Chemistry-Based Functional Proteomics Reveals Novel Members of the Deubiquitinating Enzyme Family

Borodovsky, Anna; Ovaa, Huib; Kolli, Nagamalleswari; Gan-Erdene, Tudeviin; Wilkinson, Keith D.; Ploegh, Hidde L.; Kessler, Benedikt M.

doi:10.1016/s1074-5521(02)00248-x

Cited by 531 publications

(603 citation statements)

References 57 publications

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“…A series of mutant and C-terminal truncations of USP1 were expressed in HEK293 cells and tested for DUB activity using an HA-tagged ubiquitin vinyl methylester (HA-UbVME) DUB activity probe. This probe covalently captures active DUB enzymes, as previously described 24 . Full-length exogenously expressed wild-type USP1 was labelled with the HA-UbVME DUB probe (Fig.…”

Section: Degradation Of Usp1 Requires Its Own Catalytic Activitymentioning

confidence: 99%

“…Beads were incubated overnight at 30 °C with in vitro monoubiquitinated PCNA substrate (using recombinant His-tagged PCNA, E1, RAD6, RAD18 and free ubiquitin in a ubiquitination reaction) as previously described 8 . The HA-UbVME probe reaction was performed as previously described 24 . In vitro transcription and translation was done according to the manufacturer's instructions (Promega, Madison, WI).…”

Section: Triton Extraction Crosslinking Immunoprecipitation and Immmentioning

confidence: 99%

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Regulation of monoubiquitinated PCNA by DUB autocleavage

et al. 2006

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Monoubiquitination is a reversible post-translational protein modification that has an important regulatory function in many biological processes, including DNA repair. Deubiquitinating enzymes (DUBs) are proteases that are negative regulators of monoubiquitination, but little is known about their regulation and contribution to the control of conjugated-substrate levels. Here, we show that the DUB ubiquitin specific protease 1 (USP1) deubiquitinates the DNA replication processivity factor, PCNA, as a safeguard against error-prone translesion synthesis (TLS) of DNA. Ultraviolet (UV) irradiation inactivates USP1 through an autocleavage event, thus enabling monoubiquitinated PCNA to accumulate and to activate TLS. Significantly, the site of USP1 cleavage is immediately after a conserved internal ubiquitin-like diglycine (Gly-Gly) motif. This mechanism is reminiscent of the processing of precursors of ubiquitin and ubiquitin-like modifiers by DUBs. Our results define a regulatory mechanism for protein ubiquitination that involves the signal-induced degradation of an inhibitory DUB.Monoubiquitination is a highly regulated process that is conserved in all eukaryotes 1,2 and controls a broad range of cellular functions, including DNA repair. Protein monoubiquitination is a reversible post-translational event that can be influenced by the opposing activities of a ubiquitin E3 ligase and a deubiquitinating enzyme (DUB), similar to the regulation of protein phosphorylation by kinases and phosphatases 3,4 . Protein monoubiquitination regulates the rescue of stalled DNA replication forks -an important cellular process required for cell survival 5 . The E2 ubiquitin conjugating enzyme RAD6 and the E3 ligase RAD18 are conserved in both yeast and human and they coordinate and activate the monoubiquitination of PCNA in response to UV damage or stalled replication forks [6][7][8] . Recent studies have shown that polη, a specialized TLS polymerase, is recruited to the replication fork through a specific interaction with monoubiquitinated PCNA 7 . The deployment of TLS polymerases during replication ensures timely bypass of the diverse DNA lesions encountered by the replication fork. Although many TLS polymerases are intrinsically mutagenic, polη allows replication past UV-damaged bases with high fidelity 9-11 . Thus, a model has emerged in which polη binds to monoubiquitinated PCNA and ensures accurate (error-free) replicative bypass of UV lesions. However, other (error-prone) TLS polymerases (such as polι and Rev1) have recently been shown to rely on monoubiquitinated PCNA for their function 12,13 . How cells limit PCNA monoubiquitination and the unwanted deployment of polη and/or other error-prone TLS polymerases in the absence or presence of extrinsic DNA damage during the synthesis of DNA in S phase is not known.In humans, protein deubiquitination is controlled by a family of approximately 95 distinct DUB enzymes 14,15 but the function of most of these proteins is unknown. DUBs are cysteine proteases that cleave ubiquitin fro...

