共查询到20条相似文献,搜索用时 15 毫秒
1.
Parimala R. Vajjhala Sebastian Kaiser Sarah J. Smith Qi-Rui Ong Stephanie L. Soh Katryn J. Stacey Justine M. Hill 《The Journal of biological chemistry》2014,289(34):23504-23519
Inflammasomes are macromolecular complexes that mediate inflammatory and cell death responses to pathogens and cellular stress signals. Dysregulated inflammasome activation is associated with autoinflammatory syndromes and several common diseases. During inflammasome assembly, oligomerized cytosolic pattern recognition receptors recruit procaspase-1 and procaspase-8 via the adaptor protein ASC. Inflammasome assembly is mediated by pyrin domains (PYDs) and caspase recruitment domains, which are protein interaction domains of the death fold superfamily. However, the molecular details of their interactions are poorly understood. We have studied the interaction between ASC and pyrin PYDs that mediates ASC recruitment to the pyrin inflammasome, which is implicated in the pathogenesis of familial Mediterranean fever. We demonstrate that both the ASC and pyrin PYDs have multifaceted binding modes, involving three sites on pyrin PYD and two sites on ASC PYD. Molecular docking of pyrin-ASC PYD complexes showed that pyrin PYD can simultaneously interact with up to three ASC PYDs. Furthermore, ASC PYD can self-associate and interact with pyrin, consistent with previous reports that pyrin promotes ASC clustering to form a proinflammatory complex. Finally, the effects of familial Mediterranean fever-associated mutations, R42W and A89T, on structural and functional properties of pyrin PYD were investigated. The R42W mutation had a significant effect on structure and increased stability. Although the R42W mutant exhibited reduced interaction with ASC, it also bound less to the pyrin B-box domain responsible for autoinhibition and hence may be constitutively active. Our data give new insights into the binding modes of PYDs and inflammasome architecture. 相似文献
2.
Masumoto J Zhou W Chen FF Su F Kuwada JY Hidaka E Katsuyama T Sagara J Taniguchi S Ngo-Hazelett P Postlethwait JH Núñez G Inohara N 《The Journal of biological chemistry》2003,278(6):4268-4276
The pyrin domain was identified recently in multiple proteins that are associated with apoptosis and/or inflammation, but the physiological and molecular function of these proteins remain poorly understood. We have identified Caspy and Caspy2, two zebrafish caspases containing N-terminal pyrin domains. Expression of Caspy and Caspy2 induced apoptosis in mammalian cells that were inhibited by general caspase inhibitors. Biochemical analysis revealed that both Caspy and Caspy2 are active caspases, but they exhibit different substrate specificity. Caspy, but not Caspy2, interacted with the zebrafish orthologue of ASC (zAsc), a pyrin- and caspase recruitment domain-containing protein identified previously in mammals. The pyrin domains of both Caspy and zAsc were required for their interaction. Furthermore, zAsc and Caspy co-localized to the "speck" when co-transfected into mammalian cells. Enforced oligomerization of zAsc, but not simple interaction with zAsc, induced specific proteolytic activation of Caspy and enhanced Caspy-dependent apoptosis. Injection of zebrafish embryos with a morpholino antisense oligonucleotide corresponding to caspy resulted in an "open mouth" phenotype associated with defective formation of the cartilaginous pharyngeal skeleton. These studies suggest that zAsc mediates the activation of Caspy, a caspase that plays an important role in the morphogenesis of the jaw and gill-bearing arches. 相似文献
3.
