The deubiquitinase USP7 uses a distinct ubiquitin-like domain to deubiquitinate NF-ĸB subunits

Abstract

The transcription factor NF-ĸB is a master regulator of the innate immune response and plays a central role in inflammatory diseases by mediating the expression of pro-inflammatory cytokines. Ubiquitination-triggered proteasomal degradation of DNA-bound NF-ĸB strongly limits the expression of its target genes. Conversely, USP7 (deubiquitinase ubiquitin-specific peptidase 7) opposes the activities of E3 ligases, stabilizes DNA-bound NF-ĸB, and thereby promotes NF-ĸB–mediated transcription. Using gene expression and synthetic peptide arrays on membrane support and overlay analyses, we found here that inhibiting USP7 increases NF-ĸB ubiquitination and degradation, prevents Toll-like receptor–induced pro-inflammatory cytokine expression, and represents an effective strategy for controlling inflammation. However, the broad regulatory roles of USP7 in cell death pathways, chromatin, and DNA damage responses limit the use of catalytic inhibitors of USP7 as anti-inflammatory agents. To this end, we identified an NF-ĸB–binding site in USP7, ubiquitin-like domain 2, that selectively mediates interactions of USP7 with NF-ĸB subunits but is dispensable for interactions with other proteins. Moreover, we found that the amino acids 757LDEL760 in USP7 critically contribute to the interaction with the p65 subunit of NF-ĸB. Our findings support the notion that USP7 activity could be potentially targeted in a substrate-selective manner through the development of noncatalytic inhibitors of this deubiquitinase to abrogate NF-ĸB activity. The transcription factor NF-ĸB is a master regulator of the innate immune response and plays a central role in inflammatory diseases by mediating the expression of pro-inflammatory cytokines. Ubiquitination-triggered proteasomal degradation of DNA-bound NF-ĸB strongly limits the expression of its target genes. Conversely, USP7 (deubiquitinase ubiquitin-specific peptidase 7) opposes the activities of E3 ligases, stabilizes DNA-bound NF-ĸB, and thereby promotes NF-ĸB–mediated transcription. Using gene expression and synthetic peptide arrays on membrane support and overlay analyses, we found here that inhibiting USP7 increases NF-ĸB ubiquitination and degradation, prevents Toll-like receptor–induced pro-inflammatory cytokine expression, and represents an effective strategy for controlling inflammation. However, the broad regulatory roles of USP7 in cell death pathways, chromatin, and DNA damage responses limit the use of catalytic inhibitors of USP7 as anti-inflammatory agents. To this end, we identified an NF-ĸB–binding site in USP7, ubiquitin-like domain 2, that selectively mediates interactions of USP7 with NF-ĸB subunits but is dispensable for interactions with other proteins. Moreover, we found that the amino acids 757LDEL760 in USP7 critically contribute to the interaction with the p65 subunit of NF-ĸB. Our findings support the notion that USP7 activity could be potentially targeted in a substrate-selective manner through the development of noncatalytic inhibitors of this deubiquitinase to abrogate NF-ĸB activity. The transcription factor NF-ĸB is a master regulator of inflammation and is essential for the development and homeostasis of the immune system. It is activated by most immunoreceptors including Toll-like receptors (TLRs), antigen receptors and members of the tumor necrosis factor receptor superfamily (1Mitchell J.P. Carmody R.J. NF-κB and the transcriptional control of inflammation.Int. Rev. Cell Mol. Biol. 2018; 335 (29305014): 41-8410.1016/bs.ircmb.2017.07.007Crossref PubMed Scopus (116) Google Scholar). As a consequence, NF-ĸB is critically important for the transcriptional response to infection and the development of immunity, and at least 500 genes are direct transcriptional targets of NF-κB