A ubiquitin-independent proteasome pathway controls activation of the CARD8 inflammasome

Abstract

CARD8 is a pattern-recognition receptor that forms a caspase-1-activating inflammasome. CARD8 undergoes constitutive autoproteolysis, generating an N-terminal (NT) fragment with a disordered region and a ZU5 domain and a C-terminal (CT) fragment with UPA and CARD domains. Dipeptidyl peptidase 8 and dipeptidyl peptidase 9 inhibitors, including Val-boroPro, accelerate the degradation of the NT fragment via a poorly characterized proteasome-mediated pathway, thereby releasing the inflammatory CT fragment from autoinhibition. Here, we show that the core 20S proteasome, which degrades disordered and misfolded proteins independent of ubiquitin modification, controls activation of the CARD8 inflammasome. In unstressed cells, we discovered that the 20S proteasome degrades just the NT disordered region, leaving behind the folded ZU5, UPA, and CARD domains to act as an inhibitor of inflammasome assembly. However, in Val-boroPro–stressed cells, we show the 20S proteasome degrades the entire NT fragment, perhaps due to ZU5 domain unfolding, freeing the CT fragment from autoinhibition. Taken together, these results show that the susceptibility of the CARD8 NT domain to 20S proteasome-mediated degradation controls inflammasome activation. CARD8 is a pattern-recognition receptor that forms a caspase-1-activating inflammasome. CARD8 undergoes constitutive autoproteolysis, generating an N-terminal (NT) fragment with a disordered region and a ZU5 domain and a C-terminal (CT) fragment with UPA and CARD domains. Dipeptidyl peptidase 8 and dipeptidyl peptidase 9 inhibitors, including Val-boroPro, accelerate the degradation of the NT fragment via a poorly characterized proteasome-mediated pathway, thereby releasing the inflammatory CT fragment from autoinhibition. Here, we show that the core 20S proteasome, which degrades disordered and misfolded proteins independent of ubiquitin modification, controls activation of the CARD8 inflammasome. In unstressed cells, we discovered that the 20S proteasome degrades just the NT disordered region, leaving behind the folded ZU5, UPA, and CARD domains to act as an inhibitor of inflammasome assembly. However, in Val-boroPro–stressed cells, we show the 20S proteasome degrades the entire NT fragment, perhaps due to ZU5 domain unfolding, freeing the CT fragment from autoinhibition. Taken together, these results show that the susceptibility of the CARD8 NT domain to 20S proteasome-mediated degradation controls inflammasome activation. Several intracellular danger-associated signals induce the assembly of multiprotein complexes called inflammasomes (1Broz P. Dixit V.M. Inflammasomes: mechanism of assembly, regulation and signalling.Nat. Rev. Immunol. 2016; 16: 407-420Crossref PubMed Scopus (2091) Google Scholar, 2Rathinam V.A. Fitzgerald K.A. Inflammasome complexes: emerging mechanisms and effector functions.Cell. 2016; 165: 792-800Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar). The typical process of inflammasome formation involves a pattern recognition receptor (PRR) protein detecting a specific danger signal, self-oligomerizing, and then recruiting (directly or indirectly via the adapter protein ASC) the cysteine protease caspase-1 (CASP1). CASP1 undergoes proximity-induced autoproteolysis on this platform, generating an active enzyme that cleaves and activates gasdermin D (GSDMD) and, in most cases, interleukin-1β (IL-1β) and IL-18. The N-terminal fragment of cleaved GSDMD (GSDMDp30) forms pores in the cell membrane, releasing the activated cytokines and triggering pyroptotic cell death. CARD8 is a human PRR that forms an inflammasome (3Johnson D.C. Taabazuing C.Y. Okondo M.C. Chui A.J. Rao S.D. Brown F.C. et al.DPP8/DPP9 inhibitor-induced pyroptosis for treatment of acute myeloid leukemia.Nat. Med. 2018; 24: 1151-1156Crossref PubMed Scopus (218) Google Scholar). CARD8 has an