J Masumoto S Taniguchi J Nakayama M Shiohara E Hidaka T Katsuyama S Murase J Sagara 《The journal of histochemistry and cytochemistry》2001,49(10):1269-1275
Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is a pyrin N-terminal homology domain (PYD)- and caspase recruitment domain (CARD)-containing a proapoptotic molecule. This molecule has also been identified as a target of methylation-induced silencing (TMS)-1. We cloned the ASC cDNA by immunoscreening using an anti-ASC monoclonal antibody. In this study, we determined the binding site of the anti-ASC monoclonal antibody on ASC and analyzed the expression of ASC in normal human tissues. ASC expression was observed in anterior horn cells of the spinal cord, trophoblasts of the placental villi, tubule epithelium of the kidney, seminiferous tubules and Leydig cells of the testis, hepatocytes and interlobular bile ducts of the liver, squamous epithelial cells of the tonsil and skin, hair follicle, sebaceous and eccrine glands of the skin, and peripheral blood leukocytes. In the colon, ASC was detected in mature epithelial cells facing the luminal side rather than immature cells located deeper in the crypts. These observations indicate that high levels of ASC are abundantly expressed in epithelial cells and leukocytes, which are involved in host defense against external pathogens and in well-differentiated cells, the proliferation of which is regulated. 相似文献
4.
Parimala R. Vajjhala Ruth E. Mirams Justine M. Hill 《The Journal of biological chemistry》2012,287(50):41732-41743
A key process underlying an innate immune response to pathogens or cellular stress is activation of members of the NOD-like receptor family, such as NLRP3, to assemble caspase-1-activating inflammasome complexes. Activated caspase-1 processes proinflammatory cytokines into active forms that mediate inflammation. Activation of the NLRP3 inflammasome is also associated with common diseases including cardiovascular disease, diabetes, chronic kidney disease, and Alzheimer disease. However, the molecular details of NLRP3 inflammasome assembly are not established. The adaptor protein ASC plays a key role in inflammasome assembly. It is composed of an N-terminal pyrin domain (PYD) and a C-terminal caspase recruitment domain, which are protein interaction domains of the death fold superfamily. ASC interacts with NLRP3 via a homotypic PYD interaction and recruits procaspase-1 via a homotypic caspase recruitment domain interaction. Here we demonstrate that ASC PYD contains two distinct binding sites important for self-association and interaction with NLRP3 and the modulatory protein POP1. Modeling of the homodimeric ASC PYD complex formed via an asymmetric interaction using both sites resembles a type I interaction found in other death fold domain complexes. This interaction mode also permits assembly of ASC PYDs into filaments. Furthermore, a type I binding mode is likely conserved in interactions with NLRP3 and POP1, because residues critical for interaction of ASC PYD are conserved in these PYDs. We also demonstrate that ASC PYD can simultaneously self-associate and interact with NLRP3, rationalizing the model whereby ASC self-association upon recruitment to NLRP3 promotes clustering and activation of procaspase-1. 相似文献
5.
Motani K Kushiyama H Imamura R Kinoshita T Nishiuchi T Suda T 《The Journal of biological chemistry》2011,286(39):33963-33972
The adaptor protein, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), connects pathogen/danger sensors such as NLRP3 and NLRC4 with caspases and is involved in inflammation and cell death. We have found that ASC activation induced caspase-8-dependent apoptosis or CA-074Me (cathepsin B inhibitor)-inhibitable necrosis depending on the cell type. Unlike necroptosis, another necrotic cell death, ASC-mediated necrosis, was neither RIP3-dependent nor necrostatin-1-inhibitable. Although acetyl-YVAD-chloromethylketone (Ac-YVAD-CMK) (caspase-1 inhibitor) did not inhibit ASC-mediated necrosis, comprehensive gene expression analyses indicated that caspase-1 expression coincided with the necrosis type. Furthermore, caspase-1 knockdown converted necrosis-type cells to apoptosis-type cells, whereas exogenous expression of either wild-type or catalytically inactive caspase-1 did the opposite. Knockdown of caspase-1, but not Ac-YVAD-CMK, suppressed the monocyte necrosis induced by Staphylococcus and Pseudomonas infection. Thus, the catalytic activity of caspase-1 is dispensable for necrosis induction. Intriguingly, a short period of caspase-1 knockdown inhibited IL-1β production but not necrosis, although longer knockdown suppressed both responses. Possible explanations of this phenomenon are discussed. 相似文献
6.
