Dataset Information

Deubiquitination of NF-?B by Ubiquitin-Specific Protease-7 promotes transcription.

ABSTRACT: NF-?B is the master regulator of the immune response and is responsible for the transcription of hundreds of genes controlling inflammation and immunity. Activation of NF-?B occurs in the cytoplasm through the kinase activity of the I?B kinase complex, which leads to translocation of NF-?B to the nucleus. Once in the nucleus, NF-?B transcriptional activity is regulated by DNA binding-dependent ubiquitin-mediated proteasomal degradation. We have identified the deubiquitinase Ubiquitin Specific Protease-7 (USP7) as a regulator of NF-?B transcriptional activity. USP7 deubiquitination of NF-?B leads to increased transcription. Loss of USP7 activity results in increased ubiquitination of NF-?B, leading to reduced promoter occupancy and reduced expression of target genes in response to Toll-like- and TNF-receptor activation. These findings reveal a unique mechanism controlling NF-?B activity and demonstrate that the deubiquitination of NF-?B by USP7 is critical for target gene transcription.

SUBMITTER: Colleran A

PROVIDER: S-EPMC3545798 | biostudies-other | 2013 Jan

REPOSITORIES: biostudies-other

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Deubiquitination of NF-κB by Ubiquitin-Specific Protease-7 promotes transcription.

Colleran Amy A Collins Patricia E PE O'Carroll Christine C Ahmed Abrar A Mao Xicheng X McManus Bettina B Kiely Patrick A PA Burstein Ezra E Carmody Ruaidhrí J RJ

Proceedings of the National Academy of Sciences of the United States of America 20121224 2

NF-κB is the master regulator of the immune response and is responsible for the transcription of hundreds of genes controlling inflammation and immunity. Activation of NF-κB occurs in the cytoplasm through the kinase activity of the IκB kinase complex, which leads to translocation of NF-κB to the nucleus. Once in the nucleus, NF-κB transcriptional activity is regulated by DNA binding-dependent ubiquitin-mediated proteasomal degradation. We have identified the deubiquitinase Ubiquitin Specific Pr ...[more]

PMID: 23267096

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Project description:Deubiquitinases (DUBs) are essential regulators of intracellular processes involving ubiquitin (Ub) modification. The human DUB ubiquitin-specific protease 1 (hUSP1) interacts with human USP-associated factor 1 (hUAF1), and helps to regulate processes such as DNA damage repair. Previously, we identified a chicken USP1 homologue (chUSP1) during an investigation into the properties of Marek's disease virus (MDV). However, chUSP1's deubiquitination activity, interaction with chUAF1, and substrate specificity remained unknown. In the present study, we expressed and purified both chUAF1 and chUSP1 with or without putative catalytic core mutations using the Bac-to-Bac system, before investigating their deubiquitination activity and kinetics using various substrates. chUSP1 was shown to interact with chUAF1 both in cellular assays in which the two proteins were co-expressed, and in in vitro assays using purified proteins. Heterodimerization with chUAF1 increased the deubiquitination activity of chUSP1 up to 54-fold compared with chUSP1 alone. The chUSP1 mutants C91S, H603A, and D758A reduced the deubiquitination activity of the chUSP1/chUAF1 complex by 10-, 7-, and 33-fold, respectively, while the C91A and H594A chUSP1 mutants eliminated deubiquitination activity of the chUSP1/chUAF1 complex completely. This suggests that C91 and H594, but not D758, are essential for chUSP1 deubiquitination activity, and that a nucleophilic group at position 91 is needed for the deubiquitination reaction. The chUSP1/chUAF1 complex was found to have distinct substrate preferences; efficient hydrolysis of Ub dimers with K11-, K48-, and K63-linkages was seen, with weaker hydrolysis observed with K6-, K27-, and K33-linkages and no hydrolysis seen with a K29-linkage. Furthermore, other Ub-like substrates were disfavored by the complex. No activity was seen with SUMO1-GST, SUMO2- and SUMO3-dimers, ISG15-Rho, FAT10-Rho, or Ufm1-Rho, and only weak activity was observed with NEDD8-Rho. Overall, the data presented here characterize the activity and substrate preferences of chUSP1, and thus may facilitate future studies on its in vivo role.

Dataset Information

Deubiquitination of NF-?B by Ubiquitin-Specific Protease-7 promotes transcription.

Publications

Deubiquitination of NF-κB by Ubiquitin-Specific Protease-7 promotes transcription.

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