Lysine glutarylation is a protein posttranslational modification regulated by SIRT5

Abstract

We report the identification and characterization of a five-carbon protein posttranslational 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 that 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 acidemia 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.

Figure 1. Lysine deglutarylation and glutaryl-CoA pathways

(A) Structures of glutaryl-lysine, succinyl-lysine and malonyl-lysine. (B) Illustration of glutaryl-CoA synthetic pathways.

Figure 2. Detection and verification of lysine glutarylation

(A) Specificity of the anti-K_glu antibody. Dot-blot assay was carried out using anti-K_glu antibody by incubation of the peptide libraries bearing a fixed unmodified K, K_ac, K_mal, K_succ, and K_glu, respectively. Each peptide library contains 10 residues CXXXXKXXXX, where X is a mixture of 19 amino acids (excluding cysteine), C is cysteine, and the 6^th residue is a fixed lysine residue: unmodified lysine (K), acetyl-lysine (K_ac), malonyl-lysine (K_mal), succinyl-lysine (K_succ), glutaryl-lysine (K_glu). (B) Western blot of the whole-cell lysates from bacteria E. coli, yeast S. cerevisiae, Drosophila S2, mouse MEF, human HeLa cells. Western blot was performed using the whole-cell lysates from different cells. (C) High-resolution MS/MS spectra of an E. coli tryptic peptide (SK_+114.0281DaATNLLYTR) from DNA starvation/stationary phase protection protein with a mass shift of +114.0281 Da at the lysine residue (bottom) and the synthetic SK_gluATNLLYTR (top). Inset shows the mass-to-charge ratios (m/z) of the precursor ions. (D) Extracted ion chromatograms (XICs) of the synthetic peptide SK_gluATNLLYTR (top), the in vivo-derived peptide (middle, from E. coli), and their mixture (bottom) by reversed-phase HPLC analysis.

Figure 3. SIRT5 catalyzes lysine deglutarylation reactions in vitro and in vivo

(A) Screening of HDAC lysine deglutarylation activity in vitro. Fluorometric assay to detect in vitro lysine deglutarylation activities using recombinant SIRT1-7. (B) ³²P-NAD⁺ consumption monitored by thin layer chromatography after an in vitro SIRT3, SIRT4, or SIRT5 enzymatic assay using chemically acylated BSA as a substrate; o-glutaryl-ADP ribose, OG-ADPR; o-succinyl-ADP ribose, OS-ADPR; o-acetyl-ADP ribose, OA-ADPR. (C) HPLC trace of a glutarylated peptide, VKSK_gluATNLWW, before and after in vitro deglutarylation reaction. The assays were carried out without hSIRT5, with hSIRT5, with enzymatically-inactive mutant of hSIRT5 (H158Y), without NAD⁺, with SIRT5 in the presence of nicotinamide (25 mM), or sirtinol (200 µM), or TSA (2 µM), or sodium butyrate (NaBu) (25mM). A triangle and a diamond indicate modified (HRMS, m/z, 673.8588 Da) and unmodified (HRMS, m/z, 616.8439 Da) peptides, respectively.

Figure 4. Enzymology studies on SIRT5-catalyzed deglutarylation

(A) Endpoint deacylation assay of mouse or human wild-type SIRT5 or human mutant SIRT5 (HY mutant) against acylated mono-lysine-AMC substrates; K_ac, acetyl; K_mal, malonyl; K_succ, succinyl; K_glu, glutaryl; K_adi, adipoyl. Error bars in this figure and subsequent ones represent standard error of the mean (SEM). (B) Crystal structure of SIRT5 with a glutarylated peptide superimposed in the catalytic pocket. (C) Summary of total interaction energy comprising the sum of Van der Waals and electrostatic interaction energies between SIRT5 and acylated histone peptides (shown in Figure S4D).

Figure 5. Proteomic and bioinformatic analyses of K_glu substrates in WT and SIRT5KO mouse liver

(A) Western blotting analysis of mitochondria from Sirt5^+/+ (WT) and Sirt5^−/− (KO) mouse livers. (B) Distribution of K_glu sites per protein identified by proteomic analysis. (C) Gene ontology (GO) of biological processes (BP) of proteins identified as glutarylated by proteomics.

Figure 6. Carbamoyl Phosphate Synthase 1 (CPS1) is targeted for deglutarylation by SIRT5

(A) CPS1 was immunoprecipitated from SIRT5 WT and KO mouse liver under fed and 48h fasted conditions and measured for glutarylation levels using an anti-K_glu antibody. (B) CPS1 enzymatic activity was measured in hepatic lysates in SIRT5 WT and KO mice, and presented relative to WT control mice. (C) CPS1 was immunoprecipitated from HeLa cells containing a shScramble (SCR) or shSIRT5 and measured for glutarylation levels using an anti-K_glu antibody. (D) Ammonia levels from HeLa cells containing a shScramble (SCR) or shSIRT5 after 24h of serum starvation. (E) Ornithine levels from HeLa cells containing a shScramble (SCR) or shSIRT5 after 24h of serum starvation. (F) CPS1 was transfected in HEK293 cells containing a shScramble (SCR) or shSIRT5, immunoprecipitated, and measured for glutarylation levels using an anti-K_glu antibody. (G) CPS1 was transfected in HEK293 cells and immunopurified. Glutarylation levels were measured using an anti-K_glu antibody in four samples: CPS1, glutarylated CPS1 that was chemically modified with glutaric anhydride (GA), glutarylated CPS1 after incubation with recombinant human SIRT5, glutarylated CPS1 after incubation with recombinant human SIRT5 HY mutant. (H) A homology model of human CPS1 was built based on an E. coli CarA homologous region (light gray), encompassing an inactive glutamine amidotransferase domain (teal), and an E. coli CarB homologous region (dark gray) containing two ATP grasp domains (cyan), an oligomerization domain (blue), and a NAG-binding site (magenta). Glutarylated lysine residues targeted for SIRT5 removal are labeled and highlighted in yellow.

Figure 7. Physiology and pathophysiology of lysine glutarylation

(A) Protein glutarylation was measured in hepatic whole-tissue lysates by immunoblotting with an anti-K_glu antibody in two sets (Mouse 1 and Mouse 2) of SIRT5 WT and KO mice that were fed or fasted (48h), respectively. (B) Drosophilia were fed with 0X, 1X or 2X tryptophan for 1 week and whole-cell lysates were prepared for Western blotting analysis. (C) Hepatic mitochondrial protein glutarylation and succinylation was measured by immunoblotting with anti-acyl-lysine antibodies in WT and glutaryl-CoA dehydrogenase knock-out (GCDHKO) mice. (D) CPS1 was immunoprecipitated from GCDHKO mouse liver and measured for glutarylation levels using an anti-K_glu antibody. (E) CPS1 enzymatic activity was measured in hepatic lysates from wild-type and GCDHKO mice and normalized to total CPS1 protein levels. (F) Glutaryl-CoA or glutarate was incubated with heat-inactivated hepatic mitochondrial lysates and monitored for changes in protein glutarylation using an anti-K_glu antibody.