Nucleotide-binding oligomerization domain-2 modulates specific TLR pathways for the induction of cytokine release 总被引:13,自引:0,他引:13
Netea MG Ferwerda G de Jong DJ Jansen T Jacobs L Kramer M Naber TH Drenth JP Girardin SE Kullberg BJ Adema GJ Van der Meer JW 《Journal of immunology (Baltimore, Md. : 1950)》2005,174(10):6518-6523
The recognition of peptidoglycan by cells of the innate immune system has been controversial; both TLR2 and nucleotide-binding oligomerization domain-2 (NOD2) have been implicated in this process. In the present study we demonstrate that although NOD2 is required for recognition of peptidoglycan, this leads to strong synergistic effects on TLR2-mediated production of both pro- and anti-inflammatory cytokines. Defective IL-10 production in patients with Crohn's disease bearing loss of function mutations of NOD2 may lead to overwhelming inflammation due to a subsequent Th1 bias. In addition to the potentiation of TLR2 effects, NOD2 is a modulator of signals transmitted through TLR4 and TLR3, but not through TLR5, TLR9, or TLR7. Thus, interaction between NOD2 and specific TLR pathways may represent an important modulatory mechanism of innate immune responses. 相似文献
7.
Paula M. Soriano-Teruel Guillermo GarcíaLaínez María Marco-Salvador Julin Pardo Maykel Arias Christian DeFord Irmgard Merfort María J. Vicent Pablo Pelegrín Mnica Sancho Mar Orzez 《Cell death & disease》2021,12(12)
The ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD)) protein is an scaffold component of different inflammasomes, intracellular multiprotein platforms of the innate immune system that are activated in response to pathogens or intracellular damage. The formation of ASC specks, initiated by different inflammasome receptors, promotes the recruitment and activation of procaspase-1, thereby triggering pyroptotic inflammatory cell death and pro-inflammatory cytokine release. Here we describe MM01 as the first-in-class small-molecule inhibitor of ASC that interferes with ASC speck formation. MM01 inhibition of ASC oligomerization prevents activation of procaspase-1 in vitro and inhibits the activation of different ASC-dependent inflammasomes in cell lines and primary cultures. Furthermore, MM01 inhibits inflammation in vivo in a mouse model of inflammasome-induced peritonitis. Overall, we highlight MM01 as a novel broad-spectrum inflammasome inhibitor for the potential treatment of multifactorial diseases involving the dysregulation of multiple inflammasomes.Subject terms: Small molecules, Target validation 相似文献
8.
Gaelle Spagnol Calliste Reiling Fabien Kieken Steve Caplan Paul L. Sorgen 《Biomolecular NMR assignments》2014,8(2):263-267
The C-terminal Eps15 homology (EH) domain 3 (EHD3) belongs to a eukaryotic family of endocytic regulatory proteins and is involved in the recycling of various receptors from the early endosome to the endocytic recycling compartment or in retrograde transport from the endosomes to the Golgi. EH domains are highly conserved in the EHD family and function as protein-protein interaction units that bind to Asn-Pro-Phe (NPF) motif-containing proteins. The EH domain of EHD1 was the first C-terminal EH domain from the EHD family to be solved by NMR. The differences observed between this domain and proteins with N-terminal EH domains helped describe a mechanism for the differential binding of NPF-containing proteins. Here, structural studies were expanded to include the EHD3 EH domain. While the EHD1 and EHD3 EH domains are highly homologous, they have different protein partners. A comparison of these structures will help determine the selectivity in protein binding between the EHD family members and lead to a better understanding of their unique roles in endocytic regulation. 相似文献
9.
Stahelin RV Long F Peter BJ Murray D De Camilli P McMahon HT Cho W 《The Journal of biological chemistry》2003,278(31):28993-28999
Epsin and AP180/CALM are endocytotic accessory proteins that have been implicated in the formation of clathrin-coated pits. Both proteins have phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-binding domains in their N termini, but these domains are structurally and functionally different. To understand the basis of their distinct properties, we measured the PtdIns(4,5)P2-dependent membrane binding of the epsin N-terminal homology (ENTH) domain and the AP180 N-terminal homology (ANTH) domain by means of surface plasmon resonance and monolayer penetration techniques and also calculated the effect of PtdIns(4,5)P2 on the electrostatic potential of these domains. PtdIns(4,5)P2 enhances the electrostatic membrane association of both domains; however, PtdIns(4,5)P2 binding exerts distinct effects on their membrane dissociation. Specifically, PtdIns(4,5)P2 induces the membrane penetration of the N-terminal alpha-helix of the ENTH domain, which slows the membrane dissociation of the domain and triggers the membrane deformation. These results provide the biophysical explanation for the membrane bending activity of epsin and its ENTH domain. 相似文献
10.
