Journal: iScience
Article Title: Mitochondrial glycerol 3-phosphate dehydrogenase deficiency exacerbates lipotoxic cardiomyopathy
doi: 10.1016/j.isci.2024.109796
Figure Lengend Snippet: Downregulated mGPDH/SIRT5 signaling inhibits the desuccinylation of FAO proteins and promotes ACADS degradation (A) Schematic of experimental workflow for the identification of lysine succinylation substrates by proteomics in heart lysates. (B) The distribution of the number of lysine succinylation site identifications per protein was shown. (C) Top 10 terms of KEGG pathway analysis in succinylated protein targeted by mGPDH cKO under HFD condition. (D) Schematic represents 4 steps and 15 enzymes (including 4 auxiliary enzymes) of mitochondrial FAO. 14 of them displayed an altered succinylation level after mGPDH cKO, 13 were increased (highlighted with red color) and 1 was decreased (blue). The rest one which succinylation level could not be detected was shown in white. These enzymes are showing by their gene name. (E) Heatmap shown the changes in the levels of lysine succinylation on FAO enzymes according to our quantitative proteomics. (F and G) Heart lysates from HFD-fed mGPDH cKO and Ctrl mice (F), and cell lysates from H9C2 cells with mGPDH specific siRNA transfected and PA-treated (G) were immunoprecipitated with antibodies against ACADS or ACADM, and succinylation level of these two proteins and its quantifications were shown. (H) PA-treated H9C2 cells were transfected with siRNA for mGPDH and overexpressing plasmids for SIRT5_WT or catalytically inactive SIRT5-H158Y mutant, cell lysates were immunoprecipitated with antibodies against ACADS, and succinylation level of ACADS and its quantifications were assessed. (I) PA-treated H9C2 cells were either untreated or treated with the proteasome inhibitors MG132 (MG), ACADS levels were analyzed by western blot and its quantifications were shown. (J) PA-treated H9C2 cells were transfected with siRNA for mGPDH, cell lysates were immunoprecipitated with antibodies against ACADS, and ubiquitinated ACADS levels were assessed. (K–M) PA-treated H9C2 cells were transfected with siRNA for mGPDH and overexpressing plasmids for HA-tagged ACADS_WT, ACADS-K250R and ACADS-K250E, succinylation level of ACADS (K), ACADS protein (L) and ubiquitination of ACADS (M) were assessed. Quantifications of K and L were shown. n = 3 mice per group for B-E, n = 6 mice per group for F, n = 3 per group for G-M. The data are presented as the means ± S.E.M. ∗∗∗ p < 0.001 by two-tailed unpaired Student’s t test.
Article Snippet: Rabbit polyclonal anti-ACADS antibody , Proteintech , Cat# 16623-1-AP; RRID: AB_10666165.
Techniques: Quantitative Proteomics, Transfection, Immunoprecipitation, Mutagenesis, Western Blot, Ubiquitin Proteomics, Two Tailed Test
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