N-terminal unstructured region consisting of ∼160 amino acids followed by a function-to-find domain and a caspase activation and recruitment domain (CARD) (Fig. 1A). The function-to-find domain undergoes autoproteolysis between its ZU5 (found in ZO-1 and UNC5) and UPA (conserved in UNC5, PIDD, and ankyrins) subdomains, creating N-terminal (NT) and C-terminal (CT) fragments that remain noncovalently associated (4D'Osualdo A. Weichenberger C.X. Wagner R.N. Godzik A. Wooley J. Reed J.C. CARD8 and NLRP1 undergo autoproteolytic processing through a ZU5-like domain.PLoS One. 2011; 6e27396Crossref PubMed Scopus (141) Google Scholar). The proteasome-mediated degradation of the NT fragment releases the CT fragment from autoinhibition, but the CT fragment is then captured and restrained as part of a ternary complex with one copy of full-length CARD8 and one copy of dipeptidyl peptidase 8 or 9 (DPP8/9) (5Sharif H. Hollingsworth L.R. Griswold A.R. Hsiao J.C. Wang Q. Bachovchin D.A. et al.Dipeptidyl peptidase 9 sets a threshold for CARD8 inflammasome formation by sequestering its active C-terminal fragment.Immunity. 2021; 54: 1392-1404.e10Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Stimuli that accelerate CARD8NT degradation and/or disrupt the DPP8/9-CARD8 complex enable the CARD8CT to overcome these repressive mechanisms and to self-oligomerize, recruit CASP1, and trigger pyroptosis. Two distinct danger signals have been reported to accelerate the proteasome-mediated degradation of the CARD8NT fragment. First, HIV-1 protease directly cleaves within the NT region of the CARD8FL protein, generating an unstable neo-N-terminus that is rapidly degraded by the N-end rule proteasome pathway (6Wang Q. Gao H. Clark K.M. Mugisha C.S. Davis K. Tang J.P. et al.CARD8 is an inflammasome sensor for HIV-1 protease activity.Science. 2021; 371eabe1707Crossref Scopus (61) Google Scholar). Second, DPP8/9 inhibitors, including Val-boroPro (VbP), accelerate the degradation of many disordered and misfolded proteins, including the CARD8NT (7Chui A.J. Okondo M.C. Rao S.D. Gai K. Griswold A.R. Johnson D.C. et al.N-terminal degradation activates the NLRP1B inflammasome.Science. 2019; 364: 82-85Crossref PubMed Scopus (187) Google Scholar, 8Chui A.J. Griswold A.R. Taabazuing C.Y. Orth E.L. Gai K. Rao S.D. et al.Activation of the CARD8 inflammasome requires a disordered region.Cell Rep. 2020; 33: 108264Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Notably, DPP8/9 inhibitors also destabilize the repressive DPP8/9-CARD8 ternary complex (5Sharif H. Hollingsworth L.R. Griswold A.R. Hsiao J.C. Wang Q. Bachovchin D.A. et al.Dipeptidyl peptidase 9 sets a threshold for CARD8 inflammasome formation by sequestering its active C-terminal fragment.Immunity. 2021; 54: 1392-1404.e10Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) and thereby activate the CARD8 inflammasome via two separate mechanisms. The molecular details of the homeostatic and DPP8/9-inhibition induced CARD8 degradation pathways have not been established. Intracellular proteins are often degraded by the ubiquitin-proteasome system, which involves the covalent attachment of ubiquitin to lysine residues on target proteins that mediate their recruitment to the 26S proteasome. The 26S proteasome consists of the proteolytic core 20S subunit capped at one or both ends by 19S regulatory complexes (9Voges D. Zwickl P. Baumeister W. The 26S proteasome: a molecular machine designed for controlled proteolysis.Annu. Rev. Biochem. 1999; 68: 1015-1068Crossref PubMed Scopus (1625) Google Scholar). The 19S regulatory particles recognize, deubiquitinate, and unfold target proteins, enabling their translocation into the 20S core particle for hydrolysis. In a preliminary attempt to identify sites of ubiquitination on CARD8, we previously mutated all 10 lysines within the NT fragment of CARD8FL to arginines (CARD8FL K10R). We found that CARD8FL K10R was largely, but not completely, insensitive to VbP in a reconstituted HEK 293T cell system expressing CASP1 and GSDMD (HEK 293TCASP1+GSDMD cells), suggesting that ubiquitination of the NT fragment might be important for VbP-induced degradation (8Chui A.J. Griswold A.R. Taabazuing C.Y. Orth E.L. Gai K. Rao S.D. et al.Activation of the CARD8 inflammasome requires a disordered region.Cell Rep. 2020; 33: 108264Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). However, the CARD8FL K10R protein expressed at lower levels and underwent less autoproteolysis than the CARD8FL WT protein, and these deficiencies could also account for its reduced pyroptotic activity. Here, we further investigated the molecular details of CARD8 degradation. We found that the core 20S proteasome, which degrades misfolded proteins independent of the 19S regulatory complex and ubiquitination, regulates CARD8 activation. In unstressed cells, the 20S proteasome removes the disordered region of CARD8, leaving behind the folded ZU5-UPA-CARD (ZUC) domains. This protein fragment cannot form an inflammasome but can still sequester CT fragments in the DPP8/9 ternary complex and thereby act as an inflammasome inhibitor. In VbP-stressed cells, the 20S proteasome degrades CARD8’s entire NT fragment, including the ZU5 domain, releasing the inflammatory CT fragment from autoinhibition. Collectively, these findings suggest that the propensity of the ZU5 domain to enter the 20S proteasome is a critical regulatory step that governs the activation of the CARD8 inflammasome. Before further studying VbP-induced CARD8NT degradation, we first wanted to investigate how CARD8 is processed in unstressed cells. Intriguingly, we and others have consistently observed that endogenous CARD8 in human monocytes appears as three distinct at and in CARD8CT (Fig. (3Johnson D.C. Taabazuing C.Y. Okondo M.C. Chui A.J. Rao S.D. Brown F.C. et al.DPP8/DPP9 inhibitor-induced pyroptosis for treatment of acute myeloid leukemia.Nat. Med. 2018; 24: 1151-1156Crossref PubMed Scopus (218) Google Scholar, A. Weichenberger C.X. Wagner R.N. Godzik A. Wooley J. Reed J.C. CARD8 and NLRP1 undergo autoproteolytic processing through a ZU5-like domain.PLoS One. 2011; 6e27396Crossref PubMed Scopus (141) Google Scholar, K. et NLRP1 inflammasome and via both peptidase and domain 2018; Full Text Full Text PDF PubMed Scopus Google Scholar). a of the CARD8FL undergoes autoproteolysis (4D'Osualdo A. Weichenberger C.X. Wagner R.N. Godzik A. Wooley J. Reed J.C. CARD8 and NLRP1 undergo autoproteolytic processing through a ZU5-like domain.PLoS One. 2011; 6e27396Crossref PubMed Scopus (141) Google Scholar, D.C. Okondo M.C. Orth E.L. Rao S.D. et inhibitors activate the CARD8 inflammasome in 2020; PubMed Scopus Google Scholar, A. et al.CARD8 inflammasome activation pyroptosis in human J. 2020; PubMed Scopus Google and the at and to CARD8FL and The of the at which we is is to be a as the of the the CARD8 in HEK 293T also (Fig. Notably, of the CARD8FL protein (CARD8FL (Fig. that formation of this not on We that to CARD8FL protein that was to its ZU5 domain than the ZU5-UPA-CARD domains (Fig. and and was not with CARD8NT (Fig. degradation that the N-terminal of was (Fig. and a of CARD8FL is cleaved residues the of the ZU5 domain to However, be that in we observed CARD8 fragments than suggesting that processing not at this (Fig. In also appears to further be processed into a fragment, but the and of this was not further (Fig. We previously the of CARD8 proteins to mediate VbP-induced pyroptosis in HEK 293TCASP1+GSDMD (8Chui A.J. Griswold A.R. Taabazuing C.Y. Orth E.L. Gai K. Rao S.D. et al.Activation of the CARD8 inflammasome requires a disordered region.Cell Rep. 2020; 33: 108264Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). In this we discovered a CARD8 at but not was of pyroptosis. Notably, the that at and this that cannot form an inflammasome. We wanted to this in cells, which the CARD8 inflammasome pathway and are (3Johnson D.C. Taabazuing C.Y. Okondo M.C. Chui A.J. Rao S.D. Brown F.C. et al.DPP8/DPP9 inhibitor-induced pyroptosis for treatment of acute myeloid leukemia.Nat. Med. 2018; 24: 1151-1156Crossref PubMed Scopus (218) Google Scholar). we expressed CARD8FL CARD8FL or in the with or VbP (Fig. VbP induced pyroptosis in expressing CARD8FL but not CARD8FL or The inhibitor with VbP to induce pyroptosis (7Chui A.J. Okondo M.C. Rao S.D. Gai K. Griswold A.R. Johnson D.C. et al.N-terminal degradation activates the NLRP1B inflammasome.Science. 2019; 364: 82-85Crossref PubMed Scopus (187) Google but the of VbP and still to activate (Fig. Collectively, these show that cannot form an inflammasome. The can the CARD8FL in the ternary complex and can and a CARD8CT fragment (5Sharif H. Hollingsworth L.R. Griswold A.R. Hsiao J.C. Wang Q. Bachovchin D.A. et al.Dipeptidyl peptidase 9 sets a threshold for CARD8 inflammasome formation by sequestering its active C-terminal fragment.Immunity. 2021; 54: 1392-1404.e10Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). we that which is than might as an inhibitor of inflammasome this we expressed CARD8FL with an N-terminal degradation in HEK 293TCASP1+GSDMD (Fig. (5Sharif H. Hollingsworth L.R. Griswold A.R. Hsiao J.C. Wang Q. Bachovchin D.A. et al.Dipeptidyl peptidase 9 sets a threshold for CARD8 inflammasome formation by sequestering its active C-terminal fragment.Immunity. 2021; 54: 1392-1404.e10Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, J. A. et system for and protein 2018; PubMed Scopus Google Scholar). The the degradation of proteins to and treatment of these with induced the of CARD8CT and pyroptosis. with the of in these pyroptosis degradation. these results suggest that an endogenous protease removes CARD8’s disordered region and an form of CARD8 that inflammasome activation. We wanted to the and for the of CARD8FL into We previously the protein in which the disordered region of CARD8 was by the disordered region to of (8Chui A.J. Griswold A.R. Taabazuing C.Y. Orth E.L. Gai K. Rao S.D. et al.Activation of the CARD8 inflammasome requires a disordered region.Cell Rep. 2020; 33: 108264Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Intriguingly, the of this protein in HEK 293T still a fragment (Fig. The disordered of and CARD8 not (Fig. that the protease not and a specific amino the the we the disordered region of CARD8 on the of we found that that the disordered region was to a protein with a molecular to (Fig. In we found that a to the of CARD8FL or not with (Fig. and that the protease has can disordered between In of the entire CARD8 disordered region with (Fig. Notably, an to for the in which is and but not from the protein (Fig. to be that the protease the disordered region from these proteins to fragments to the domains but that fragments with disordered region often observed as (Fig. and these that the protease removes disordered in a independent including disordered between two domains. Intriguingly, the proteolytic 20S core of the proteasome, which often of the 19S in cells, degrades unstructured of amino including between domains of the PubMed Scopus Google Scholar, can a of proteins independent of PubMed Scopus Google Scholar, J. is not for the degradation of proteins by Full Text Full Text PDF PubMed Scopus Google Scholar). we that the 20S proteasome the disordered from CARD8 and the this process is also account for the molecular fragments that we observed in the fragments to proteins that not this we HEK 293T with a CARD8FL with and with of the proteasome inhibitor and for by (Fig. We observed that the of that the proteasome was for this The 26S proteasome requires the covalent attachment of ubiquitin to proteins their degradation, the 20S proteasome directly degrades misfolded proteins ubiquitination of the PubMed Scopus Google Scholar, can a of proteins independent of PubMed Scopus Google Scholar, J. is not for the degradation of