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

Abstract

Tumor necrosis factor receptor-associated protein-1 (TRAP1) is a mitochondrial (MITO) antiapoptotic heat-shock protein. The information available on the TRAP1 pathway describes just a few well-characterized functions of this protein in mitochondria. However, our group's use of mass-spectrometric analysis identified TBP7, an AAA-ATPase of the 19S proteasomal subunit, as a putative TRAP1-interacting protein. Surprisingly, TRAP1 and TBP7 colocalize in the endoplasmic reticulum (ER), as demonstrated by biochemical and confocal/electron microscopic analyses, and interact directly, as confirmed by fluorescence resonance energy transfer analysis. This is the first demonstration of TRAP1's presence in this cellular compartment. TRAP1 silencing by short-hairpin RNAs, in cells exposed to thapsigargin-induced ER stress, correlates with upregulation of BiP/Grp78, thus suggesting a role of TRAP1 in the refolding of damaged proteins and in ER stress protection. Consistently, TRAP1 and/or TBP7 interference enhanced stress-induced cell death and increased intracellular protein ubiquitination. These experiments led us to hypothesize an involvement of TRAP1 in protein quality control for mistargeted/misfolded mitochondria-destined proteins, through interaction with the regulatory proteasome protein TBP7. Remarkably, expression of specific MITO proteins decreased upon TRAP1 interference as a consequence of increased ubiquitination. The proposed TRAP1 network has an impact in vivo, as it is conserved in human colorectal cancers, is controlled by ER-localized TRAP1 interacting with TBP7 and provides a novel model of the ER-mitochondria crosstalk. Tumor necrosis factor receptor-associated protein-1 (TRAP1) was initially identified as a TNF-receptor-associated protein and is a member of the heat-shock protein-90 (HSP90) chaperone family. 1,2 Through an mRNA-differential display analysis between oxidant-adapted and control osteosarcoma cells, our group identified, among other proteins, TRAP1, whose expression was highly induced upon oxidant adaptation. 3 Furthermore, TRAP1 showed antioxidant and antiapoptotic functions, 4 while an involvement of this mitochondrial (MITO) chaperone in the multi-drug resistance of human colorectal carcinoma (CRC) cells was also established. 5 Little is known about TRAP1 signal transduction: the first most important finding on TRAP1 function came from studies by the Altieri's group, which identified TRAP1 as a member of a cytoprotective network selectively active in the mitochondria of tumor tissues. 6 The same group has recently proposed TRAP1 as a novel molecular target in localized and metastatic prostate cancer, 7 and is now involved in a promising preclinical characterization of mitochondria-targeted smallmolecule HSP90 inhibitors. 8,9 Besides some well-characterized TRAP1 functions in mitochondria, during preparation of this manuscript it was reported that interference by HSP90 chaperones triggers an unfolded protein response (UPR) and activates autophagy in the mitochondria of tumor cells. 10 A put...

ER stress protection in cancer cells: the multifaceted role of the heat shock protein TRAP1

“…Several lines of evidence suggest that Sorcin is up-regulated in human malignancies, where it is involved in cytoprotective functions and drug resistance. The potential involvement of Sorcin in cytoprotection is consistent with the evidence that the isoform B, whose expression is restricted to mitochondria, is a TRAP1-binding protein [4,19]. Remarkably, TRAP1 regulates, together with TBP7, the ubiquitination of Sorcin isoform B; consequently, Sorcin expression levels are decreased upon TRAP1 interference, as a consequence of increased ubiquitination [4].…”

“…The potential involvement of Sorcin in cytoprotection is consistent with the evidence that the isoform B, whose expression is restricted to mitochondria, is a TRAP1-binding protein [4,19]. Remarkably, TRAP1 regulates, together with TBP7, the ubiquitination of Sorcin isoform B; consequently, Sorcin expression levels are decreased upon TRAP1 interference, as a consequence of increased ubiquitination [4]. The 22kDa isoform of Sorcin, which is the most abundant cellular isoform and is up-regulated in about 50% of human CRCs, is not a TRAP1 interacting protein [19]; however, in human CRCs, TRAP1 expression significantly correlates with the protein levels of 22kDa Sorcin [20].…”

“…In this scenario, our recent evidence demonstrating that TRAP1 is involved in the crosstalk between mitochondria and Endoplasmic Reticulum (ER) and in ER stress protection of tumor cells appear remarkable: in fact, our group, for the first time, reported a role of TRAP1 in protein quality control, due to its interaction with the proteasomal particle TBP7 outside of mitochondria, namely on the outer side of the ER [4]. Given the ER localization of TRAP1 and TBP7, it became important to assess the involvement of these two proteins in ER homeostasis.…”

“…Accordingly, this phenotype is rescued upon TRAP1 re-addition and/or transfection of a TRAP1 deletion mutant (Δ1-59-TRAP1), which lacks the mitochondrial targeting sequence and is therefore unable to enter mitochondria; conversely, another deletion mutant with mitochondrial localization (Δ101-221-TRAP1) not only is unable to counteract ER stress, but even further increased BiP/Grp78 levels. The interference of either TRAP1 or TBP7 proteins results in the induction of apoptosis both in response to drug-induced ER stress and in increased intracellular protein ubiquitination, which is selectively rescued by the re-addition of TRAP1 [4]. The elimination of misfolded proteins represents an important mechanism to maintain cell viability.…”

TRAP1 regulates cell cycle and apoptosis in thyroid carcinoma cells

“…Indeed, TRAP1 multifaceted functions in protection from apoptosis, regulation of cell cycle, cell metabolism and protein homeostasis have been widely demonstrated in several human malignancies (Kang et al 2007, Amoroso et al 2012, Condelli et al 2014, Rasola et al 2014. However, controversial data have been reported on whether TRAP1 behaves as oncogene or oncosuppressor, being its expression levels increased in the majority of human malignancies (Costantino et al 2009, Agorreta et al 2014, Rasola et al 2014, but downregulated along with increased tumor grading in specific human tumors (i.e.…”