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• W2514355059 abstract "•CYLD recruitment to LUBAC and the TNF receptor 1 complex is mediated by SPATA2•SPATA2 forms a high-affinity complex with CYLD and stimulates CYLD’s activity•SPATA2, like OTULIN, uses a conserved PIM to dock to the HOIP PUB domain•SPATA2 limits ubiquitination of LUBAC substrates to regulate inflammatory signaling The linear ubiquitin chain assembly complex (LUBAC) regulates immune signaling, and its function is regulated by the deubiquitinases OTULIN and CYLD, which associate with the catalytic subunit HOIP. However, the mechanism through which CYLD interacts with HOIP is unclear. We here show that CYLD interacts with HOIP via spermatogenesis-associated protein 2 (SPATA2). SPATA2 interacts with CYLD through its non-canonical PUB domain, which binds the catalytic CYLD USP domain in a CYLD B-box-dependent manner. Significantly, SPATA2 binding activates CYLD-mediated hydrolysis of ubiquitin chains. SPATA2 also harbors a conserved PUB-interacting motif that selectively docks into the HOIP PUB domain. In cells, SPATA2 is recruited to the TNF receptor 1 signaling complex and is required for CYLD recruitment. Loss of SPATA2 increases ubiquitination of LUBAC substrates and results in enhanced NOD2 signaling. Our data reveal SPATA2 as a high-affinity binding partner of CYLD and HOIP, and a regulatory component of LUBAC-mediated NF-κB signaling. The linear ubiquitin chain assembly complex (LUBAC) regulates immune signaling, and its function is regulated by the deubiquitinases OTULIN and CYLD, which associate with the catalytic subunit HOIP. However, the mechanism through which CYLD interacts with HOIP is unclear. We here show that CYLD interacts with HOIP via spermatogenesis-associated protein 2 (SPATA2). SPATA2 interacts with CYLD through its non-canonical PUB domain, which binds the catalytic CYLD USP domain in a CYLD B-box-dependent manner. Significantly, SPATA2 binding activates CYLD-mediated hydrolysis of ubiquitin chains. SPATA2 also harbors a conserved PUB-interacting motif that selectively docks into the HOIP PUB domain. In cells, SPATA2 is recruited to the TNF receptor 1 signaling complex and is required for CYLD recruitment. Loss of SPATA2 increases ubiquitination of LUBAC substrates and results in enhanced NOD2 signaling. Our data reveal SPATA2 as a high-affinity binding partner of CYLD and HOIP, and a regulatory component of LUBAC-mediated NF-κB signaling. Modification of proteins with ubiquitin (Ub) constitutes a versatile posttranslational modification that regulates a variety of cellular processes, including receptor signaling, cell cycle progression, and DNA damage responses. Ub signaling controls activation of nuclear factor-κB (NF-κB) and innate immune responses downstream of pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs), nucleotide-oligomerization domain (NOD)-like receptors, and cytokine receptors, such as tumor necrosis factor (TNF) receptor 1 (TNFR1) (Fiil and Gyrd-Hansen, 2014Fiil B.K. Gyrd-Hansen M. Met1-linked ubiquitination in immune signalling.FEBS J. 2014; 281: 4337-4350Crossref PubMed Scopus (51) Google Scholar, Jiang and Chen, 2011Jiang X. Chen Z.J. The role of ubiquitylation in immune defence and pathogen evasion.Nat. Rev. Immunol. 