CIPER, a novel NF kappaB-activating protein containing a caspase recruitment domain with homology to Herpesvirus-2 protein E10 总被引:5,自引:0,他引:5
Koseki T Inohara N Chen S Carrio R Merino J Hottiger MO Nabel GJ Núñez G 《The Journal of biological chemistry》1999,274(15):9955-9961
We have identified and characterized CIPER, a novel protein containing a caspase recruitment domain (CARD) in its N terminus and a C-terminal region rich in serine and threonine residues. The CARD of CIPER showed striking similarity to E10, a product of the equine herpesvirus-2. CIPER formed homodimers via its CARD and interacted with viral E10 but not with several apoptosis regulators containing CARDs including ARC, RAIDD, RICK, caspase-2, caspase-9, or Apaf-1. Expression of CIPER induced NF-kappaB activation, which was inhibited by dominant-negative NIK and a nonphosphorylable IkappaB-alpha mutant but not by dominant-negative RIP. Mutational analysis revealed that the N-terminal region of CIPER containing the CARD was sufficient and necessary for NF-kappaB-inducing activity. Point mutations in highly conserved residues in the CARD of CIPER disrupted the ability of CIPER to activate NF-kappaB and to form homodimers, indicating that the CARD is essential for NF-kappaB activation and dimerization. We propose that CIPER acts in a NIK-dependent pathway of NF-kappaB activation. 相似文献
11.
Role of charged and hydrophobic residues in the oligomerization of the PYRIN domain of ASC 总被引:2,自引:0,他引:2
Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor protein composed of two homophilic protein-protein interaction domains, a PYRIN domain (PYD) and a caspase recruitment domain. PYD-dependent oligomerization of ASC is thought to play a crucial role in formation of a molecular platform, the inflammasome, which activates caspase-1. When expressed in cells, the PYD of ASC was shown to form cytoplasmic filaments through self-association. Over 70 single point mutants were analyzed for filament formation in cells expressing the mutant proteins. The set of mutations comprised every single amino acid residue with a charged side chain (Arg, Lys, Asp, and Glu) and a large hydrophobic side chain (Ile, Leu, Met, Phe, Pro, and Val). Filament formation of the ASC PYD was prevented by mutation of Lys21, Leu25, Lys26, Pro40, Arg41, Asp48, and Asp51 of helices 2, 3, and 4. These data identify a coherent interaction surface, establishing a molecular model of PYD-PYD complexes with an important role for charge-charge interactions. 相似文献
12.
Yu JW Wu J Zhang Z Datta P Ibrahimi I Taniguchi S Sagara J Fernandes-Alnemri T Alnemri ES 《Cell death and differentiation》2006,13(2):236-249
Mutations in cryopyrin and pyrin proteins are responsible for several autoinflammatory disorders in humans, suggesting that these proteins play important roles in regulating inflammation. Using a HEK293 cell-based reconstitution system that stably expresses ASC and procaspase-1 we demonstrated that neither cryopyrin nor pyrin or their corresponding disease-associated mutants could significantly activate NF-kappaB in this system. However, both cryopyrin and two disease-associated cryopyrin mutants induced ASC oligomerization and ASC-dependent caspase-1 activation, with the disease-associated mutants being more potent than the wild-type (WT) cryopyrin, because of increased self-oligomerization. Contrary to the proposed anti-inflammatory activity of WT pyrin, our results demonstrated that pyrin, like cryopyrin, can also assemble an inflammasome complex with ASC and procaspase-1 leading to ASC oligomerization, caspase-1 activation and interleukin-1beta processing. Thus, we propose that pyrin could function as a proinflammatory molecule. 相似文献
13.
