Biology subjects

Hochheiser, I. V.

Publications and source records attributed to Hochheiser, I. V..

3 recordsLinked to original sources

bioRxiv2023

Non-decameric NLRP3 forms an MTOC-independent inflammasome

NLRP3 is an inflammasome-forming protein that plays a key role in conditions ranging from infections to Alzheimers disease. NLRP3 is activated by many potassium (K+)-dependent or - independent stimuli related to pathogens or sterile insults. Molecularly, human NLRP3 assembles into a decameric cage and its interaction with both the trans-Golgi network (TGN) and the microtubule organization centre (MTOC) has been proposed as critical for NLRP3 activation. However, the relative mechanistic requirement for K+-dependent versus - independent stimuli is unclear. Using spatially and dynamically high-resolution microscopy in human macrophages, we found that K+-dependent NLRP3 stimulation triggers two distinct activation pathways: (i) the decameric cage-dependent pathway, which was also exclusively required for the K+-independent stimulus, imiquimod; and (ii) a cage- and TGN/MTOC-independent pathway that was fully functional in an NLRP3 protein engineered to form monomers and unable to interact with membrane lipids. Collectively, our results delineate two parallel yet biologically distinct NLRP3 activation pathways.

molecular biology↗

bioRxiv2021

Directionality of PYD filament growth determined by the transition of NLRP3 nucleation seeds to ASC elongation

Inflammasomes sense intrinsic and extrinsic danger signals to trigger inflammatory responses and pyroptotic cell death. Homotypic pyrin domain (PYD) interactions of inflammasome forming Nod-like receptors with the adaptor protein ASC mediate oligomerization into helical filamentous assemblies. These supramolecular organizing centers recruit and activate caspase-1, which results in IL-1{beta} family cytokine maturation and pyroptotic cell death. The molecular details of the critical step in signal transduction of inflammasome signaling, however, remain ill-defined. Here, we describe the cryo-EM structure of the human NLRP3 PYD filament at 3.6 [A] resolution. We identify a unique pattern of highly polar interface residues that form the homomeric interactions leading to characteristic filament ends that we designate as A- and B-end, respectively. Coupling a titration polymerization assay to cryo-EM, we demonstrate that the ASC adaptor protein elongation on NLRP3 PYD filament seeds is unidirectional, associating exclusively to the B-end of the NLRP3 filament. Notably, NLRP3 and ASC PYD filaments exhibit the same symmetry in rotation and axial rise per subunit, allowing for a continuous transition between NLRP3 as the nucleation seed and ASC as the elongator. Integrating the directionality of filament growth, we present a molecular model of the ASC speck consisting of active NLRP3-NEK7, ASC, and Caspase-1 proteins.

immunology↗

bioRxiv2021

Cryo-EM structure of the NLRP3 decamer bound to the cytokine release inhibitory drug CRID3

NLRP3 is an intracellular sensor protein whose activation by a broad spectrum of exogenous and endogenous stimuli leads to inflammasome formation and pyroptosis. The mechanisms leading to NLRP3 activation and the way how antagonistic small molecules function remain poorly understood. Here we report the cryo-electron microscopy structures of full-length NLRP3 in its native form and complexed with the inhibitor CRID3 (also named MCC950). Inactive, ADP-bound NLRP3 is a decamer composed of homodimers of intertwined LRR domains that assemble back-to-back as pentamers with the NACHT domain located at the apical axis of this spherical structure. Molecular contacts between the concave sites of two opposing LRRs are mediated by an acidic loop extending from an LRR transition segment. Binding of CRID3 significantly stabilizes the NACHT and LRR domains relative to each other, allowing structural resolution of 3.9-4.2 [A]. CRID3 binds into a cleft, connecting four subdomains of the NACHT with the transition LRR. Its central sulfonylurea group interacts with the Walker A motif of the NLRP3 nucleotide-binding domain and is sandwiched between two arginines from opposing sites, explaining the specificity of NLRP3 for this chemical entity. With the determination of the binding site of this lead therapeutic, specific targeting of NLRP3 for the treatment of autoinflammatory and autoimmune diseases and rational drug optimization are within reach.

immunology↗