2011; 12: 35-48PubMed Google Scholar). Stimulation of these receptors triggers assembly of multi-protein signaling complexes where Ub ligases and deubiquitinases (DUBs) coordinate the deposition of Ub chains linked via lysine 63 (Lys63-Ub) and methionine 1 (Met1-Ub) on protein substrates to orchestrate activation of the TAB-TAK1 and NEMO-IKKα/β kinase complexes, respectively. Activation of IKK is required for productive signaling and NF-κB-mediated transcriptional responses, and its activation depends on the binding of Met1-Ub by the IKK subunit NEMO (also known as IKKγ) (Fiil and Gyrd-Hansen, 2014Fiil B.K. Gyrd-Hansen M. Met1-linked ubiquitination in immune signalling.FEBS J. 2014; 281: 4337-4350Crossref PubMed Scopus (51) Google Scholar, Jiang and Chen, 2011Jiang X. Chen Z.J. The role of ubiquitylation in immune defence and pathogen evasion.Nat. Rev. Immunol. 2011; 12: 35-48PubMed Google Scholar). Met1-Ub is conjugated by the linear ubiquitin chain assembly complex (LUBAC), composed of HOIP, HOIL-1, and SHARPIN, which has emerged as an important Ub ligase activity in innate immune signaling and immune regulation (Boisson et al., 2012Boisson B. Laplantine E. Prando C. Giliani S. Israelsson E. Xu Z. Abhyankar A. Israël L. Trevejo-Nunez G. Bogunovic D. et al.Immunodeficiency, autoinflammation and amylopectinosis in humans with inherited HOIL-1 and LUBAC deficiency.Nat. Immunol. 2012; 13: 1178-1186Crossref PubMed Scopus (325) Google Scholar, Boisson et al., 2015Boisson B. Laplantine E. Dobbs K. Cobat A. Tarantino N. Hazen M. Lidov H.G. Hopkins G. Du L. Belkadi A. et al.Human HOIP and LUBAC deficiency underlies autoinflammation, immunodeficiency, amylopectinosis, and lymphangiectasia.J. Exp. Med. 2015; 212: 939-951Crossref PubMed Scopus (169) Google Scholar, Damgaard et al., 2012Damgaard R.B. Nachbur U. Yabal M. Wong W.W. Fiil B.K. Kastirr M. Rieser E. Rickard J.A. Bankovacki A. Peschel C. et al.The ubiquitin ligase XIAP recruits LUBAC for NOD2 signaling in inflammation and innate immunity.Mol. Cell. 2012; 46: 746-758Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, Gerlach et al., 2011Gerlach B. Cordier S.M. Schmukle A.C. Emmerich C.H. Rieser E. Haas T.L. Webb A.I. Rickard J.A. Anderton H. Wong W.W. et al.Linear ubiquitination prevents inflammation and regulates immune signalling.Nature. 2011; 471: 591-596Crossref PubMed Scopus (684) Google Scholar, Ikeda et al., 2011Ikeda F. Deribe Y.L. Skånland S.S. Stieglitz B. Grabbe C. Franz-Wachtel M. van Wijk S.J. Goswami P. Nagy V. Terzic J. et al.SHARPIN forms a linear ubiquitin ligase complex regulating NF-κB activity and apoptosis.Nature. 2011; 471: 637-641Crossref PubMed Scopus (550) Google Scholar, Kirisako et al., 2006Kirisako T. Kamei K. Murata S. Kato M. Fukumoto H. Kanie M. Sano S. Tokunaga F. Tanaka K. Iwai K. A ubiquitin ligase complex assembles linear polyubiquitin chains.EMBO J. 2006; 25: 4877-4887Crossref PubMed Scopus (569) Google Scholar, Tokunaga et al., 2011Tokunaga F. Nakagawa T. Nakahara M. Saeki Y. Taniguchi M. Sakata S. Tanaka K. Nakano H. Iwai K. SHARPIN is a component of the NF-κB-activating linear ubiquitin chain assembly complex.Nature. 2011; 471: 633-636Crossref PubMed Scopus (476) Google Scholar). In cells, LUBAC function is regulated by at least two associated DUBs, OTULIN and CYLD, which serve both overlapping and unique roles. OTULIN exclusively hydrolyzes Met1-Ub, prevents spurious accumulation of Met1-Ub on LUBAC components under basal conditions, and restricts ubiquitination of LUBAC substrates such as RIPK2 after NOD2 stimulation (Fiil et al., 2013Fiil B.K. Damgaard R.B. Wagner S.A. Keusekotten K. Fritsch M. Bekker-Jensen S. Mailand N. Choudhary C. Komander D. Gyrd-Hansen M. OTULIN restricts Met1-linked ubiquitination to control innate immune signaling.Mol. Cell. 2013; 50: 818-830Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, Keusekotten et al., 2013Keusekotten K. Elliott P.R. Glockner L. Fiil B.K. Damgaard R.B. Kulathu Y. Wauer T. Hospenthal M.K. Gyrd-Hansen M. Krappmann D. et al.OTULIN antagonizes LUBAC signaling by specifically hydrolyzing Met1-linked polyubiquitin.Cell. 