ASC, a novel 22-kDa protein, aggregates during apoptosis of human promyelocytic leukemia HL-60 cells

The cytoskeletal and/or nuclear matrix molecules responsible for morphological changes associated with apoptosis were identified using monoclonal antibodies (mAbs). We developed mAbs against Triton X-100-insoluble components of HL-60 cells pretreated with all-trans retinoic acid. In particular, one mAb recognized a 22-kDa protein that exhibited intriguing behavior by forming an aggregate and appearing as a speck during apoptosis induced by retinoic acid and other anti-tumor drugs. Cloning and sequencing of its cDNA revealed that this protein comprises 195 amino acids and that its C-terminal half has a caspase recruitment domain (CARD) motif, characteristic of numerous proteins involved in apoptotic signaling. We referred to this protein as ASC (apoptosis-associated speck-like protein containing a CARD). The ASC gene was mapped on chromosome 16p11.2-12. The antisense oligonucleotides of ASC were found to reduce the expression of ASC, and consequently, etoposide-mediated apoptosis of HL-60 cells was suppressed. Our results indicate that ASC is a novel member of the CARD-containing adaptor protein family.     

Apoptosis-associated speck-like protein containing a caspase recruitment domain is a regulator of procaspase-1 activation

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)/target of methylation-induced silencing/PYCARD represents one of only two proteins encoded in the human genome that contains a caspase recruitment domain (CARD) together with a pyrin, AIM, ASC, and death domain-like (PAAD)/PYRIN/DAPIN domain. CARDs regulate caspase family proteases. We show here that ASC binds by its CARD to procaspase-1 and to adapter proteins involved in caspase-1 activation, thereby regulating cytokine pro-IL-1beta activation by this protease in THP-1 monocytes. ASC enhances IL-1beta secretion into the cell culture supernatants, at low concentrations, while suppressing at high concentrations. When expressed in HEK293 cells, ASC interferes with Cardiak/Rip2/Rick-mediated oligomerization of procaspase-1 and suppresses activation this protease, as measured by protease activity assays. Moreover, ASC also recruits procaspase-1 into ASC-formed cytosolic specks, separating it from Cardiak. We also show that expression of the PAAD/PYRIN family proteins pyrin or cryopyrin/PYPAF1/NALP3 individually inhibits IL-1beta secretion but that coexpression of ASC with these proteins results in enhanced IL-1beta secretion. However, expression of ASC uniformly interferes with caspase-1 activation and IL-1beta secretion induced by proinflammatory stimuli such as LPS and TNF, suggesting pathway competition. Moreover, LPS and TNF induce increases in ASC mRNA and protein expression in cells of myeloid/monocytic origin, revealing another level of cross-talk of cytokine-signaling pathways with the ASC-controlled pathway. Thus, our results suggest a complex interplay of the bipartite adapter protein ASC with PAAD/PYRIN family proteins, LPS (Toll family receptors), and TNF in the regulation of procaspase-1 activation, cytokine production, and control of inflammatory responses.     

Murine ortholog of ASC, a CARD-containing protein, self-associates and exhibits restricted distribution in developing mouse embryos

ASC (apoptosis-associated speck-like protein containing a CARD) was first identified as a cytosolic soluble protein that forms insoluble aggregates and enhances etoposide-induced apoptosis. We have cloned a murine ortholog of ASC (mASC) comprising 193 amino acids with a well-conserved pyrin N-terminal homology domain and caspase recruitment domain (CARD). mASC fused with green fluorescent protein appeared as a speck in transfected COS-7 cells and showed self-association. We analyzed mASC gene expression in developing embryos by in situ hybridization and found it to have a restricted distribution in mouse embryos. At E9.5, mASC was strongly expressed in the telencephalon, thalamic areas of the diencephalon, heart, and liver. Northern blotting analysis revealed that the mASC gene was expressed ubiquitously in multiple organs in adult mice. These findings indicate that mASC shows conservation of not only the primary structure of human ASC but also the ability to aggregate and has some similarity in its distribution to other CARD-containing molecules, including the apoptosis regulator Apaf-1.     

