Journal: The Journal of Biological Chemistry

Article Title: Deglutarylation of glutaryl-CoA dehydrogenase by deacylating enzyme SIRT5 promotes lysine oxidation in mice

doi: 10.1016/j.jbc.2022.101723

Figure Lengend Snippet: Characterization of the SIRT5 crKO cell line. A , schematic of the CRISPR-Cas9 system used to generate the SIRT5 crKO cell line. B , schematic of the human SIRT5 gene showing the sites of crRNA targets within Exon5/4 the four known SIRT5 coding transcripts in humans. C , immunoblot for SIRT5 protein from HEK293T crWT ( circles ) clone and crKO ( blank ) clone (using CRISPR guide E, Table S1 ). D – F , immunoblots of SIRT5 crWT and crKO whole-cell lysates blotted using antibodies against glutaryl-lysine ( D ), glutaryl-succinyl-lysine ( E ), and malonyl-lysine ( F ). G – I , relative quantification of acylated proteins normalized to β-actin levels: glutarylation ( G ), succinylation ( H ), and malonylation ( I ) (mean ± SD, n = 3, ∗ p -value ≤ 0.05). crRNA, crispr RNA; PAM, protospacer adjacent motif; SIRT5, sirtuin 5; tracrRNA, trans-activating crispr RNA.

Article Snippet: Antibodies: anti-MG-lysine (Millipore-Sigma # ABS2120-25UG), anti-SIRT5 (Millipore-Sigma #HPA022002 [for human SIRT5] and Cell Signaling Technologies #8782 [for mouse SIRT5]), anti-GCDH (Millipore-Sigma # HPA048492), mouse anti-β-actin (Cell Signaling Technologies, #3700), rabbit anti-β-actin (Cell Signaling Technologies #8457), anti-glutaryl-lysine (Cell Signaling Technologies, noncommercial #14943MF and PTM-Biolabs #1151), anti-succinyl-lysine (Cell Signaling Technologies, noncommercial # 13599 and PTM-Biolabs #401), anti-malonyl-lysine (Cell Signaling Technologies #14942), anti-acetyl-lysine (Cell Signaling Technologies, #9441), IRDye 680RD donkey anti-mouse IgG (LI-COR #926-68072), and IRDye 800CW donkey anti-rabbit IgG (LI-COR #926-32213).

Techniques: CRISPR, Western Blot

Journal: Cell metabolism

Article Title: A Class of Reactive Acyl-CoA Species Reveals the Non-Enzymatic Origins of Protein Acylation

doi: 10.1016/j.cmet.2017.03.006

Figure Lengend Snippet: Protein acylation by four- and five-carbon dicarboxylic acyl-CoAs. The terminal carboxylic acids in succinyl-CoA, glutaryl-CoA, HMG-CoA, and 3-methylglutaryl-CoA facilitate non-enzymatic lysine acylation through intramolecular general base catalysis-mediated formation of a highly reactive cyclic anhydride intermediate. In a mouse model of the human disorder HMG-CoA lyase deficiency, HMG-CoA and 3-methylglutaryl-CoA accumulate and, consequently, protein lysine HMGylation and 3-MGylation are elevated. In a mouse model of the human disorder glutaric academia (GA I), glutaryl-CoA accumulates and, consequently, protein lysine glutarylation is elevated.

Article Snippet: Antibodies used were a polyclonal acetyl-lysine and monoclonal glutaryl-lysine antibodies (Cell Signaling, non-commercial), polyclonal succinyl-lysine, butyryl-lysine, and malonyl-lysine antibodies (PTM Biolabs, Inc.), and custom-made polyclonal glutaryl-lysine, succinyl-lysine, hydroxylmethylglutaryl-lysine (custom made from Yenzym Antibodies) and anti-myc (Santa Cruz Biotechnology).

Techniques: