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• W2013497239 abstract "The molecular mechanism by which mutations in the cytoskeleton-organizing protein PSTPIP1 cause the autoinflammatory PAPA syndrome is still elusive. Here, we demonstrate that PSTPIP1 requires the familial Mediterranean fever protein pyrin to assemble the ASC pyroptosome, a molecular platform that recruits and activates caspase-1. We provide evidence that pyrin is a cytosolic receptor for PSTPIP1. Pyrin exists as a homotrimer in an autoinhibited state due to intramolecular interactions between its pyrin domain (PYD) and B-box. Ligation by PSTPIP1, which is also a homotrimer, activates pyrin by unmasking its PYD, thereby allowing it to interact with ASC and facilitate ASC oligomerization into an active ASC pyroptosome. Because of their high binding affinity to pyrin's B-box, PAPA-associated PSTPIP1 mutants were found to be more effective than WT PSTPIP1 in inducing pyrin activation. Therefore, constitutive ligation and activation of pyrin by mutant PSTPIP1 proteins explain the autoinflammatory phenotype seen in PAPA syndrome. The molecular mechanism by which mutations in the cytoskeleton-organizing protein PSTPIP1 cause the autoinflammatory PAPA syndrome is still elusive. Here, we demonstrate that PSTPIP1 requires the familial Mediterranean fever protein pyrin to assemble the ASC pyroptosome, a molecular platform that recruits and activates caspase-1. We provide evidence that pyrin is a cytosolic receptor for PSTPIP1. Pyrin exists as a homotrimer in an autoinhibited state due to intramolecular interactions between its pyrin domain (PYD) and B-box. Ligation by PSTPIP1, which is also a homotrimer, activates pyrin by unmasking its PYD, thereby allowing it to interact with ASC and facilitate ASC oligomerization into an active ASC pyroptosome. Because of their high binding affinity to pyrin's B-box, PAPA-associated PSTPIP1 mutants were found to be more effective than WT PSTPIP1 in inducing pyrin activation. Therefore, constitutive ligation and activation of pyrin by mutant PSTPIP1 proteins explain the autoinflammatory phenotype seen in PAPA syndrome. Caspase-1 plays a pivotal role in innate immunity and host defense against pathogenic infections. Caspase-1 is activated by intracellular assemblies, called inflammasomes, in response to diverse pathogenic infections and cellular stresses (Mariathasan and Monack, 2007Mariathasan S. Monack D.M. Inflammasome adaptors and sensors: intracellular regulators of infection and inflammation.Nat. Rev. Immunol. 2007; 7: 31-40Crossref PubMed Scopus (691) Google Scholar). The activated caspase-1 cleaves the inactive pro-IL-1β and pro-IL-18 to produce the active proinflammatory cytokines IL-1β and IL-18, respectively, which are potent proinflammatory cytokines. Caspase-1 also plays an important role in an inflammatory form of cell death called pyroptosis (Fernandes-Alnemri et al., 2007Fernandes-Alnemri T. Wu J. Yu J.-W. Datta P. Miller B. Jankowski W. Rosenberg S. Zhang J. Alnemri E.S. The pyroptosome: a supramolecular assembly of ASC dimers mediating inflammatory cell death via caspase-1 activation.Cell Death Differ. 2007; 14: 1590-1604Crossref PubMed Scopus (717) Google Scholar, Fink and Cookson, 2005Fink S.L. Cookson B.T. Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells.Infect. Immun. 2005; 73: 1907-1916Crossref PubMed Scopus (1477) Google Scholar). During pyroptosis, caspase-1 is activated by a unique supramolecular platform called the pyroptosome, which is composed of oligomerized dimers of the adaptor protein ASC (Fernandes-Alnemri et al., 2007Fernandes-Alnemri T. Wu J. Yu J.