Legionella pneumophila induces IFNbeta in lung epithelial cells via IPS-1 and IRF3, which also control bacterial replication

Legionella pneumophila, a Gram-negative facultative intracellular bacterium, causes severe pneumonia (Legionnaires' disease). Type I interferons (IFNs) were so far associated with antiviral immunity, but recent studies also indicated a role of these cytokines in immune responses against (intracellular) bacteria. Here we show that wild-type L. pneumophila and flagellin-deficient Legionella, but not L. pneumophila lacking a functional type IV secretion system Dot/Icm, or heat-inactivated Legionella induced IFNbeta expression in human lung epithelial cells. We found that factor (IRF)-3 and NF-kappaB-p65 translocated into the nucleus and bound to the IFNbeta gene enhancer after L. pneumophila infection of lung epithelial cells. RNA interference demonstrated that in addition to IRF3, the caspase recruitment domain (CARD)-containing adapter molecule IPS-1 (interferon-beta promoter stimulator 1) is crucial for L. pneumophila-induced IFNbeta expression, whereas other CARD-possessing molecules, such as RIG-I (retinoic acid-inducible protein I), MDA5 (melanoma differentiation-associated gene 5), Nod27 (nucleotide-binding oligomerization domain protein 27), and ASC (apoptosis-associated speck-like protein containing a CARD) seemed not to be involved. Finally, bacterial multiplication assays in small interfering RNA-treated cells indicated that IPS-1, IRF3, and IFNbeta were essential for the control of intracellular replication of L. pneumophila in lung epithelial cells. In conclusion, we demonstrated a critical role of IPS-1, IRF3, and IFNbeta in Legionella infection of lung epithelium.     

Pyrin N-terminal homology domain- and caspase recruitment domain-dependent oligomerization of ASC

ASC was first identified as a caspase recruitment domain (CARD)-containing proapoptotic molecule that forms insoluble aggregates during apoptosis. Here, we report both the pyrin N-terminal homology domain (PYD) and CARD domains are involved in the aggregation of ASC. Preliminary experiments indicated that overexpression of ASC formed filament-like aggregates in COS-7 cells. Expression experiments using green fluorescent protein (GFP) constructs showed that not only the GFP-ASC-CARD but also the GFP-ASC-PYD formed filament-like aggregates in COS-7 cells. We confirmed these filament-like aggregates of both the ASC-PYD and the ASC-CARD due to homophilic interaction by immunoprecipitation method. We also demonstrated that the ASC-PYD associated with the ASC-CARD by heterophilic interaction. These observations suggest that the dimerization of the PYD as well as the CARD plays an important role in the oligomerization of ASC as an adaptor molecule.     

TMS1/ASC: The cancer connection

TMS1/ASC is a bipartite protein comprising two protein-protein interaction domains, a pyrin domain (PYD) and a caspase recruitment domain (CARD). Proteins containing these domains play pivotal roles in regulating apoptosis and immune response pathways, and mutations in a number of PYD- and CARD-containing proteins have been linked to autoinflammatory diseases and cancer. Indeed, one of the ways in which TMS1/ASC was identified was as a target of methylation-mediated silencing in breast cancer cells. This review discusses the mounting evidence supporting a correlation between the silencing of TMS1/ASC expression and cancer. In addition, it addresses the reported functions of TMS1/ASC that include apoptosis, activation of inflammatory caspases and regulation of NF-kappa B, and discusses the potential ways in which loss of TMS1/ASC contributes to carcinogenesis.     

凋亡相关斑点样蛋白的研究进展

凋亡相关斑点样蛋白（apoptosis-associated speck-like protein containing a CARD,ASC）是一种含有N端热蛋白样结构域和C端胱天氨酸募集结构域的接头分子。ASC可以通过它含有的同源蛋白互作结构域PYD和CARD的寡聚化来募集上下游与其含有同源结构域的其他蛋白,从而参与多条信号转导途径,在炎症反应、肿瘤发生、细胞凋亡和NF-κB信号通路的调节方面发挥重要的生物学作用。     

ASC,which is composed of a PYD and a CARD,is up-regulated by inflammation and apoptosis in human neutrophils

ASC is an adaptor protein that is composed of two protein-protein interaction domains, a PYRIN domain (PYD), and a caspase-recruitment domain (CARD). Recently, ASC was identified as a binding partner of pyrin, which is the product of MEFV, a gene causing familial Mediterranean fever (FMF). Mutations in MEFV result in defects in control of neutrophil-mediated inflammation. Thus we focused on the expression of ASC in neutrophils. Immunohistochemical study showed that ASC is increased in neutrophils in severe inflammatory sites of gangrenous appendicitis. We, then, tested whether proinflammatory mediators induce ASC using peripheral blood neutrophils in vitro. ASC expression was transiently up-regulated by IL-1alpha, IL-1beta, IFN-alpha, IFN-gamma, TNFalpha, and LPS. ASC was also increased by incubation with either anti-Fas antibody or recombinant soluble Fas ligand. The Fas-mediated induction of ASC was inhibited by a general caspase inhibitor, z-VAD-fmk, and an immunocytochemical study showed that ASC was increased in neutrophils exhibiting characteristic phenotypes for apoptosis. These findings suggest that up-regulation of ASC is closely associated with inflammation and apoptosis in neutrophils.     