including cytokines, chemokines, regulators of antigen presentation and cell adhesion, as well as genes that control cell survival, proliferation, and differentiation (1Mitchell J.P. Carmody R.J. NF-κB and the transcriptional control of inflammation.Int. Rev. Cell Mol. Biol. 2018; 335 (29305014): 41-8410.1016/bs.ircmb.2017.07.007Crossref PubMed Scopus (116) Google Scholar). The functional nature of these transcriptional targets makes NF-κB a central factor in the pathology of a number of important human diseases including chronic inflammatory disease (e.g. arthritis, autoimmunity), atherosclerosis, cancer, and neurodegeneration (1Mitchell J.P. Carmody R.J. NF-κB and the transcriptional control of inflammation.Int. Rev. Cell Mol. Biol. 2018; 335 (29305014): 41-8410.1016/bs.ircmb.2017.07.007Crossref PubMed Scopus (116) Google Scholar). In many of these diseases NF-ĸB is inappropriately activated or active at elevated levels, establishing it as a factor with significant therapeutic potential. NF-ĸB is in fact a family of transcription factors formed by the dimerization of five subunits; RelA (p65), c-Rel, RelB, p50, and p52. In resting cells, NF-κB is sequestered in the cytoplasm through interaction with the inhibitor of NF-κB (IκB) proteins, of which IκBα is the archetypal member. Activation of the IĸB kinase (IKK) complex (which contains the IKKα and IKKβ kinases and the scaffold protein NEMO) leads to IκBα degradation and the nuclear translocation of NF-κB. Once in the nucleus, NF-κB binds to specific DNA sequences (ĸB sites) to promote transcription (1Mitchell J.P. Carmody R.J. NF-κB and the transcriptional control of inflammation.Int. Rev. Cell Mol. Biol. 2018; 335 (29305014): 41-8410.1016/bs.ircmb.2017.07.007Crossref PubMed Scopus (116) Google Scholar). The primary mechanism for terminating NF-κB activity is a negative feedback loop involving NF-κB–directed resynthesis of IκBα, which relocates NF-κB from the nucleus to the cytoplasm. There is also a critical IκBα-independent mechanism to limit NF-κB activity that requires the ubiquitination and proteasomal degradation of NF-κB itself (2Saccani S. Marazzi I. Beg A.A. Natoli G. Degradation of promoter-bound p65/RelA is essential for the prompt termination of the nuclear factor κB response.J. Exp. Med. 2004; 200 (15226358): 107-11310.1084/jem.20040196Crossref PubMed Scopus (202) Google Scholar, 3Natoli G. Chiocca S. Nuclear ubiquitin ligases, NF-κB degradation, and the control of inflammation.Sci. Signal. 2008; 1 (18270169): pe110.1126/stke.11pe1Crossref PubMed Scopus (86) Google Scholar, 4Collins P.E. Mitxitorena I. Carmody R.J. The ubiquitination of NF-κB subunits in the control of transcription.Cells. 2016; 5 (27187478): 2310.3390/cells5020023Crossref Google Scholar). Ubiquitination of NF-κB occurs in the nucleus and depends on the binding of NF-κB to DNA (2Saccani S. Marazzi I. Beg A.A. Natoli G. Degradation of promoter-bound p65/RelA is essential for the prompt termination of the nuclear factor κB response.J. Exp. Med. 2004; 200 (15226358): 107-11310.1084/jem.20040196Crossref PubMed Scopus (202) Google Scholar, 5Carmody R.J. Ruan Q. Palmer S. Hilliard B. Chen Y.H. Negative regulation of toll-like receptor signaling by NF-κB p50 ubiquitination blockade.Science. 2007; 317 (17673665): 675-67810.1126/science.1142953Crossref PubMed Scopus (171) Google Scholar). NF-κB ubiquitination is predominantly composed of K48-linked polyubiquitin chains that trigger proteasomal mediated degradation, leading to reduced NF-κB promoter occupancy and inhibition of transcription (4Collins P.E. Mitxitorena I. Carmody R.J. The ubiquitination of NF-κB subunits in the control of transcription.Cells. 2016; 5 (27187478): 2310.3390/cells5020023Crossref Google Scholar). The ubiquitination of NF-ĸB with nondegradative ubiquitin linkages has also been described (6Li H. Wittwer T. Weber A. Schneider H. Moreno R. Maine G.N. Kracht M. Schmitz M.L. Burstein E. Regulation of NF-κB activity by competition between RelA acetylation and ubiquitination.Oncogene. 