-W. Datta P. Miller B. Jankowski W. Rosenberg S. Zhang J. Alnemri E.S. The pyroptosome: a supramolecular assembly of ASC dimers mediating inflammatory cell death via caspase-1 activation.Cell Death Differ. 2007; 14: 1590-1604Crossref PubMed Scopus (717) Google Scholar). Deregulated activation of caspase-1 is responsible for a number of systemic autoinflammatory diseases in humans (Ting et al., 2006Ting J.P. Kastner D.L. Hoffman H.M. CATERPILLERs, pyrin and hereditary immunological disorders.Nat. Rev. Immunol. 2006; 6: 183-195Crossref PubMed Scopus (277) Google Scholar). These diseases represent a group of inherited disorders characterized by recurrent episodes of inflammation and fever without an apparent stimulus, or a major involvement of autoantibodies or autoreactive T cells (Galeazzi et al., 2006Galeazzi M. Gasbarrini G. Ghirardello A. Grandemange S. Hoffman H.M. Manna R. Podswiadek M. Punzi L. Sebastiani G.D. Touitou I. Doria A. Autoinflammatory syndromes.Clin. Exp. Rheumatol. 2006; 24: S79-S85PubMed Google Scholar, Hull et al., 2003Hull K.M. Shoham N. Chae J.J. Aksentijevich I. Kastner D.L. The expanding spectrum of systemic autoinflammatory disorders and their rheumatic manifestations.Curr. Opin. Rheumatol. 2003; 15: 61-69Crossref PubMed Scopus (203) Google Scholar). Mutations in basic components of the inflammasomes appear to be responsible for some of these autoinflammatory diseases. For example, mutations in the CIAS1 gene which encodes cryopyrin cause three autoinflammatory diseases in humans (Feldmann et al., 2002Feldmann J. Prieur A.M. Quartier P. Berquin P. Certain S. Cortis E. Teillac-Hamel D. Fischer A. de Saint Basile G. Chronic infantile neurological cutaneous and articular syndrome is caused by mutations in CIAS1, a gene highly expressed in polymorphonuclear cells and chondrocytes.Am. J. Hum. Genet. 2002; 71: 198-203Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar, Hoffman et al., 2001Hoffman H.M. Mueller J.L. Broide D.H. Wanderer A.A. Kolodner R.D. Mutation of a new gene encoding a putative pyrin-like protein causes familial cold autoinflammatory syndrome and Muckle-Wells syndrome.Nat. Genet. 2001; 29: 301-305Crossref PubMed Scopus (1290) Google Scholar). Our recent studies suggest that some of these mutations increase self-self interactions and oligomerization of the mutant cryopyrin proteins, thereby causing more oligomerization of ASC and activation of caspase-1 (Yu et al., 2006Yu J.W. Wu J. Zhang Z. Datta P. Ibrahimi I. Taniguchi S. Sagara J. Fernandes-Alnemri T. Alnemri E.S. Cryopyrin and pyrin activate caspase-1, but not NF-kappaB, via ASC oligomerization.Cell Death Differ. 2006; 13: 236-249Crossref PubMed Scopus (282) Google Scholar). Likewise, mutations in the MEFV gene that encodes pyrin are associated with the most common autoinflammatory disease, familial Mediterranean fever (FMF) in humans (Consortium, 1997aConsortium T.F.F. A candidate gene for familial Mediterranean fever. The French FMF Consortium.Nat. Genet. 1997; 17: 25-31Crossref PubMed Scopus (1276) Google Scholar, Consortium, 1997bConsortium T.I.F. Ancient missense mutations in a new member of the RoRet gene family are likely to cause familial Mediterranean fever. The International FMF Consortium.Cell. 1997; 90: 797-807Abstract Full Text Full Text PDF PubMed Scopus (1331) Google Scholar). Pyrin interacts with ASC, and like cryopyrin, triggers ASC oligomerization, activation of procaspase-1, and IL-1β processing (Yu et al., 2006Yu J.W. Wu J. Zhang Z. Datta P. Ibrahimi I. Taniguchi S. Sagara J. Fernandes-Alnemri T. Alnemri E.S. Cryopyrin and pyrin activate caspase-1, but not NF-kappaB, via ASC oligomerization.Cell Death Differ. 