Fig 2: The dysregulation of TRAP1 resulted in a defective ETC, resulting in electron leakage and ROS production. (A) The mitochondrial and total ROS were assessed by flow cytometry in A549 cells transfected with WT and variants (A64T, E93Q, and A64T+E93Q). Representative histograms of flow cytometric analysis of MitoSOX and H2DCFDA fluorescence in different experimental groups (n=3). The bar graphs show the percentage of cells positive for mitochondrial ROS and total ROS production. The significance of the results was determined by ANOVA. * p ≤ 0.05. (B) The level of mitochondrial and total ROS in PBMCs from controls and the patient was measured by flow cytometry. Results are expressed as means ± standard error of the mean (SEM). n=3 per group. * p ≤ 0.05 (C) Oxygen consumption rate (OCR) trace of the Seahorse Mito Stress assay on A549 transfected with different Trap1 variants. (n = 3 repeats, 5 technical replicates each repeat). (D) Maximal glycolytic rate of A549 transfected with different Trap1 variants. (n = 2 repeats, 5 technical replicates each repeat). Results are expressed as means ± standard error of the mean (SEM). The significance of the results was determined by ANOVA (**p ≤ 0.01, *** p≤ 0.001, **** p ≤ 0.0001). ETC, Electron transport chain; ROS, Reactive Oxygen Species.

Fig 3: Circp53–209aa-R175 is identified as the site-specific target of CypD to trigger mPTP opening in MM cells.a, GO analysis of RNA-sequencing data revealed that the circp53–209aa is highly associated with mitochondrial pathways. b, Go analysis of RNA-sequencing data revealed that the circp53–209aa is highly associated with apoptotic process signaling pathways. c, A graphic illustration of circp53–209aa interacting with CypD and releasing CypD from the CypD/TRAP1/HSP90 complex, thereby activating its isomerase activity and inducing mPTP opening. d, A Co-IP assay confirmed the direct interaction between circp53–209aa and CypD in MM cells. e,Representative confocal images of HA and CypD demonstrate the interaction between circp53–209aa and CypD. f, Statistical analysis of the colocation of circp53-209aa and CypD in H929 and XG1 cells. g, A Co-IP assay showed a weaker interaction between CypD and HSP90 in circp53-OE MM cells compared with Ctrl cells. h, Distribution of calcein in H929 and MM1.S cells. i, Distribution of calcein in XG1 and RPMI 8226 cells. j, IOD quantitative statistics of Calcein in Ctrl and circp53–209aa-OE MM cells. k, ATP levels were significantly decreased in circp53–209aa-OE cells. l, Statistical analysis of ATP levels in different cells (P < 0.001). m, A model showing the high confidence interval of the CypD protein. n, A model of the circp53–209aa-CypD complex. R175 of circp53–209aa binds to E43, E34 and Q163 of CypD through hydrogen bonds and/or ionic interactions. o, Circp53–209aa-R175 is involved in hydrogen-bond interactions with CypD, and these interactions are disrupted upon mutation of arginine to a histidine, as demonstrated by Co-IP using an HA antibody as bait in circp53-OE cells, followed by WB analysis. p, Circp53–209aa-R175H-OE MM cells were constructed successfully, as shown by WB analysis. q, Circp53–209aa-R175H did not influence the proliferation of MM cells. r, Circp53–209aa interacts with CypD and releases it from the CypD/TRAP1/HSP90 complex, as evidenced by Co-IP. s, Colocalization dynamics of CypD/HSP90 in H929 and MM1.S cells. t, Colocalization dynamics of CypD/HSP90 in XG1 and 8226 cells. u, Statistical analysis of the colocation of CypD and HSP90 in H929 and MM1.S cells. v, Statistical analysis of the colocation of CypD and HSP90 in XG1 and RPMI 8226 cells. w, IOD quantitative statistics of CypD and HSP90 in MM cells. x, Distribution of calcein in circp53-OE and circp53–209aa-R175 MM cells. y, The mPTP was not opened in the circp53–209aa-R175H group by quantitative statistics. The data are presented as mean ± s.d. (n = 3 cultures for each group, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, no statistical significance).

Fig 4: TRAP1 inhibits KANSL3 acetylation to mediate mitophagy of cardiomyocytes under diabetic conditions. A RT‒qPCR was used to measure the mRNA level of KANSL3 in AC16 cells from different groups. B AC16 cells were treated as indicated for 24 h. Western blots of WCLs from AC16 cells were performed with the indicated antibodies. C Western blots of mitochondria isolated from AC16 cells were performed with the indicated antibodies. D Representative images of immunofluorescence staining showing KANSL3 (red) and TOM20 (green) colocalization in AC16 cells. Scale bar: 50 µm. E Immunohistochemical staining of KANSL3 in cardiac tissue sections under different conditions. Scale bar: 250 µm. F Immunoblot analysis of WT AC16 cells overexpressing HA-tagged KANSL3. G Coimmunoprecipitation of HA-KANSL3 and acetylated proteins under HG/PA conditions. H Coimmunoprecipitation of KANSL3 and acetylated proteins in Flag-tagged TRAP1-overexpressing AC16 cells under HG/PA conditions. For comparisons between two groups, statistical significance was determined using a two-tailed unpaired Student’s t test. Group comparisons were performed by one-way analysis of variance followed by Tukey’s post hoc test. *p < 0.05. **p < 0.01. ns, not significant

Fig 5: TRAP1 is a molecular chaperone that specifically interacts with KANSL3, and KANSL3 is required for mitophagy. A Silver staining of TRAP1 immunoprecipitates. B LC‒MS/MS of TRAP1 immunoprecipitates. C Representative fragmentation spectrum of the identified KANSL3 peptides. D Immunoblotting analysis of TRAP1 and KANSL3 expression in a co-IP assay performed in AC16 cells with anti-TRAP1 magnetic beads from different groups. E Western blots of IP samples from AC16 cells performed with the indicated antibodies. F Western blots of WCLs from AC16 cells at 7 days post transduction with the indicated shRNAs targeting KANSL3. G AC16 cells were starved for 6 h and costained with MitoTracker Green and LysoTracker Red. Images were acquired by fluorescence microscopy. Scale bar = 10 µm. H Representative images of immunofluorescence staining showing LC3B (red) and TOM20 (green) colocalization in AC16 cells following 6 h of starvation. Scale bar = 10 µm