The ASC inflammasome adapter governs SAA-derived protein aggregation in inflammatory amyloidosis
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ABSTRACT:
SUBMITTER: Marco Losa
PROVIDER: S-SCDT-10_1038-S44321-024-00107-0 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:ASC (apoptosis-associated speck-like protein containing a CARD) is a modular protein that functions as an adapter of the inflammasome, a multi-protein complex that triggers the inflammatory response in the presence of infection or cell damage. ASC bridges the inflammasome components (PYD-containing sensors and procaspase 1) via homotypic interactions mediated by its two death domains, PYD and CARD. The self-assembly and oligomerization of multiple copies of these three proteins result in the activation of procaspase 1, in turn rendering different cytokines functional. An in-depth understanding of ASC binding capabilities is crucial to decipher the molecular mechanisms of its role in inflammasome formation. In this chapter, we discuss the use of solution NMR to identify specific interacting surfaces of the inflammasome adapter ASC, and describe detailed protocols to perform NMR titrations with Death Domains to obtain apparent dissociation constants of the resulting complexes. The incorporation of NMR restraints in molecular docking to obtain models of these protein assemblies is presented.
| S-EPMC8455076 | biostudies-literature
Project description:The inflammasome is a multiprotein complex necessary for the onset of inflammation. The adapter protein ASC assembles inflammasome components by acting as a molecular glue between danger-signal sensors and procaspase-1. The assembly is mediated by ASC self-association and protein interactions via its two Death Domains, PYD and CARD. Truncated versions of ASC have been shown to form filaments, but information on the filaments formed by full-length ASC is needed to construct a meaningful model of inflammasome assembly. To gain insights into this system, we used a combination of transmission EM, NMR, and computational analysis to investigate intact ASC structures. We show that ASC forms ∼6-7-nm-wide filaments that stack laterally to form bundles. The structural characteristics and dimensions of the bundles indicate that both PYD and CARD are integral parts of the filament. A truncated version of ASC with only the CARD domain (ASCCARD) forms different filaments (∼3-4-nm width), providing further evidence that both domains work in concert in filament assembly. Ring-shaped protein particles bound to pre-existing filaments match the size of ASC dimer structures generated by NMR-based protein docking, suggesting that the ASC dimer could be a basic building block for filament formation. Solution NMR binding studies identified the protein surfaces involved in the ASCCARD-ASCCARD interaction. These data provide new insights into the structural underpinnings of the inflammasome and should inform future efforts to interrogate this important biological system.
| S-EPMC6333874 | biostudies-literature
Project description:Recently, studies of protein/peptide aggregation, particularly the amyloidosis, have attracted considerable attention in discussions of the pathological mechanisms of most neurodegenerative diseases. The protein/peptide aggregation processes often occur at the membrane-cytochylema interface in vivo and behave differently from those occurring in bulk solution, which raises great interest to investigate how the interfacial properties of artificial biomaterials impact on protein aggregation. From the perspective of bionics, current progress in this field has been obtained mainly from four aspects: (1) hydrophobic-hydrophilic interfaces; (2) charged surface; (3) chiral surface; and (4) biomolecule-related interfaces. The specific physical and chemical environment provided by these interfaces is reported to strongly affect the adsorption of proteins, transition of protein conformation, and diffusion of proteins on the biointerface, all of which are ultimately related to protein assembly. Meanwhile, these compelling results of in vitro experiments can greatly promote the development of early diagnostics and therapeutics for the relevant neurodegenerative diseases. This paper presents a brief review of these appealing studies, and particular interests are placed on weak interactions (i.e., hydrogen bonding and stereoselective interactions) that are also non-negligible in driving amyloid aggregation at the interfaces. Moreover, this paper also proposes the future perspectives, including the great opportunities and challenges in this field as well.
| S-EPMC5457079 | biostudies-other
Project description:Members of the IFN-inducible PYHIN protein family, such as absent in melanoma-2 and IFN-?-inducible protein (IFI)16, bind dsDNA and form caspase-1-activating inflammasomes that are important in immunity to cytosolic bacteria, DNA viruses, or HIV. IFI16 has also been shown to regulate transcription of type I IFNs during HSV infection. The role of other members of the PYHIN protein family in the regulation of immune responses is much less clear. In this study, we identified an immune-regulatory function for a member of the murine PYHIN protein family, p205 (also called Ifi205). Examination of immune responses induced by dsDNA and other microbial ligands in bone marrow-derived macrophages lacking p205 revealed that inflammasome activation by dsDNA, as well as ligands that engage the NLRP3 inflammasome, was severely compromised in these cells. Further analysis revealed that p205-knockdown cells showed reduced expression of apoptosis-associated speck-like molecule containing CARD domain (Asc) at the protein and RNA levels. p205 knockdown resulted in reduced binding of actively transcribing RNA polymerase II to the endogenous Asc gene, resulting in decreased transcription and processing of Asc pre-mRNA. Deletion of p205 in B16 melanoma cells using CRISPR/Cas9 showed a similar loss of Asc expression. Ectopic expression of p205 induced expression of an Asc promoter-luciferase reporter gene. Together, these findings suggest that p205 controls expression of Asc mRNA to regulate inflammasome responses. These findings expand on our understanding of immune-regulatory roles for the PYHIN protein family.
| S-EPMC5929989 | biostudies-literature
Project description:The breach of proteostasis, leading to the accumulation of protein aggregates, is a hallmark of ageing and age-associated disorders, up to now well-established in neurodegeneration. Few studies have addressed the issue of dysfunctional cell response to protein deposition also for the cardiovascular system. However, the molecular basis of proteostasis decline in vascular cells, as well as its relation to ageing, are not understood. Recent studies have indicated the associations of Nrf2 transcription factor, the critical modulator of cellular stress-response, with ageing and premature senescence. In this report, we outline the significance of protein aggregation in physiological and premature ageing of murine and human endothelial cells (ECs). Our study shows that aged donor-derived and prematurely senescent Nrf2-deficient primary human ECs, but not those overexpressing dominant-negative Nrf2, exhibit increased accumulation of protein aggregates. Such phenotype is also found in the aortas of aged mice and young Nrf2 tKO mice. Ageing-related loss of proteostasis in ECs depends on Keap1, well-known repressor of Nrf2, recently perceived as a key independent regulator of EC function and protein S-nitrosation (SNO). Deposition of protein aggregates in ECs is associated with impaired autophagy. It can be counteracted by Keap1 depletion, S-nitrosothiol reductant or rapamycin treatment. Our results show that Keap1:Nrf2 protein balance and Keap1-dependent SNO predominate Nrf2 transcriptional activity-driven mechanisms in governing proteostasis in ageing ECs.
| S-EPMC7327977 | biostudies-literature
Project description:The inflammasomes are multiprotein complexes that activate caspase-1 in response to infections and stress, resulting in the secretion of pro-inflammatory cytokines. Here we report that I?B kinase ? (IKK?) is a critical negative regulator of apoptosis-associated specklike protein containing a C-terminal caspase-activation-andrecruitment (CARD) domain (ASC)-dependent inflammasomes. IKK? controls the inflammasome at the level of the adaptor ASC, which interacts with IKK? in the nucleus of resting macrophages in an IKK? kinase-dependent manner. Loss of IKK? kinase activity results in inflammasome hyperactivation. Mechanistically, the downstream nuclear effector IKK-related kinase (IKKi) facilitates translocation of ASC from the nucleus to the perinuclear area during inflammasome activation. ASC remains under the control of IKK? in the perinuclear area following translocation of the ASC/IKK? complex. Signal 2 of NLRP3 activation leads to inhibition of IKK? kinase activity through the recruitment of PP2A, allowing ASC to participate in NLRP3 inflammasome assembly. Taken together, these findings reveal a IKKi-IKK?-ASC axis that serves as a common regulatory mechanism for ASC-dependent inflammasomes.
| S-EPMC4298287 | biostudies-literature
Project description:Apoptosis-associated Speck-like protein containing a CARD (caspase activation and recruitment domain) (ASC), also called PYCARD/Target of Methylation-induced Silencing-1 (TMS1), was originally discovered as a protein that forms aggregates ("specks") in human leukemia cells treated with chemotherapeutic agents. Its expression was found to be silenced by methylation in many human tumors, preventing tumor cells from undergoing apoptosis and supporting its role as a tumor suppressor. Subsequently, ASC was also identified as a central adaptor molecule of the inflammasome complex, which mediates the secretion of inflammatory cytokines (i.e., IL-1? and IL-18). Inflammatory cytokines have been shown to mediate tumor-promoting functions. Thus, in the context of cancer development and progression, ASC may exert opposing functions, i.e., be either tumor-suppressing by inducing tumor cell apoptosis, or tumor-promoting by favoring secretion of inflammatory cytokines (by tumor cells and/or tumor infiltrating myeloid cells) within the tumor microenvironment. Here, we report and discuss this dual role of ASC by also considering the final contribution of each of its two main functions in several cancer types, taking into consideration the correlation between ASC expression, clinical correlates, and patients' survival. ASC and inflammasome targeting strategies are being developed. However, before the use of such treatments in clinical practice, it is fundamental to better dissect the role of ASC in different tumors, in order to privilege or avoid their use in those tumors in which ASC exerts an anti-tumor or pro-tumor function, respectively.
