2E1 from degradation in vivo.

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Two Ubiquitin-Conjugating Enzymes, UbcP1/Ubc4 and UbcP4/Ubc11, Have Distinct Functions for Ubiquitination of Mitotic Cyclin

Molecular and Cellular Biology ◽

10.1128/mcb.23.10.3497-3505.2003 ◽

2003 ◽

Vol 23 (10) ◽

pp. 3497-3505 ◽

Cited By ~ 23

Author(s):

Hiroaki Seino ◽

Tsutomu Kishi ◽

Hideo Nishitani ◽

Fumiaki Yamao

Keyword(s):

Fission Yeast ◽

Ubiquitin Proteasome System ◽

High Expression ◽

Mitotic Cyclin ◽

Ubiquitin Conjugating Enzyme ◽

Conjugating Enzymes ◽

Ubiquitin Conjugating Enzymes ◽

Mutant Cells ◽

Conjugating Enzyme

ABSTRACT Cell cycle events are regulated by sequential activation and inactivation of Cdk kinases. Mitotic exit is accomplished by the inactivation of mitotic Cdk kinase, which is mainly achieved by degradation of cyclins. The ubiquitin-proteasome system is involved in this process, requiring APC/C (anaphase-promoting complex/cyclosome) as a ubiquitin ligase. In Xenopus and clam oocytes, the ubiquitin-conjugating enzymes that function with APC/C have been identified as two proteins, UBC4 and UBCx/E2-C. Previously we reported that the fission yeast ubiquitin-conjugating enzyme UbcP4/Ubc11, a homologue of UBCx/E2-C, is required for mitotic transition. Here we show that the other fission yeast ubiquitin-conjugating enzyme, UbcP1/Ubc4, which is homologous to UBC4, is also required for mitotic transition in the same manner as UbcP4/Ubc11. Both ubiquitin-conjugating enzymes are essential for cell division and directly required for the degradation of mitotic cyclin Cdc13. They function nonredundantly in the ubiquitination of CDC13 because a defect in ubcP1/ubc4 + cannot be suppressed by high expression of UbcP4/Ubc11 and a defect in ubcP4/ubc11 + cannot be suppressed by high expression of UbcP1/Ubc4. In vivo analysis of the ubiquitinated state of Cdc13 shows that the ubiquitin chains on Cdc13 were short in ubcP1/ubc4 mutant cells while ubiquitinated Cdc13 was totally reduced in ubcP4/ubc11 mutant cells. Taken together, these results indicate that the two ubiquitin-conjugating enzymes play distinct and essential roles in the degradation of mitotic cyclin Cdc13, with the UbcP4/Ubc11-pathway initiating ubiquitination of Cdc13 and the UbcP1/Ubc4-pathway elongating the short ubiquitin chains on Cdc13.

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Arabidopsis COP10 forms a complex with DDB1 and DET1 in vivo and enhances the activity of ubiquitin conjugating enzymes

Genes & Development ◽

10.1101/gad.1229504 ◽

2004 ◽

Vol 18 (17) ◽

pp. 2172-2181 ◽

Cited By ~ 119

Author(s):

Y. Yanagawa

Keyword(s):

Conjugating Enzymes ◽

Ubiquitin Conjugating Enzymes

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Characterization of OTUB1 activation and inhibition by different E2 enzymes

10.1101/847806 ◽

2019 ◽

Author(s):

Lauren T. Que ◽

Marie E. Morrow ◽

Cynthia Wolberger

Keyword(s):

Deubiquitinating Enzyme ◽

Polyubiquitin Chains ◽

Kinetics And Thermodynamics ◽

Conjugating Enzymes ◽

E2 Enzymes ◽

Ubiquitin Conjugating Enzymes ◽

Stimulation Of

AbstractOTUB1 is a highly expressed cysteine protease that specifically cleaves K48-linked polyubiquitin chains. This unique deubiquitinating enzyme (DUB) can bind to a subset of E2 ubiquitin conjugating enzymes, forming complexes in which the two enzymes can regulate one another’s activity. OTUB1 can non-catalytically suppress the ubiquitin conjugating activity of its E2 partners by sequestering the charged E2~Ub thioester and preventing ubiquitin transfer. The same E2 enzymes, when uncharged, can stimulate the DUB activity of OTUB1 in vitro, although the importance of OTUB1 stimulation in vivo remains unclear. In order to assess the potential balance between these activities that might occur in cells, we characterized the kinetics and thermodynamics governing the formation and activity of OTUB1:E2 complexes. We show that both stimulation of OTUB1 by E2 enzymes and noncatalytic inhibition of E2 enzymes by OTUB1 occur at physiologically relevant concentrations of both partners. Whereas E2 partners differ in their ability to stimulate OTUB1 activity, we find that this variability is not correlated with the affinity of each E2 for OTUB1. In addition to UBE2N and the UBE2D isoforms, we find that OTUB1 inhibits polyubiquitination activity of all three UBE2E enzymes, UBE2E1, UBE2E2, and UBE2E3. Interestingly, although OTUB1 also inhibits the autoubiquitination activity of UBE2E1 and UBE2E2, it is unable to suppress autoubiquitination by UBE2E3.

