Mechanism of filament formation in UPA-promoted CARD8 and NLRP1 inflammasomes

L Robert Hollingsworth,Daniel A Bachovchin,Andrew R Griswold,Humayun Sharif,Tian-Min Fu,Hao Wu,Pietro Fontana,Yang Li,Liron David,Jianbin Ruan,Elizabeth L Orth-He

doi:10.1038/s41467-020-20320-y

Abstract

NLRP1 and CARD8 are related cytosolic sensors that upon activation form supramolecular signalling complexes known as canonical inflammasomes, resulting in caspase−1 activation, cytokine maturation and/or pyroptotic cell death. NLRP1 and CARD8 use their C-terminal (CT) fragments containing a caspase recruitment domain (CARD) and the UPA (conserved in UNC5, PIDD, and ankyrins) subdomain for self-oligomerization, which in turn form the platform to recruit the inflammasome adaptor ASC (apoptosis-associated speck-like protein containing a CARD) or caspase-1, respectively. Here, we report cryo-EM structures of NLRP1-CT and CARD8-CT assemblies, in which the respective CARDs form central helical filaments that are promoted by oligomerized, but flexibly linked, UPAs surrounding the filaments. Through biochemical and cellular approaches, we demonstrate that the UPA itself reduces the threshold needed for NLRP1-CT and CARD8-CT filament formation and signalling. Structural analyses provide insights on the mode of ASC recruitment by NLRP1-CT and the contrasting direct recruitment of caspase-1 by CARD8-CT. We also discover that subunits in the central NLRP1CARD filament dimerize with additional exterior CARDs, which roughly doubles its thickness and is unique among all known CARD filaments. Finally, we engineer and determine the structure of an ASCCARD–caspase-1CARD octamer, which suggests that ASC uses opposing surfaces for NLRP1, versus caspase-1, recruitment. Together these structures capture the architecture and specificity of the active NLRP1 and CARD8 inflammasomes in addition to key heteromeric CARD-CARD interactions governing inflammasome signalling.

Highlights

NLRP1 and CARD8 are related cytosolic sensors that upon activation form supramolecular signalling complexes known as canonical inflammasomes, resulting in caspase−1 activation, cytokine maturation and/or pyroptotic cell death
To elucidate if and how CARD8-CT and NLRP1-CT form filaments, we expressed these CTs in fusion with an N-terminal maltosebinding protein (MBP) tag separated by a linker cleavable by the human rhinovirus (HRV) 3C protease
We showed that NLRP1-CT and CARD8-CT use their caspase recruitment domain (CARD) to assemble filamentous structures for inflammasome signaling

Summary

Introduction

NLRP1 and CARD8 are related cytosolic sensors that upon activation form supramolecular signalling complexes known as canonical inflammasomes, resulting in caspase−1 activation, cytokine maturation and/or pyroptotic cell death. Nucleotide binding domains (NBDs) often drive this self-oligomerization to cluster death-fold domains[5,6], which in turn recruit downstream adapter and effector molecules These death-fold domains, including pyrin domains (PYD) and caspase recruitment domains (CARD), participate in homotypic (CARD–CARD or PYD–PYD) interactions that lead to polymerization of caspase-1 into filaments[3,4,7,8,9]. Keratinocytes constitutively express high levels of NLRP1 and germline mutations of NLRP1 in humans lead to a number of skin-related inflammatory diseases, including multiple self-healing palmoplantar carcinoma (MSPC), familial keratosis lichenoides chronica (FKLC)[13], vitiligo[14,15], autoinflammation with arthritis and dyskeratosis (AIADK)[16,17], and juvenile-onset recurrent respiratory papillomatosis (JRRP)[18] These mutations cause constitutive NLRP1 activation and downstream pyroptosis[13,16], leading to damaging inflammation. Activated CARD8 and NLRP1 inflammasomes are distinctive in that they are only composed of UPA and CARD, unlike others such as NLRC4 and NLRP3 inflammasomes which use NBDs and leucine-rich repeats (LRRs) to facilitate oligomerization and inflammasome activation[4,5,6,30]

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Results

Conclusion