proteins by Full Text Full Text PDF PubMed Scopus Google Scholar). formation involves lysine ubiquitination, we mutated all lysines to arginines within the NT fragment (CARD8FL or the entire protein (CARD8FL be that the CARD8FL protein is of including its C-terminal and We observed that both proteins still fragments in HEK 293T and that still the formation of these (Fig. and results suggest that the 20S proteasome removes the disordered region of CARD8 through a proteasome might indirectly formation in cells, we to that the 20S proteasome directly removes CARD8’s disordered region to in We a CARD8FL protein from HEK 293T and then this protein with 20S We found that the 20S proteasome degraded CARD8FL into but that the and CARD8CT from degradation (Fig. be that the 20S proteasome a the as the one from constitutive processing by endogenous that the 20S proteasome in cells. In the 20S proteasome also CARD8 in this with the that degrades CARD8 in an to the proteasome inhibitors and the in of (Fig. and we found that the was to 20S proteasome-mediated degradation for at (Fig. the 20S proteasome has previously been to disordered but domains of the PubMed Scopus Google Scholar, can a of proteins independent of PubMed Scopus Google Scholar). the 20S proteasome could process the proteins we of these proteins from HEK 293T and with 20S we found that the 20S proteasome the CARD8 disordered region to the of (Fig. as as the disordered region to the of (Fig. as the 20S proteasome generating a was to the of CARD8FL (Fig. In a protein that the disordered region, was to 20S degradation (Fig. 20S processed the CARD8FL K10R and proteins into in with the of the 20S to lysines or ubiquitination (Fig. the 20S proteasome not to of from CARD8FL in and and suggesting that degradation through the ZU5 domain in VbP not induce ubiquitination of CARD8 by (Fig. (3Johnson D.C. Taabazuing C.Y. Okondo M.C. Chui A.J. Rao S.D. Brown F.C. et al.DPP8/DPP9 inhibitor-induced pyroptosis for treatment of acute myeloid leukemia.Nat. Med. 2018; 24: 1151-1156Crossref PubMed Scopus (218) Google Scholar, H. Hollingsworth L.R. Griswold A.R. Hsiao J.C. Wang Q. Bachovchin D.A. et al.Dipeptidyl peptidase 9 sets a threshold for CARD8 inflammasome formation by sequestering its active C-terminal fragment.Immunity. 2021; 54: 1392-1404.e10Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, A.J. Okondo M.C. Rao S.D. Gai K. Griswold A.R. Johnson D.C. et al.N-terminal degradation activates the NLRP1B inflammasome.Science. 2019; 364: 82-85Crossref PubMed Scopus (187) Google Scholar, 8Chui A.J. Griswold A.R. Taabazuing C.Y. Orth E.L. Gai K. Rao S.D. et al.Activation of the CARD8 inflammasome requires a disordered region.Cell Rep. 2020; 33: 108264Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, K. et NLRP1 inflammasome and via both peptidase and domain 2018; Full Text Full Text PDF PubMed Scopus Google Scholar). We wanted to the 20S proteasome also VbP-induced degradation of the entire CARD8NT fragment, the process that releases the pyroptotic CARD8CT fragment from autoinhibition. further this we investigated the of CARD8FL K10R to pyroptosis. we previously found that CARD8FL K10R was in VbP-induced pyroptosis in the reconstituted HEK 293TCASP1+GSDMD system (8Chui A.J. Griswold A.R. Taabazuing C.Y. Orth E.L. Gai K. Rao S.D. et al.Activation of the CARD8 inflammasome requires a disordered region.Cell Rep. 2020; 33: 108264Abstract Full Text Full Text PDF PubMed Scopus (24) Google but this was perhaps due to autoproteolysis or Here, we expressed CARD8FL K10R in the cells. We observed that and the of VbP and induced as by and GSDMD in expressing CARD8FL K10R (Fig. we found that caspase-1 inhibitor CARD8FL that the CARD8NT fragment was degraded by the proteasome. the CARD8FL the fragment of CARD8FL K10R In that VbP the ubiquitination of the CT fragment of the CARD8FL K10R protein and that this ubiquitination is to recruit CARD8 to the 26S proteasome and