Caspase-1 protein induces apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)-mediated necrosis independently of its catalytic activity

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.     

Role of charged and hydrophobic residues in the oligomerization of the PYRIN domain of ASC

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.     

Crystal structure of NALP3 protein pyrin domain (PYD) and its implications in inflammasome assembly

NALP3 inflammasome, composed of the three proteins NALP3, ASC, and Caspase-1, is a macromolecular complex responsible for the innate immune response against infection with bacterial and viral pathogens. Formation of the inflammasome can lead to the activation of inflammatory caspases, such as Caspase-1, which then activate pro-inflammatory cytokines by proteolytic cleavage. The assembly of the NALP3 inflammasome depends on the protein-interacting domain known as the death domain superfamily. NALP3 inflammasome is assembled via a pyrin domain (PYD)/PYD interaction between ASC and NALP3 and a caspase recruitment domain/caspase recruitment domain interaction between ASC and Caspase-1. As a first step toward elucidating the molecular mechanisms of inflammatory caspase activation by formation of inflammasome, we report the crystal structure of the PYD from NALP3 at 1.7-Å resolution. Although NALP3 PYD has the canonical six-helical bundle structural fold similar to other PYDs, the high resolution structure reveals the possible biologically important homodimeric interface and the dynamic properties of the fold. Comparison with other PYD structures shows both similarities and differences that may be functionally relevant. Structural and sequence analyses further implicate conserved surface residues in NALP3 PYD for ASC interaction and inflammasome assembly. The most interesting aspect of the structure was the unexpected disulfide bond between Cys-8 and Cys-108, which might be important for regulation of the activity of NALP3 by redox potential.     

The death-domain fold of the ASC PYRIN domain, presenting a basis for PYRIN/PYRIN recognition

The PYRIN domain is a conserved sequence motif identified in more than 20 human proteins with putative functions in apoptotic and inflammatory signalling pathways. The three-dimensional structure of the PYRIN domain from human ASC was determined by NMR spectroscopy. The structure determination reveals close structural similarity to death domains, death effector domains, and caspase activation and recruitment domains, although the structural alignment with these other members of the death-domain superfamily differs from previously predicted amino acid sequence alignments. Two highly positively and negatively charged surfaces in the PYRIN domain of ASC result in a strong electrostatic dipole moment that is predicted to be present also in related PYRIN domains. These results suggest that electrostatic interactions play an important role for the binding between PYRIN domains. Consequently, the previously reported binding between the PYRIN domains of ASC and ASC2/POP1 or between the zebrafish PYRIN domains of zAsc and Caspy is proposed to involve interactions between helices 2 and 3 of one PYRIN domain with helices 1 and 4 of the other PYRIN domain, in analogy to previously reported homophilic interactions between caspase activation and recruitment domains.     

CXCR3-dependent plasma blast migration to the central nervous system during viral encephalomyelitis

Immunoglobulin in cerebral spinal fluid and antibody secreting cells (ASC) within the central nervous system (CNS) parenchyma are common hallmarks of microbial infections and autoimmune disorders. However, the signals directing ASC migration into the inflamed CNS are poorly characterized. This study demonstrates that CXCR3 mediates CNS accumulation of ASC during neurotropic coronavirus-induced encephalomyelitis. Expansion of CXCR3-expressing ASC in draining lymph nodes prior to accumulation within the CNS was consistent with their recruitment by sustained expression of CXCR3 ligands during viral persistence. Both total and virus-specific ASC were reduced greater than 80% in the CNS of infected CXCR3(-/-) mice. Similar T cell CNS recruitment and local T cell-dependent antiviral activity further indicated that the ASC migration defect was T cell independent. Furthermore, in contrast to the reduction of ASC in the CNS, neither virus-specific ASC trafficking to bone marrow nor antiviral serum antibody was reduced relative to levels in control mice. Impaired ASC recruitment into the CNS of infected CXCR3(-/-) mice coincided with elevated levels of persisting viral RNA, sustained infectious virus, increased clinical disease, and mortality. These results demonstrate that CXCR3 ligands are indispensable for recruitment of activated ASC into the inflamed CNS and highlight their local protective role during persistent infection.     

Production of a monoclonal antibody reacting with vimentin, a structural protein of intermediate filaments

This paper reports on the production of a monoclonal antibody (i-18) reacting with vimentin, the major structural component of intermediate filaments in cells of mesenchymal origin. The antibody was obtained following immunization with hamster fibroblasts and was selected for its ability to bind to the cytoskeleton fraction of the aforementioned cells. It decorated a perinuclear filamentous network characteristic of vimentin filaments in cells of mesenchymal origin of avian through human species. The specificity of the reagent was further ascertained on the basis of the sensitivity of the decorated filaments to colcemide. The strict antibody specificity for cells of mesenchymal versus epithelial origin was confirmed also in vivo on histological specimens from solid tissue. The i-18 monoclonal antibody precipitated a molecule of about 57 Kd from metabolically labelled cellular extracts. The broad cross-reactivity of this monoclonal antibody among different animal species, as well as its strict in vivo mesenchymal tissue specificity makes this antibody a useful reagent for both experimental and diagnostic purposes.     

