Fig. 1: Multiple phosphorylation sites of CypD and mPTP regulation.
a Table of the specific-GSK3β kinase post-translational phosphorylation site of CypD from the amino acid sequence Human PPIF (Uniprot #P30405)50. NetPhos 3.1 score of potential serine phosphorylation sites of CypD, and HTP (High Throughput papers) score from PhosphoSitePlus: number of records in which this modification site was assigned using only proteomic discovery mass spectrometry. Non applicable (na), phosphorylation (p). b Top: representative western blot of the mitochondrial fraction of HEK cells overexpressing FLAG-tagged mutants of CypD probed for phospho-serine (P-Ser) and FLAG. Merge was included to show that the P-Ser band is at the same level than the CypD-FLAG band. Bottom: Quantification of P-Ser/Flag intensities (mean ± SD, n = 4/group independent experiments) (*p < 0.05 vs. WT). Differences in means among multiple groups were analyzed using one-way ANOVA with a Tukey′s post hoc test. c Left: typical curves of calcium retention capacity (CRC) in HEK CypD-KO cells rescued with different CypD mutants. Right: Quantification of CRC in different CypD mutants normalized to the protein content and maximum protection afforded by 1 µM CsA. Differences in means among multiple groups were analyzed using one-way ANOVA with a Tukey′s post hoc test. (Mean ± SD, n = 3 independent experiments) (*p < 0.05 vs. KO, †p < 0.05 vs. WT, and ‡p < 0.05 vs. S191A). CypD can be phosphorylated at multiple serine residues, but only the CypD phosphorylation at S191 seems to impact the ability of CypD to regulate the mPTP opening. Differences in means among multiple groups were analyzed using one-way ANOVA with a Bonferroni′s or Tukey′s post hoc test.
