Proteome integral solubility alteration high-throughput proteomics assay identifies Collectin-12 as a non-apoptotic microglial caspase-3 substrate.

ABSTRACT: Caspases are a family of proteins mostly known for their role in the activation of the apoptotic pathway leading to cell death. In the last decade, caspases have been found to fulfil other tasks regulating the cell phenotype independently to cell death. Microglia are the immune cells of the brain responsible for the maintenance of physiological brain functions but can also be involved in disease progression when overactivated. We have previously described non30 apoptotic roles of caspase-3 (CASP3) in the regulation of the inflammatory phenotype of microglial cells or pro-tumoral activation in the context of brain tumors. CASP3 can regulate protein functions by cleavage of their target and therefore could have multiple substrates. So far, identification of CASP3 substrates has been performed mostly in apoptotic conditions where CASP3 activity is highly upregulated and these approaches do not have the capacity to uncover CASP3 substrates at the physiological level. In our study, we aim at discovering highaffinity substrates of CASP3 involved in the normal regulation of the cell. We used an unconventional approach by chemically reducing the basal level activity of CASP3 (by DEVD38 fmk treatment) coupled to a Mass Spectrometry screen (PISA) to identify stabilized, and consequently, non-cleaved proteins in microglia cells. PISA identified several significantly stabilized proteins after DEVD-fmk treatment, including a few already known CASP3substrates which validated our approach. Among them, we focused on the Collectin12 (COLEC12 or CL-P1) transmembrane receptor and uncovered a potential role for CASP3 cleavage of COLEC12 in the regulation of the phagocytic capacity of microglial cells. Taken together, these findings suggest a new way to uncover non-apoptotic high-affinity substrates of CASP3 important for the modulation of microglia cell physiology.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Microglial Cell Line

SUBMITTER: Massimiliano Gaetani  

LAB HEAD: Massimiliano Gaetani

PROVIDER: PXD032835 | Pride | 2023-05-10

REPOSITORIES: Pride