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 AIM2PYDfilament reveals that the architecture of the upstream filament is essentially identical to that of the adaptor ASCPYDfilament. 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.
Upon sensing cytosolic- and/or viral double-stranded (ds)DNA, absent-in-melanoma-2 (AIM2)-like-receptors (ALRs) assemble into filamentous signaling platforms to initiate inflammatory responses. The versatile yet critical roles of ALRs in host innate defense are increasingly appreciated; however, the mechanisms by which AIM2 and its related IFI16 specifically recognize dsDNA over other nucleic acids remain poorly understood (i.e. single-stranded (ss)DNA, dsRNA, ssRNA and DNA:RNA hybrid). Here, we find that although AIM2 can interact with various nucleic acids, it preferentially binds to and assembles filaments faster on dsDNA in a duplex length-dependent manner. Moreover, AIM2 oligomers assembled on nucleic acids other than dsDNA not only display less ordered filamentous structures, but also fail to induce the polymerization of downstream ASC. Likewise, although showing broader nucleic acid selectivity than AIM2, IFI16 binds to and oligomerizes most readily on dsDNA in a duplex length-dependent manner. Nevertheless, IFI16 fails to form filaments on single-stranded nucleic acids and does not accelerate the polymerization of ASC regardless of bound nucleic acids. Together, we reveal that filament assembly is integral to nucleic acid distinction by ALRs.
more » « less- Award ID(s):
- 1845003
- PAR ID:
- 10400078
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 51
- Issue:
- 6
- ISSN:
- 0305-1048
- Format(s):
- Medium: X Size: p. 2574-2585
- Size(s):
- p. 2574-2585
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
Abstract -
more » « less
Abstract Pathogenic dsDNA prompts AIM2 assembly leading to the formation of the inflammasome, a multimeric complex that triggers the inflammatory response. The recognition of foreign dsDNA involves AIM2 self-assembly concomitant with dsDNA binding. However, we lack mechanistic and kinetic information on the formation and propagation of the assembly, which can shed light on innate immunity’s time response and specificity. Combining optical traps and confocal fluorescence microscopy, we determine here the association and dissociation rates of the AIM2-DNA complex at the single molecule level. We identify distinct mechanisms for oligomer growth via the binding of incoming AIM2 molecules to adjacent dsDNA or direct interaction with bound AIM2 assemblies, resembling primary and secondary nucleation. Through these mechanisms, the size of AIM2 oligomers can increase fourfold in seconds. Finally, our data indicate that single AIM2 molecules do not diffuse/scan along the DNA, suggesting that oligomerization depends on stochastic encounters with DNA and/or DNA-bound AIM2.
-
more » « less
Abstract AIM2 is a cytosolic innate immune receptor which recognizes double‐stranded DNA (dsDNA) released during cellular perturbation and pathogenic assault. AIM2 recognition of dsDNA leads to the assembly of a large multiprotein oligomeric complex termed the inflammasome. This inflammasome assembly leads to the secretion of bioactive interleukin‐1β (IL‐1β) and IL‐18 and induction of an inflammatory form of cell death called pyroptosis. Sensing of dsDNA by AIM2 in the cytosol is crucial to mediate protection against the invading pathogens including bacteria, virus, fungi and parasites. AIM2 also responds to dsDNA released from damaged host cells, resulting in the secretion of the effector cytokines thereby driving the progression of sterile inflammatory diseases such as skin disease, neuronal disease, chronic kidney disease, cardiovascular disease and diabetes. Additionally, the protection mediated by AIM2 in the development of colorectal cancer depends on its ability to regulate epithelial cell proliferation and gut microbiota in maintaining intestinal homeostasis independently of the effector cytokines. In this review, we will highlight the recent progress on the role of the AIM2 inflammasome as a guardian of cellular integrity in modulating chronic inflammatory diseases, cancer and infection.
-
more » « less
Abstract Poly (ADP)-ribose polymerase 1 (PARP1) is an abundant nuclear protein well-known for its role in DNA repair yet also participates in DNA replication, transcription, and co-transcriptional splicing, where DNA is undamaged. Thus, binding to undamaged regions in DNA and RNA is likely a part of PARP1’s normal repertoire. Here we describe analyses of PARP1 binding to two short single-stranded DNAs, a single-stranded RNA, and a double stranded DNA. The investigations involved comparing the wild-type (WT) full-length enzyme with mutants lacking the catalytic domain (∆CAT) or zinc fingers 1 and 2 (∆Zn1∆Zn2). All three protein types exhibited monomeric characteristics in solution and formed saturated 2:1 complexes with single-stranded T20and U20oligonucleotides. These complexes formed without accumulation of 1:1 intermediates, a pattern suggestive of positive binding cooperativity. The retention of binding activities by ∆CAT and ∆Zn1∆Zn2 enzymes suggests that neither the catalytic domain nor zinc fingers 1 and 2 are indispensable for cooperative binding. In contrast, when a double stranded 19mer DNA was tested, WT PARP1 formed a 4:1 complex while the ∆Zn1Zn2 mutant binding saturated at 1:1 stoichiometry. These deviations from the 2:1 pattern observed with T20and U20oligonucleotides show that PARP’s binding mechanism can be influenced by the secondary structure of the nucleic acid. Our studies show that PARP1:nucleic acid interactions are strongly dependent on the nucleic acid type and properties, perhaps reflecting PARP1’s ability to respond differently to different nucleic acid ligands in cells. These findings lay a platform for understanding how the functionally versatile PARP1 recognizes diverse oligonucleotides within the realms of chromatin and RNA biology.
-
more » « less
Inflammasomes are filamentous signaling platforms essential for host defense against various intracellular calamities such as pathogen invasion and genotoxic stresses. However, dysregulated inflammasomes cause an array of human diseases including autoinflammatory disorders and cancer. It was recently identified that endogenous pyrin-only-proteins (POPs) regulate inflammasomes by directly inhibiting their filament assembly. Here, by combining Rosetta in silico, in vitro, and in cellulo methods, we investigate the target specificity and inhibition mechanisms of POPs. We find here that POP1 is ineffective in directly inhibiting the central inflammasome adaptor ASC. Instead, POP1 acts as a decoy and targets the assembly of upstream receptor pyrin-domain (PYD) filaments such as those of AIM2, IFI16, NLRP3, and NLRP6. Moreover, not only does POP2 directly suppress the nucleation of ASC, but it can also inhibit the elongation of receptor filaments. In addition to inhibiting the elongation of AIM2 and NLRP6 filaments, POP3 potently suppresses the nucleation of ASC. Our Rosetta analyses and biochemical experiments consistently suggest that a combination of favorable and unfavorable interactions between POPs and PYDs is necessary for effective recognition and inhibition. Together, we reveal the intrinsic target redundancy of POPs and their inhibitory mechanisms.