2013; 153: 1312-1326Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). CYLD, a bona fide tumor suppressor and negative regulator of NF-κB signaling (Harhaj and Dixit, 2012Harhaj E.W. Dixit V.M. Regulation of NF-κB by deubiquitinases.Immunol. Rev. 2012; 246: 107-124Crossref PubMed Scopus (217) Google Scholar), disassembles both Met1-Ub and Lys63-Ub (Komander et al., 2009Komander D. Reyes-Turcu F. Licchesi J.D. Odenwaelder P. Wilkinson K.D. Barford D. Molecular discrimination of structurally equivalent Lys 63-linked and linear polyubiquitin chains.EMBO Rep. 2009; 10: 466-473Crossref PubMed Scopus (450) Google Scholar, Ritorto et al., 2014Ritorto M.S. Ewan R. Perez-Oliva A.B. Knebel A. Buhrlage S.J. Wightman M. Kelly S.M. Wood N.T. Virdee S. Gray N.S. et al.Screening of DUB activity and specificity by MALDI-TOF mass spectrometry.Nat. Commun. 2014; 5: 4763Crossref PubMed Scopus (214) Google Scholar, Sato et al., 2015Sato Y. Goto E. Shibata Y. Kubota Y. Yamagata A. Goto-Ito S. Kubota K. Inoue J. Takekawa M. Tokunaga F. Fukai S. Structures of CYLD USP with Met1- or Lys63-linked diubiquitin reveal mechanisms for dual specificity.Nat. Struct. Mol. Biol. 2015; 22: 222-229Crossref PubMed Scopus (91) Google Scholar). CYLD is recruited with LUBAC to TNFR1 and NOD2 signaling complexes and trims Ub chains on LUBAC substrates (Draber et al., 2015Draber P. Kupka S. Reichert M. Draberova H. Lafont E. de Miguel D. Spilgies L. Surinova S. Taraborrelli L. Hartwig T. et al.LUBAC-recruited CYLD and A20 regulate gene activation and cell death by exerting opposing effects on linear ubiquitin in signaling complexes.Cell Rep. 2015; 13: 2258-2272Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, Hrdinka et al., 2016Hrdinka M. Fiil B.K. Zucca M. Leske D. Bagola K. Yabal M. Elliott P.R. Damgaard R.B. Komander D. Jost P.J. Gyrd-Hansen M. CYLD limits Lys63- and Met1-linked ubiquitin at receptor complexes to regulate innate immune signaling.Cell Rep. 2016; 14: 2846-2858Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, Takiuchi et al., 2014Takiuchi T. Nakagawa T. Tamiya H. Fujita H. Sasaki Y. Saeki Y. Takeda H. Sawasaki T. Buchberger A. Kimura T. Iwai K. Suppression of LUBAC-mediated linear ubiquitination by a specific interaction between LUBAC and the deubiquitinases CYLD and OTULIN.Genes Cells. 2014; 19: 254-272Crossref PubMed Scopus (93) Google Scholar). Both CYLD and OTULIN associate with LUBAC via an N-terminal peptide:N-glycanase/UBA- or UBX-containing protein (PUB) domain in the catalytic subunit HOIP (Draber et al., 2015Draber P. Kupka S. Reichert M. Draberova H. Lafont E. de Miguel D. Spilgies L. Surinova S. Taraborrelli L. Hartwig T. et al.LUBAC-recruited CYLD and A20 regulate gene activation and cell death by exerting opposing effects on linear ubiquitin in signaling complexes.Cell Rep. 2015; 13: 2258-2272Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, Elliott et al., 2014Elliott P.R. Nielsen S.V. Marco-Casanova P. Fiil B.K. Keusekotten K. Mailand N. Freund S.M. Gyrd-Hansen M. Komander D. Molecular basis and regulation of OTULIN-LUBAC interaction.Mol. Cell. 2014; 54: 335-348Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, Hrdinka et al., 2016Hrdinka M. Fiil B.K. Zucca M. Leske D. Bagola K. Yabal M. Elliott P.R. Damgaard R.B. Komander D. Jost P.J. Gyrd-Hansen M. CYLD limits Lys63- and Met1-linked ubiquitin at receptor complexes to regulate innate immune signaling.Cell Rep. 2016; 14: 2846-2858Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, Schaeffer et al., 2014Schaeffer V. Akutsu M. Olma M.H. Gomes L.C. Kawasaki M. Dikic I. Binding of OTULIN to the PUB domain of HOIP controls NF-κB signaling.Mol. Cell. 2014; 54: 349-361Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, Takiuchi et al., 2014Takiuchi T. Nakagawa T. Tamiya H. Fujita H. Sasaki Y. Saeki Y. Takeda H. Sawasaki T. Buchberger A. Kimura T. Iwai K. Suppression of LUBAC-mediated linear ubiquitination by a specific interaction between LUBAC and the deubiquitinases CYLD and OTULIN.Genes Cells. 