2006; 13: 236-249Crossref PubMed Scopus (282) Google Scholar). How the FMF-associated mutations lead to increased inflammation is not yet clear, but it is likely that these mutations alter the activity of pyrin, leading to increased ASC oligomerization in FMF patients. Some human autoinflammatory diseases are caused by mutations in upstream regulatory components of the inflammasomes (Galeazzi et al., 2006Galeazzi M. Gasbarrini G. Ghirardello A. Grandemange S. Hoffman H.M. Manna R. Podswiadek M. Punzi L. Sebastiani G.D. Touitou I. Doria A. Autoinflammatory syndromes.Clin. Exp. Rheumatol. 2006; 24: S79-S85PubMed Google Scholar, Hull et al., 2003Hull K.M. Shoham N. Chae J.J. Aksentijevich I. Kastner D.L. The expanding spectrum of systemic autoinflammatory disorders and their rheumatic manifestations.Curr. Opin. Rheumatol. 2003; 15: 61-69Crossref PubMed Scopus (203) Google Scholar, Ting et al., 2006Ting J.P. Kastner D.L. Hoffman H.M. CATERPILLERs, pyrin and hereditary immunological disorders.Nat. Rev. Immunol. 2006; 6: 183-195Crossref PubMed Scopus (277) Google Scholar). In particular, two missense mutations in a protein called proline serine threonine phosphatase-interacting protein 1 (PSTPIP1) (also known as CD2-binding protein 1, CD2BP1) have been reported in patients with the dominantly inherited autoinflammatory syndrome of pyogenic arthritis, pyoderma gangrenosum, acne (PAPA) (Wise et al., 2002Wise C.A. Gillum J.D. Seidman C.E. Lindor N.M. Veile R. Bashiardes S. Lovett M. Mutations in CD2BP1 disrupt binding to PTP PEST and are responsible for PAPA syndrome, an autoinflammatory disorder.Hum. Mol. Genet. 2002; 11: 961-969Crossref PubMed Scopus (373) Google Scholar). PSTPIP1 interacts with pyrin (Shoham et al., 2003Shoham N.G. Centola M. Mansfield E. Hull K.M. Wood G. Wise C.A. Kastner D.L. Pyrin binds the PSTPIP1/CD2BP1 protein, defining familial Mediterranean fever and PAPA syndrome as disorders in the same pathway.Proc. Natl. Acad. Sci. USA. 2003; 100: 13501-13506Crossref PubMed Scopus (388) Google Scholar), and also associates with actin and plays an important role in the organization of the cytoskeleton (Badour et al., 2003Badour K. Zhang J. Shi F. McGavin M.K. Rampersad V. Hardy L.A. Field D. Siminovitch K.A. The Wiskott-Aldrich syndrome protein acts downstream of CD2 and the CD2AP and PSTPIP1 adaptors to promote formation of the immunological synapse.Immunity. 2003; 18: 141-154Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, Cote et al., 2002Cote J.F. Chung P.L. Theberge J.F. Halle M. Spencer S. Lasky L.A. Tremblay M.L. PSTPIP is a substrate of PTP-PEST and serves as a scaffold guiding PTP-PEST toward a specific dephosphorylation of WASP.J. Biol. Chem. 2002; 277: 2973-2986Crossref PubMed Scopus (107) Google Scholar, Spencer et al., 1997Spencer S. Dowbenko D. Cheng J. Li W. Brush J. Utzig S. Simanis V. Lasky L.A. PSTPIP: a tyrosine phosphorylated cleavage furrow-associated protein that is a substrate for a PEST tyrosine phosphatase.J. Cell Biol. 1997; 138: 845-860Crossref PubMed Scopus (152) Google Scholar). The two PAPA-associated mutations appear to diminish the interaction of PSTPIP1 with PEST-type protein tyrosine phosphatase PTP-PEST (Wise et al., 2002Wise C.A. Gillum J.D. Seidman C.E. Lindor N.M. Veile R. Bashiardes S. Lovett M. Mutations in CD2BP1 disrupt binding to PTP PEST and are responsible for PAPA syndrome, an autoinflammatory disorder.Hum. Mol. Genet. 