2012; 31 (21706061): 611-62310.1038/onc.2011.253Crossref PubMed Scopus (53) Google Scholar); however, at present the functional consequences, if any, are unknown. At least six different E3 ligases for NF-κB have been identified that appear to regulate NF-κB transcriptional activity in a gene selective manner (4Collins P.E. Mitxitorena I. Carmody R.J. The ubiquitination of NF-κB subunits in the control of transcription.Cells. 2016; 5 (27187478): 2310.3390/cells5020023Crossref Google Scholar, 7Ryo A. Suizu F. Yoshida Y. Perrem K. Liou Y.C. Wulf G. Rottapel R. Yamaoka S. Lu K.P. Regulation of NF-κB signaling by Pin1-dependent prolyl isomerization and ubiquitin-mediated proteolysis of p65/RelA.Mol. Cell. 2003; 12 (14690596): 1413-142610.1016/S1097-2765(03)00490-8Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar, 8Tanaka T. Grusby M.J. Kaisho T. PDLIM2-mediated termination of transcription factor NF-κB activation by intranuclear sequestration and degradation of the p65 subunit.Nat. Immunol. 2007; 8 (17468759): 584-59110.1038/ni1464Crossref PubMed Scopus (219) Google Scholar, 9Shin C. Ito Y. Ichikawa S. Tokunaga M. Sakata-Sogawa K. Tanaka T. MKRN2 is a novel ubiquitin E3 ligase for the p65 subunit of NF-κB and negatively regulates inflammatory responses.Sci. Rep. 2017; 7 (28378844): 4609710.1038/srep46097Crossref PubMed Scopus (23) Google Scholar, 10Hou Y. Moreau F. Chadee K. PPARγ is an E3 ligase that induces the degradation of NFκB/p65.Nat. Commun. 2012; 3 (23250430): 130010.1038/ncomms2270Crossref PubMed Scopus (147) Google Scholar, 11Hou Y. Zhang Z. Xu Q. Wang H. Xu Y. Chen K. Inhibitor of growth 4 induces NFκB/p65 ubiquitin-dependent degradation.Oncogene. 2014; 33 (23624912): 1997-200310.1038/onc.2013.135Crossref PubMed Scopus (37) Google Scholar, 12Chang M. Jin W. Chang J.H. Xiao Y. Brittain G.C. Yu J. Zhou X. Wang Y.H. Cheng X. Li P. Rabinovich B.A. Hwu P. Sun S.C. The ubiquitin ligase Peli1 negatively regulates T cell activation and prevents autoimmunity.Nat. Immunol. 2011; 12 (21874024): 1002-100910.1038/ni.2090Crossref PubMed Scopus (117) Google Scholar). The apparent nonredundant roles for these E3 ligases in the regulation of NF-ĸB suggest a complex mechanism for the control of NF-ĸB transcriptional activity by ubiquitination, which remains largely undefined. The importance of ubiquitination in regulating NF-ĸB transcriptional activity was fully recognized following the identification of USP7 (ubiquitin-specific protease 7) as a deubiquitinase of NF-κB (13Colleran A. Collins P.E. O'Carroll C. Ahmed A. Mao X. McManus B. Kiely P.A. Burstein E. Carmody R.J. Deubiquitination of NF-κB by ubiquitin-specific protease-7 promotes transcription.Proc. Natl. Acad. Sci. U.S.A. 2013; 110 (23267096): 618-62310.1073/pnas.1208446110Crossref PubMed Scopus (88) Google Scholar). USP7 directly counteracts the activity of E3 ligases by removing polyubiquitin chains from NF-κB, thereby stabilizing NF-ĸB and promoting the transcription of target genes (13Colleran A. Collins P.E. O'Carroll C. Ahmed A. Mao X. McManus B. Kiely P.A. Burstein E. Carmody R.J. Deubiquitination of NF-κB by ubiquitin-specific protease-7 promotes transcription.Proc. Natl. Acad. Sci. U.S.A. 2013; 110 (23267096): 618-62310.1073/pnas.1208446110Crossref PubMed Scopus (88) Google Scholar). Blocking USP7-mediated deubiquitination of NF-ĸB inhibits NF-ĸB transcriptional activity while inhibiting the E3 ligases of NF-ĸB or the proteasome leads to increased transcription of NF-ĸB target genes (4Collins P.E. Mitxitorena I. Carmody R.J. The ubiquitination of NF-κB subunits in the control of transcription.Cells. 