2014; 19: 254-272Crossref PubMed Scopus (93) Google Scholar). OTULIN harbors a PUB-interacting motif (PIM) that inserts into a PIM binding pocket in the HOIP PUB domain to create a high-affinity interaction important for its ability to counteract LUBAC auto-ubiquitination (Elliott et al., 2014Elliott P.R. Nielsen S.V. Marco-Casanova P. Fiil B.K. Keusekotten K. Mailand N. Freund S.M. Gyrd-Hansen M. Komander D. Molecular basis and regulation of OTULIN-LUBAC interaction.Mol. Cell. 2014; 54: 335-348Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, Schaeffer et al., 2014Schaeffer V. Akutsu M. Olma M.H. Gomes L.C. Kawasaki M. Dikic I. Binding of OTULIN to the PUB domain of HOIP controls NF-κB signaling.Mol. Cell. 2014; 54: 349-361Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The association of CYLD with LUBAC and its recruitment to receptor complexes also involves the PIM binding pocket in the HOIP PUB domain (Draber et al., 2015Draber P. Kupka S. Reichert M. Draberova H. Lafont E. de Miguel D. Spilgies L. Surinova S. Taraborrelli L. Hartwig T. et al.LUBAC-recruited CYLD and A20 regulate gene activation and cell death by exerting opposing effects on linear ubiquitin in signaling complexes.Cell Rep. 2015; 13: 2258-2272Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, Hrdinka et al., 2016Hrdinka M. Fiil B.K. Zucca M. Leske D. Bagola K. Yabal M. Elliott P.R. Damgaard R.B. Komander D. Jost P.J. Gyrd-Hansen M. CYLD limits Lys63- and Met1-linked ubiquitin at receptor complexes to regulate innate immune signaling.Cell Rep. 2016; 14: 2846-2858Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, Takiuchi et al., 2014Takiuchi T. Nakagawa T. Tamiya H. Fujita H. Sasaki Y. Saeki Y. Takeda H. Sawasaki T. Buchberger A. Kimura T. Iwai K. Suppression of LUBAC-mediated linear ubiquitination by a specific interaction between LUBAC and the deubiquitinases CYLD and OTULIN.Genes Cells. 2014; 19: 254-272Crossref PubMed Scopus (93) Google Scholar), but the molecular basis for the interaction is not understood. Here, we show that CYLD does not interact directly with HOIP and identify the uncharacterized protein spermatogenesis-associated protein 2 (SPATA2) as the factor that bridges CYLD and HOIP. SPATA2 contains a PIM that binds the PUB domain in HOIP, but not other PUB domains. SPATA2 binds the USP domain of CYLD via its PUB domain, but in a PIM-independent manner. Interestingly, this interaction also activates CYLD. Functionally, SPATA2 mediates the recruitment of CYLD to the TNFR1 signaling complex and supports CYLD-dependent regulation of LUBAC-mediated NF-κB signaling. In cells, CYLD interaction with HOIP depends on the PIM-binding pocket within the HOIP PUB domain (Draber et al., 2015Draber P. Kupka S. Reichert M. Draberova H. Lafont E. de Miguel D. Spilgies L. Surinova S. Taraborrelli L. Hartwig T. et al.LUBAC-recruited CYLD and A20 regulate gene activation and cell death by exerting opposing effects on linear ubiquitin in signaling complexes.Cell Rep. 2015; 13: 2258-2272Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, Hrdinka et al., 2016Hrdinka M. Fiil B.K. Zucca M. Leske D. Bagola K. Yabal M. Elliott P.R. Damgaard R.B. Komander D. Jost P.J. Gyrd-Hansen M. CYLD limits Lys63- and Met1-linked ubiquitin at receptor complexes to regulate innate immune signaling.Cell Rep. 2016; 14: 2846-2858Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, Takiuchi et al., 2014Takiuchi T. Nakagawa T. Tamiya H. Fujita H. Sasaki Y. Saeki Y. Takeda H. Sawasaki T. Buchberger A. Kimura T. Iwai K. Suppression of LUBAC-mediated linear ubiquitination by a specific interaction between LUBAC and the deubiquitinases CYLD and OTULIN.Genes Cells. 