Dataset Information

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

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			Action	DRS
	20181212_BertrandJ_chemprot_F01.raw	Raw
	20181212_BertrandJ_chemprot_F02.raw	Raw
	20181212_BertrandJ_chemprot_F03.raw	Raw
	20181212_BertrandJ_chemprot_F04.raw	Raw
	20181212_BertrandJ_chemprot_F05.raw	Raw

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

Grabert Kathleen K Engskog-Vlachos Pinelopi P Škandík Martin M Vazquez-Cabrera Guillermo G Murgoci Adriana-Natalia AN Keane Lily L Gaetani Massimiliano M Joseph Bertrand B Cheray Mathilde M

Cell death & disease 20230311 3

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 fulfill 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 non-apoptotic roles of caspase-3 (CASP3 ...[more]

PMID: 36906641

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Project description:Background: Despite current therapeutic treatments including surgery, chemotherapy, radiotherapy and recently immunotherapy, the mortality rate of lung cancer stays high. Regarding lung cancer, epigenetic modifications altering cell cycle, angiogenesis and programmed cancer cell death are therapeutic targets to combine with immunotherapy to improve treatment success. In a recent study, we uncovered a dual function of a molecule called QAPHA ((E)-3-(5-((2-cyanoquinolin-4-yl)(methyl)amino)-2-methoxyphenyl)-N-hydroxyacrylamide) as both a tubulin polymerization and HDAC inhibitors. Here, we investigate the impact of this novel dual inhibitor on the lung cancer immune response. Methods: To elucidate the mechanism of action of QAPHA, we conducted a chemical proteomics analysis. Using an in vivo mouse model of lung cancer (TC-1 tumor cells), we assessed the effects of QAPHA on tumor regression. Tumor infiltrating immune cells were characterize by flow cytometry. Results In this study, we first show that QAPHA can inhibit HDAC6, leading to upregulation of HSP90, cytochrome C and caspases, found by proteomic analysis. We also confirm that QAPHA induces immunogenic cell death (ICD) by upregulating HMGB1 in vitro and demonstrated its effectiveness as a vaccine in vivo. Remarkably, even at a low concentration (0.5mg/kg), QAPHA achieved complete tumor regression in approximately 60% of intratumorally treated mice, establishing a long-lasting anticancer immune response. Moreover, QAPHA treatment promoted infiltration of neutrophils in the treated mice, reflecting inflammatory cell death promoted. Very interestingly, we show that QAPHA is also able to upregulate MHC-II expression on TC-1 tumor cells in vitro and in vivo. This process participates to the recruitment of CD4+ NKG2D+ CRTAM+ Perforin+ T cells in tumor infiltrate, defined as cytotoxic CD4+ T cells (CD4+ CTL). Finally, we show that tumor regression is strongly correlated to MHC-II expression level on tumor cell and CD4+ CTL infiltrate. Conclusion Collectively, our findings shed light on the discovery of a new multi-target inhibitor able to induce ICD and MHC-II upregulation in TC-1 tumor cell. These two processes participate to enhance a specific CD4+ cytotoxic T cell-mediated anti-tumor response in vivo in our model of lung cancer. This breakthrough suggests the potential of QAPHA as a promising therapeutic agent for cancer treatment.

Dataset Information

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

Dataset's files

Publications

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

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