Boughan PK Argent RH Body-Malapel M Park JH Ewings KE Bowie AG Ong SJ Cook SJ Sorensen OE Manzo BA Inohara N Klein NJ Nuñez G Atherton JC Bajaj-Elliott M 《The Journal of biological chemistry》2006,281(17):11637-11648
Host-pathogen interactions that allow Helicobacter pylori to survive and persist in the stomach of susceptible individuals remain unclear. Human beta-defensins (hBDs), epithelial-derived antimicrobial peptides are critical components of host-defense at mucosal surfaces. The role of H. pylori-mediated NF-kappaB and epidermal growth factor receptor (EGFR) activation on beta-defensin expression was investigated. Transient transfection studies utilizing beta-defensin promoter constructs were conducted in gastric cells with contribution of individual signaling events evaluated by the addition of specific inhibitors, small interference nucleotide-binding oligomerization domain 1 (NOD1) RNA or plasmids encoding Vaccinia virus proteins that interrupt interleukin-1 and Toll-like receptor signaling. The role of individual MAPK pathways was further delineated in HEK-293 cells expressing conditional MAPK mutants. We found hBD2 expression exclusively dependent on the presence of the bacterial cag pathogenicity island, with NOD1 a critical host sensor. Impairment of murinebeta-defensin 4 (an orthologue of hBD2) expression in NOD1-deficient mice 7-days post-infection further confirmed the role of this cytoplasmic pattern-recognition receptor in eliciting host innate immunity. In contrast to hBD2, hBD3 expression was NOD1-independent but EGFR and ERK pathway-dependent. Importantly, Toll-like receptor signaling was not implicated in H. pylori-mediated hBD2 and hBD3 gene expression. The divergent signaling events governing hBD2 and hBD3 expression suggest temporal functional variation, such that hBD2 may contribute to antimicrobial barrier function during the inflammatory phase with hBD3 playing a greater role during the repair, wound healing phase of infection. 相似文献
14.
Signaling and function of caspase and c-jun N-terminal kinase in cisplatin-induced apoptosis 总被引:2,自引:0,他引:2
Caspases and c-Jun N-terminal kinase (JNK) are activated in tumor cells during induction of apoptosis. We investigated the signaling cascade and function of these enzymes in cisplatin-induced apoptosis. Treatment of Jurkat T-cells with cisplatin induced cell death with DNA fragmentation and activation of caspase and JNK. Bcl-2 overexpression suppressed activation of both enzymes, whereas p35 and CrmA inhibited only the DEVDase (caspase-3-like) activity, indicating that the activation of these enzymes may be differentially regulated. Cisplatin induced apoptosis with the cytochrome c release and caspase-3 activation in both wild-type and caspase-8-deficient JB-6 cells, while the Fas antibody induced these apoptotic events only in wild-type cells. This indicates that caspase-8 activation is required for Fas-mediated apoptosis, but not cisplatin-induced cell death. On the other hand, cisplatin induced the JNK activation in both the wild-type and JB-6 cells, and the caspase-3 inhibitor Z-DEVD-fmk did not inhibit this activation. The JNK overexpression resulted in a higher JNK activity, AP-1 DNA binding activity, and metallothionein expression than the empty vector-transfected cells following cisplatin treatment. It also partially protected the cells from cisplatin-induced apoptosis by decreasing DEVDase activity. These data suggest that the cisplatin-induced apoptotic signal is initiated by the caspase-8-independent cytochrome c release, and the JNK activation protects cells from cisplatin-induced apoptosis via the metallothionein expression. 相似文献
15.
