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Summary Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by loss of motor neurons. With conformation specific antibodies, we now demonstrate that misfolded mutant SOD1 binds directly to the voltage-dependent anion channel (VDAC1), an integral membrane protein imbedded in the outer mitochondrial membrane. This interaction is found on isolated spinal cord mitochondria and can be reconstituted with purified components in vitro. ADP passage through the outer membrane is diminished in spinal mitochondria from mutant SOD1-expressing ALS rats. Direct binding of mutant SOD1 to VDAC1 inhibits conductance of individual channels when reconstituted in a lipid bilayer. Reduction of VDAC1 activity with targeted gene disruption is shown to diminish survival by accelerating onset of fatal paralysis in mice expressing the ALS-causing mutation SOD1G37R. Taken together, our results establish a direct link between misfolded mutant SOD1 and mitochondrial dysfunction in this form of inherited ALS.

The antiapoptotic proteins of the Bcl-2 family are expressed at high levels in many types of cancer. However, the mechanism by which Bcl-2 family proteins regulate apoptosis is not fully understood. Here, we demonstrate the interaction of Bcl-2 with the outer mitochondrial membrane protein, voltage-dependent anion channel 1 (VDAC1). A direct interaction of Bcl-2 with bilayer-reconstituted purified VDAC was demonstrated, with Bcl-2 decreasing channel conductance. Expression of Bcl-2-GFP prevented apoptosis in cells expressing native but not certain VDAC1 mutants. VDAC1 sequences and amino acid residues important for interaction with Bcl-2 were defined through site-directed mutagenesis. Synthetic peptides corresponding to the VDAC1 N-terminal region and selected sequences bound specifically, in a concentration-and time-dependent manner, to immobilized Bcl-2, as revealed by the real-time surface plasmon resonance. Moreover, expression of the VDAC1-based peptides in cells over-expressing Bcl-2 prevented Bcl-2-mediated protection against staurosporine-induced apoptotic cell death. Similarly, a cell-permeable VDAC1-based synthetic peptide was also found to prevent Bcl-2-GFP-mediated protection against apoptosis. These results point to Bcl-2 as promoting tumor cell survival through binding to VDAC1, thereby inhibiting cytochrome c release and apoptotic cell death. Moreover, these findings suggest that interfering with the binding of Bcl-2 to mitochondria by VDAC1-based peptides may serve to potentiate the efficacy of conventional chemotherapeutic agents.Mitochondria-mediated apoptosis can occur in response to conditions that result in an increase in outer mitochondrial membrane permeability, allowing for the release of apoptogenic protein to the cytosol including cytochrome c, apoptosis inducing factor, and Smac/Diablo (1, 2), in a manner controlled by pro-and antiapoptotic proteins of the Bcl-2 family. Members of the Bcl-2 family of proteins are central regulators of apoptosis and include proteins essential for cell survival, as well as those required to initiate cell death (3-5). The Bcl-2 family comprises antiapoptotic members, such as Bcl-2, Mcl-1, Bcl-x L , and Bcl2A1; multidomain proapoptotic members, such as Bax and Bak; and proapoptotic BH3-only proteins, including Bad, Bim, Puma, Bid, Bik, Noxa, and Bmf (3, 6 -9). Whereas antiapoptotic proteins, such as Bcl-2, are localized to several intracellular membranes, including those of the mitochondria, endoplasmic reticulum, and the nuclear envelope (4, 10), proapoptotic members of the Bcl-2 family, such as Bax, translocate to mitochondria upon sensing a death signal (11). Antiapoptotic members of the Bcl-2-family also contribute to tumor initiation, disease progression, and drug resistance. Indeed, high expression levels of antiapoptotic Bcl-2 family members were shown to be associated with resistance of many tumors to chemotherapy (6, 12, 13).The mechanisms by which Bcl-2 family proteins regulate apoptosis are still not fully understood, yet it is well established ...

Background: Bcl-xL is overexpressed in cancer, contributing to resistance to chemotherapy. Results: Bcl-xL directly interacts with VDAC1 to mediate its antiapoptotic activity, activity that can be prevented by VDAC1-based peptides. Conclusion: Bcl-xL regulates apoptosis through direct interaction with VDAC1. Significance: Interfering with the interaction of Bcl-xL with VDAC1 can lead to apoptosis and potentiate the efficacy of conventional chemotherapeutics.