trigger its degradation in an to this we investigated the of the CARD8FL protein to mediate pyroptosis. We found that VbP the of VbP and induced pyroptosis in expressing CARD8FL (Fig. and However, this protein was for at two First, CARD8FL was in autoproteolytic (Fig. and and of in CARD8FL WT and are and Second, the CT fragment of this lysines mutated to was in its to into a as of this CT fragment to the WT CT fragment was to induce GSDMD in HEK 293TCASP1+GSDMD (Fig. these CARD8FL still to and as an inflammasome inhibitor in the ternary complex (Fig. and Collectively, these results suggest that the of CARD8FL to mediate pyroptosis is not due to a of ubiquitination this protein is for is not to that VbP-induced NT degradation is independent of lysine We previously discovered that the enzyme inhibitor A.J. et inhibitor of enzyme as a to PubMed Scopus Google Scholar) VbP-induced CARD8 activation (8Chui A.J. Griswold A.R. Taabazuing C.Y. Orth E.L. Gai K. Rao S.D. et al.Activation of the CARD8 inflammasome requires a disordered region.Cell Rep. 2020; 33: 108264Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). for their and we that a might Notably, the of is the that that CARD8’s degradation requires Here, we found that VbP-induced pyroptosis in expressing CARD8FL K10R (Fig. this protein has lysines on its we that treatment not CARD8 inflammasome activation by directly CARD8 we that its be due to an of 20S proteasome activity. the ubiquitination of many proteins that be (Fig. to the of proteins that and thereby with CARD8 degradation. this we found that reduced the formation of that indirectly with 20S proteasome in (Fig. that CARD8FL K10R is with the of VbP-induced ubiquitination, that the 20S proteasome VbP-induced pyroptosis independent of the ubiquitin-proteasome The VbP-induced proteasome pathway that degrades CARD8 has not been established. We previously that VbP activates that the CARD8NT fragment, thereby to the 26S proteasome for (8Chui A.J. Griswold A.R. Taabazuing C.Y. Orth E.L. Gai K. Rao S.D. et al.Activation of the CARD8 inflammasome requires a disordered region.Cell Rep. 2020; 33: 108264Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). However, that the ubiquitin-proteasome system is in this degradation process has Here, we show that the core 20S proteasome, which or misfolded proteins independent of ubiquitin of the PubMed Scopus Google Scholar, can a of proteins independent of PubMed Scopus Google controls CARD8 inflammasome activation. However, we that degradation in might also be in CARD8 proteasome complexes in to the 20S core proteasome can proteins independent of ubiquitin the 20S proteasome degradation PubMed Scopus Google Scholar, degradation of proteins by the PubMed Scopus Google and we that these particles might be in CARD8 activation. we that CARD8FL K10R the to mediate pyroptotic cell is that the NT fragment is on a than lysine A. mechanisms and J. Biochem. PubMed Scopus Google Scholar). as is that CARD8 CT ubiquitination is for activation. are to rule these mechanisms. for the regulation of CARD8 by the 20S proteasome is in In unstressed cells, the CARD8 ZU5, UPA, and CARD domains but the N-terminal disordered region The 20S in these this disordered However, the ZU5 domain is to enter the 20S and the 20S proteasome the fragment. cannot form an inflammasome but can sequester CT fragments in the DPP8/9 ternary further unstressed CARD8 inflammasome activation. In or VbP cells), the ZU5 domain is with the disordered region, to the of the inflammatory CARD8CT fragment. The molecular mechanisms that accelerate the degradation of the ZU5 domain are but we that involves the of the ZU5 domain and/or of the 20S proteasome that activate the 20S proteasome by protein and reduced protein Med. PubMed Scopus Google Scholar). that disordered the of domains J. 1999; PubMed Scopus Google and we that CARD8’s disordered region, which is for inflammasome activation (8Chui A.J. Griswold