Abstract
The protein substrates of sirtuin 5-regulated lysine malonylation (Kmal) remain unknown, hindering its functional analysis. In this study, we carried out proteomic screening, which identified 4042 Kmal sites on 1426 proteins in mouse liver and 4943 Kmal sites on 1822 proteins in human fibroblasts. Increased malonyl-CoA levels in malonyl-CoA decarboxylase (MCD)-deficient cells induces Kmal levels in substrate proteins. We identified 461 Kmal sites showing more than a 2-fold increase in response to MCD deficiency as well as 1452 Kmal sites detected only in MCD-/- fibroblast but not MCD+/+ cells, suggesting a pathogenic role of Kmal in MCD deficiency. Cells with increased lysine malonylation displayed impaired mitochondrial function and fatty acid oxidation, suggesting that lysine malonylation plays a role in pathophysiology of malonic aciduria. Our study establishes an association between Kmal and a genetic disease and offers a rich resource for elucidating the contribution of the Kmal pathway and malonyl-CoA to cellular physiology and human diseases.
Highlights
Reversible acetylation at lysine residues in proteins has been extensively studied over the past few decades [1, 2]
Four hundred sixty-one Kmal sites on 339 proteins showed a 2-fold or greater increase in MCDϪ/Ϫ cells relative to MCDϩ/ϩ cells, and 1452 Kmal sites on 822 proteins were only detected in MCDϪ/Ϫ cells, suggesting that malonyl-CoA decarboxylase (MCD) activity has a profound impact on Kmal levels and distribution
Kmal Is Affected by SIRT5 and MCD—Our previous studies showed that SIRT5 can catalyze removal of malonyl groups from malonylated lysine residues both in vitro and in vivo [15]
Summary
Lysine malonylation; CPT, carnitine palmitoyltransferase; GO, gene ontology; Kac, lysine acetylation; Ksucc, lysine succinylation; Kglu, lysine glutarylation; MCD, malonyl-CoA decarboxylase; MCDϩ/ϩ, MCD wild type; MCDϪ/Ϫ, MCD-deficient; PTM, post-translational modification; KO, knock-out; SILAC, stable isotope labeling by amino acids in cell culture; SIRT, sirtuin; OCR, oxygen consumption rate; VLCAD, very long-chain acylCoA dehydrogenase; LCHAD, long-chain 3-hydroxyacyl-CoA dehydrogenase; KEGG, Kyoto Encyclopedia of Genes and Genomes; adj, adjusted. Diverse symptoms are observed among malonic aciduria patients including delayed development, seizures, diarrhea, vomiting, low blood sugar (hypoglycemia), and cardiomyopathy [22] It appears that inhibition of fatty acid catabolism caused by a high level of malonyl-CoA is at least partially responsible for the manifestations of disease. Our proteomics data illuminate the landscape of the Kmal modification in mammalian cells, offer a valuable resource for studying its biology, and propose possible roles of Kmal in diseases associated with dysregulation of malonyl-CoA homeostasis
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