The Ubiquitin Binding Domain ZnF UBP Recognizes the C-Terminal Diglycine Motif of Unanchored Ubiquitin

Reyes‐Turcu, Francisca E.; Horton, J.R.; Mullally, James E.; Héroux, A.; Cheng, Xiaodong; Wilkinson, Keith D.

doi:10.1016/j.cell.2006.02.038

Cited by 300 publications

(421 citation statements)

References 51 publications

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“…The polyubiquitin ladder generated by gp78C⌬L appeared to be unanchored chains, as judged by the size increment of Ϸ9 kDa, which corresponded to the size of Flag-ubiquitin. Consistent with this interpretation, these chains could be rapidly disassembled by isopeptidase T, a deubiquitinating enzyme that only disassembles unanchored chains (25,26). Together, these results indicate that the monomeric gp78C mutant cannot promote Ube2g2 active siteassociated polyubiquitination although it retains ligase activity.…”

Section: Results Gp78 Contains 2 Oligomerization Sites One Of Which supporting

confidence: 72%

Mechanistic insights into active site-associated polyubiquitination by the ubiquitin-conjugating enzyme Ube2g2

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

115

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Lys-48-linked polyubiquitination regulates a variety of cellular processes by targeting ubiquitinated proteins to the proteasome for degradation. Although polyubiquitination had been presumed to occur by transferring ubiquitin molecules, one at a time, from an E2 active site to a substrate, we recently showed that the endoplasmic reticulum-associated RING finger ubiquitin ligase gp78 can mediate the preassembly of Lys-48-linked polyubiquitin chains on the catalytic cysteine of its cognate E2 Ube2g2 and subsequent transfer to a substrate. Active site-linked polyubiquitin chains are detected in cells on Ube2g2 and its yeast homolog Ubc7p, but how these chains are assembled is unclear. Here, we show that gp78 forms an oligomer via 2 oligomerization sites, one of which is a hydrophobic segment located in the gp78 cytosolic domain. We further demonstrate that a gp78 oligomer can simultaneously associate with multiple Ube2g2 molecules. This interaction is mediated by a novel Ube2g2 surface distinct from the predicted RING binding site. Our data suggest that a large gp78 -Ube2g2 heterooligomer brings multiple Ube2g2 molecules into close proximity, allowing ubiquitin moieties to be transferred between neighboring Ube2g2s to form active site-linked polyubiquitin chains.crystallography ͉ endoplasmic reticulum-associated protein degradation ͉ gp78 ͉ polyubiquitin chain C ovalent attachment of the 76-residue ubiquitin to polypeptides regulates the stability, localization, or activity of the modified proteins (1-3). This modification requires concerted actions of 3 types of enzymes: an activating enzyme (E1) that forms a thioester linkage (designated herein as ''ϳ'') between its catalytic cysteine and the carboxyl group of the Gly-76 in ubiquitin; a conjugating enzyme (E2) that receives ubiquitin from an E1; and an ubiquitin ligase (E3) that catalyzes the transfer of ubiquitin from the E2 active-site cysteine to a substrate (4). To date, 2 E1s and dozens of E2 enzymes have been identified in mammals, whereas the number of E3s is in the range of several hundreds (5-8). Many E3 enzymes contain a RING finger domain that interacts transiently with a cognate E2 to mediate polyubiquitination reactions (4, 9-12).Ubiquitination often occurs in the form of a polymer whereby the C-terminal Gly-76 in an ubiquitin moiety is linked to a lysine residue in another ubiquitin molecule. It had been presumed that polyubiquitin chains are formed by conjugating ubiquitin moieties, one at a time, first to a lysine residue in a substrate, and then to a lysine in the previously-attached ubiquitin molecule. This appears to be the case for some E2s (13-15). However, we and several other groups recently found that ubiquitin chains can also be preassembled on the catalytic cysteine of the E2 enzyme Ube2g2 and its yeast homologue Ubc7p both in vitro and in vivo (16)(17)(18)(19). These ubiquitin-conjugating enzymes are associated with endoplasmic reticulum (ER) membranes to modify misfolded polypeptides that have been exported from the ER lumen in a proc...

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Section: Results Gp78 Contains 2 Oligomerization Sites One Of Which supporting

confidence: 72%

Mechanistic insights into active site-associated polyubiquitination by the ubiquitin-conjugating enzyme Ube2g2

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

115

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“…Asterisks mark two similar cases where the preferred accession shown is not the UniProt reference genome for D. rerio. selectivity for unanchored ubiquitin and is necessary for optimal catalytic activity (207), whilst in combination with other ubiquitin binding domains it contributes to a high avidity for tetra-ubiquitin (208). However, it is possible that the ZnF-UBP domain also presents a protein interaction module for the 72 other human proteins which possess COOH-terminal di-Gly motifs.…”

Section: A Usp Familymentioning

confidence: 99%

“…Multiple USPs carry a ZnF-UBP binding domain, of which a subset (USP3, USP5, USP13, USP16, USP44, USP45, USP49), have been shown to (or by homology are predicted to) specifically recognize the free COOH terminus Gly-Gly motif of ubiquitin (21,183,193,207). This interaction underpins the central function of the abundant USP5 (isopeptidase T) in processing newly synthesized linear polyubiquitin chains.…”