2016; 5 (27187478): 2310.3390/cells5020023Crossref Google Scholar). Thus, the transcriptional activity of NF-κB is determined by a balance of ubiquitination and deubiquitination. E3 ligases and USP7 target DNA-bound, transcriptionally active NF-κB and do not control the upstream activation of NF-ĸB. As such, the ubiquitination of NF-ĸB represents an unexploited avenue for the therapeutic control of inflammatory disease. We previously demonstrated that the inhibition of USP7 leads to the inhibition of NF-ĸB directed transcription of pro-inflammatory cytokines such as Il1b, Tnf, and Il6, identifying USP7 as a potential therapeutic target in inflammatory disease (13Colleran A. Collins P.E. O'Carroll C. Ahmed A. Mao X. McManus B. Kiely P.A. Burstein E. Carmody R.J. Deubiquitination of NF-κB by ubiquitin-specific protease-7 promotes transcription.Proc. Natl. Acad. Sci. U.S.A. 2013; 110 (23267096): 618-62310.1073/pnas.1208446110Crossref PubMed Scopus (88) Google Scholar). However, USP7 deubiquitinates a growing number of substrate proteins, many with important roles in the regulation of key cellular processes such as cell cycle, differentiation, apoptosis, DNA replication, and transcription (14Pozhidaeva A. Bezsonova I. USP7: Structure, substrate specificity, and inhibition.DNA Repair (Amst.). 2019; 76 (30807924): 30-3910.1016/j.dnarep.2019.02.005Crossref PubMed Scopus (28) Google Scholar, 15Rawat R. Starczynowski D.T. Ntziachristos P. Nuclear deubiquitination in the spotlight: the multifaceted nature of USP7 biology in disease.Curr. Opin. Cell Biol. 2019; 58 (30897496): 85-9410.1016/j.ceb.2019.02.008Crossref PubMed Scopus (17) Google Scholar). Indeed, the importance of USP7 in cancer, and has to significant to USP7 The inhibitors of USP7 target the catalytic activity of USP7 and the activity of USP7 its including and NF-ĸB S. M. in and other 2018; 3 PubMed Scopus Google Scholar). the of USP7 inhibitors on cell and cell death is in the of such broad activity is to be in an anti-inflammatory In this we the of USP7 in the interaction with the p65 subunit of NF-ĸB, peptide and We the ubiquitin-like domain of USP7 as critical for the interaction and deubiquitination of p65 but dispensable for the interaction of USP7 with the USP7 and The domain of USP7 is also for interaction with the subunit but not the subunit of NF-ĸB. we the domain of USP7 is not for USP7-mediated deubiquitination of The findings described here suggest that noncatalytic of USP7 substrate-selective inhibition of USP7 activity. this we a site in the domain of USP7 that the selective inhibition of USP7-mediated deubiquitination of We have previously demonstrated that USP7 deubiquitinates NF-ĸB p65 to promote NF-ĸB transcriptional responses by tumor necrosis factor and (13Colleran A. Collins P.E. O'Carroll C. Ahmed A. Mao X. McManus B. Kiely P.A. Burstein E. Carmody R.J. Deubiquitination of NF-κB by ubiquitin-specific protease-7 promotes transcription.Proc. Natl. Acad. Sci. U.S.A. 2013; 110 (23267096): 618-62310.1073/pnas.1208446110Crossref PubMed Scopus (88) Google Scholar). Our that inhibition of USP7 by the inhibitor or the expression of key inflammatory cytokines including and To the of USP7 inhibition on transcriptional we a of with the in the or of the USP7 inhibitor F. E. X. C. C. S. J. J. A. C. W. G. inhibitor of ubiquitin protease stabilizes and in 8 PubMed Scopus Google Scholar). of genes demonstrated that the USP7 inhibitor reduced the expression of a significant number of genes in including the NF-ĸB target genes inflammatory cytokines Tnf, Il6, Il1b, and To the of USP7 inhibition on NF-ĸB–mediated transcriptional we the promoter sequences of genes by USP7 inhibitor for the of transcription a significant of in genes by USP7 inhibitor promoter also in binding for and response transcription factors that with NF-ĸB to promote the expression of pro-inflammatory genes that USP7 inhibition is an effective to NF-ĸB–mediated inflammatory However, the of identified USP7 that the inhibition of