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Distinct axial and lateral interactions within homologous filaments dictate the signaling specificity and order of the AIM2-ASC inflammasome.

ABSTRACT: Inflammasomes are filamentous signaling platforms integral to innate immunity. Currently, little is known about how these structurally similar filaments recognize and distinguish one another. A cryo-EM structure of the AIM2^PYD filament reveals that the architecture of the upstream filament is essentially identical to that of the adaptor ASC^PYD filament. In silico simulations using Rosetta and molecular dynamics followed by biochemical and cellular experiments consistently demonstrate that individual filaments assemble bidirectionally. By contrast, the recognition between AIM2 and ASC requires at least one to be oligomeric and occurs in a head-to-tail manner. Using in silico mutagenesis as a guide, we also identify specific axial and lateral interfaces that dictate the recognition and distinction between AIM2 and ASC filaments. Together, the results here provide a robust framework for delineating the signaling specificity and order of inflammasomes.

SUBMITTER: Matyszewski M

PROVIDER: S-EPMC8115694 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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Distinct axial and lateral interactions within homologous filaments dictate the signaling specificity and order of the AIM2-ASC inflammasome.

Matyszewski Mariusz M Zheng Weili W Lueck Jacob J Mazanek Zachary Z Mohideen Naveen N Lau Albert Y AY Egelman Edward H EH Sohn Jungsan J

Nature communications 20210512 1

Inflammasomes are filamentous signaling platforms integral to innate immunity. Currently, little is known about how these structurally similar filaments recognize and distinguish one another. A cryo-EM structure of the AIM2<sup>PYD</sup> filament reveals that the architecture of the upstream filament is essentially identical to that of the adaptor ASC<sup>PYD</sup> filament. In silico simulations using Rosetta and molecular dynamics followed by biochemical and cellular experiments consistently d ...[more]

PMID: 33980849

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Project description:Macroautophagy/autophagy plays an important role in regulating cellular homeostasis and influences the pathogenesis of degenerative diseases. Tendinopathy is characterized by tendon degeneration and inflammation. However, little is known about the role of selective autophagy in tendinopathy. Here, we find that pristimerin (PM), a quinone methide triterpenoid, is more effective in treating tendinopathy than the first-line drug indomethacin. PM inhibits the AIM2 inflammasome and alleviates inflammation during tendinopathy by promoting the autophagic degradation of AIM2 through a PYCARD/ASC-dependent manner. A mechanistic study shows that PM enhances the K63-linked ubiquitin chains of PYCARD/ASC at K158/161, which serves as a recognition signal for SQSTM1/p62-mediated autophagic degradation of the AIM2-PYCARD/ASC complex. We further identify that PM binds the Cys53 site of deubiquitinase USP50 through the Michael-acceptor and blocks the binding of USP50 to PYCARD/ASC, thereby reducing USP50-mediated cleavage of K63-linked ubiquitin chains of PYCARD/ASC. Finally, PM treatment in vivo generates an effect comparable to inflammasome deficiency in alleviating tendinopathy. Taken together, these findings demonstrate that PM alleviates the progression of tendinopathy by modulating AIM2-PYCARD/ASC stability via SQSTM1/p62-mediated selective autophagic degradation, thus providing a promising autophagy-based therapeutic for tendinopathy.Abbreviations: 3-MA: 3-methyladenine; AIM2: absent in melanoma 2; AT: Achilles tenotomy; ATP: adenosine triphosphate; BMDMs: bone marrow-derived macrophages; CHX: cycloheximide; Col3a1: collagen, type III, alpha 1; CQ: chloroquine; Cys: cysteine; DARTS: drug affinity responsive target stability; DTT: dithiothreitol; DUB: deubiquitinase; gDNA: genomic DNA; GSH: glutathione; His: histidine; IL1B/IL-1β: interleukin 1 beta; IND: indomethacin; IP: immunoprecipitation; LPS: lipopolysaccharide; MMP: mitochondrial membrane potential; NLRP3: NLR family, pyrin domain containing 3; PM: pristimerin; PYCARD/ASC: PYD and CARD domain containing; SN: supernatants; SOX9: SRY (sex determining region Y)-box 9; SQSTM1: sequestosome 1; Tgfb: transforming growth factor, beta; TIMP3: tissue inhibitor of metalloproteinase 3; TNMD: tenomodulin; TRAF6: TNF receptor-associated factor 6; Ub: ubiquitin; USP50: ubiquitin specific peptidase 50; WCL: whole cell lysates.

Project description:BackgroundAIM2, a cytosolic DNA sensor, plays an important role during infection caused by pathogens with double-stranded DNA; however, its role in human cytomegalovirus (HCMV) infection remains unclear. Previously, we showed an increase in AIM2 protein levels during the early stage of HCMV infection and a decrease 24 h post infection. Because HCMV has developed a variety of strategies to evade host immunity, we speculated that this decline might be attributed to a viral immune escape mechanism. The tegument protein pUL83 is an important immune evasion protein and several studies have reported that pUL83 binds to specific cellular proteins, such as AIM2-like receptor IFI16, to affect their functions. To determine whether pUL83 contributes to the variation in AIM2 levels during HCMV infection, we investigated the pUL83/AIM2 interaction and its impact on the AIM2 inflammasome activation.MethodsWe constructed plasmids expressing recombinant pUL83 and AIM2 proteins for two-hybrid and chemiluminescence assays. Using co-immunoprecipitation and immunofluorescent co-localization, we confirmed the interaction of pUL83/AIM2 in THP-1-derived macrophages infected with HCMV AD169 strain. Furthermore, by investigating the expression and cleavage of inflammasome-associated proteins in recombinant HEK293T cells expressing AIM2, apoptosis-associated speck-like protein (ASC), pro-caspase-1 and pro-IL-1β, we evaluated the effect of pUL83 on the AIM2 inflammasome.ResultsAn interaction between pUL83 and AIM2 was detected in macrophages infected with HCMV as well as in transfected HEK293T cells. Moreover, transfection of the pUL83 expression vector into recombinant HEK293T cells stimulated by poly(dA:dT) resulted in reduced expression and activation of AIM2 inflammasome-associated proteins, compared with the absence of pUL83.ConclusionsOur data indicate that pUL83 interacts with AIM2 in the cytoplasm during the early stages of HCMV infection. The pUL83/AIM2 interaction deregulates the activation of AIM2 inflammasome. These findings reveal a new strategy of immune evasion developed by HCMV, which may facilitate latent infection.

Dataset Information

Distinct axial and lateral interactions within homologous filaments dictate the signaling specificity and order of the AIM2-ASC inflammasome.

Publications

Distinct axial and lateral interactions within homologous filaments dictate the signaling specificity and order of the AIM2-ASC inflammasome.

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