Influence of the naturally occurring human CASP1 variant L265S on subcellular distribution and pyroptosis

Introduction

Patients with unexplained recurrent febrile episodes and CASP1 variants suffer from systemic sterile inflammation despite altered enzymatic activity of procaspase-1 and reduced IL-1β release. Most recent findings from our group indicate that the proinflammatory effects of CASP1 variants with reduced or abrogated enzymatic activity could be due to receptor interacting protein kinase 2 (RIP2) mediated increase of NF-kB activation. These findings are additionally supported by a trend to elevated IL-6 and TNF-α expression in patients with CASP1 variants.

Objectives

The objective of this project is the identification of possible subcellular mechanisms how the CASP1-L265S variant influences proinflammatory cell death (pyroptosis) and IL-1β secretion.

Methods

We established an in vitro model of a virally transduced human monocytic cell line (THP-1 with shRNA knock-down of endogenous procaspase-1), expressing either wild type or enzymatically inactive (L265S) procaspase-1 fusion-reporter proteins and characterized them after NLRP3-inflammasome stimulation. Using confocal microscopy and in vivo live cell imaging we analyzed the subcellular distribution of fluorescently labeled procaspase-1 wildtype and variant as well as the interaction with ASC.

Results

First results revealed a disturbed nuclear localization of the CASP1-L265S variant compared to procaspase-1 wildtype. Furthermore, CASP1-L265S variant revealed a strongly decreased pyroptosome formation and a less intense interaction with ASC (apoptosis-associated speck-like protein containing a CARD) after NLRP3-stimulation. Variant procaspase-1 L265S and ASC formed smaller pyroptosomes than wildtype procaspase-1and ASC.

Conclusion

Those findings suggest a model, in which variant procaspase-1 L265S impairs nuclear localization, pyroptosome formation and ASC-interaction, leading all together to reduced IL-1β production and secretion.