catalytic activity not a selective on NF-ĸB activity. that USP7 activity in a manner are which a of the of p65 by Our that the of USP7, five is essential for interaction with the domain is dispensable (13Colleran A. Collins P.E. O'Carroll C. Ahmed A. Mao X. McManus B. Kiely P.A. Burstein E. Carmody R.J. Deubiquitination of NF-κB by ubiquitin-specific protease-7 promotes transcription.Proc. Natl. Acad. Sci. U.S.A. 2013; 110 (23267096): 618-62310.1073/pnas.1208446110Crossref PubMed Scopus (88) Google To the of USP7 that interaction with we the of specific of To do we a of USP7 in which of the five or in cell from with p65 that the and of USP7 dispensable for interaction with Thus, the of and or not the interaction of USP7 with p65 2, and or of the between and also not interaction with p65 the of not USP7 interaction with p65 However, of or in with the of and the interaction of USP7 with p65 2, and that the domain of USP7 is essential for the interaction with To the of the domain to the interaction of USP7 with other we USP7 binding to by with a of USP7 The of these demonstrated that the of not the interaction of USP7 with or and findings support that the domain of USP7 mediates interaction with and Y. T. C. interaction of the ubiquitin-specific Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, M. Li P. Li M. Li W. T. W. Y. of a in and in complex with ubiquitin Full Text Full Text PDF PubMed Scopus Google Scholar). We also the of to with a substrate for which the of the of USP7 to the interaction is J. M. X. is by and Commun. PubMed Scopus Google Scholar). to and with and that the of not the interaction of USP7 and In we also the for in the interaction of USP7 with the NF-ĸB subunits and RelB, which significant and with to the interaction of with USP7 requires the domain of USP7 however, and in to and the of not the interaction of USP7 with that the domain of USP7 is selectively for the interaction of USP7 with its including members of the NF-ĸB family of transcription We previously demonstrated that p65 is a direct substrate of USP7 and that interaction of p65 with USP7 is for USP7-mediated deubiquitination of p65 (13Colleran A. Collins P.E. O'Carroll C. Ahmed A. Mao X. McManus B. Kiely P.A. Burstein E. Carmody R.J. Deubiquitination of NF-κB by ubiquitin-specific protease-7 promotes transcription.Proc. Natl. Acad. Sci. U.S.A. 2013; 110 (23267096): 618-62310.1073/pnas.1208446110Crossref PubMed Scopus (88) Google Scholar). We the of to p65 and in an ubiquitination The with USP7 or and or or p65 and with that USP7 but not p65 with the of to with p65 In USP7 and to RelB, that is dispensable for USP7 interaction with and deubiquitination of the identification of the domain as essential for the interaction of USP7 with we to the specific of USP7 that interaction with To do this we a peptide in which an peptide the of USP7 was with a of amino acids in the of USP7 acids was synthetic peptide arrays on membrane support on to USP7 arrays The peptide arrays with or and interaction with specific was as previously described P.E. Kiely P.A. Carmody R.J. of transcription by cell 3 protein requires interaction with nuclear factor κB Biol. 2014; Full Text Full Text PDF PubMed Scopus Google Scholar). a of that strongly with but not to sequences in of the and of USP7, potentially broad with p65 the of USP7 and To specific amino acids the identified of USP7 that be important for interaction with we arrays of domain and the peptide The in these arrays such that amino acids with and from the peptide by amino As peptide arrays with and binding was with to the peptide on the with binding of the peptide as important amino acids for mediating the interaction of the peptide with identified amino acids in peptide that for interaction with the of amino acids in a peptide amino acids the domain of USP7 interaction with p65 identified the 757LDEL760 of USP7 as