2014; 19: 254-272Crossref PubMed Scopus (93) Google Scholar). Mutational analysis of CYLD showed that the interaction is mediated by the CYLD USP domain and depends on the CYLD B-box (Takiuchi et al., 2014Takiuchi T. Nakagawa T. Tamiya H. Fujita H. Sasaki Y. Saeki Y. Takeda H. Sawasaki T. Buchberger A. Kimura T. Iwai K. Suppression of LUBAC-mediated linear ubiquitination by a specific interaction between LUBAC and the deubiquitinases CYLD and OTULIN.Genes Cells. 2014; 19: 254-272Crossref PubMed Scopus (93) Google Scholar) (Figures 1A and 1B ). Deletion of the CYLD B-box impaired the ability of CYLD to antagonize LUBAC-mediated NF-κB activity, suggesting that this region in CYLD regulates LUBAC function (Figures 1C and S1A, available online). However, CYLD does not contain a discernible PIM within this region and there was no obvious binding between the CYLD USP and HOIP PUB domain, as determined by size-exclusion chromatography (SEC), where CYLD and HOIP eluted in separate fractions (Figure 1D), in vitro pull-downs (Figure S1B), or nuclear magnetic resonance (NMR) spectroscopy (data not shown) using purified proteins. This prompted us to search for a protein that would mediate the interaction between CYLD and HOIP. For this, we purified FLAG-tagged wild-type (WT) CYLD and CYLD with deletion of the B-box (ΔB-box) from CYLD knockout (KO) U2OS/NOD2 cells (Figure 1B) and subjected the purified material to liquid chromatography-tandem mass spectrometry (LC-MS/MS). Among the detected proteins were previously described CYLD interactors such as TAK1 (Reiley et al., 2007Reiley W.W. Jin W. Lee A.J. Wright A. Wu X. Tewalt E.F. Leonard T.O. Norbury C.C. Fitzpatrick L. Zhang M. Sun S.C. Deubiquitinating enzyme CYLD negatively regulates the ubiquitin-dependent kinase Tak1 and prevents abnormal T cell responses.J. Exp. Med. 2007; 204: 1475-1485Crossref PubMed Scopus (198) Google Scholar), TNF receptor-associated factor 2 (TRAF2) (Kovalenko et al., 2003Kovalenko A. Chable-Bessia C. Cantarella G. Israël A. Wallach D. Courtois G. The tumour suppressor CYLD negatively regulates NF-kappaB signalling by deubiquitination.Nature. 2003; 424: 801-805Crossref PubMed Scopus (849) Google Scholar), and also the known CYLD interactors SPATA2 and SPATA2-like (SPATA2L) (Sowa et al., 2009Sowa M.E. Bennett E.J. Gygi S.P. Harper J.W. Defining the human deubiquitinating enzyme interaction landscape.Cell. 2009; 138: 389-403Abstract Full Text Full Text PDF PubMed Scopus (1180) Google Scholar). Strikingly, SPATA2 was the most highly enriched protein in the CYLD WT sample relative to the CYLD ΔB-box sample, indicating that the interaction depends on the CYLD B-box (Figure 1E; Table S1). Also, SPATA2L was preferentially enriched by CYLD WT whereas TAK1-TAB components and TRAF2 were co-purified similarly with CYLD WT and CYLD ΔB-box (Figure 1E; Table S1). The interaction between SPATA2 and CYLD was confirmed in cells by co-immunoprecipitation of ectopic or endogenous proteins (Figures 1F and 1G). To ensure that SPATA2 was detected by the SPATA2 antibody in the CYLD immunoprecipitation, the specificity of the antibody was carefully characterized in cells where SPATA2 was depleted by RNAi-mediated silencing and in cells where SPATA2 had been genetically deleted by CRISPR/Cas9 genome editing (Figures 1H and S1C–S1E). This confirmed that the antibody detected SPATA2 in cell lysates and in CYLD