Hsp90 inhibition decreases mitochondrial protein turnover.

BACKGROUND: Cells treated with hsp90 inhibitors exhibit pleiotropic changes, including an expansion of the mitochondrial compartment, accompanied by mitochondrial fragmentation and condensed mitochondrial morphology, with ultimate compromise of mitochondrial integrity and apoptosis. FINDINGS: We identified several mitochondrial oxidative phosphorylation complex subunits, including several encoded by mtDNA, that are upregulated by hsp90 inhibitors, without corresponding changes in mRNA abundance. Post-transcriptional accumulation of mitochondrial proteins observed with hsp90 inhibitors is also seen in cells treated with proteasome inhibitors. Detailed studies of the OSCP subunit of mitochondrial F1F0-ATPase revealed the presence of mono- and polyubiquitinated OSCP in mitochondrial fractions. We demonstrate that processed OSCP undergoes retrotranslocation to a trypsin-sensitive form associated with the outer mitochondrial membrane. Inhibition of proteasome or hsp90 function results in accumulation of both correctly targeted and retrotranslocated mitochondrial OSCP. CONCLUSIONS: Cytosolic turnover of mitochondrial proteins demonstrates a novel connection between mitochondrial and cytosolic compartments through the ubiquitin-proteasome system. Analogous to defective protein folding in the endoplasmic reticulum, a mitochondrial unfolded protein response may play a role in the apoptotic effects of hsp90 and proteasome inhibitors.

2-Methoxy antimycin reveals a unique mechanism for Bcl-x(L) inhibition.

Overexpression of Bcl-x(L) in multiple cancers correlates with resistance to chemotherapy and radiation therapy, and provides a rationale for development of small-molecule Bcl-x(L) inhibitors. Based on knockout studies, nonneoplastic cells also require Bcl-x(L) survival functions, particularly when challenged with cytotoxic agents. We analyze the selective cytotoxicity of one Bcl-x(L) inhibitor, 2-methoxy antimycin A, toward cells with excess exogenous Bcl-x(L) in isogenic cell line pairs. This selectivity, characteristic of a gain-of-function mechanism, is not shared by other known Bcl-x(L) inhibitors, including BH3I-2, HA14-1, ABT-737, gossypol, or the stapled BH3 helical peptide SAHB-BID. We show that Bcl-x(L) overexpression induces a shift in energy metabolism from oxidative phosphorylation to glycolysis. Treatment with 2-methoxy antimycin A acutely reverses the metabolic effects of Bcl-x(L), causing mitochondrial hyperpolarization and a progressive increase in mitochondrial NAD(P)H. We identify an additional small-molecule Bcl-x(L) inhibitor, NSC 310343, establishing a class of Bcl-x(L) inhibitors with gain-of-function activity. In contrast to other Bcl-x(L) inhibitors, combining gain-of-function Bcl-x(L) inhibitors with a standard inducer of apoptosis, staurosporine, enhances selective cytotoxicity toward Bcl-x(L)-overexpressing cells. These results provide an example of the intersection of bioenergetic metabolism and Bcl-x(L) functions and suggest a metabolic basis for the gain-of-function mechanism of Bcl-x(L) inhibitors.