Feng S Okenka GM Bai CX Streets AJ Newby LJ DeChant BT Tsiokas L Obara T Ong AC 《The Journal of biological chemistry》2008,283(42):28471-28479
Autosomal dominant polycystic kidney disease (ADPKD), the most common inherited cause of kidney failure, is caused by mutations in either PKD1 (85%) or PKD2 (15%). The PKD2 protein, polycystin-2 (PC2 or TRPP2), is a member of the transient receptor potential (TRP) superfamily and functions as a non-selective calcium channel. PC2 has been found to form oligomers in native tissues suggesting that it may form functional homo- or heterotetramers with other subunits, similar to other TRP channels. Our experiments unexpectedly revealed that PC2 mutant proteins lacking the known C-terminal dimerization domain were still able to form oligomers and co-immunoprecipitate full-length PC2, implying the possible existence of a proximal dimerization domain. Using yeast two-hybrid and biochemical assays, we have mapped an alternative dimerization domain to the N terminus of PC2 (NT2-1-223, L224X). Functional characterization of this domain demonstrated that it was sufficient to induce cyst formation in zebrafish embryos and inhibit PC2 surface currents in mIMCD3 cells probably by a dominant-negative mechanism. In summary, we propose a model for PC2 assembly as a functional tetramer which depends on both C- and N-terminal dimerization domains. These results have significant implications for our understanding of PC2 function and disease pathogenesis in ADPKD and provide a new strategy for studying PC2 function. 相似文献
16.
Tötemeyer S Sheppard M Lloyd A Roper D Dowson C Underhill D Murray P Maskell D Bryant C 《Journal of immunology (Baltimore, Md. : 1950)》2006,176(8):4804-4810
Pattern recognition receptors are central to the responsiveness of various eukaryotic cell types when they encounter pathogen-associated molecular patterns. IFN-gamma is a cytokine that is elevated in humans and other animals with bacterial infection and enhances the LPS-induced production of antibacterial mediators by macrophages. Mice lacking the pattern recognition receptor, TLR4, respond very poorly to stimulation by LPS, but administration of IFN-gamma has been described as restoring apparent sensitivity to this stimulatory ligand. In this study, we show that IFN-gamma primes murine macrophages stimulated by crude LPS preparations to produce the antibacterial mediator NO, a proportion of which is independent of TLRs 2 and 4. This response is lost in tlr4-/- IFN-gamma-primed murine macrophages when the LPS preparation is highly purified. NO is also induced if chemically synthesized muramyl dipeptide, an intermediate in the biosynthesis of peptidoglycan, is used to stimulate macrophages primed with IFN-gamma. This is absolutely dependent on the presence of a functional nucleotide oligomerization domain-2 (NOD-2) protein. IFN-gamma increases NOD-2 expression and dissociates this protein from the actin cytoskeleton within the cell. IFN-gamma priming of macrophages therefore reveals a key proinflammatory role for NOD-2. This study also shows that the effect of IFN-gamma in restoring inflammatory responses to gram-negative bacteria or bacterial products in mice with defective TLR4 signaling is likely to be due to a response to peptidoglycan, not LPS. 相似文献
17.
Receptor interacting protein-2, RIP2, is a serine/threonine kinase and has sequence homology to RIP. It functions as an adaptor molecule for some members from the tumor necrosis factor receptor family and mediates divergent signaling pathways including NF-κB activation and cell death. RIP2 contains an N-terminal kinases domain and a C-terminal caspase activation and recruitment domain (CARD). The apoptotic activity of RIP2 is restricted to its C-terminal CARD domain while NF-κB activation requires the intact RIP2 for binding. RIP2 CARD involved homotypic or heterotypic interactions with members of the death domains superfamily. Here I report backbone and sidechain 1H, 13C and 15N resonance assignments of soluble RIP2 CARD as a basis for further structural and functional studies. 相似文献
18.
The mechanism(s) by which heat shock protein 25 (hsp25) protects cells from stress may involve one or more of the biochemical properties attributed to hsp25 and other small M(r) hsp. In this report, structural and functional properties of an N-terminal 33 amino acid deletion variant of hsp25 (termed hsp25.c) were considered by comparison with hsp25. 6-His tagged recombinant hsp25 and hsp25.c (termed (H6)hsp25.a and (H6)hsp25.c) were expressed and purified. Oligomeric proteins formed and possessed properties previously attributed to hsp25. The 33 amino acid deletion represented by hsp27.c did not affect the ability of the recombinant protein to act as an inhibitor of elastase, as a molecular chaperone in the refolding of denatured citrate synthase, or as an actin-binding protein. The overexpression of either hsp25 or hsp25.c, enhanced the stress resistance of stable transformed eukaryotic cells. This N-terminal variant protein may be used in further cellular and biochemical assessment of hsp25 oligomerization and function. 相似文献
19.
