scholarly journals Regulation of p53 Localization and Activity by Ubc13

2006 ◽  
Vol 26 (23) ◽  
pp. 8901-8913 ◽  
Author(s):  
Aaron Laine ◽  
Ivan Topisirovic ◽  
Dayong Zhai ◽  
John C. Reed ◽  
Katherine L. B. Borden ◽  
...  
Keyword(s):  
Dna Damage ◽  
Transcriptional Activity ◽  
Ubiquitin Ligases ◽  
Wild Type ◽  
Terminal Domain ◽  
Ubiquitin Conjugating Enzyme ◽  
P53 Stability ◽  
Conjugating Enzyme

ABSTRACT The abundance and activity of p53 are regulated largely by ubiquitin ligases. Here we demonstrate a previously undisclosed regulation of p53 localization and activity by Ubc13, an E2 ubiquitin-conjugating enzyme. While increasing p53 stability, Ubc13 decreases p53 transcriptional activity and increases its localization to the cytoplasm, changes that require its ubiquitin-conjugating activity. Ubc13 elicits K63-dependent ubiquitination of p53, which attenuates Hdm2-induced polyubiquitination of p53. Ubc13 association with p53 requires an intact C-terminal domain of p53 and is markedly stronger with a p53 mutant that cannot tetramerize. Expression of Ubc13 in vivo increases the pool of monomeric p53, indicating that Ubc13 affects tetramerization of p53. Significantly, wild-type but not mutant Ubc13 is associated with polysomes and enriches p53 within this fraction. In response to DNA damage, Ubc13 is no longer capable of facilitating p53 monomerization, in part due to a decrease in its own levels which is p53 dependent. Our findings point to a newly discerned mechanism important in the regulation of p53 organization, localization, and activity by Ubc13.

scholarly journals Recombinant human phenylalanine hydroxylase is a substrate for the ubiquitin-conjugating enzyme system

1996 ◽  
Vol 319 (3) ◽  
pp. 941-945 ◽  
Author(s):  
Anne P. DØSKELAND ◽  
Torgeir FLATMARK
Keyword(s):  
Phenylalanine Hydroxylase ◽  
Enzyme System ◽  
Wild Type ◽  
Human Phenylalanine Hydroxylase ◽  
Ubiquitin Conjugating Enzyme ◽  
Mutant Forms ◽  
Tetrameric Form ◽  
Synthesis And Degradation ◽  
Conjugating Enzyme

Mammalian phenylalanine hydroxylase (PAH) catalyses the conversion of L-phenylalanine to L-tyrosine in the presence of dioxygen and tetrahydrobiopterin; it is a highly regulated enzyme. Little is known about the rates of synthesis and degradation of PAH in vivo. The enzyme has been reported to have a half-life of approx. 2 days in rat liver and 7–8 h in rat hepatoma cells, but the mechanism of its degradation is not known. In the present study it is shown that the tetrameric form of the recombinant wild-type human enzyme is a substrate for the ubiquitin-conjugating enzyme system in the cytosolic fraction of rat testis. Our findings support the conclusion that multi-/poly-ubiquitination of human PAH plays a key role in the turnover of this cytosolic liver enzyme and provides a mechanism for the increased turnover observed for a number of recombinant mutant forms of the enzyme related to the metabolic disorder phenylketonuria, when expressed in eukaryotic cells.

Effect of Arabidopsis COP10 ubiquitin E2 enhancement activity across E2 families and functional conservation among its canonical homologues

2009 ◽  
Vol 418 (3) ◽  
pp. 683-690 ◽  
Author(s):  
On Sun Lau ◽  
Xing Wang Deng
Keyword(s):  
Dna Damage ◽  
Arabidopsis Thaliana ◽  
Plant Development ◽  
Functional Conservation ◽  
Human Ubiquitin ◽  
Ubiquitin Conjugating Enzyme ◽  
Variant Protein ◽  
Conjugating Enzyme

