Fig. 1: Ischemic TA muscle Cytc is acetylated on lysine 39 and acetylmimetic K39Q and acetylated Cytc increases COX activity and decreases caspase-3 activity.

A Representative mass spectrum of Cytc peptide K(acetyl)TGQAPGFSYTDANKNK identifying Cytc K39 acetylation in ischemic, but not control, samples (n = 12: 6 control TA (3 male and 3 female) and 6 ischemic TA (3 male and 3 female with 1 male and 2 female samples demonstrating K39 acetylation); ProteomeXchange Consortium, PRIDE: PXD040915). B Cytc is acetylated in ischemic but not control TA muscle as shown after immunoprobing for acetyl-lysine and Cytc (n = 3). C Representative IP-Immunoblot experiment of porcine TA muscle showing acetylation state of Cytc at 0, 15, 30, 45, and 60 min of ischemia (n = 3). D Representative in vitro deacetylation assay showing that sirtuin5 removes acetylation from Cytc (n = 3). E Representative LabSafe GEL Blue stained 10% tris-tricine SDS-PAGE gel showing purity of the recombinant Cytc purified from bacteria after overexpression (n = 3). F Reduced Cytc spectra and oxidized Cytc spectra (inset) of recombinant Cytc indicate correct folding of the proteins (the reduced K39Q spectrum overlaps with the reduced K39R spectrum). Oxygen consumption rate of 26.7 nM pig heart COX was measured using an Oxygraph+ system at 25 °C. G The recombinant WT, K39R, K39Q, and K39E proteins (n = 3) or H control and ischemic purified pig TA muscle Cytc samples (n = 5) were titrated at concentrations of 0, 2, 5, 10, 15, 20, and 25 μM or injected at 5 μM, respectively. For G, a one-way ANOVA comparing the mean of each mutant with the mean of the control mutant (WT) at the 25 μM condition with the Dunnett post-hoc test was used. For H, a student’s two-tailed t-test assuming equal variance was used. I Cytosolic extracts from Cytc knockout embryonic fibroblasts were incubated with the recombinant WT, K39R, K39Q, and K39E proteins (n = 5 for WT, K39Q; n = 4 for K39R, K39E) or (J) control and ischemic purified pig TA muscle Cytc samples (n = 4) for 2.5 h at 37 °C. Rhodamine fluorescence that resulted from caspase-3 mediated cleavage of Z-DEVD-R110 was used as a measure of caspase-3 activity. For I, a one-way ANOVA comparing the mean of each mutant with the mean of the control mutant (WT) with the Dunnett post-hoc test was used. For J, a student’s two-tailed t-test assuming equal variance was used. Data are represented as means ± standard deviation.