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Regulation of p53 by the Ubiquitin-conjugating Enzymes UbcH5B/C in Vivo

Journal of Biological Chemistry ◽

10.1074/jbc.m403362200 ◽

2004 ◽

Vol 279 (40) ◽

pp. 42169-42181 ◽

Cited By ~ 95

Author(s):

Mark K. Saville ◽

Alison Sparks ◽

Dimitris P. Xirodimas ◽

Julie Wardrop ◽

Lauren F. Stevenson ◽

...

Keyword(s):

Conjugating Enzymes ◽

Ubiquitin Conjugating Enzymes

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UbcH5A, a member of human E2 ubiquitin-conjugating enzymes, is closely related to SFT, a stimulator of iron transport, and is up-regulated in hereditary hemochromatosis

Blood ◽

10.1182/blood-2002-07-2192 ◽

2003 ◽

Vol 101 (8) ◽

pp. 3288-3293 ◽

Cited By ~ 8

Author(s):

Sven G. Gehrke ◽

Hans-Dieter Riedel ◽

Thomas Herrmann ◽

Boris Hadaschik ◽

Karin Bents ◽

...

Keyword(s):

Mrna Expression ◽

Mammalian Cells ◽

Transmembrane Protein ◽

Hereditary Hemochromatosis ◽

Regulatory Region ◽

Iron Chelation ◽

Conjugating Enzymes ◽

Ubiquitin Conjugating Enzymes

Abstract SFT, a stimulator of iron (Fe) transport, has been described as a transmembrane protein that facilitates the uptake of ferrous and ferric iron in mammalian cells. This study was initiated to investigate the 5′ regulatory region of SFT and its role in the etiology of hereditary hemochromatosis. Sequence analyses of the putative 5′ regulatory region revealed that the SFT cDNA sequence corresponds to intron 6/exon 7 of UbcH5A, a member of E2 ubiquitin-conjugating enzymes, which is involved in the iron-dependent ubiquitination of the hypoxia-inducible factor (HIF) by the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase complex. Further mRNA expression studies using a sequence-specific reverse transcriptase–polymerase chain reaction (RT-PCR) assay showed that UbcH5A is significantly up-regulated in the liver of iron-overloaded patients with hereditary hemochromatosis, as previously published for SFT. However, in vitro studies on HepG2 cells failed to demonstrate any significant UbcH5A regulation in response to iron loading or iron chelation. In conclusion, in vivo mRNA expression data previously obtained for SFT might be attributed to UbcH5A. The role of UbcH5A and the ubiquitination pathway in the etiology of hereditary hemochromatosis remains to be elucidated further.

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Human Cdc34 and Rad6B Ubiquitin-Conjugating Enzymes Target Repressors of Cyclic AMP-Induced Transcription for Proteolysis

Molecular and Cellular Biology ◽

10.1128/mcb.19.7.5001 ◽

1999 ◽

Vol 19 (7) ◽

pp. 5001-5013 ◽

Cited By ~ 57

Author(s):

Debananda Pati ◽

Marvin L. Meistrich ◽

Sharon E. Plon

Keyword(s):

Cyclic Amp ◽

Germ Cells ◽

Mammalian Cells ◽

Mitotic Cell ◽

Dominant Negative ◽

Testicular Development ◽

Developmentally Regulated ◽

Conjugating Enzymes ◽

Ubiquitin Conjugating Enzymes

ABSTRACT Ubiquitin-mediated proteolysis controls diverse physiological processes in eukaryotes. However, few in vivo targets of the mammalian Cdc34 and Rad6 ubiquitin-conjugating enzymes are known. A yeast-based genetic assay to identify proteins that interact with human Cdc34 resulted in three cDNAs encoding bZIP DNA binding motifs. Two of these interactants are repressors of cyclic AMP (cAMP)-induced transcription: hICERIIγ, a product of the CREM gene, and hATF5, a novel ATF homolog. Transfection assays with mammalian cells demonstrate both hCdc34- and hRad6B-dependent ubiquitin-mediated proteolysis of hICERIIγ and hATF5. This degradation requires an active ubiquitin-conjugating enzyme and results in abrogation of ICERIIγ- and ATF5-mediated repression of cAMP-induced transcription. Consistent with these results, the endogenous ICER protein is elevated in cells which are null for murine Rad6B (mHR6B −/−) or transfected with dominant negative and antisense constructs of human CDC34. Based on the requirement for CREM/ICER and Rad6B proteins in spermatogenesis, we determined expression of Cdc34, Rad6B, CREM/ICER isoforms, and the Skp1–Cullin–F-box ubiquitin protein ligase subunits Cul-1 and Cul-2, which are associated with Cdc34 activity during murine testicular development. Cdc34, Rad6B, and the Cullin proteins are expressed in a developmentally regulated manner, with distinctly different patterns for Cdc34 and the Cullin proteins in germ cells. The Cdc34 and Rad6B proteins are significantly elevated in meiotic and postmeiotic haploid germ cells when chromatin modifications occur. Thus, the stability of specific mammalian transcription factors is the result of complex targeting by multiple ubiquitin-conjugating enzymes and may have an impact on cAMP-inducible gene regulation during both meiotic and mitotic cell cycles.

