No abstract

Glycolysis and apoptosis are considered major but independent pathways that are critical for cell survival. The activity of BAD, a pro-apoptotic BCL-2 family member, is regulated by phosphorylation in response to growth/survival factors. Here we undertook a proteomic analysis to assess whether BAD might also participate in mitochondrial physiology. In liver mitochondria, BAD resides in a functional holoenzyme complex together with protein kinase A and protein phosphatase 1 (PP1) catalytic units, Wiskott-Aldrich family member WAVE-1 as an A kinase anchoring protein, and glucokinase (hexokinase IV). BAD is required to assemble the complex in that Bad-deficient hepatocytes lack this complex, resulting in diminished mitochondria-based glucokinase activity and blunted mitochondrial respiration in response to glucose. Glucose deprivation results in dephosphorylation of BAD, and BAD-dependent cell death. Moreover, the phosphorylation status of BAD helps regulate glucokinase activity. Mice deficient for BAD or bearing a non-phosphorylatable BAD(3SA) mutant display abnormal glucose homeostasis including profound defects in glucose tolerance. This combination of proteomics, genetics and physiology indicates an unanticipated role for BAD in integrating pathways of glucose metabolism and apoptosis.

The antibiotic lactacystin was reported to covalently modify ␤-subunit X of the mammalian 20 S proteasome and inhibit several of its peptidase activities. However, we demonstrate that [ 3 H]lactacystin treatment modifies all the proteasome's catalytic ␤-subunits. Lactacystin and its more potent derivative ␤-lactone irreversibly inhibit protein breakdown and the chymotryptic, tryptic, and peptidylglutamyl activities of purified 20 S and 26 S particles, although at different rates. Exposure to these agents for 1 to 2 h reduced the degradation of short-and long-lived proteins in four different mammalian cell lines. Unlike peptide aldehyde inhibitors, lactacystin and the ␤-lactone do not inhibit lysosomal degradation of an endocytosed protein. These agents block class I antigen presentation of a model protein, ovalbumin (synthesized endogenously or loaded exogenously), but do not affect presentation of the peptide epitope SIINFEKL, which does not require proteolysis for presentation. Generation of most peptides required for formation of stable class I heterodimers is also inhibited. Because these agents inhibited protein breakdown and antigen presentation similarly in interferon-␥-treated cells (where proteasomes contain LMP2 and LMP7 subunits in place of X and Y), all ␤-subunits must be affected similarly. These findings confirm our prior conclusions that proteasomes catalyze the bulk of protein breakdown in mammalian cells and generate the majority of class I-bound epitopes for immune recognition.

The degradation of most cellular proteins starts with their covalent conjugation with ubiquitin. This labels the proteins for rapid hydrolysis to oligopeptides by a (26S) proteolytic complex containing a (20S) degradative particle called the proteasome. Some system in the cytosol also generates antigenic peptides from endogenously synthesized cellular and viral proteins. These peptides bind to newly synthesized class I major histocompatibility complex molecules in the endoplasmic reticulum and peptide/class I complexes are then transported to the cell surface for presentation to cytotoxic T cells. How these peptides are produced is unknown, although a modification that promotes ubiquitin-dependent degradation of a viral protein enhances its presentation with class I13 and indirect evidence suggests a role for proteolytic particles closely resembling and perhaps identical to the proteasome. Using cells that exhibit a temperature-sensitive defect in ubiquitin conjugation, we report here that non-permissive temperature inhibited class I-restricted presentation of ovalbumin introduced into the cytosol, but did not affect presentation of an ovalbumin peptide synthesized from a minigene. These results implicate the ubiquitin-dependent proteolytic pathway in the production of antigenic peptides.