Calpain-5 regulates inflammatory gene expression through IRX3

Abstract

Purpose : Hyperactive mutants of the cysteine protease Calpain-5 (CAPN5) cause Neovascular Inflammatory Vitreoretinopathy (NIV), an inherited retinal disease. The molecular mechanism(s) by which CAPN5 alters the expression of inflammatory genes remains unknown.

Methods : In vitro co-immunoprecipitation using inactive CAPN5 as a probe, followed by shotgun mass-spectrometry, was conducted to identify potential substrates of CAPN5. The interacting proteins were tested using proteolytic assays, including in vitro enzyme-substrate kinetic assays and proteolysis in cultured cells. Subcellular fractionization characterized changes in the nuclear localization of transcription factors before and after CAPN5 proteolysis. The downstream inflammatory genes were profiled in a cellular luciferase reporter system.

Results : Co-immunoprecipitation with a CAPN5 antibody identified 25 potential substrates of CAPN5, of which 8 (32%) were associated with gene transcription. IRX3 was the only transcription factor. In vitro proteolytic assays confirmed IRX3 was a proteolytic target of CAPN5. In cells, the CAPN5-processed IRX3 translocated to the nucleus. A luciferase reporter system suggested that IRX3 regulated IL-6 and IL-6RA.

Conclusions : CAPN5 proteolysis of IRX3 influences its nuclear localization, and this may regulate expression of NIV-associated inflammatory genes.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.