title: Bcl-2 is a novel interacting partner for the 2-oxoglutarate carrier and a key regulator of mitochondrial glutathione.
abstract: Despite making up only a minor fraction of the total cellular glutathione, recent studies indicate that the mitochondrial glutathione pool is essential for cell survival. Selective depletion of mitochondrial glutathione is sufficient to sensitize cells to mitochondrial oxidative stress (MOS) and intrinsic apoptosis. Glutathione is synthesized exclusively in the cytoplasm and must be actively transported into mitochondria. Therefore, regulation of mitochondrial glutathione transport is a key factor in maintaining the antioxidant status of mitochondria. Bcl-2 resides in the outer mitochondrial membrane where it acts as a central regulator of the intrinsic apoptotic cascade. In addition, Bcl-2 displays an antioxidant-like function that has been linked experimentally to the regulation of cellular glutathione content. We have previously demonstrated a novel interaction between recombinant Bcl-2 and reduced glutathione (GSH), which was antagonized by either Bcl-2 homology-3 domain (BH3) mimetics or a BH3-only protein, recombinant Bim. These previous findings prompted us to investigate if this novel Bcl-2/GSH interaction might play a role in regulating mitochondrial glutathione transport. Incubation of primary cultures of cerebellar granule neurons (CGNs) with the BH3 mimetic HA14-1 induced MOS and caused specific depletion of the mitochondrial glutathione pool. Bcl-2 was coimmunoprecipitated with GSH after chemical cross-linking in CGNs and this Bcl-2/GSH interaction was antagonized by preincubation with HA14-1. Moreover, both HA14-1 and recombinant Bim inhibited GSH transport into isolated rat brain mitochondria. To further investigate a possible link between Bcl-2 function and mitochondrial glutathione transport, we next examined if Bcl-2 associated with the 2-oxoglutarate carrier (OGC), an inner mitochondrial membrane protein known to transport glutathione in liver and kidney. After cotransfection of CHO cells, Bcl-2 was coimmunoprecipitated with OGC and this novel interaction was significantly enhanced by glutathione monoethyl ester. Similarly, recombinant Bcl-2 interacted with recombinant OGC in the presence of GSH. Bcl-2 and OGC cotransfection in CHO cells significantly increased the mitochondrial glutathione pool. Finally, the ability of Bcl-2 to protect CHO cells from apoptosis induced by hydrogen peroxide was significantly attenuated by the OGC inhibitor phenylsuccinate. These data suggest that GSH binding by Bcl-2 enhances its affinity for the OGC. Bcl-2 and OGC appear to act in a coordinated manner to increase the mitochondrial glutathione pool and enhance resistance of cells to oxidative stress. We conclude that regulation of mitochondrial glutathione transport is a principal mechanism by which Bcl-2 suppresses MOS.
authors: Department of Biological Sciences and Eleanor Roosevelt Institute, University of Denver, Denver, CO 80208, USA.
authors: Heather M
authors: HM
authors: Wilkins
authors: Kristin
authors: K
authors: Marquardt
authors: Lawrence H
authors: LH
authors: Lash
authors: Daniel A
authors: DA
authors: Linseman
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001578:Benzopyrans / Q000494:pharmacology; D016466:CHO Cells; D002531:Cerebellum / Q000166:cytology; D006224:Cricetinae; D005978:Glutathione / Q000378:metabolism* / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008315:Malondialdehyde / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress; D011485:Protein Binding; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D001578:Benzopyrans; D026901:Membrane Transport Proteins; D009570:Nitriles; D016877:Oxidants; D019253:Proto-Oncogene Proteins c-bcl-2; C456800:ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate; C021723:oxoglutarate translocator; D008315:Malondialdehyde; D006861:Hydrogen Peroxide; D005978:Glutathione