2002; 11: 961-969Crossref PubMed Scopus (373) Google Scholar), but markedly increase the binding of PSTPIP1 to pyrin (Shoham et al., 2003Shoham N.G. Centola M. Mansfield E. Hull K.M. Wood G. Wise C.A. Kastner D.L. Pyrin binds the PSTPIP1/CD2BP1 protein, defining familial Mediterranean fever and PAPA syndrome as disorders in the same pathway.Proc. Natl. Acad. Sci. USA. 2003; 100: 13501-13506Crossref PubMed Scopus (388) Google Scholar). Based on these observations, it was suggested that these PSTPIP1 mutants exert a dominant-negative effect on pyrin and inhibit its anti-inflammatory activity, leading to increased production of IL-1β in PAPA patients (Shoham et al., 2003Shoham N.G. Centola M. Mansfield E. Hull K.M. Wood G. Wise C.A. Kastner D.L. Pyrin binds the PSTPIP1/CD2BP1 protein, defining familial Mediterranean fever and PAPA syndrome as disorders in the same pathway.Proc. Natl. Acad. Sci. USA. 2003; 100: 13501-13506Crossref PubMed Scopus (388) Google Scholar). However, based on our recent observations that pyrin could assemble an inflammasome and could promote ASC oligomerization and caspase-1 activation (Yu et al., 2006Yu J.W. Wu J. Zhang Z. Datta P. Ibrahimi I. Taniguchi S. Sagara J. Fernandes-Alnemri T. Alnemri E.S. Cryopyrin and pyrin activate caspase-1, but not NF-kappaB, via ASC oligomerization.Cell Death Differ. 2006; 13: 236-249Crossref PubMed Scopus (282) Google Scholar), we propose that binding of the PSTPIP1 mutants to pyrin increases its ability to assemble the ASC pyroptosome rather than inhibits its anti-inflammatory activity. In this report, we investigated the role of pyrin in the mechanism of activation of caspase-1 by the PAPA-associated PSTPIP1 mutants in THP-1 monocytes and an HEK293T cell-based reconstitution system. In support of our hypothesis, we found that pyrin is required for formation of the ASC pyroptosome and caspase-1 activation by the autoinflammatory PSTPIP1 mutants. We have also elucidated the mechanism of activation of pyrin by PSTPIP1. Our results show that both pyrin and PSTPIP1 are homotrimers and that pyrin is a cytosolic receptor for PSTPIP1. The pyrin homotrimer is not fully active due to the masking of its PYD by its B-box. Binding of PSTPIP1 to the B-box of pyrin unmasks the PYD, which then interacts with ASC, thereby causing oligomerization of ASC and formation of the ASC pyroptosome that recruits and activates procaspase-1. Our findings therefore provide the biochemical basis for understanding how pyrin activity is regulated, and shed light on pyrin as a direct activator of the ASC pyroptosome in innate immunity. PAPA syndrome, like FMF, is associated with increased generation of IL-1β and is responsive to treatment with the IL-1 receptor antagonist anakinra (Chae et al., 2006Chae J.J. Wood G. Masters S.L. Richard K. Park G. Smith B.J. Kastner D.L. The B30.2 domain of pyrin, the familial Mediterranean fever protein, interacts directly with caspase-1 to modulate IL-1beta production.Proc. Natl. Acad. Sci. USA. 2006; 103: 9982-9987Crossref PubMed Scopus (445) Google Scholar, Dierselhuis et al., 2005Dierselhuis M.P. Frenkel J. Wulffraat N.M. Boelens J.J. Anakinra for flares of pyogenic arthritis in PAPA syndrome.Rheumatology (Oxford). 2005; 44: 406-408Crossref PubMed Scopus (152) Google Scholar), suggesting that the underlying cause of this disease is likely attributed to an excessive activation of caspase-1 by the mutant PSTPIP1 proteins. To test this hypothesis, we examined the effect of retrovirus-mediated transient expression of PAPA-associated PSTPIP1 mutants in THP-1 monocytes on caspase-1 activation and IL-1β generation. Indeed, expression of the A230T or E250Q PSTPIP1 mutants in THP-1 cells resulted in substantially more caspase-1 activation and IL-1β secretion compared with expression of WT PSTPIP1 or an empty vector control (Figures 1A and 1B). While studying the effect of retrovirus-mediated expression of PSTPIP1 variants in THP-1 cells, we noticed that retroviral infection, even with an empty retroviral vector, causes a dramatic increase in the expression of endogenous pyrin and pro-IL-1β proteins in the infected cells 16–24 hr postinfection (Figure 1C). Therefore, we became intrigued by the possibility that