Crystal Structure of a UBP-Family Deubiquitinating Enzyme in Isolation and in Complex with Ubiquitin Aldehyde

Hu, Min; Li, Pingwei; Li, Muyang; Li, Wenyu; Yao, Tingting; Wu, Jing; Gu, Wei; Cohen, Robert E.; Shi, Yigong

doi:10.1016/s0092-8674(02)01199-6

Cited by 546 publications

(764 citation statements)

References 51 publications

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“…Vitro-Previous reports have demonstrated that USP7 has ubiquitin-specific protease activity in vitro, both on model ␣-peptide-linked ubiquitin substrates and on isopeptide-linked p53 ubiquitin conjugates (32)(33)(34). We expressed and purified a His-tagged version of wild type USP7 (Fig.…”

Section: Enzymatic Activity Of Purified Usp7 On Model Substrates Inmentioning

confidence: 99%

“…has been shown to bind to and cleave ubiquitinated forms of both p53 and Epstein-Barr virus regulatory protein EBNA-1 (32,34); although in the case of p53, the K m value was estimated to be greater than 50 M (33). As a control for the intrinsic activity of our preparations of USP7, we generated ubiquitinated p53 in reactions using ICP0 as the E3 ubiquitin ligase (14).…”

Section: Enzymatic Activity Of Purified Usp7 On Model Substrates Inmentioning

confidence: 99%

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A RING Finger Ubiquitin Ligase Is Protected from Autocatalyzed Ubiquitination and Degradation by Binding to Ubiquitin-specific Protease USP7

Canning

Boutell

Parkinson

et al. 2004

Journal of Biological Chemistry

127

122

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Herpes simplex virus type 1 immediate-early regulatory protein ICP0 stimulates lytic infection and reactivation from latency, processes that require the ubiquitin E3 ligase activity mediated by the RING finger domain in the N-terminal portion of the protein. ICP0 stimulates the production of polyubiquitin chains by the ubiquitin-conjugating enzymes UbcH5a and UbcH6 in vitro, and in infected and transfected cells it induces the proteasome-dependent degradation of a number of cellular proteins including PML, the major constituent protein of PML nuclear bodies. However, ICP0 binds strongly to the cellular ubiquitin-specific protease USP7, a member of a family of proteins that cleave polyubiquitin chains and/or ubiquitin precursors. The region of ICP0 that is required for its interaction with USP7 has been mapped, and mutations in this domain reduce the functionality of ICP0. These findings pose the question: why does ICP0 include domains that are associated with the potentially antagonistic functions of ubiquitin conjugation and deconjugation? Here we report that although neither protein affected the intrinsic activities of the other in vitro, USP7 protected ICP0 from autoubiquitination in vitro, and their interaction can greatly increase the stability of ICP0 in vivo. These results demonstrate that RING finger-mediated autoubiquitination of ICP0 is biologically relevant and can be regulated by interaction with USP7. This principle may extend to a number of cellular RING finger E3 ubiquitin ligase proteins that have analogous interactions with ubiquitin-specific cleavage enzymes.Herpes simplex virus type 1 (HSV-1) 1 is a common human pathogen that can establish a lifelong quiescent infection in sensory neurons following primary infection of epithelial cells. Environmental stimuli such as stress and sunlight trigger periodic recurrences of lytic infection, causing cold sores and genital lesions. HSV-1 expresses three broad groups of temporally regulated genes during lytic infection, termed immediate-early (IE), early, and late (for reviews, see Refs. 1 and 2). The IE protein ICP0 has an important role in the mechanisms that govern the switch between lytic and latent infection (reviewed in Refs. 3-5). Although not essential for viral replication, ICP0 increases the probability of the virus entering lytic infection, particularly after low multiplicity infection of human fibroblasts, and in its absence, viral genomes are more likely to become repressed and establish a quiescent infection (5-7). ICP0 stimulates the expression of all three classes of viral genes by as yet uncertain mechanisms that correlate with its ability to induce the degradation of a number of cellular proteins (8 -12). ICP0 includes a zinc-binding RING finger domain in its N-terminal portion, and in its C-terminal third lie a nuclear localization signal and motifs required for self-multimerization and efficient localization at specific nuclear substructures known as ND10 or PML nuclear bodies. Consistent with its ability to induce the degradation of...

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Section: Enzymatic Activity Of Purified Usp7 On Model Substrates Inmentioning

confidence: 99%

Section: Enzymatic Activity Of Purified Usp7 On Model Substrates Inmentioning

confidence: 99%

A RING Finger Ubiquitin Ligase Is Protected from Autocatalyzed Ubiquitination and Degradation by Binding to Ubiquitin-specific Protease USP7

Canning

Boutell

Parkinson

et al. 2004

Journal of Biological Chemistry

127

122

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“…In recent years, several general principles have emerged for protein-protein interactions in UBL transfer cascades [15][16][17][18][19][20][21][22][23][24][25][26] . Nonetheless, the selective coordination of enzymes within a particular UBL's cascade remains incompletely understood.…”

Section: Introductionmentioning

confidence: 99%

A unique E1-E2 interaction required for optimal conjugation of the ubiquitin-like protein NEDD8