Regulation of c-Fes tyrosine kinase and biological activities by N-terminal coiled-coil oligomerization domains
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The cytoplasmic protein-tyrosine kinase Fes has been implicated in cytokine signal transduction, hematopoiesis, and embryonic development. Previous work from our laboratory has shown that active Fes exists as a large oligomeric complex in vitro. However, when Fes is expressed in mammalian cells, its kinase activity is tightly repressed. The Fes unique N-terminal sequence has two regions with strong homology to coiled-coil-forming domains often found in oligomeric proteins. Here we show that disruption or deletion of the first coiled-coil domain upregulates Fes tyrosine kinase and transforming activities in Rat-2 fibroblasts and enhances Fes differentiation-inducing activity in myeloid leukemia cells. Conversely, expression of a Fes truncation mutant consisting only of the unique N-terminal domain interfered with Rat-2 fibroblast transformation by an activated Fes mutant, suggesting that oligomerization is essential for Fes activation in vivo. Coexpression with the Fes N-terminal region did not affect the transforming activity of v-Src in Rat-2 cells, arguing against a nonspecific suppressive effect. Taken together, these findings suggest a model in which Fes activation may involve coiled-coil-mediated interconversion of monomeric and oligomeric forms of the kinase. Mutation of the first coiled-coil domain may activate Fes by disturbing intramolecular coiled-coil interaction, allowing for oligomerization via the second coiled-coil domain. Deletion of the second coiled-coil domain blocks fibroblast transformation by an activated form of c-Fes, consistent with this model. These results provide the first evidence for regulation of a nonreceptor protein-tyrosine kinase by coiled-coil domains. 相似文献
20.
M G Sanders M J Parsons A G A Howard J Liu S R Fassio J A Martinez L Bouchier-Hayes 《Cell death & disease》2015,6(7):e1813
The human inflammatory caspases, including caspase-1, -4, -5 and -12, are considered as key regulators of innate immunity protecting from sepsis and numerous inflammatory diseases. Caspase-1 is activated by proximity-induced dimerization following recruitment to inflammasomes but the roles of the remaining inflammatory caspases in inflammasome assembly are unclear. Here, we use caspase bimolecular fluorescence complementation to visualize the assembly of inflammasomes and dimerization of inflammatory caspases in single cells. We observed caspase-1 dimerization induced by the coexpression of a range of inflammasome proteins and by lipospolysaccharide (LPS) treatment in primary macrophages. Caspase-4 and -5 were only dimerized by select inflammasome proteins, whereas caspase-12 dimerization was not detected by any investigated treatment. Strikingly, we determined that certain inflammasome proteins could induce heterodimerization of caspase-1 with caspase-4 or -5. Caspase-5 homodimerization and caspase-1/-5 heterodimerization was also detected in LPS-primed primary macrophages in response to cholera toxin subunit B. The subcellular localization and organization of the inflammasome complexes varied markedly depending on the upstream trigger and on which caspase or combination of caspases were recruited. Three-dimensional imaging of the ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain)/caspase-1 complexes revealed a large spherical complex of ASC with caspase-1 dimerized on the outer surface. In contrast, NALP1 (NACHT leucine-rich repeat protein 1)/caspase-1 complexes formed large filamentous structures. These results argue that caspase-1, -4 or -5 can be recruited to inflammasomes under specific circumstances, often leading to distinctly organized and localized complexes that may impact the functions of these proteases.Correct assembly and regulation of inflammasomes is critical for mediating inflammation and preventing uncontrolled inflammation under infectious and sterile conditions. These supramolecular structures converge on the activation of caspase-1. Upon activation, caspase-1 cleaves the proinflammatory cytokines interleukin1β (IL-1β) and IL-18 to their active mature forms,1, 2 which are then released from the cell to direct the immediate removal of pathogens.Caspase-1 is