pyrin might play an important role in the robust caspase-1 activation and IL-1β generation by the autoinflammatory PSTPIP1 mutants, especially because pyrin has been shown to interact with PSTPIP1 (Shoham et al., 2003Shoham N.G. Centola M. Mansfield E. Hull K.M. Wood G. Wise C.A. Kastner D.L. Pyrin binds the PSTPIP1/CD2BP1 protein, defining familial Mediterranean fever and PAPA syndrome as disorders in the same pathway.Proc. Natl. Acad. Sci. USA. 2003; 100: 13501-13506Crossref PubMed Scopus (388) Google Scholar) and also to induce caspase-1 activation (Yu et al., 2006Yu J.W. Wu J. Zhang Z. Datta P. Ibrahimi I. Taniguchi S. Sagara J. Fernandes-Alnemri T. Alnemri E.S. Cryopyrin and pyrin activate caspase-1, but not NF-kappaB, via ASC oligomerization.Cell Death Differ. 2006; 13: 236-249Crossref PubMed Scopus (282) Google Scholar). To test this possibility, we examined the effect of retroviral infection on caspase-1 activation in THP-1 cells that stably express WT PSTPIP1 (THP-1-WT) or the two PSTPIP1 mutants A230T (THP-1-A230T) or E250Q (THP-1-E250Q). As shown in Figure 1D, expression of endogenous pyrin was substantially increased in the retrovirus-infected cells (5th to 8th lanes) compared with the uninfected cells (1st to 4th lanes). There was also noticeable caspase-1 activation (Figure 1D, 5th to 8th lanes) and IL-1β generation (Figure 1E, 5th to 8th columns) in the infected cells compared to uninfected cells. Significantly, retroviral infection caused more caspase-1 activation (Figure 1D, 7th and 8th lanes) and IL-1β generation (Figure 1E, 7th and 8th columns) in the mutant PSTPIP1-expressing cells compared to the empty vector or the WT PSTPIP1-expressing cells (Figure 1D, 5th and 6th lanes; Figure 1E, 5th and 6th columns). Combined, these results reveal that pyrin is induced by retroviral infection, and its induction is associated with increased caspase-1 activation and IL-1β generation in cells expressing the PAPA-associated PSTPIP1 mutants. After establishing that retroviral infection causes induction of pyrin and more activation of caspase-1 in the mutant PSTPIP1-expressing cells, we next examined the effect of siRNA-mediated knockdown of pyrin on retrovirus-induced IL-1β generation in the stable THP-1-A230T cells. These cells were transiently transfected with control or pyrin-specific siRNAs, and 48 hr after transfection the cells were infected with a GFP-encoding retrovirus for an additional 24 hr. As shown in Figure 1F, knocking down pyrin significantly reduced retrovirus-induced IL-1β secretion from these cells. Taken together, our results indicate that pyrin plays an important role in the activation of caspase-1 by the autoinflammatory PSTPIP1 mutants, because its induction increases caspase-1 activation and IL-1β generation and its knockdown has an opposite effect. To investigate in more detail the role of pyrin in the mechanism of caspase-1 activation by the autoinflammatory PSTPIP1 mutants, we used an HEK293 cell-based reconstitution system. HEK293 cells do not normally express PSTPIP1, pyrin, caspase-1, or the adaptor protein ASC (Yu et al., 2006Yu J.W. Wu J. Zhang Z. Datta P. Ibrahimi I. Taniguchi S. Sagara J. Fernandes-Alnemri T. Alnemri E.S. Cryopyrin and pyrin activate caspase-1, but not NF-kappaB, via ASC oligomerization.Cell Death Differ. 