Branched Chain Amino Acid Metabolism

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In addition to their role as protein building blocks, amino acids have a significant role as nutritional signals. The BCAA leucine is a nutrient signal that stimulates increased protein synthesis in target tissues, and it Js our hypothesis that leucine signaling is linked to leucine metabolism. The first step in the catabolic pathway is reversible transamination catalyzed by the branched chain aminotransferase isozymes (BCAT). We have solved several structures of the human mitochondrial BCAT (hBCATm) and discovered that it contains a redox-active dithiol/disulfide center. The redox-linked regulation of activity by the CXXC center demonstrates that the cysteine residues are part of a peroxide-sensing mechanism that may have an in vivo role. We have discovered that hBCATm can associate with the BCKD complex and that this interaction is redox sensitive. The association between hBCATm and the mitochondrial BCKD complex suggests that BCAA catabolic enzymes form supramolecular complex called a metabolon. We will test the hypothesis that a protein web controls BCAA metabolism using the BCAT proteins as "bait" to identify protein components of the BCAA metabolon. Determination of the protein web for the BCAT, particularly of the neuron-specific cytosolic isozyme (BCATc) and a newly recognized alternatively spliced form of human BCATm (hBCATm- sp), is likely to allow identification of new functions for these proteins. The rate-limiting step in BCAA oxidation is catalyzed in mitochondria by the branched-chain alpha-keto acid dehydrogenase enzyme complex (BCKD). BCKD activity is regulated by phosphorylation and dephosphorylation of a specific serine residue on the Ela subunit. We will determine the role of BCATm in leucine signaling, that is, whether leucine or KIC is the signal that activates the mTOR-signaling pathway in leucine-sensitive tissues in vivo in the rat. Using a newly developed rapid and simple test system to measure acute regulation of BCKD, the role of BCATm and link between leucine activation of the mTOR signaling pathway and BCKD complex (metabolism) will be determined in vivo and in a 13-cell model. It is our hope that the ideas put forth in this proposal will bring new ways of thinking into the field of amino acid metabolism and will lead to exciting new research directions.