Section: A Usp Familymentioning

confidence: 99%

Deubiquitylases From Genes to Organism

Clague

Barsukov

Coulson

et al. 2013

Physiological Reviews

375

384

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Ubiquitylation is a major posttranslational modification that controls most complex aspects of cell physiology. It is reversed through the action of a large family of deubiquitylating enzymes (DUBs) that are emerging as attractive therapeutic targets for a number of disease conditions. Here, we provide a comprehensive analysis of the complement of human DUBs, indicating structural motifs, typical cellular copy numbers, and tissue expression profiles. We discuss the means by which specificity is achieved and how DUB activity may be regulated. Generically DUB catalytic activity may be used to 1) maintain free ubiquitin levels, 2) rescue proteins from ubiquitin-mediated degradation, and 3) control the dynamics of ubiquitin-mediated signaling events. Functional roles of individual DUBs from each of five subfamilies in specific cellular processes are highlighted with an emphasis on those linked to pathological conditions where the association is supported by whole organism models. We then specifically consider the role of DUBs associated with protein degradative machineries and the influence of specific DUBs upon expression of receptors and channels at the plasma membrane.

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“…This protease shares a 54.8% identity with its isoform USP5 (18,19). Both proteins contain four ubiquitin-associated (UBA) 2 domains and a ubiquitin-specific processing protease (UBP) domain.…”

mentioning

confidence: 99%

“…Whereas USP5 is implicated as a specialized deubiquitinating enzyme responsible for disassembly of unanchored polyubiquitin chains in vivo (19), the function of USP13 is unknown. Although USP13 contains all four putative ubiquitin-binding domains present in USP5, it has not been shown to hydrolyze polyubiquitin (21) but rather serves as a protease for ISG15, whereby it reduces ISGylation (21).…”

mentioning

confidence: 99%

USP13 Enzyme Regulates Siah2 Ligase Stability and Activity via Noncatalytic Ubiquitin-binding Domains

Scortegagna

Subtil

et al. 2011

Journal of Biological Chemistry

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The RING finger E3 ubiquitin ligase Siah2 is implicated in control of diverse cellular biological events, including MAPK signaling and hypoxia. Here we demonstrate that Siah2 is subject to regulation by the deubiquitinating enzyme USP13. Overexpression of USP13 increases Siah2 stability by attenuating its autodegradation. Consequently, the ability of Siah2 to target its substrates prolyl hydroxylase 3 and Spry2 (Sprouty2) for ubiquitin-mediated proteasomal degradation is attenuated. Conversely, inhibition of USP13 expression with corresponding shRNA decreases the stability of both Siah2 and its substrate Spry2. Thus, USP13 limits Siah2 autodegradation and its ubiquitin ligase activity against its target substrates. Strikingly, the effect of USP13 on Siah2 is not mediated by its isopeptidase activity: mutations in its ubiquitin-binding sequences positioned within the ubiquitin-specific processing protease and ubiquitin-binding domains, but not within putative catalytic sites, abolish USP13 binding to and effect on Siah2 autodegradation and targeted ubiquitination. Notably, USP13 expression is attenuated in melanoma cells maintained under hypoxia, thereby relieving Siah2 inhibition and increasing its activity under low oxygen levels. Significantly, on melanoma tissue microarray, high nuclear expression of USP13 coincided with high nuclear expression of Siah2. Overall, this study identifies a new layer of Siah2 regulation mediated by USP13 binding to ubiquitinated Siah2 protein with a concomitant inhibitory effect on its activity under normoxia.Siah proteins are RING finger E3 ubiquitin ligases implicated in the ubiquitination and proteasome-dependent degradation of substrate molecules, which also limit their own availability through self-ubiquitination and degradation (1-3). Siah was first identified in Drosophila melanogaster as seven in absentia (sina), which regulates formation of the R7 photoreceptor through control of tramtrack stability (4, 5). Three murine Siah genes (Siah1a, Siah1b, and Siah2) share significant homology with the two human (SIAH1 and SIAH2) orthologues (6, 7).Siah2 is an important regulator of pathways activated under stress and hypoxia. Among substrates reportedly regulated by Siah under these conditions are TRAF2, ␣-ketoglutarate dehydrogenase, Spry2 (Sprouty2), and two of the three prolyl hydroxylases (8 -10). Given the role of PHD proteins as oxygen sensors and regulators of HIF1␣, the master transcription factor responding to hypoxia, Siah is implicated in the control of hypoxia, particularly within the range of 2-6% oxygen at which PHD proteins retain sufficient activity (11,12). The role of Siah2 in tumor development has been extensively studied. Inhibition of Siah reportedly attenuates breast, pancreatic, lung, prostate, and melanoma tumors (13-16). Mechanistically, Siah2 contributes to tumor development and metastasis via its control of diverse substrates, as shown in a melanoma model (14).A search for novel Siah2 interacting factors led to identification of the isopeptidase USP13 ...

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The Ubiquitin Binding Domain ZnF UBP Recognizes the C-Terminal Diglycine Motif of Unanchored Ubiquitin

Cited by 300 publications

References 51 publications

Mechanistic insights into active site-associated polyubiquitination by the ubiquitin-conjugating enzyme Ube2g2

Mechanistic insights into active site-associated polyubiquitination by the ubiquitin-conjugating enzyme Ube2g2

Deubiquitylases From Genes to Organism

USP13 Enzyme Regulates Siah2 Ligase Stability and Activity via Noncatalytic Ubiquitin-binding Domains

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