immunoprecipitation experiments, but it also showed that the antibody detected several unrelated proteins in lysates, some of which migrated at a similar molecular weight (MW) as SPATA2 (Figures 1H and S1C–S1F). We then analyzed which region of SPATA2 was responsible for CYLD binding. This showed that the SPATA2 N-terminal PUB domain mediates CYLD interaction in cells (Figures 1I and 1J). Indeed, the CYLD USP domain (aa 583–956) and SPATA2 PUB domain (aa 1–241) formed an SEC-stable complex (Figure 1K), confirming a direct interaction. It was striking that while CYLD was unable to form a stable complex with the PUB domain of HOIP, it instead interacted with the PUB domain in SPATA2 (Figures 1D, 1K, and S1B). A crystal structure of the SPATA2 PUB domain (aa 7–219) at 1.45 Å resolution (Figures 2A–2D, S2A, and S2B; Table 1) revealed a fold most similar to that of the extended PUB domain in HOIP (root-mean-square deviation [RMSD] 2.4 Å) (Elliott et al., 2014Elliott P.R. Nielsen S.V. Marco-Casanova P. Fiil B.K. Keusekotten K. Mailand N. Freund S.M. Gyrd-Hansen M. Komander D. Molecular basis and regulation of OTULIN-LUBAC interaction.Mol. Cell. 2014; 54: 335-348Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) (Figures 2A–2C) and the smaller PUB domain of PNGase (RMSD 3.2 Å) (Zhao et al., 2007Zhao G. Zhou X. Wang L. Li G. Schindelin H. Lennarz W.J. Studies on peptide:N-glycanase-p97 interaction suggest that p97 phosphorylation modulates endoplasmic reticulum-associated degradation.Proc. Natl. Acad. Sci. USA. 2007; 104: 8785-8790Crossref PubMed Scopus (76) Google Scholar) (Figure 2D). Species conservation of SPATA2 mapped onto the surface of the PUB domain reveals that while the PIM pocket is highly conserved (Figures 2E, 2F, S2A, and S2C), this interaction site is also very different from canonical PUB domains. The previously mapped PUB-PIM interactions include a conserved Asp-Leu/Met-Tyr (see below), in which Leu and Tyr occupy a deep, hydrophobic gorge on the PUB surface, the PIM pocket. In SPATA2, this pocket is significantly different from both HOIP as well as PNGase structures, and modeling of interactions with PIM peptides derived from OTULIN or p97 would generate steric clashes (Figures 2E and 2F). Consistently, the SPATA2 PUB domain does not bind PIM peptides (see below), suggesting an interaction motif in CYLD may need to display distinct properties. Nonetheless, the high conservation in this area did suggest that this surface may mediate CYLD interactions, and single amino acid mutations in or near the SPATA2 PIM pocket interfered with CYLD binding (Figure 2G). In particular, mutations in the “lower wall” of the SPATA2 PIM pocket (Y114A, T115N, and T115A) decreased CYLD interactions, while mutation of residues in the “upper wall” of the pocket (N98A and T94K) did not have strong effects on CYLD binding. The strongest effect on CYLD binding was observed when we mutated Tyr114, which points away from the PIM pocket (Figures 2E and 2G), supporting that CYLD binds the SPATA2 PUB domain in a PIM-independent manner.Table 1Data Collection and Refinement StatisticsSPATA2 7–219HOIP 5–180 + SPATA2 334–344Data CollectionBeamlineDiamond I02Diamond I02Space groupP 21P 43a, b, c (Å)43.48, 51.14, 56.2989.04, 89.04, 53.56α, β, γ (°)90.00, 105.97, 90.0090.00, 90.00, 90.00Wavelength0.97940.9795Resolution (Å)54.12–1.45 (1.48–1.45)62.96–2.70 (2.83–2.70)Rmerge3.7 (32.7)7.2 (67.9)< I / σI >12.1 (2.2)9.5 (2.0)CC(1/2)0.99 (0.91)0.99 (0.61)Completeness (%)90.2 (81.2)97.7 (99.7)Redundancy3.0 (2.9)2.7 (2.7)RefinementResolution (Å)54.11–1.4562.96–2.70No. reflections37,45811,425Rwork / Rfree18.2/22.523.3/28.1No. AtomsProtein1,7222,695Ligand/ion1215Water308–B FactorsWilson