Lysine Glutarylation Is a Protein Posttranslational Modification Regulated by SIRT5

“Bioinformatic Analyses of Kglu Substrates To identify the possible association of Kglu substrates with cellular functions, we carried out gene ontology (GO) enrichment analysis using DAVID bioinformatics resources (Huang et al., 2009).”

“In addition to histones, PTM-regulatory enzymes, such as AMP-activated protein kinase (AMPK) and lysine deacetylases (KDACs or HDACs), can control activity of their substrates (Chalkiadaki and Guarente, 2012; Hardie et al., 2012), many of which are metabolic enzymes.”

“For example, acetyl-CoA, SAM, NAD+, and a-ketoglutarate are donors/substrates or cofactors for histone lysine-modifying enzymes, i.e., those modulating levels of acetylation and methylation (Imai et al., 2000; Takahashi et al., 2006; Tsukada et al., 2006; Wellen et al., 2009).”

“These PTM enzymes have important roles in modulation of chromatin structure and transcriptional regulation (Berger, 2007; Chi et al., 2010).”

“For example, acetyl-CoA, SAM, NAD+, and a-ketoglutarate are donors/substrates or cofactors for histone lysine-modifying enzymes, i.e., those modulating levels of acetylation and methylation (Imai et al., 2000; Takahashi et al., 2006; Tsukada et al., 2006; Wellen et al., 2009).”

“For example, acetyl-CoA, SAM, NAD+, and a-ketoglutarate are donors/substrates or cofactors for histone lysine-modifying enzymes, i.e., those modulating levels of acetylation and methylation (Imai et al., 2000; Takahashi et al., 2006; Tsukada et al., 2006; Wellen et al., 2009).”

“Our previous studies identified new lysine acylation modifications on histones (Chen et al., 2007; Tan et al., 2011; Xie et al., 2012), including lysine crotonylation and succinylation, revealing potential crosstalk between cellular physiology and gene transcription.”

“coli and HeLa cells, we used an affinity enrichment-based proteomics approach as previously described (Chen et al., 2012; Kim et al., 2006).”

“In addition to these metabolites, NAD+ is required for SIRT5-dependent desuccinylation and demalonylation reactions (Du et al., 2011; Peng et al., 2011).”

“SIRT5was originally annotated as a deacetylase (Frye, 2000) and was predicted to have deacetylase activity, but appears to preferentially deacylate negatively charged modifications, including malonylation, succinylation, and glutarylation.”

“In contrast, SIRT5 has little influence on global lysine acetylation (Lombard et al., 2007).”

“These PTM enzymes have important roles in modulation of chromatin structure and transcriptional regulation (Berger, 2007; Chi et al., 2010).”

“Succinate and malonate can be used by cells to generate lysine succinylation and malonylation, respectively, presumably by generating succinyl-CoA and malonyl-CoA intermediary metabolites (Peng et al., 2011; Zhang et al., 2011).”

“Together, association of three key cellular metabolites in these pathways strongly suggests that succinylation and malonylation PTMs have roles in metabolism (Park et al., 2013).”

“Because CPS1 was the protein identified with the highest level of glutarylation, and SIRT5 has been previously described to regulate CPS1 (Nakagawa et al., 2009), we sought to determine if glutarylation had a functional effect on CPS1 enzymatic activity and was regulated by SIRT5.”

“In addition, lysine succinylation is an abundant protein PTM in diverse model organisms (Weinert et al., 2013).”

“Our previous studies identified new lysine acylation modifications on histones (Chen et al., 2007; Tan et al., 2011; Xie et al., 2012), including lysine crotonylation and succinylation, revealing potential crosstalk between cellular physiology and gene transcription.”

“Succinate and malonate can be used by cells to generate lysine succinylation and malonylation, respectively, presumably by generating succinyl-CoA and malonyl-CoA intermediary metabolites (Peng et al., 2011; Zhang et al., 2011).”

“…substrates containing 7-amino-4-methylcoumarin (AMC) were evaluated: one series based on a simple a-N-acetylated lysine (Figure 4A) and one containing the tripeptide sequence, Ac-Leu-Gly-Lys(acyl), which is based on H4K12—a common substrate for deacylation (Bradner et al., 2010) (Figure S4A).”

“Indeed, several mechanisms have been described bywhich cells adapt to such environmental changes, such as transcriptional regulation by alterations of epigenetic marks, modulation of metabolic enzyme activities by cellular metabolites, and protein posttranslational modifications (PTMs) (Furuya and Uyeda, 1980; Lu and…”

“Lysine acetylation levels are coupled to metabolism and play key roles in regulating mitochondrial enzymes, chromatin biology, and other cellular processes (Chalkiadaki andGuarente, 2012; Verdin et al., 2010; Xiong and Guan, 2012).”