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Section: Degradation Of Usp1 Requires Its Own Catalytic Activitymentioning

confidence: 99%

Section: Triton Extraction Crosslinking Immunoprecipitation and Immmentioning

confidence: 99%

Regulation of monoubiquitinated PCNA by DUB autocleavage

et al. 2006

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“…Protein bands were visualized by staining with GelCode Blue (Pierce). Individual Coomassie blue-stained bands were excised from SDS gels, destained, and subjected to enzyme digestion as described previously (61). The peptides were separated using a Nanoflow Liquid Coupled Chromatography System (Waters Cap LC), and amino acid sequences were determined by tandem mass spectrometer (Q-TOF micro).…”

Section: Immunoprecipitation and Protein Identification By Tandem Masmentioning

confidence: 99%

Activation of the Lectin Pathway by Natural IgM in a Model of Ischemia/Reperfusion Injury

Zhang

Takahashi

Alicot

et al. 2006

The Journal of Immunology

128

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Reperfusion of ischemic tissues elicits an acute inflammatory response involving serum complement, which is activated by circulating natural IgM specific to self-Ags exposed by ischemia. Recent reports demonstrating a role for the lectin pathway raise a question regarding the initial events in complement activation. To dissect the individual roles of natural IgM and lectin in activation of complement, mice bearing genetic deficiency in early complement, IgM, or mannan-binding lectin were characterized in a mesenteric model of ischemia reperfusion injury. The results reveal that IgM binds initially to ischemic Ag providing a binding site for mannan-binding lectin which subsequently leads to activation of complement and injury.

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“…17)), suggesting that this protein may be regulated by deubiquitination during adipogenesis. To identify specific dominant deubiquitinating enzyme (DUB) activities during adipogenesis, we made use of an HA-tagged probe that covalently binds active DUBs 20 . Incubation of 3T3-L1 lysates with this probe followed by western blotting (WB) with an HA antibody revealed only two major DUB activities during adipogenesis (Fig.…”

Section: Resultsmentioning

confidence: 99%

Early adipogenesis is regulated through USP7-mediated deubiquitination of the histone acetyltransferase TIP60

et al. 2013

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Transcriptional coregulators, including the acetyltransferase Tip60, have a key role in complex cellular processes such as differentiation. Whereas post-translational modifications have emerged as an important mechanism to regulate transcriptional coregulator activity, the identification of the corresponding demodifying enzymes has remained elusive. Here we show that the expression of the Tip60 protein, which is essential for adipocyte differentiation, is regulated through polyubiquitination on multiple residues. USP7, a dominant deubiquitinating enzyme in 3T3-L1 adipocytes and mouse adipose tissue, deubiquitinates Tip60 both in intact cells and in vitro and increases Tip60 protein levels. Furthermore, inhibition of USP7 expression and activity decreases adipogenesis. Transcriptome analysis reveals several cell cycle genes to be co-regulated by both Tip60 and USP7. Knockdown of either factor results in impaired mitotic clonal expansion, an early step in adipogenesis. These results reveal deubiquitination of a transcriptional coregulator to be a key mechanism in the regulation of early adipogenesis.

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Chemistry-Based Functional Proteomics Reveals Novel Members of the Deubiquitinating Enzyme Family

Cited by 531 publications

References 57 publications

Regulation of monoubiquitinated PCNA by DUB autocleavage

Regulation of monoubiquitinated PCNA by DUB autocleavage

Activation of the Lectin Pathway by Natural IgM in a Model of Ischemia/Reperfusion Injury

Early adipogenesis is regulated through USP7-mediated deubiquitination of the histone acetyltransferase TIP60

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