B15.7357.1Protein25.161.3Ligand/ion21.885.0Water38.0–RMSDsBond lengths (Å)0.0050.002Bond angles (°)0.7720.526Ramachandran statistics (outliers, allowed, favored)0.0, 1.4, 98.60.0, 2.6, 97.4Related to Figures 2 and 5. Values in parentheses are for the highest-resolution shell. Datasets were collected and structures determined from a single crystal. Open table in a new tab Related to Figures 2 and 5. Values in parentheses are for the highest-resolution shell. Datasets were collected and structures determined from a single crystal. The B-box dependence of the CYLD-SPATA2 interaction (Figures 1B, 1C, and 1F) could suggest a direct interaction between the B-box and the SPATA2 PUB domain. Surprisingly, NMR titration experiments with an isolated 15N-labeled B-box domain (aa 778–855) and unlabeled SPATA2 PUB domain revealed no signs of an interaction (Figure S3A). This contrasts the formation of a stable complex on gel filtration between the SPATA PUB domain and the CYLD USP domain (Figure 1K). Further studies using SEC coupled to multi-angle light scattering (SEC-MALS) revealed that the intact CYLD USP domain (aa 583–956, including the B-box, 43 kDa) eluted as a dimer (86 kDa), while CYLD ΔB-box (35 kDa) eluted as a monomer (34 kDa) (Figures 3A and S3B). Furthermore, the isolated B-box domain (aa 778–855, 9.1 kDa) eluted as a dimer (17.1 kDa) (Figure 3A). Strikingly, the SPATA2 PUB domain (aa 1–241, 27.6 kDa), a monomer on its own (26.6 kDa), formed a 2:2 complex with dimeric CYLD USP domain of 136 kDa (calculated 140 kDa) in SEC-MALS (Figure 3A). This was independently confirmed by equilibrium analytical ultracentrifugation (Figure S3C). Thus, nicely consistent with the earlier results from mass spectrometry (Figure 1E), this complex forms in a B-box-dependent manner (Figures 3A and S3B). Previous structural analysis of the CYLD USP domain (Komander et al., 2008Komander D. Lord C.J. Scheel H. Swift S. Hofmann K. Ashworth A. Barford D. The structure of the CYLD USP domain explains its specificity for Lys63-linked polyubiquitin and reveals a B box module.Mol. Cell. 2008; 29: 451-464Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar) suggested how the B-box domain might mediate CYLD dimerization. A conserved B-box surface forms a hydrophobic interface across a crystallographic symmetry axis, which orients the two catalytic domains such that both can access polyUb without steric hindrance (Komander et al., 2008Komander D. Lord C.J. Scheel H. Swift S. Hofmann K. Ashworth A. Barford D. The structure of the CYLD USP domain explains its specificity for Lys63-linked polyubiquitin and reveals a B box module.Mol. Cell. 2008; 29: 451-464Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, Sato et al., 2015Sato Y. Goto E. Shibata Y. Kubota Y. Yamagata A. Goto-Ito S. Kubota K. Inoue J. Takekawa M. Tokunaga F. Fukai S. Structures of CYLD USP with Met1- or Lys63-linked diubiquitin reveal mechanisms for dual specificity.Nat. Struct. Mol. Biol. 2015; 22: 222-229Crossref PubMed Scopus (91) Google Scholar) (Figure 3B). Mutation of Ile790 (I790D) within the B-box dimerization interface generated a monomeric B-box (9 kDa) and monomeric CYLD USP domain (44 kDa) (Figure 3A). Interestingly, the I790D dimerization mutant still formed a 1:1 complex with the SPATA2 PUB domain of 65 kDa that was less stable on SEC-MALS (Figure 3A, blue profile). These results were corroborated by in vitro pull-downs and surface plasmon resonance (SPR), which revealed the CYLD-SPATA2 interaction to be high affinity (96 nM), and also showed no binding of the isolated B-box domain to SPATA2 (Figures 3C, 3D, S3D, and S3E). CYLD I790D affinity for SPATA2 was still respectable (518 nM), but a higher