Localization of MRP‐1 to the outer mitochondrial membrane by the chaperone protein HSP90β

Abstract

Overexpression of plasma membrane multidrug resistance-associated protein 1 (MRP-1) in Ewing's sarcoma (ES) predicts poor outcome. MRP-1 is also expressed in mitochondria, and we have examined the submitochondrial localization of MRP-1 and investigated the mechanism of MRP-1 transport and role of this organelle in the response to doxorubicin. The mitochondrial localization of MRP-1 was examined in ES cell lines by differential centrifugation and membrane solubilization by digitonin. Whether MRP-1 is chaperoned by heat shock proteins (HSPs) was investigated by immunoprecipitation, immunofluorescence microscopy, and HSP knockout using small hairpin RNA and inhibitors (apoptozole, 17-AAG, and NVPAUY). The effect of disrupting mitochondrial MRP-1-dependent efflux activity on the cytotoxic effect of doxorubicin was investigated by counting viable cell number. Mitochondrial MRP-1 is glycosylated and localized to the outer mitochondrial membrane, where it is coexpressed with HSP90. MRP-1 binds to both HSP90 and HSP70, although only inhibition of HSP90β decreases expression of MRP-1 in the mitochondria. Disruption of mitochondrial MRP-1-dependent efflux significantly increases the cytotoxic effect of doxorubicin (combination index, <0.9). For the first time, we have demonstrated that mitochondrial MRP-1 is expressed in the outer mitochondrial membrane and is a client protein of HSP90β, where it may play a role in the doxorubicin-induced resistance of ES.-Roundhill, E., Turnbull, D., Burchill, S. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β.

RNA sequencing and functional studies of patient-derived cells reveal that neurexin-1 and regulators of this pathway are associated with poor outcomes in Ewing sarcoma

“…On the other hand, although low levels of ALDH were detected by RNA sequencing in the patient-derived ES cells, its high expression did not predict outcome in the GSE17618 dataset. In contrast, read counts and prognostic significance of the ABC transporter proteins P-glycoprotein (Pgp) and multi-drug resistance protein-1 (MRP1) (Additional file 6; Table S3), were consistent with previous observations reporting the prognostic significance of high levels of MRP1 [37,46], but not Pgp [46] in ES. These observations emphasise the importance of comparing the prognostic and functional significance of candidate biomarkers of risk with established clinical risk factors at diagnosis, to develop a high performance prognostic model combining biology and clinical risk factors to predict patient outcomes to meet personalised medical needs.…”

“…2.6 Knock-down of neurexin-1 by shRNA and siRNA ES cells were infected with neurexin-1 (sc-42,050-V) short hairpin RNA (shRNA) lentiviral particles targeting both neurexin-1α and -β isoforms or scrambled sequence controls (sc-108,080; Santa Cruz Biotechnology, Inc.; each shRNA pool containing 3-5 constructs [37]). Infected cells were selected in puromycin (0.5 μg/μl; Sigma-Aldrich) for 10 days before placing them in normal growth media.…”

Novel function of ceramide for regulation of mitochondrial ATP release in astrocytes

“…S4A and B). For the coimmunoprecipitation experiment, digitonin (1%) was used as a detergent because it is known to preserve the membrane environment in ceramide biosynthesis and it is commonly used for solubilization of proteins from the OMM (79)(80)(81). Figure 9G shows that ceramide depletion reduced the amount of coimmunoprecipitated VDAC1 (non-treated cells and nonspecific mouse IgG was used as controls) consistent with decrease of the number of PLA signals by guest, on May 11, 2018 www.jlr.org…”

Anti‐tumor effects of nitidine on camptothecin‐resistant A549 cells in a xenograft mouse model

“…This phenomenon was thought to contribute to the suppression of apoptosis induced by anti‐cancer drugs in these cells. Interestingly, it has also been noted that ABC transporters in the mitochondrial membrane are involved in the drug response . We observed a dissipation of mitochondrial membrane potential accompanied by NTD accumulation (@@@Iwasaki et al , unpubl.…”