Fig. 6: Detection of a truncated form of CyPD in cells and role of calpain-1 in CyPD cleavage.

a Western blot analysis of CyPD and F-ATP synthase α-subunit in lysates from different murine tissues. For each well, 30 μg of proteins were loaded. b Western blot analysis of CyPD and F-ATP synthase α-subunit in total lysates (Lysate, 10 μg and 20 μg in the first and second lane, respectively) or crude mitochondrial preparations (Mito, 5 μg and 10 μg in the third and fourth lane, respectively) from primary human skin fibroblasts. The cytosolic marker glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was almost undetectable in the mitochondrial fraction. c Mass spectrometry analysis of the upper band at ~19 kDa and of the lower band at ~18 kDa from murine heart lysates detected by anti-CyPD antibodies (left panel) or human skin fibroblasts (right panel). Peptides were sequenced and matched against murine (left panel) or human (right panel) mature CyPD sequence. The univocally sequenced and matched peptides are shown in red, while residues in black represent non-detected ones (representative experiment out of five). For murine samples, sequence coverages were 51% ± 9% for the 19-kDa band and 37% ± 21% for the 18-kDa band. For human samples, sequence coverages were 39% ± 16% for the 19-kDa band and 30% ± 15% for the 18-kDa band. d Workflow for the interrogation of the MEROPS database and its analysis. The murine (NS) and human (SS) cleavage sites were used as a query in the search for proteases. Then, among the different hits revealed by the database, a manual inspection was performed. Proteins of the calpain family were found as the most interesting hits. e Results for the GPS-CCD 1.0 analysis for mouse and human mature CyPD sequence. Each table shows the most probable target sites for calpain 1, assigning to each putative cleavage site a score. Letters in bold-red show the actual cleavage sites. f SDS-PAGE/Coomassie staining following the incubation of FL-CyPD or ΔN-CyPD with Calpain 1 (CPN1) at different times (0 min, 5 min, 15 min). On the right, a magnification of the enzymatic products of FL-CyPD+CPN1 after 15 minutes is shown. The two bands were manually excised for mass spectrometry analysis. The N-terminal peptide detected in each band is shown. g Western blot analysis of CyPD in total lysates from HEK293 cells treated for 30 min with 50 μM of the broad-spectrum calpain inhibitor PD150606 or with the DMSO concentration as vehicle control. The ratio between the lower and the higher band was quantified by densitometry and is reported as mean ± S.D. of three independent experiments. Data were analysed according to the one-sample t-test (*p = 0.044).