Biology subjects

Fontana, P.

Publications and source records attributed to Fontana, P..

3 recordsLinked to original sources

bioRxiv2020

DPP9 directly sequesters the NLRP1 C-terminus to repress inflammasome activation

NLRP1 is a cytosolic inflammasome sensor that mediates activation of caspase-1, which in turn induces cytokine maturation and pyroptotic cell death1-6. Gain-of-function NLPR1 mutations cause skin inflammatory diseases including carcinoma, keratosis, and papillomatosis7-14. NLRP1 contains a unique function-to-find domain (FIIND) that autoproteolyzes into noncovalently associated subdomains15-18. Proteasomal degradation of the autoinhibitory N-terminal fragment (NT) activates NLRP1 by releasing the inflammatory C-terminal fragment (CT)19,20. Cytosolic dipeptidyl peptidases 8 and 9 (DPP8/9) interact with NLRP1, and small-molecule DPP8/9 inhibitors activate NLRP1 by poorly characterized mechanisms11,19,21. Here, we report cryo-EM structures of the human NLRP1-DPP9 complex, alone and in complex with the DPP8/9 inhibitor Val-boroPro (VbP). Surprisingly, the NLRP1-DPP9 complex is a ternary complex comprised of DPP9, one intact FIIND of a non-degraded full-length NLRP1 (NLRP1-FL) and one NLRP1-CT freed by NT degradation. The N-terminus of the NLRP1-CT unfolds and inserts into the DPP9 active site but is not cleaved by DPP9, and this binding is disrupted by VbP. Structure-based mutagenesis reveals that the binding of NLRP1-CT to DPP9 requires NLRP1-FL and vice versa, and inflammasome activation by ectopic NLRP1-CT expression is rescued by co-expressing autoproteolysis-deficient NLRP1-FL. Collectively, these data indicate that DPP9 functions as a "bomb-diffuser" to prevent NLRP1-CTs from inducing inflammation during homeostatic protein turnover.

immunology↗

bioRxiv2020

Novel mode of filament formation in UPA-promoted CARD8 and NLRP1 Inflammasomes

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 subdomain of a function-to-find domain (FIIND) for self-oligomerization and recruitment of the inflammasome adaptor ASC and/or caspase-1. 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. We 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. The thick NLRP1 filament only forms with the presence of UPA, which we hypothesize drives the intrinsic propensity for NLRP1CARD dimerization. Structural analyses provide insights on the requirement of ASC for NLRP1-CT signalling and the contrasting direct recruitment of caspase-1 by CARD8-CT. Additionally, we present a low-resolution 4 ASCCARD–4 caspase-1CARD octamer structure, illustrating that ASC uses opposing surfaces for NLRP1, versus caspase-1, recruitment. These structures capture the architecture and specificity of CARD inflammasome polymerization in NLRP1 and CARD8.Competing Interest StatementH.W. is a co-founder of Ventus Therapeutics. The other authors declare no competing financial interests.View Full Text

immunology↗

bioRxiv2020

A new perspective on the evolution of the interaction between the Vg/VGLL1-3 proteins and the TEAD transcription factors

The most downstream elements of the Hippo pathway, the TEAD transcription factors, are regulated by several cofactors, such as Vg/VGLL1-3. Earlier findings on human VGLL1 and here on human VGLL3 show that these proteins interact with TEAD via a conserved amino acid motif called the TONDU domain. Surprisingly, our studies reveal that the TEAD-binding domain of Drosophila Vg and of human VGLL2 is more complex and contains an additional structural element, an {Omega}-loop, that contributes to TEAD binding and in vivo function. To explain this unexpected structural difference between proteins from the same family, we propose that, after the genome-wide duplications at the origin of vertebrates, the {Omega}-loop present in an ancestral VGLL gene has been lost in some VGLL variants. These findings illustrate how structural and functional constraints can guide the evolution of transcriptional cofactors to preserve their ability to compete with other cofactors for binding to transcription factors.

biochemistry↗