A.R. Taabazuing C.Y. Orth E.L. Gai K. Rao S.D. et al.Activation of the CARD8 inflammasome requires a disordered region.Cell Rep. 2020; 33: 108264Abstract Full Text Full Text PDF PubMed Scopus (24) Google might a in the of the ZU5 NLRP1 is an PRR that is to CARD8 C.Y. Griswold A.R. Bachovchin D.A. The NLRP1 and CARD8 Rev. 2020; PubMed Scopus Google Scholar). NLRP1 and CARD8 a ZU5-UPA-CARD region, but NLRP1 has N-terminal and domains of a disordered We have that NLRP1 and CARD8 both the in cell (8Chui A.J. Griswold A.R. Taabazuing C.Y. Orth E.L. Gai K. Rao S.D. et al.Activation of the CARD8 inflammasome requires a disordered region.Cell Rep. 2020; 33: 108264Abstract Full Text Full Text PDF PubMed Scopus (24) Google but that which a inflammatory than CARD8 Taabazuing C.Y. Griswold A.R. Orth E.L. Rao S.D. et is for 2020; PubMed Scopus Google its NT domains to further its activation Wang Wang Q. Griswold A.R. Rao S.D. et the NLRP1 2021; Scopus Google Scholar). on findings we that the NLRP1 NT domains in the of NT fragment by the 20S proteasome. The between the 20S proteasome and NLRP1 further the of the CARD8 inflammasome has not been established. The core 20S proteasome rapidly misfolded and disordered proteins but proteins and thereby a critical in findings that the 20S proteasome controls CARD8 that this inflammasome sensor its to and are to this between protein and is by the GSDMD CARD8CT CARD8NT 20S and for and Val-boroPro and protease inhibitor 20S are from and and from and from HEK 293T in with and and in with and at in a for the for full-length and CARD8 as previously (8Chui A.J. Griswold A.R. Taabazuing C.Y. Orth E.L. Gai K. Rao S.D. et al.Activation of the CARD8 inflammasome requires a disordered region.Cell Rep. 2020; 33: 108264Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). constitutive CARD8 into that have been to N-terminal or or C-terminal of CARD8 was with a that to within CARD8 the the the of the was as the to in CARD8FL that has been with a followed by a and two The of the was by and into the the of an assembly was to CARD8’s to the of the was The of CARD8 lysine CARD8 K10R and by and into the or The CARD8 K10R have all 10 lysines to the CARD8 autoproteolysis mutated to arginines and for the CARD8 the of the lysines that CARD8 lysine and their controls a C-terminal followed by an and two to the the C-terminal CARD8’s not for HEK 293T in at in The the to a of as the in and to the of was constitutive the for an their the with at with The for an their CARD8 in cells, expressing (3Johnson D.C. Taabazuing C.Y. Okondo M.C. Chui A.J. Rao S.D. Brown F.C. et al.DPP8/DPP9 inhibitor-induced pyroptosis for treatment of acute myeloid leukemia.Nat. Med. 2018; 24: 1151-1156Crossref PubMed Scopus (218) Google Scholar) with in HEK 293T and of the and with cell by and by CARD8FL WT into in HEK 293T as be that all CARD8 into have at residues and by the of then with the and with HEK 293T and as in at and with as for levels the levels to a in of a cell and at for The and with of for at or for at with three of of proteins by with of or of for at of was to the and at for 10 with two of in that with protease by and by at for 10 and the The by and the HEK 293T with of the the with or with the to for the expressed The is of 10 and In the with the three with or of 20S In the to a step the protein with protein CARD8 was then with 20S 20S at for at with to HEK 293T with a CARD8FL WT the with the was with and and the protein was by The was then a and with The of and to the Davis for in and and D and less than and of a of three or independent The not in all was in this are within the and/or from the on This The that have of with the of this We the Davis for the D. A. D. A. J. A. A. J. A. H. D. P. Q. and H. D. A. and J. H. This was by the and to D. A. to and to A. to the A. and the for and The for of A. and the A. The is the of the and not the of the of

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