koff likely affects stability of the complex when CYLD is not dimeric (Figures 3C, 3D, and S3E). Surface conservation depicted on the CYLD dimer revealed that while exposed areas of the B-box were not conserved, a highly conserved surface exists on the solvent-exposed side of the CYLD palm domain not involved in Ub interactions (Figure 3B). Mutation of conserved surface residues revealed that Leu622 was essential for SPATA2 interaction (Figure S3D). Leu622 is 45 Å away from the B-box domain, indicating that the catalytic USP core of CYLD mediates SPATA2 binding. Hence, CYLD and SPATA2 form a highly stable heterotetramer in vitro and likely in cells, which is destabilized when the core dimerization domain, the B-box of CYLD, is deleted or disrupted. A number of USP domains are activated allosterically by binding partners (Sahtoe and Sixma, 2015Sahtoe D.D. Sixma T.K. Layers of DUB regulation.Trends Biochem. Sci. 2015; 40: 456-467Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Recent structural insights into the USP46-UAF1 complex and the USP12-UAF1-WDR20 complex revealed that the activators interact with surfaces remote from the catalytic center and mediate activation via long-range allosteric mechanisms (Li et al., 2016Li H. Lim K.S. Kim H. Hinds T.R. Jo U. Mao H. Weller C.E. Sun J. Chatterjee C. D’Andrea A.D. Zheng N. Allosteric activation of ubiquitin-specific proteases by β-propeller proteins UAF1 and WDR20.Mol. Cell. 2016; 63: 249-260Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, Yin et al., 2015Yin J. Schoeffler A.J. Wickliffe K. Newton K. Starovasnik M.A. Dueber E.C. Harris S.F. Structural insights into WD-repeat 48 activation of ubiquitin-specific protease 46.Structure. 2015; 23: 2043-2054Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). SPATA2 also has a reproducible, yet moderate, activating effect on CYLD. Hydrolysis of Met1- or Lys63-linked tetraUb is enhanced in presence of SPATA2 (Figures 3E and S3F). Quantification of this effect employing fluorescent Met1/Lys63-linked diUb substrates (Keusekotten et al., 2013Keusekotten K. Elliott P.R. Glockner L. Fiil B.K. Damgaard R.B. Kulathu Y. Wauer T. Hospenthal M.K. Gyrd-Hansen M. Krappmann D. et al.OTULIN antagonizes LUBAC signaling by specifically hydrolyzing Met1-linked polyubiquitin.Cell. 2013; 153: 1312-1326Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar) reveals an ∼2-fold increase in kcat/KM in presence of SPATA2 (Figures S3G and S3H). Deletion of the B-box does not affect the catalytic activity or structure of the CYLD USP domain (Komander et al., 2008Komander D. Lord C.J. Scheel H. Swift S. Hofmann K. Ashworth A. Barford D. The structure of the CYLD USP domain explains its specificity for Lys63-linked polyubiquitin and reveals a B box module.Mol. Cell. 2008; 29: 451-464Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, Sato et al., 2015Sato Y. Goto E. Shibata Y. Kubota Y. Yamagata A. Goto-Ito S. Kubota K. Inoue J. Takekawa M. Tokunaga F. Fukai S. Structures of CYLD USP with Met1- or Lys63-linked diubiquitin reveal mechanisms for dual specificity.Nat. Struct. Mol. Biol. 2015; 22: 222-229Crossref PubMed Scopus (91) Google Scholar), but SPATA2-mediated CYLD activation is lost in CYLD ΔB-box (Figure S3I) or in CYLD L622D (Figure 3F), as these CYLD variants no longer bind SPATA2. Likewise, mutations of SPATA2 residues in the CYLD interface decrease or abolish its ability to activate CYLD (Figures S3J–S3L). SPATA2 does not affect CYLD specificity, which remains Lys63 and Met1 specific at the diUb level (Figure S3M), and still does not significantly cleave Lys48-tetraUb (data not shown). Together" @default.
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