activated by proximity-induced dimerization upon recruitment to inflammasomes, which are multiprotein signaling complexes that act as activation platforms.3 Many distinct inflammasomes exist, and each inflammasome includes a sensor protein (e.g., NALP1 (NACHT leucine-rich repeat protein 1)/NLRP1 (NOD-like receptor protein 1), NALP3/NLRP3, AIM2 (absent in melanoma 2) or IPAF (ICE protease-activating factor)/NLRC4 (NLR family CARD domain-containing protein 4)), which is activated by specific proinflammatory molecules. These include pathogen-derived stimuli, known as pathogen-associated molecular patterns (PAMPs), or non-pathogenic inflammatory stimuli, known as damage-associated molecular patterns.4 Inflammasome assembly is governed by a series of homotypic interactions, which are mediated by specific protein:protein interaction domains, such as the pyrin domain (PYD) and the caspase recruitment domain (CARD).5, 6 For example, NALP1 and NALP3 both contain a PYD at their C terminus that binds to the PYD in the adaptor protein, ASC (apoptosis-associated speck-like protein containing a CARD).7, 8 ASC also contains a CARD,9 which binds to the CARD in the prodomain of caspase-1 (C1-Pro), resulting in caspase-1 dimerization and activation.10 Inflammasomes can also be ASC-independent, such as IPAF, which interacts directly with caspase-1.11 Interestingly, ASC can enhance IPAF-induced caspase-1 activation,12, 13 which indicates that more complex interactions between these proteins may exist.Caspase-1 is one of the inflammatory caspases, including the human caspases (caspase-1, -4, -5 and -12) and murine caspase-11.14 The roles of caspase-4, -5, and -12 in caspase-1 activation and inflammasome pathways are not clear. Full-length caspase-12, which is only expressed by ~20% of people of African descent, can inhibit caspase-1 activity.15 Most of all human populations express the short form of caspase-12 (caspase-12 S or C12S), which arose from a point mutation leading to a premature stop codon just after the prodomain. This truncated form of caspase-12 is associated with increased resistance to sepsis,15 indicating that caspase-12 has an important role in inflammation. Caspase-11 mediates caspase-1 activation in response to Escherichia coli and Citrobacter rodentium in mice.16 Caspase-11 also triggers an inflammatory form of cell death, known as pyroptosis, independent of caspase-1, ASC and NALP3. This is known as the noncanonical inflammasome pathway.16 Humans do not express caspase-11 and express caspase-4 and -5 instead. Recent evidence indicates that caspase-4 and -5 act as direct intracellular sensors for lipopolysaccharide (LPS) to induce pyroptosis, independent of any additional inflammasome proteins.17 However, this does not rule out the possibility that caspase-4 and -5 can be recruited to inflammasomes under certain circumstances.The inflammatory caspases are considered to be initiator caspases, based on structural similarities between them and caspase-2, -8 and -9.18 We previously reported the use of caspase bimolecular fluorescence complementation (BiFC) to measure induced proximity of the initiator caspase, caspase-2.19 We adapted BiFC, where non-fluorescent fragments of the yellow fluorescent protein, Venus (‘split Venus''), can associate to reform the fluorescent complex when fused to interacting proteins. When caspase-2 was fused to each half of split Venus, the recruitment of caspase-2 to its activation platform and the subsequent induced proximity resulted in association of the two Venus halves. This resulted in an increase in fluorescence that represents caspase dimerization. Our caspase BiFC method facilitates specific analysis of caspase interactions at the level of the activation platform. Importantly, caspase BiFC can reveal the structural organization and localization of activation platforms in living cells.Here, we extend the caspase BiFC approach to interrogate the inflammatory caspases. We show that there are considerable differences in the organization and distribution of different inflammasomes depending on the upstream signals and on which inflammatory caspase is recruited. Our studies reveal unexpected heterodimerization interactions between caspase-1 and additional inflammatory caspases, presenting a new outcome of inflammasome assembly. 相似文献