Supplementary Materials Supplemental Data supp_14_1_227__index. metabolism. In addition, the biological part

Supplementary Materials Supplemental Data supp_14_1_227__index. metabolism. In addition, the biological part of lysine malonylation was validated in an enzyme of the glycolysis pathway. Collectively, our findings support a potential part of protein lysine malonylation in type 2 diabetes with possible implications for its therapy in the future. Post-translational modifications (PTMs)1 have been recognized as a common feature of proteins (1C3). More than 300 types of PTMs have been identified according to the Swiss-Prot database (4, 5). Most of them use small molecular compounds as group donors. For BEZ235 ic50 example, adenosine-triphosphate (ATP) is used in phosphorylation, S-adenosylmethionine (SAM) in methylation, and acetyl-CoA in acetylation. Lysine acylations including malonylation (6), succinylation (7), butyrylation (8), propionylation (9), and crotonylation (10) represent a group of PTMs that use intermediates of energy metabolism like malonyl-CoA, succinyl-CoA, butyryl-CoA, propionyl-CoA, and crotonyl-CoA as group donors. Among the lysine acylations, lysine malonylation was first identified in (and 17 proteins in HeLa cells. Using a novel chemical fluorescent probe, another group identified more than 300 malonylated protein candidates in HeLa cells (11). Despite the rapid progress in detection technologies and tools, functional studies of lysine malonylation and its role in human diseases have been lagging behind. Type 2 diabetes is characterized by hyperglycemia and production of glycated proteins. For example, glycated hemoglobin A1c (HbA1c) has been clinically used as diagnostic criteria for diabetes. In addition to glycation, the role of other types of PTMs in type 2 diabetes remains to be revealed. In fact, elevated malonyl-CoA levels have been found in type 2 diabetic patients (12), and prediabetic rats (13). And hepatic overexpression of malonyl-CoA decarboxylase (MCD) decreased malonyl-CoA and reversed insulin resistance (14). Given the use of malonyl-CoA as malonyl donor in lysine malonylation, lysine malonylation is therefore anticipated to BEZ235 ic50 be of functional significance in the pathogenesis of type 2 diabetes. In the present study, we observed elevated lysine malonylation in liver tissues of mice after unbiased screening seven types of lysine acylations. We then detected elevated levels of lysine malonylation in liver tissues of more and mice. Using an immunoaffinity based proteomic method, we identified a total of 573 malonylated lysine sites from 268 proteins in liver tissues of wt and mice. Elevation of lysine malonylation in five proteins was confirmed by immunoprecipitation coupled with Western blot analysis. Functional analysis of the malonylated proteins showed an apparent enrichment in metabolic pathways, especially those involved in the glucose and BEZ235 ic50 fatty acid metabolism. Our study indicates the putative association between protein lysine malonylation and type 2 diabetes. EXPERIMENTAL PROCEDURES Reagents Anti-malonyllysine (PTM-901), anti-acetyllysine (PTM-105), anti-1,2-dimethyllysine (PTM-602), anti-succinyllysine (PTM-401), anti-butyryllysine (PTM-301), anti-propionyllysine (PTM-201), anti-crotonyllysine (PTM-502) antibodies, and anti-malonyllysine conjugated agarose beads were purchased from PTM Biolabs (Chicago, IL). G6PI (ab86950), LDHA (ab47010), FBP1 (ab109732), and 10-FTHFDH/ALDH1L1 (ab56777) antibodies were purchased from Abcam (Cambridge, MA). Glutathione (GSH) agarose beads were purchased from GE Healthcare Life Sciences (Uppsala, Sweden). Sequencing-grade trypsin was purchased from Promega (Madison, WI), and C18 ZipTips had been bought from Millipore (Billerica, MA). A summary of other chemicals useful for LC-MS/MS comes in previously released study (15). Pets and Examples C57 BLKS mice (= 5) and wt mice (= 5) had been provided PPP2R1B by the pet Middle of Peking College or university. C57 BL/6J-mice (= 4) and wt mice (= 4) had been supplied by the Institute of Lab Animal Technology, CAMS (Chinese language Academy of Medical Technology). Mouse research were authorized by the pet Experimentation Ethics Committee (Institute of Biophysics, CAS) as well as the National Health insurance and Medical Study Council of China Recommendations on Pet Experimentation. Cells Lysate Planning Mice had been sacrificed by cervical dislocation. Cells had been homogenized in 1 ml lysis buffer (50 mm Tris-Cl, BEZ235 ic50 pH 7.4, 150 mm NaCl, 0.5 mm EDTA, 1 mm DTT, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS). The homogenized cells had been centrifuged at 12,000 rpm as well as the supernatant including the cells lysates was held for later measures. Traditional western blotting and Coomassie Blue Staining Traditional western blot and Coomassie blue staining evaluation were completed as referred to previously (16). In Vitro Malonylation Response and ALDOB Activity Assay malonylation was performed as previously referred to (17). Quickly, FLAG-tagged ALDOB was overexpressed in.