2006; 13: 236-249Crossref PubMed Scopus (282) Google Scholar), which makes them an ideal system to reconstitute the PSTPIP1-pyrin complex to study how PSTPIP1 interacts with pyrin to induce caspase-1 activation. Therefore, we generated stable HEK293T cell lines (293-C1AP) that express physiological levels of procaspase-1, ASC, and pyrin. Other cell lines that stably express procaspase-1, ASC, and cryopyrin (293-C1AC); procaspase-1 and pyrin (293-casp1-pyrin); or procaspase-1 and ASC (293-casp1-ASC) were also produced to use as controls. Next, we transfected these cell lines with WT PSTPIP1 or PAPA-associated PSTPIP1 mutants and assayed the activation of caspase-1 by western blot analysis and IL-1β processing. As shown in Figure 2A, right panels, expression of WT PSTPIP1 or PAPA-associated mutants in 293-C1AP cell line, which expresses procaspase-1, ASC and pyrin, resulted in caspase-1 activation and IL-1β processing. As observed in THP-1 cells, the PAPA-associated mutants induced more caspase-1 activation and IL-1β processing than the WT PSTPIP1 protein (3rd and 4th lanes). In contrast, expression of these PSTPIP1 proteins in the 293-casp1-ASC cells that express procaspase-1 and ASC without pyrin did not induce caspase-1 activation (Figure 2A, left panels), indicating that pyrin is required for PSTPIP1-induced caspase-1 activation. Similarly, expression of the PSTPIP1 proteins in the 293-casp1-pyrin cell line that expresses pyrin and procaspase-1 without ASC also did not induce caspase-1 activation (Figure 2A, middle panels), indicating that ASC is also required in addition to pyrin for PSTPIP1 to induce caspase-1 activation. To further demonstrate that pyrin is specifically required for PSTPIP1-induced caspase-1 activation, we compared the effect of expression of PSTPIP1 variants on caspase-1 activation in the pyrin-expressing 293-C1AP and the cryopyrin-expressing 293-C1AC cell lines. In contrast to 293-C1AP, ectopic expression of WT or the PAPA-associated PSTPIP1 mutants in the cryopyrin-expressing 293-C1AC cells did not induce caspase-1 activation or IL-1β processing (Figure 2B). These results indicate that pyrin, but not cryopyrin, is required for PSTPIP1 to induce caspase-1 activation and that the disease-associated PSTPIP1 mutants are more potent than the WT PSTPIP1 in inducing pyrin-dependent caspase-1 activation. Consistent with the above results, the ability of PSTPIP1 to activate caspase-1 was dependent on the level of pyrin in the cell. When the PSTPIP1 A230T mutant protein was expressed in two stable 293-C1AP cell lines having different levels of pyrin, it activated more caspase-1 in the higher pyrin-containing cells than in the lower pyrin-containing cells (Figure 2C). Taken together, our results demonstrate that pyrin is absolutely required for caspase-1 activation by PSTPIP1 and that the ability of PSTPIP1 to activate caspase-1 is enhanced at higher pyrin concentrations. Diverse proinflammatory stimuli trigger the assembly of an ASC pyroptosome in monocytes and macrophages by inducing ASC dimerization (Fernandes-Alnemri et al., 2007Fernandes-Alnemri T. Wu J. Yu J.-W. Datta P. Miller B. Jankowski W. Rosenberg S. Zhang J. Alnemri E.S. The pyroptosome: a supramolecular assembly of ASC dimers mediating inflammatory cell death via caspase-1 activation.Cell Death Differ. 2007; 14: 1590-1604Crossref PubMed Scopus (717) Google Scholar). ASC pyroptosome assembly can be observed in live cells using a THP-1 cell line (THP-1-ASC-GFP cells) that stably expresses an ASC-GFP fusion protein (see Movies S1 and S2 in the Supplemental Data available with this article online). Considering that ASC is also important for PSTPIP1-induced caspase-1 activation, we examined the effect of retrovirus-mediated ectopic expression of the A230T PSTPIP1 mutant on ASC-GFP in the THP-1-ASC-GFP cells. As shown in Figure 3A, infection with an empty retroviral vector induced a small amount of pyroptosome formation in these cells. In contrast, infection with a retrovirus encoding the A230T PSTPIP1 mutant induced substantially more ASC pyroptosome formation compared to the empty vector control. These results indicate that PSTPIP1 induces