Arabidopsis thaliana COP10 (constitutive photomorphogenic 10) is a UEV [Ub (ubiquitin)-conjugating enzyme (E2) variant protein] that is required for repression of seedling photomorphogenesis in darkness. COP10 forms a complex {the CDD complex [COP10–DET1 (de-etiolated 1)–DDB1 (DNA damage binding protein 1) complex]} with DET1 and DDB1a in vivo and can enhance the activity of Ub-conjugating enzyme (E2) in vitro. To investigate whether COP10 might act as a general regulator of E2s, we tested the specificity of COP10 E2 enhancement activity across E2 families of Arabidopsis. We found that COP10 is capable of enhancing members of four E2 subgroups significantly, while having a milder effect on another. Surprisingly, we found that close canonical E2 homologues of COP10, such as UbcH5a (human ubiquitin-conjugating enzyme 5), are also capable of enhancing E2s. Furthermore, we detected direct interactions between COP10 and three of the enhanced E2s, hinting at a possible mechanism for the enhancements. The present study suggests that some E2s, including the generic Ubc4/5p families involved in many processes, might possess dual activities: the formation of the classic E2–Ub thiol ester and the previously unknown E2 enhancement activity. Therefore COP10, despite being a catalytically inactive E2, might still enhance a variety of E2s and regulate numerous aspects of plant development.

scholarly journals RuvAB and RecG Are Not Essential for the Recovery of DNA Synthesis Following UV-Induced DNA Damage in Escherichia coli

Genetics ◽  
2004 ◽  
Vol 166 (4) ◽  
pp. 1631-1640 ◽  
Author(s):  
Janet R Donaldson ◽  
Charmain T Courcelle ◽  
Justin Courcelle
Keyword(s):  
Dna Damage ◽  
Dna Synthesis ◽  
Dna Lesions ◽  
Replication Forks ◽  
Wild Type ◽  
Replication Machinery ◽  
Dna Junctions ◽  
Fork Regression

Abstract Ultraviolet light induces DNA lesions that block the progression of the replication machinery. Several models speculate that the resumption of replication following disruption by UV-induced DNA damage requires regression of the nascent DNA or migration of the replication machinery away from the blocking lesion to allow repair or bypass of the lesion to occur. Both RuvAB and RecG catalyze branch migration of three- and four-stranded DNA junctions in vitro and are proposed to catalyze fork regression in vivo. To examine this possibility, we characterized the recovery of DNA synthesis in ruvAB and recG mutants. We found that in the absence of either RecG or RuvAB, arrested replication forks are maintained and DNA synthesis is resumed with kinetics that are similar to those in wild-type cells. The data presented here indicate that RecG- or RuvAB-catalyzed fork regression is not essential for DNA synthesis to resume following arrest by UV-induced DNA damage in vivo.

DNA Damage-Induced Foci of E2 Ubiquitin-Conjugating Enzyme are Detectable upon Co-transfection with an Interacting E3 Ubiquitin Ligase

2015 ◽  
Vol 54 (2) ◽  
pp. 147-157 ◽  
Author(s):  
Degui Wang ◽  
Yingxia Tian ◽  
Dong Wei ◽  
Yuhong Jing ◽  
Haitao Niu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzyme

scholarly journals In vivo repair of alkylating and oxidative DNA damage in the mitochondrial and nuclear genomes of wild-type and glycosylase-deficient Caenorhabditis elegans

DNA Repair ◽  
2012 ◽  
Vol 11 (11) ◽  
pp. 857-863 ◽  
Author(s):  
Senyene E. Hunter ◽  
Margaret A. Gustafson ◽  
Kathleen M. Margillo ◽  
Sean A. Lee ◽  
Ian T. Ryde ◽  
...  
Keyword(s):  
Dna Damage ◽  
Caenorhabditis Elegans ◽  
Oxidative Dna Damage ◽  
Wild Type ◽  
Nuclear Genomes

scholarly journals OTUB1 non-catalytically stabilizes the E2 ubiquitin-conjugating enzyme UBE2E1 by preventing its autoubiquitination

2018 ◽  
Vol 293 (47) ◽  
pp. 18285-18295 ◽  
Author(s):  
Nagesh Pasupala ◽  
Marie E. Morrow ◽  
Lauren T. Que ◽  
Barbara A. Malynn ◽  
Averil Ma ◽  
...  
Keyword(s):  
Polyubiquitin Chains ◽  
Protein Ubiquitination ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzymes ◽  
E2 Enzymes ◽  
Ubiquitin Conjugating Enzymes ◽  
In Cells ◽  
Conjugating Enzyme

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.