| S-EPMC7010858 | biostudies-literature
Project description:BackgroundDysregulated activation of the inflammasome is involved in various human diseases including acute cerebral ischemia, multiple sclerosis and sepsis. Though many inflammasome inhibitors targeting NOD-like receptor protein 3 (NLRP3) have been designed and developed, none of the inhibitors are clinically available. Growing evidence suggests that targeting apoptosis-associated speck-like protein containing a CARD (ASC), the oligomerization of which is the key event for the assembly of inflammasome, may be another promising therapeutic strategy. Lonidamine (LND), a small-molecule inhibitor of glycolysis used as an antineoplastic drug, has been evidenced to have anti-inflammation effects. However, its anti-inflammatory mechanism is still largely unknown.MethodsMiddle cerebral artery occlusion (MCAO), experimental autoimmune encephalomyelitis (EAE) and LPS-induced sepsis mice models were constructed to investigate the therapeutic and anti-inflammasome effects of LND. The inhibition of inflammasome activation and ASC oligomerization by LND was evaluated using western blot (WB), immunofluorescence (IF), quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA) in murine bone marrow-derived macrophages (BMDMs). Direct binding of LND with ASC was assessed using molecular mock docking, surface plasmon resonance (SPR), and drug affinity responsive target stability (DARTS).ResultsHere, we find that LND strongly attenuates the inflammatory injury in experimental models of inflammasome-associated diseases including autoimmune disease-multiple sclerosis (MS), ischemic stroke and sepsis. Moreover, LND blocks diverse types of inflammasome activation independent of its known targets including hexokinase 2 (HK2). We further reveal that LND directly binds to the inflammasome ligand ASC and inhibits its oligomerization.ConclusionsTaken together, our results identify LND as a broad-spectrum inflammasome inhibitor by directly targeting ASC, providing a novel candidate drug for the treatment of inflammasome-driven diseases in clinic.
| S-EPMC9798610 | biostudies-literature
Project description:NOD-like receptor family protein 3 (NLRP3)-mediated inflammasome activation promotes caspase-1-dependent production of interleukin-1β (IL-1β) and requires the adaptor protein ASC. Compared with the priming and activation mechanisms of the inflammasome signaling pathway, post-translational ubiquitination/deubiquitination mechanisms controlling inflammasome activation have not been clearly addressed. We here demonstrate that the deubiquitinating enzyme USP50 binds to the ASC protein and subsequently regulates the inflammasome signaling pathway by deubiquitinating the lysine 63-linked polyubiquitination of ASC. USP50 knockdown in human THP-1 cells and mouse bone marrow-derived macrophages shows a significant decrease in procaspase-1 cleavage, resulting in a reduced secretion of IL-1β and interleukin-18 (IL-18) upon treatment with NLRP3 stimuli and a reduction in ASC speck formation and oligomerization. Thus, we elucidate a novel regulatory mechanism of the inflammasome signaling pathway mediated by the USP50 deubiquitinating enzyme.
| S-EPMC5324553 | biostudies-literature
Project description:The inflammasome adaptor protein, ASC, contributes to both innate immune responses and inflammatory diseases via self-oligomerization, which leads to the activation of the protease, caspase-1. Here, we report that the cytosolic tyrosine kinases, FAK and Pyk2, are differentially involved in NLRP3 and AIM2 inflammasome activation. The inhibition of FAK and Pyk2 with RNA interference or chemical inhibitors dramatically abolished ASC oligomerization, caspase-1 activation, and IL-1? secretion in response to NLRP3 or AIM2 stimulation. Pyk2 is phosphorylated by the kinase Syk and relocalizes to the ASC specks upon NLRP3 inflammasome activation. Pyk2, but not FAK, could directly phosphorylate ASC at Tyr146, and only the phosphorylated ASC could participate in speck formation and trigger IL-1? secretion. Moreover, the clinical-trial-tested Pyk2/FAK dual inhibitor PF-562271 reduced monosodium urate-mediated peritonitis, a disease model used for studying the consequences of NLRP3 activation. Our results suggest that although Pyk2 and FAK are involved in inflammasome activation, only Pyk2 directly phosphorylates ASC and brings ASC into an oligomerization-competent state by allowing Tyr146 phosphorylation to participate ASC speck formation and subsequent NLRP3 inflammation.
| S-EPMC5087076 | biostudies-literature