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Control of ubiquitination of proteins in rat tissues by ubiquitin conjugating enzymes and isopeptidases

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00511.2001 ◽

2002 ◽

Vol 282 (4) ◽

pp. E739-E745 ◽

Cited By ~ 27

Author(s):

Venkatesh Rajapurohitam ◽

Nathalie Bedard ◽

Simon S. Wing

Keyword(s):

Rat Tissues ◽

Deubiquitinating Enzymes ◽

Ubiquitinated Proteins ◽

Tissue Extracts ◽

Ubiquitin Conjugation ◽

Tissue Differences ◽

Conjugating Enzymes ◽

Rat Tissue ◽

Ubiquitin Conjugating Enzymes

The activity of the ubiquitin-dependent proteolytic system in differentiated tissues under basal conditions remains poorly explored. We measured rates of ubiquitination in rat tissue extracts. Accumulation of ubiquitinated proteins increased in the presence of ubiquitin aldehyde, indicating that deubiquitinating enzymes can regulate ubiquitination. Rates of ubiquitination varied fourfold, with the highest rate in the testis. We tested whether ubiquitin-activating enzyme (E1) or ubiquitin-conjugating enzymes (E2s) could be limiting for conjugation. Immunodepletion of the E2s UBC2 or UBC4 lowered rates of conjugation similarly. Supplementation of extracts with excess UBC2 or UBC4, but not E1, stimulated conjugation. However, UBC2-stimulated rates of ubiquitination still differed among tissues, indicating that tissue differences in E3s or substrate availability may also be rate controlling. UBC2 and UBC4 stimulated conjugation half-maximally at concentrations of 10–50 and 28–44 nM, respectively. Endogenous tissue levels of UBC2, but not UBC4, appeared saturating for conjugation, suggesting that in vivo modulation of UBC4 levels can likely control ubiquitin conjugation. Thus the pool of ubiquitin conjugates and therefore the rate of degradation of proteins by this system may be controlled by E2s, E3s, and isopeptidases. The regulation of the ubiquitin pathway appears complex, but precise.

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Conserved α-helix-3 is crucial for structure and functions of Rad6 E2 ubiquitin-conjugating enzymes

10.1101/2021.10.12.464121 ◽

2021 ◽

Author(s):

Prakash K. Shukla ◽

Dhiraj Sinha ◽

Andrew M. Leng ◽

Jesse E. Bissell ◽

Shravya Thatipamula ◽

...

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulations ◽

Biological Functions ◽

Conjugating Enzymes ◽

Α Helix ◽

Dynamics Simulations ◽

Ubiquitin Conjugating Enzymes ◽

Structure And Activity

AbstractRad6, an E2 ubiquitin-conjugating enzyme conserved from yeast to humans, functions in transcription, genome maintenance and proteostasis. The contributions of many conserved secondary structures of Rad6 and its human homologs UBE2A and UBE2B to their biological functions are not understood. A mutant RAD6 allele with a missense substitution at alanine-126 (A126) of α-helix-3 that causes defects in telomeric gene silencing, DNA repair and protein degradation was reported over two decades ago. Here, using a combination of genetics, biochemical, biophysical, and computational approaches, we discovered that α-helix-3 A126 mutations compromise the ability of Rad6 to ubiquitinate target proteins without disrupting interactions with partner E3 ubiquitin-ligases that are required for their various biological functions in vivo. Explaining the defective in vitro or in vivo ubiquitination activities, molecular dynamics simulations and NMR showed that α-helix-3 A126 mutations cause local disorder of the catalytic pocket of Rad6, and also disorganize the global structure of the protein to decrease its stability in vivo. We further demonstrate that α-helix-3 A126 mutations deform the structures of UBE2A and UBE2B, the human Rad6 homologs, and compromise the in vitro ubiquitination activity and folding of UBE2B. Molecular dynamics simulations and circular dichroism spectroscopy along with functional studies further revealed that cancer-associated mutations in α-helix-3 of UBE2A or UBE2B alter both structure and activity, providing an explanation for their pathogenicity. Overall, our studies reveal that the conserved α-helix-3 is a crucial structural constituent that controls the organization of catalytic pockets and biological functions of the Rad6-family E2 ubiquitin-conjugating enzymes.HighlightsContributions of the conserved α-helix-3 to the functions of E2 enzymes is not known.Mutations in alanine-126 of α-helix-3 impair in vitro enzymatic activity and in vivo biological functions of Rad6.Mutations in alanine-126 of α-helix-3 disorganize local or global protein structure, compromise folding or stability, and impair the catalytic activities of yeast Rad6 and its human homologs UBE2A and UBE2B.Cancer-associated mutations in α-helix-3 of human UBE2A or UBE2B alter protein flexibility, structure, and activity.α-helix-3 is a key structural component of yeast and human Rad6 E2 ubiquitin-conjugating enzymes.

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