a potential binding site for In the of amino acids in of USP7 in the other of the protein also found to interaction with p65 and a number of amino acids in to the as well as the peptide the from USP7 that these are not essential for the interaction of USP7 with p65 it is that potential of interaction in these also contribute to the interaction with through of The peptide identified USP7 amino acids and as that interaction of USP7 with To this we these amino acids in USP7 and the binding to p65 The with p65 and USP7 or a and USP7 to and by The of these that the of and of USP7 reduced interaction with p65 Thus, these a site involving of USP7 that mediates the selective binding to The importance of NF-ĸB in the regulation of pro-inflammatory gene expression has it as a therapeutic target of significant potential Carmody R.J. of NF-κB signaling as a therapeutic target in 2016; PubMed Scopus Google Scholar). to NF-ĸB largely on the development of inhibitors of the key of the NF-ĸB the development of selective inhibitors of the kinases was therapeutic use was of in from the of the kinases A. The of subunits in and Cell. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, the therapeutic potential of the NF-ĸB remains and inhibiting the kinases are The proteasomal degradation of the NF-ĸB subunits is an important mechanism for the termination of NF-ĸB transcriptional activity (13Colleran A. Collins P.E. O'Carroll C. Ahmed A. Mao X. McManus B. Kiely P.A. Burstein E. Carmody R.J. Deubiquitination of NF-κB by ubiquitin-specific protease-7 promotes transcription.Proc. Natl. Acad. Sci. U.S.A. 2013; 110 (23267096): 618-62310.1073/pnas.1208446110Crossref PubMed Scopus (88) Google Scholar). The of pro-inflammatory gene transcription is by the balance between the of NF-ĸB E3 ligases and the NF-ĸB deubiquitinase The inhibition of USP7 leads to ubiquitination and proteasomal degradation of NF-ĸB in the inhibition of pro-inflammatory gene expression (13Colleran A. Collins P.E. O'Carroll C. Ahmed A. Mao X. McManus B. Kiely P.A. Burstein E. Carmody R.J. Deubiquitination of NF-κB by ubiquitin-specific protease-7 promotes transcription.Proc. Natl. Acad. Sci. U.S.A. 2013; 110 (23267096): 618-62310.1073/pnas.1208446110Crossref PubMed Scopus (88) Google Scholar). we have the of the of USP7 inhibition in with the a significant and selective inhibition of NF-ĸB target gene expression following activation and strongly the inhibition of USP7 deubiquitination of NF-ĸB as a potential therapeutic the importance of USP7 in promoting NF-ĸB–mediated inflammatory responses is (13Colleran A. Collins P.E. O'Carroll C. Ahmed A. Mao X. McManus B. Kiely P.A. Burstein E. Carmody R.J. Deubiquitination of NF-κB by ubiquitin-specific protease-7 promotes transcription.Proc. Natl. Acad. Sci. U.S.A. 2013; 110 (23267096): 618-62310.1073/pnas.1208446110Crossref PubMed Scopus (88) Google USP7 catalytic activity is to inhibitors have is the USP7 has with important roles in a broad of cellular including cell cycle, DNA damage and cell the therapeutic of USP7 control of NF-ĸB a substrate-selective The in this a this p65 that not USP7 is has a and has reduced transcriptional activity (13Colleran A. Collins P.E. O'Carroll C. Ahmed A. Mao X. McManus B. Kiely P.A. Burstein E. Carmody R.J. Deubiquitination of NF-κB by ubiquitin-specific protease-7 promotes transcription.Proc. Natl. Acad. Sci. U.S.A. 2013; 110 (23267096): 618-62310.1073/pnas.1208446110Crossref PubMed Scopus (88) Google Scholar). Thus, with USP7 and p65 interaction a substrate-selective inhibition of mediated activity to the expression of pro-inflammatory genes. Our identified the domain of USP7 as a critical for interaction with p65 (13Colleran A. Collins P.E. O'Carroll C. Ahmed A. Mao X. McManus B. Kiely P.A. Burstein E. Carmody R.J. Deubiquitination of NF-κB by ubiquitin-specific protease-7 promotes