caspase-1 activation by triggering the formation of the ASC pyroptosome in THP-1 monocytes. To reconstitute the signaling pathway that leads to the formation of the ASC pyroptosome in response to PSTPIP1, we used a 293T-based ASC pyroptosome assembly assay similar to the THP-1-based assay. As shown in Figures 3B and 3C, expression of the WT PSTPIP1 or the PAPA-associated PSTPIP1 mutants with pyrin in a stable 293-ASC-EGFP-N1 cell line, which expresses an ASC-GFP fusion protein, induced substantially more ASC pyroptosome formation than expression of pyrin alone. Consistent with the caspase-1 activation results (Figure 2), the PSTPIP1 mutants induced more ASC pyroptosomes than WT PSTPIP1 in these cells (Figures 3B and 3C). No significant change in ASC pyroptosome formation was observed when PSTPIP1 proteins were expressed without pyrin, or when coexpressed with cryopyrin, indicating that pyrin is specifically required for PSTPIP1-induced ASC pyroptosome formation (Figure 3B). To provide additional evidence on the critical role of pyrin in PSTPIP1-induced ASC pyroptosome formation, we examined the effect of ectopic expression of WT or mutant PSTPIP1 proteins on ASC pyroptosome formation in an HEK293 cell line that expresses caspase-1, pyrin, and an ASC-GFP fusion protein (designated 293-C1P-ASC-EGFP-N1 cells) and a control HEK293 cell line that expresses only caspase-1 and ASC-GFP without pyrin (designated 293-C1-ASC-EGFP-N1 cells). As in the 293-ASC-EGFP-N1 cells, the ASC-GFP in the 293-C1P-ASC-EGFP-N1 cells was evenly distributed in the entire cytoplasm and nucleus, indicating that stable coexpression of pyrin and caspase-1 together with ASC-GFP does not affect its distribution. Consistent with the above results, the PAPA-associated PSTPIP1 mutants induced dramatic ASC pyroptosome formation in the pyrin-expressing 293-C1P-ASC-EGFP-N1 cells (Figure 3D, right panels). In contrast, these PSTPIP1 mutants were not able to induce ASC pyroptosome formation in the control HEK293-C1-ASC-EGFP-N1 cells, which do not express pyrin (Figure 3D, left panels). These results underscore the essential role of pyrin in mutant PSTPIP1-induced ASC oligomerization. Our results also suggest that the engagement of pyrin by the mutant PSTPIP1 proteins generates the molecular signal necessary for ASC oligomerization. To determine how engagement of pyrin by mutant PSTPIP1 induces more ASC oligomerization, we measured the interaction of pyrin with ASC in the presence or absence of PSTPIP1. As shown in Figure 3E, the interaction between pyrin and ASC was enhanced by coexpression of pyrin with WT PSTPIP1 and further enhanced by coexpression with the disease-associated PSTPIP1 mutants. These results indicate that PSTPIP1 induces ASC oligomerization by increasing the interaction of pyrin with ASC. The ability of pyrin to induce ASC oligomerization suggests that pyrin itself is an oligomer or it oligomerizes before it engages ASC. To examine the first possibility, we performed chemical crosslinking analyses with ethylene glycol bis (succinimidylsuccinate) (EGS) to determine the oligomeric state of full-length pyrin. As shown in Figure 4A, treatment of full-length pyrin from pyrin-transfected 293T cells or THP-1 cells, or purified from bacteria with low concentrations of EGS, produced a major crosslinked species with apparent molecular mass of ∼300 kDa. Because monomeric pyrin has an apparent molecular mass of ∼100 kDa in SDS-PAGE, this indicates that native pyrin is a homotrimer. These results were confirmed by gel filtration on Superdex 200, which also revealed that the native form of pyrin is indeed a homotrimer (Figure S1E). Human pyrin contains four distinct domains: the N-terminal PYD (residues 1–92) followed by the B-box (BB) domain (residues 370–412), the coiled-coil (CC) domain (residues 420–582), and the SPRY domain (residues 597–781) (Figure 4B and Figure S2). Between the PYD and B-box, pyrin contains a 278 amino acid-long linker region with no homology to any known domains. The PYD of pyrin is required for pyrin-induced ASC oligomerization because PYD mutations that abolish its interaction with ASC or deletion of the PYD of pyrin inhibit pyrin-induced ASC oligomerization (Yu et al., 2006Yu J.W. Wu J. Zhang Z. Datta P. Ibrahimi I. Taniguchi S. Sagara J. Fernandes-Alnemri T. Alnemri E.S. Cryopyrin and pyrin activate caspase-1, but not NF-kappaB, via ASC oligomerization.Cell Death Differ. 2006; 13: 236-249Crossref PubMed Scopus (282) Google Scholar and data not shown). To identify the exact region in pyrin that mediates its homotrimerization, we first determined the oligomeric state of a truncated pyrin mutant lacking the SPRY domain (pyrin-1–580). As shown in Figure 4C, left panel, treatment of pyrin-1–580, which has an apparent molecular mass of ∼75 kDa in SDS-PAGE, with low concentrations of EGS yielded a major crosslinked species with apparent molecular mass of ∼220 kDa corresponding to a trimeric form of pyrin. This indicates that deletion of the SPRY domain of pyrin does not affect the trimeric state of pyrin. Next, we deleted the N-terminal PYD and the C-terminal SPRY domain and determined the oligomeric state of this truncated pyrin mutant (designated pyrin-LN-BB-CC) (Figure 4B). Treatment of this truncated pyrin mutant with low concentrations of EGS also yielded a distinct trimeric species (Figure 4C, middle panel, 2nd and 3rd lanes). This indicates that the remaining linker region, B-box, or coiled-coil domain mediates pyrin homotrimerization. To test this possibility, we generated two additional truncated pyrin mutants: one containing the linker region and the B-box (pyrin-LN-BB), and the other containing only the linker region (pyrin-LN). In contrast to the pyrin-LN-BB-CC, the pyrin-LN-BB and pyrin-LN mutants, which lack the coiled-coil domain, were no longer able to form any higher molecular mass crosslinked species and migrated in SDS-PAGE as monomers (Figure 4C, middle panel, 4th to 6th lanes; right panel, 1st to 3rd lanes). Collectively, these results indicate that pyrin is a homotrimer and that its trimeric state is maintained by self-association of its coiled-coil domain. To determine whether homotrimerization of pyrin is critical for its activity, we examined the effect of different deletions that remove the SPRY domain, SPRY plus coiled-coil domains, or SPRY, coiled-coil plus B-box domains (Figure 4D) on the ability of pyrin to induce ASC pyroptosome formation and caspase-1 activation in the 293-ASC-EGFP-N1 and 293-casp1-ASC cells, respectively. As shown in Figures 4E and 4F, deletion of the SPRY domain of pyrin did not affect the basal or PSTPIP1-induced activities of pyrin, as similar amounts of ASC pyroptosome formation and caspase-1 activation were observed with pyrin-1–580 compared with the full-length protein. In contrast, further deletion of the coiled-coil or the coiled-coil plus B-box domains impaired both the basal and PSTPIP1-induced activities of pyrin. These results indicate that the coiled-coil domain of pyrin is critical for its basal and PSTPIP1-induced activities, whereas the SPRY domain is not essential for either activity. Considering that deletion of the coiled-coil domain inactivates pyrin by abrogating its homotrimerization, we asked whether restoring homotrimerization with a homologous domain from the related family member Trim5α (Javanbakht et al., 2006Javanbakht H. Yuan W. Yeung D.F. Song B. Di" @default.
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