“Our bioinformatic analyses showed that Kglu is highly enriched with metabolic processes, such as cellular respiration, and fatty acid and amino acid metabolism, similar to what has been observed for acetylation and succinylation (Chen et al., 2012; Hebert et al., 2013; Park et al., 2013).”

“Together, association of three key cellular metabolites in these pathways strongly suggests that succinylation and malonylation PTMs have roles in metabolism (Park et al., 2013).”

“M.D.H. and his laboratory are supported by the American Heart Association grants 12SDG8840004 and 12IRG9010008, The Edward Mallinckrodt Jr. Foundation, The Ellison Medical Foundation, the National Institutes of Health (AA022146 and AG045351), and the Duke O’Brien Center for Kidney Research ell Metabolism 19, 605–617,…”

“For example, acetyl-CoA, SAM, NAD+, and a-ketoglutarate are donors/substrates or cofactors for histone lysine-modifying enzymes, i.e., those modulating levels of acetylation and methylation (Imai et al., 2000; Takahashi et al., 2006; Tsukada et al., 2006; Wellen et al., 2009).”

“In addition to histones, PTM-regulatory enzymes, such as AMP-activated protein kinase (AMPK) and lysine deacetylases (KDACs or HDACs), can control activity of their substrates (Chalkiadaki and Guarente, 2012; Hardie et al., 2012), many of which are metabolic enzymes.”

“Lysine acetylation levels are coupled to metabolism and play key roles in regulating mitochondrial enzymes, chromatin biology, and other cellular processes (Chalkiadaki andGuarente, 2012; Verdin et al., 2010; Xiong and Guan, 2012).”

“coli and HeLa cells, we used an affinity enrichment-based proteomics approach as previously described (Chen et al., 2012; Kim et al., 2006).”

“We used strict manual verification criteria to filter out false positives, as previously described (Chen et al., 2005), to ensure bona fide identification of glutarylated lysines (Table S1).”

“For example, sirtuin family HDACs (or class III HDACs) can be regulated by the cellular NAD levels (Imai et al., 2000; Schmidt et al., 2004).”

“C pathways, and GA patients as well as GCDHKO mice display increased levels of glutaryl-CoA (Goodman and Frerman, 2001; Koeller et al., 2002), we predicted loss of GCDH would result in increased protein glutarylation.”

“CPS1 requires N-acetylglutamate (NAG) as an obligate allosteric activator (Hall et al., 1958).”

“CPS1 is present as a homodimer and is active in its monomeric form (Guthöhrlein and Knappe, 1968).”

“To compare the enzymatic efficiencies against different acyl groups, we then performed detailed kinetic studies using the tripeptide substrates containing malonyllysine (Kmal), succinyl-lysine (Ksucc), and glutaryl-lysine (Kglu) by measuring initial rate velocities as a function of substrate concentration and fitting…”

“CPS1 Activity Assay CPS1 activity was assessed by measuring converted citrulline by a colorimetric method with modification (Chan et al., 2009; Pierson, 1980).”

“Indeed, several mechanisms have been described bywhich cells adapt to such environmental changes, such as transcriptional regulation by alterations of epigenetic marks, modulation of metabolic enzyme activities by cellular metabolites, and protein posttranslational modifications (PTMs) (Furuya and Uyeda, 1980; Lu and…”

“This region has been predicted to be important for CPS1 structure (Lopes-Marques et al., 2012); alternatively, these sites could be important for CPS1-interacting proteins, such as with glutaminase (Meijer, 1985).”

“We predicted that glutaryl-CoA could serve as the donor molecule for the Kglu reaction (Koeppen et al., 1979; Mitzen and Koeppen, 1984), analogous to a role for acetyl-CoA in lysine acetylation.”

“We predicted that glutaryl-CoA could serve as the donor molecule for the Kglu reaction (Koeppen et al., 1979; Mitzen and Koeppen, 1984), analogous to a role for acetyl-CoA in lysine acetylation.”

“CPS1 Activity Assay CPS1 activity was assessed by measuring converted citrulline by a colorimetric method with modification (Chan et al., 2009; Pierson, 1980).”

“This region has been predicted to be important for CPS1 structure (Lopes-Marques et al., 2012); alternatively, these sites could be important for CPS1-interacting proteins, such as with glutaminase (Meijer, 1985).”