Interaction between pyrin and the apoptotic speck protein (ASC) modulates ASC-induced apoptosis

Patients with familial Mediterranean fever suffer sporadic inflammatory attacks characterized by fever and intense pain (in joints, abdomen, or chest). Pyrin, the product of the MEFV locus, is a cytosolic protein whose function is unknown. Using pyrin as a "bait" to probe a yeast two-hybrid library made from neutrophil cDNA, we isolated apoptotic speck protein containing a caspase recruitment domain (CARD) (ASC), a proapoptotic protein that induces the formation of large cytosolic "specks" in transfected cells. We found that when HeLa cells are transfected with ASC, specks are formed. After co-transfection of cells with ASC plus wild type pyrin, an increase in speck-positive cells is found, and speck-positive cells show increased survival. Immunofluorescence studies show that pyrin co-localizes with ASC in specks. Speck localization requires exon 1 of pyrin, but exon 1 alone of pyrin does not result in an increase in the number of specks. Exon 1 of pyrin and exon 1 of ASC show 42% sequence similarity and resemble death domain-related structures in modeling studies. These findings link pyrin to apoptosis pathways and suggest that the modulation of cell survival may be a component of the pathophysiology of familial Mediterranean fever.     

Potential role of CD34 in cerebral vasospasm after experimental subarachnoid hemorrhage in rats

Inflammatory responses have been implicated in the elaboration of several forms of central nervous system injury, including cerebral vasospasm after subarachnoid hemorrhage (SAH). A critical event participating in such responses is the recruitment of circulating leukocytes into the inflammatory site. CD34 is a key adhesion molecule responsible for recruitment of monocytes/macrophages and the attachment of leukocytes to endothelial cells. However, it has not been investigated whether, and to what degree, CD34 is induced by SAH and also the role of CD34 in the pathogenesis of cerebral vasospasm following SAH remains unknown. Experiment 1 aimed to investigate the timecourse of the CD34 expression in the basilar artery after SAH. In experiment 2, we chose the maximum time point of vasospasm (day 3) and assessed the effect of monoclonal antibody against CD34 on regulation of cerebral vasospasm. As a result, the elevated expression of CD34 was detected in the basilar artery after SAH and peaked on day 3. After intracisternal administration of CD34 monoclonal antibody, the vasospasm was markedly attenuated after blood injection on day 3. Our results suggest that CD34 is increasingly expressed in a parallel time course to the development of cerebral vasospasm in a rat experimental model of SAH and administration of the specific CD34 antibody could prevent or reduce cerebral vasospasm caused by SAH.     

Multiple Binding Sites on the Pyrin Domain of ASC Protein Allow Self-association and Interaction with NLRP3 Protein

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.     

Mapping of POP1-binding site on pyrin domain of ASC

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an essential adaptor protein in the formation of a multiprotein complex that activates procaspase-1. ASC is also known as a modulator of NF-kappaB activation pathways. ASC has a bipartite domain structure, consisting of an N-terminal pyrin domain (PYD) and a C-terminal caspase-recruitment domain. The PYD of ASC (ASC_PYD) is known to interact with various PYD-containing intracellular danger signal sensors and PYD-only proteins. Using purified proteins, we characterized the in vitro interaction of ASC_PYD with PYD-only protein 1 (POP1). POP1 specifically interacts with ASC_PYD with a dissociation constant of 4.08 +/- 0.52 microm but does not interact with Cryopyrin. NMR and mutagenesis experiments show that a negative electrostatic potential surface patch (EPSP) on ASC_PYD, consisting of the first (H1) and fourth (H4) helices, is essential in the interaction with POP1. A positive EPSP on POP1, consisting of the second (H2) and third (H3) helices, is a counterpart of this interaction. The interaction between ASC_PYD and POP1 is similar to the interaction between caspase recruitment domains of Apaf-1 and procaspase-9. In addition, we present evidence that conformational changes at the long loop of ASC_PYD between the H2 and H3 helices can affect its interaction with POP1. Based on our observations, we propose that the positive EPSP of ASC_PYD, including the H2 and H3 helices, may be the binding site for Cryopyrin, and the interaction with Cryopyrin may induce the dissociation of POP1 from ASC_PYD.