transcription.Proc. Natl. Acad. Sci. U.S.A. 2013; 110 (23267096): 618-62310.1073/pnas.1208446110Crossref PubMed Scopus (88) Google Scholar). In this we have the domain of USP7 as essential for the interaction of USP7 with In we that the domain is also essential for the interaction of USP7 with the subunit of NF-ĸB but not the The that different of USP7 are important for the of such as the NF-ĸB subunits is and that substrate by USP7 occurs through proteins. also that the domain of USP7 is not for the deubiquitination of by USP7, that the domain in substrate to catalytic activity. have the domain to be important for USP7 interaction with and Y. T. C. interaction of the ubiquitin-specific Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Y. Zhang G. B. Z. of is critical for deubiquitination activity and contains a binding PubMed Scopus Google Scholar, A. E. J. and of USP7 targets in Rep. 2018; 8 PubMed Scopus Google the domain is essential for USP7 interaction with Q. J. G. A.A. USP7 deubiquitinase promotes ubiquitin-dependent DNA damage signaling by stabilizing PubMed Scopus Google Scholar). number of USP7 do not the of USP7 for including and Indeed, a to that most interactions through the domain of USP7 A. E. J. and of USP7 targets in Rep. 2018; 8 PubMed Scopus Google Scholar). with this these that interactions for the selective inhibition of USP7 activity. the we have identified the amino acids 757LDEL760 as important to the interaction with of a the of USP7 that is important for the interaction with binding site is but a binding site for and formed by amino acids with the suggest that the binding site in USP7 for the inhibition of p65 leading to increased proteasomal degradation and reduced transcriptional the activity of USP7 to other substrate interaction of USP7 to the identification of binding that be selectively targeted to substrate-selective inhibition of USP7-mediated deubiquitination. an of USP7 substrate interaction the selective of to such as NF-ĸB and other transcription in and as previously described R.J. Ruan Q. Palmer S. Hilliard B. Chen Y.H. Negative regulation of toll-like receptor signaling by NF-κB p50 ubiquitination blockade.Science. 2007; 317 (17673665): 675-67810.1126/science.1142953Crossref PubMed Scopus (171) Google Scholar). was from and at and in with and with for 7 with 4 in with 5 and in and the on The at in a with in with the of the of for the of 1 to the was for of to the expression for USP7, c-Rel, RelB, and following and of the or was a from was from was by gene was the to the was the with and the with for 200 of was with was to arrays and with for was in The including B. Y. W. expression for and PubMed Scopus Google Scholar). was to an and of The are at the site was of with The with and for 3 with at The in and by and to was and in and reduced of USP7 by as previously described P.E. Kiely P.A. Carmody R.J. of transcription by cell 3 protein requires interaction with nuclear factor κB Biol. 2014; Full Text Full Text PDF PubMed Scopus Google Scholar). The interaction of and with the peptide was by the peptide membrane with of protein at 4 was by with and to arrays the and protein was and a cell from in 1 1 1 1 1 and 1 on and to and with specific was from and and was from and and and by of cell for at 4 with protein and with specific at 4 The in and by in 200 of by Ubiquitination as previously described R.J. Ruan Q. Palmer S. Hilliard B. Chen Y.H. Negative regulation of toll-like receptor signaling by NF-κB p50 ubiquitination blockade.Science. 2007; 317 (17673665): 675-67810.1126/science.1142953Crossref PubMed Scopus (171) Google Scholar). the with for and in with The in for 5 and in with was as and by to The have been in the with the following are the We for the of the with Toll-like receptor synthetic peptide arrays on membrane support IĸB kinase ubiquitin-like

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