We report the identification and characterization of a five-carbon protein post-translational modification (PTM) called lysine glutarylation (Kglu). This protein modification was detected by immunoblot and mass spectrometry (MS), and then comprehensively validated by chemical and biochemical methods. We demonstrated that the previously annotated deacetylase, sirtuin 5 (SIRT5), is a lysine deglutarylase. Proteome-wide analysis identified 683 Kglu sites in 191 proteins and showed Kglu is highly enriched on metabolic enzymes and mitochondrial proteins. We validated carbamoyl phosphate synthase 1 (CPS1), the rate-limiting enzyme in urea cycle, as a glutarylated protein and demonstrated that CPS1 is targeted by SIRT5 for deglutarylation. We further showed that glutarylation suppresses CPS1 enzymatic activity in cell lines, mice, and a model of glutaric academia type I disease, the last of which has elevated glutaric acid and glutaryl-CoA. This study expands the landscape of lysine acyl modifications and increases our understanding of the deacylase SIRT5.

Highlights d Pro-ferroptotic stimuli induce expression of the pentaspanin protein Prominin2 d Prominin2 facilitates ferroptosis resistance in epithelial and carcinoma cells d Prominin2 promotes formation of ferritin-containing multivesicular bodies and exosomes d Exosomal transport of ferritin out of the cell inhibits ferroptosis

Understanding how cancer cells resist ferroptosis is a significant problem that impacts ongoing efforts to stimulate ferroptosis as a therapeutic strategy. We reported that prominin2 is induced by ferroptotic stimuli and functions to resist ferroptotic death. Although this finding has significant implications for therapy, specific prominin2 inhibitors are not available. We rationalized that the mechanism by which prominin2 expression is induced by ferroptotic stress could be targeted, expanding the range of options to overcome ferroptosis resistance. Here, we show that that 4‐hydroxynonenal (4HNE), a specific lipid metabolite formed from the products of lipid peroxidation stimulates PROM2 transcription by a mechanism that involves p38 MAP kinase‐mediated activation of HSF1 and HSF1‐dependent transcription of PROM2. HSF1 inhibitors sensitize a wide variety of resistant cancer cells to drugs that induce ferroptosis. Importantly, the combination of a ferroptosis‐inducing drug and an HSF1 inhibitor causes the cytostasis of established tumors in mice, although neither treatment alone is effective. These data reveal a novel approach for the therapeutic induction of ferroptosis in cancer.