Mitochondrial Chaperone Trap1 and the Calcium Binding Protein Sorcin Interact and Protect Cells against Apoptosis Induced by Antiblastic Agents

Abstract

TRAP1, a mitochondrial chaperone (Hsp75) with antioxidant and antiapoptotic functions, is involved in multidrug resistance in human colorectal carcinoma cells. Through a proteomic analysis of TRAP1 coimmunoprecipitation complexes, the Ca 2+ -binding protein Sorcin was identified as a new TRAP1 interactor. This result prompted us to investigate the presence and role of Sorcin in mitochondria from human colon carcinoma cells. Using fluorescence microscopy and Western blot analysis of purified mitochondria and submitochondrial fractions, we showed the mitochondrial localization of an isoform of Sorcin with an electrophoretic motility lower than 20 kDa that specifically interacts with TRAP1. Furthermore, the effects of overexpressing or downregulating Sorcin and/or TRAP1 allowed us to demonstrate a reciprocal regulation between these two proteins and to show that their interaction is required for Sorcin mitochondrial localization and TRAP1 stability. Indeed, the depletion of TRAP1 by short hairpin RNA in colorectal carcinoma cells lowered Sorcin levels in mitochondria, whereas the depletion of Sorcin by small interfering RNA increased TRAP1 degradation. We also report several lines of evidence suggesting that intramitochondrial Sorcin plays a role in TRAP1 cytoprotection. Finally, preliminary evidence that TRAP1 and Sorcin are both implicated in multidrug resistance and are coupregulated in human colorectal carcinomas is provided. These novel findings highlight a new role for Sorcin, suggesting that some of its previously reported cytoprotective functions may be explained by involvement in mitochondrial metabolism through the TRAP1 pathway.

TRAP1 and the proteasome regulatory particle TBP7/Rpt3 interact in the endoplasmic reticulum and control cellular ubiquitination of specific mitochondrial proteins

“…Accordingly, the protein levels of p18 Sorcin, another MITO protein, recently identified by our group as a novel MITO Sorcin isoform interacting with TRAP1, 12 decreased upon TRAP1 interference (Figure 5b, arrow). Of note, under the same experimental conditions, no differences were observed in the protein levels of the higher mobility p22 Sorcin isoform, which shares high homology with p18 Sorcin, but is not a MITO protein, neither is a TRAP1 'partner' (Figure 5b).…”

“…Of note, under the same experimental conditions, no differences were observed in the protein levels of the higher mobility p22 Sorcin isoform, which shares high homology with p18 Sorcin, but is not a MITO protein, neither is a TRAP1 'partner' (Figure 5b). 12 Therefore, we hypothesize that the decreased expressions of F1ATPase and p18 Sorcin in the mitochondria of TRAP1-interferred cells were dependent on increased ubiquitination. To this aim, the respective ubiquitination levels in scrambled-and TRAP1-interfered cells were analyzed.…”

“…The 'fishing for partners' strategy combined with mass spectrometric procedures performed to identify TRAP1 protein partners has already been described elsewhere. 12 Among the putative TRAP1-binding partners, we focused our attention on a protein of about 50 kDa identified by our LC-MS/MS analysis as S6/TBP7/ATPase-4/Rpt3. 19 To confirm that this proteasome subunit was indeed a TRAP1-interacting protein, we performed western blot (WB) and co-IP analyses in total extracts from HCT116 colon carcinoma cells ( Figure 1A).…”

“…This unchanged overall protein stability led us to hypothesize that TRAP1 could be involved in the control of protein folding/stability for selective proteins, likely those directed to mitochondria. To evaluate this hypothesis, the levels of F1ATPase, a nuclear-encoded MITO protein and a potential TRAP1 interactor as suggested by MS analysis, 12 were analyzed in cells in which TRAP1 expression was lowered by short-hairpin RNA (shRNA) interference. Interestingly, protein levels decreased in TRAP1 stably interfered cells (Figure 5b and Supplementary Figure 2), whereas a rescue was obtained upon re-addition of the D1-59 TRAP1 mutant but not by the D101-221 TRAP1 mutant (Supplementary Figure 3).…”

“…Among several other proteins, a novel MITO isoform of Sorcin, a calcium-binding protein, was identified as a new TRAP1 'ligand' and a cytoprotective function against apoptosis induced by antiblastic agents was recently demonstrated for this protein by our group. 12 In the present paper, we characterize another new interaction of TRAP1 with TBP7/ATPase-4/Rpt3, an S6 ATPase protein of the proteasome regulatory subunit. 13,14 TBP7 was first identified as a novel synphilin-1-interacting protein, 13 so a functional role in Parkinson's disease was proposed for this protein.…”