Lysine Glutarylation Is a Protein Posttranslational Modification Regulated by SIRT5

Abstract

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.

Mitochondrial Sirtuins and Their Relationships with Metabolic Disease and Cancer

“…The preference of SIRT5 for negatively charged acyl groups was further corroborated by another recent study (156). Tan et al identified and validated lysine glutarylation as an evolutionary conserved posttranslational modification; similar to lysine succinylation, this modification was also regulated by SIRT5 (156). Proteomic screening of liver extracts from Sirt5 KO mice revealed hyperglutarylation of 683 lysine sites on 191 proteins; more than three quarters of these proteins were found to be mitochondrial (156).…”

“…Similarly, Rardin et al reported that SIRT5 deficiency resulted in hypersuccinylation of 32% sites in 56% of mitochondrial proteins overall (127). The preference of SIRT5 for negatively charged acyl groups was further corroborated by another recent study (156). Tan et al identified and validated lysine glutarylation as an evolutionary conserved posttranslational modification; similar to lysine succinylation, this modification was also regulated by SIRT5 (156).…”

“…However, recently SIRT5 has been shown to preferentially deacylate negatively charged modifications: malonylation, succinylation, and glutarylation (34,116,121,127,156). Du et al showed that SIRT5 possesses minimal deacetylase activity compared with SIRT1 and SIRT3 (34); however, the catalytic efficiency of SIRT5 for demalonylation and desuccinylation was much higher than for deacetylation (34,121).…”

“…In recent years, protein posttranslational modifications (PTMs) such as ADP-ribosylation and lysine acetylation, succinylation, malonylation, and glutarylation on diverse mitochondrial proteins have emerged as a novel mechanism of mitochondrial regulation. These modifications are directly regulated by members of the sirtuin enzyme family (2,34,91,121,156).…”

“…However, certain sirtuins are now known to possess alternative catalytic functions-for example, deacylase, decrotonylase, desuccinylase, demalonylase, deglutarylase, and ADP-ribosyltransferase activities (Fig. 1)-that play crucial roles in the regulation of diverse cellular processes (2,10,32,34,36,48,65,72,121,156).…”