Huang

Miller

Mathew

et al. 2004

Nat Struct Mol Biol

130

109

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Ubiquitin-like proteins (UBLs), such as NEDD8, are transferred to their targets by distinct, parallel, multi-enzyme cascades that involve the sequential action of E1, E2, and E3 enzymes. How do enzymes within a particular UBL conjugation cascade interact with each other? We report here that the unique N-terminal sequence of NEDD8's E2, Ubc12, selectively recruits NEDD8's E1 to promote Ubc12~NEDD8 thioester formation. A peptide corresponding to Ubc12's N-terminus (Ubc12N26) specifically binds and inhibits NEDD8's E1, the heterodimeric APPBP1-UBA3 complex. The structure of APPBP1-UBA3-Ubc12N26 reveals conserved Ubc12 residues docking in a groove generated by loops conserved in UBA3s but not other E1s, explaining why this interaction is unique to the NEDD8 pathway. These studies define a novel mechanism for E1-E2 interaction and show how enzymes within a particular UBL conjugation cascade can be tethered together by unique protein-protein interactions emanating from their common structural scaffolds.

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“…In addition, DUBs are responsible for the normal processing and maturation of the genome-encoded ubiquitin precursors, all of which are linear fusion proteins. Structural studies and X-ray crystallographic analysis of UBPs and UCHs have demonstrated that substrate recognition by these enzymes relies on extensive contacts with structural features located on the surface of the ubiquitin/UBL molecules themselves [39,45,46]. This type of interaction takes place regardless of the nature of the linkage between the C terminus of ubiquitin/SUMO and the target protein.…”

Section: Discussionmentioning

confidence: 99%

Small ubiquitin-like modifying protein isopeptidase assay based on poliovirus RNA polymerase activity

Arnold

Bernal

Uche

et al. 2006

Analytical Biochemistry

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The ubiquitin-proteasome pathway is the major nonlysosomal proteolytic system in eukaryotic cells responsible for regulating the level of many key regulatory molecules within the cells. Modification of cellular proteins by ubiquitin and ubiquitin-like proteins, such as small ubiquitin-like modifying protein (SUMO), plays an essential role in a number of biological schemes, and ubiquitin pathway enzymes have become important therapeutic targets. Ubiquitination is a dynamic reversible process; a multitude of ubiquitin ligases and deubiquitinases (DUBs) are responsible for the wide-ranging influence of this pathway as well as its selectivity. The DUB enzymes serve to maintain adequate pools of free ubiquitin and regulate the ubiquitination status of cellular proteins. Using SUMO fusions, a novel assay system, based on poliovirus RNA-dependent RNA polymerase activity, is described here. The method simplifies the isopeptidase assay and facilitates high-throughput analysis of these enzymes. The principle of the assay is the dependence of the viral polymerase on a free N terminus for activity; accordingly, the polymerase is inactive when fused at its N terminus to SUMO or any other ubiquitin-like protein. The assay is sensitive, reproducible, and adaptable to a highthroughput format for use in screens for inhibitors/activators of clinically relevant SUMO proteases and deubiquitinases. Keywords Isopeptidases; Protein degradation; N terminus; 3D polymeraseThe ubiquitin-proteasomal pathway regulates the cellular content and/or compartmentalization of many proteins [1,2] and is a promising, albeit underexploited, area for drug discovery. The therapeutic value of this pathway was recently validated by the introduction and clinical success of Velcade, a proteasome inhibitor, for the treatment of refractory relapsed multiple myeloma [3]. Other ubiquitin-like proteins (UBLs) 1 have recently been shown to contribute similarly to the cellular regulation of proteins, with the most extensively characterized UBL being small ubiquitin-like modifying protein (SUMO) [4].Destruction of a targeted protein via the ubiquitin system initially involves recognition by a multienzyme system that attaches ubiquitin to the target protein. Energy is required to activate ubiquitin at its carboxy terminus. Activation is catalyzed by the enzyme E1, which links the ubiquitin C terminus to a cysteine side chain of the enzyme. This activated ubiquitin is * Corresponding author. Fax: +1 610 644 8616., E-mail address: mattern@progenra.com (M.R. Mattern).. 1 Abbreviations used: UBL, ubiquitin-like protein; SUMO, small ubiquitin-like modifying protein; UBP/USP, ubiquitin-specific protease; UCH, ubiquitin terminal hydrolase; DUB, deubiquitinating enzyme; Ub-AMC, Ub-7-amino-4-methylcoumarin; EDTA, ethylenediaminetetraacetic acid; IPTG, isopropyl-β-D-thiogalactopyranoside; PMSF, phenylmethylsulfonylfluoride; GFP, green fluorescent protein; DTT, dithiothreitol. transferred to a second enzyme of the series, E2 (ubiquitin-conjugating protein), as a thioe...

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Crystal Structure of a UBP-Family Deubiquitinating Enzyme in Isolation and in Complex with Ubiquitin Aldehyde

Cited by 546 publications

References 51 publications

A RING Finger Ubiquitin Ligase Is Protected from Autocatalyzed Ubiquitination and Degradation by Binding to Ubiquitin-specific Protease USP7

A RING Finger Ubiquitin Ligase Is Protected from Autocatalyzed Ubiquitination and Degradation by Binding to Ubiquitin-specific Protease USP7

A unique E1-E2 interaction required for optimal conjugation of the ubiquitin-like protein NEDD8

Small ubiquitin-like modifying protein isopeptidase assay based on poliovirus RNA polymerase activity

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