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

2003 ◽  
Vol 23 (10) ◽  
pp. 3497-3505 ◽  
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.

scholarly journals In Vivo Action of the HRD Ubiquitin Ligase Complex: Mechanisms of Endoplasmic Reticulum Quality Control and Sterol Regulation

2001 ◽  
Vol 21 (13) ◽  
pp. 4276-4291 ◽  
Author(s):  
Richard G. Gardner ◽  
Alexander G. Shearer ◽  
Randolph Y. Hampton
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Ubiquitin Ligase ◽  
Sequence Similarity ◽  
High Specificity ◽  
Specific Sequence ◽  
Ubiquitin Ligase Complex ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzyme

ABSTRACT Ubiquitination is used to target both normal proteins for specific regulated degradation and misfolded proteins for purposes of quality control destruction. Ubiquitin ligases, or E3 proteins, promote ubiquitination by effecting the specific transfer of ubiquitin from the correct ubiquitin-conjugating enzyme, or E2 protein, to the target substrate. Substrate specificity is usually determined by specific sequence determinants, or degrons, in the target substrate that are recognized by the ubiquitin ligase. In quality control, however, a potentially vast collection of proteins with characteristic hallmarks of misfolding or misassembly are targeted with high specificity despite the lack of any sequence similarity between substrates. In order to understand the mechanisms of quality control ubiquitination, we have focused our attention on the first characterized quality control ubiquitin ligase, the HRD complex, which is responsible for the endoplasmic reticulum (ER)-associated degradation (ERAD) of numerous ER-resident proteins. Using an in vivo cross-linking assay, we directly examined the association of the separate HRDcomplex components with various ERAD substrates. We have discovered that the HRD ubiquitin ligase complex associates with both ERAD substrates and stable proteins, but only mediates ubiquitin-conjugating enzyme association with ERAD substrates. Our studies with the sterol pathway-regulated ERAD substrate Hmg2p, an isozyme of the yeast cholesterol biosynthetic enzyme HMG-coenzyme A reductase (HMGR), indicated that the HRD complex discerns between a degradation-competent “misfolded” state and a stable, tightly folded state. Thus, it appears that the physiologically regulated, HRD-dependent degradation of HMGR is effected by a programmed structural transition from a stable protein to a quality control substrate.

Ubiquitination of the Ubiquitin-Conjugating Enzyme UbcH10: in Vivo Characterization Following Arterial Injury

2011 ◽  
Vol 165 (2) ◽  
pp. 343
Author(s):  
N.D. Tsihlis ◽  
A.K. Vavra ◽  
C.S. Oustwani ◽  
J. Martinez ◽  
M.R. Kibbe
Keyword(s):  
Arterial Injury ◽  
Ubiquitin Conjugating Enzyme ◽  
In Vivo Characterization ◽  
Conjugating Enzyme

scholarly journals Regulation of NAD Synthesis by the Trifunctional NadR Protein of Salmonella enterica

2005 ◽  
Vol 187 (8) ◽  
pp. 2774-2782 ◽  
Author(s):  
Julianne H. Grose ◽  
Ulfar Bergthorsson ◽  
John R. Roth
Keyword(s):  
Missense Mutations ◽  
Wild Type ◽  
Central Domain ◽  
Terminal Domain ◽  
Binding Residue ◽  
Nicotinamide Mononucleotide ◽  
Nad Synthesis ◽  
Default State ◽  
Regulatory Effects

ABSTRACT The three activities of NadR were demonstrated in purified protein and assigned to separate domains by missense mutations. The N-terminal domain represses transcription of genes for NAD synthesis and salvage. The C-terminal domain has nicotinamide ribose kinase (NmR-K; EC 2.7.1.22) activity, which is essential for assimilation of NmR, converting it internally to nicotinamide mononucleotide (NMN). The central domain has a weak adenylyltransferase (NMN-AT; EC 2.7.7.1) activity that converts NMN directly to NAD but is physiologically irrelevant. This central domain mediates regulatory effects of NAD on all NadR activities. In the absence of effectors, pure NadR protein binds operator DNA (the default state) and is released by ATP (expected to be present in vivo). NAD allows NadR to bind DNA in the presence of ATP and causes repression in vivo. A superrepressor mutation alters an ATP-binding residue in the central (NMN-AT) domain. This eliminates NMN-AT activity and places the enzyme in its default (DNA binding) state. The mutant protein shows full NmR kinase activity that is 10-fold more sensitive to NAD inhibition than the wild type. It is proposed that NAD and the superrepressor mutation exert their effects by preventing ATP from binding to the central domain.

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