PW03-027 - CASP1 variants and live cell imaging
Pediatric Rheumatology volume 11, Article number: A253 (2013)
Patients with unexplained recurrent febrile episodes and CASP1 variants suffer from systemic sterile inflammation despite reduced secretion of IL-1ß. As previously demonstrated by our group CASP1 variants lead to reduced enzymatic activity of procaspase-1 by destabilizing the tertiary structure of the caspase-1 tetramer. A possible explanation for an alternative pro-inflammatory pathway has been provided by Lamkanfi and colleagues indicating an association between enzymatically inactive procaspase-1 and receptor interacting protein kinase 2 (RIP2) leading to NF-kB activation.
The objective of this project is the identification of possible subcellular mechanisms how CASP1 variants interfere with the IL-1β production or release and lead to the activation of alternative pro-inflammatory pathways.
Using confocal microscopy, in vivo live cell imaging and an in situ proximity ligation assay we analyzed the subcellular distribution of procaspase-1 wildtype and mutants as well as the interaction with RIP2 in naïve or virally transduced THP-1 cells.
THP-1 cells were virally transduced with GFP- or mCherry fusion proteins of procaspase-1 wildtype and variant CASP1-L 265S. Procaspase-1 activation, initiated by the assembly of multiprotein complexes (inflammasomes), was induced by stimulation with LPS and Nigericin. First results suggest disturbed microvesicle shedding from CASP1-L 265S expressing cells after administration of Nigericin. In addition to live cell imaging, the interaction of procaspase-1 and RIP2 has been studied in vitro in naïve THP-1 cells using antibody labeling and proximity ligation assay showing a time dependency after LPS stimulation.
This result suggests a possible influence of procaspase-1 variants on plasma membrane properties, pyroptosis and the release of microvesicles.
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Rabe, S., Heymann, M., Russ, S. et al. PW03-027 - CASP1 variants and live cell imaging. Pediatr Rheumatol 11 (Suppl 1), A253 (2013). https://doi.org/10.1186/1546-0096-11-S1-A253