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Abstract

The branched chain amino acid transaminase 1 (BCAT1) catalyzes the first step in the catabolism of the branched chain amino acids (BCAAs), a reaction that utilizes alpha-ketoglutarate (α-KG) as an acceptor of the BCAA alpha-amino group to generate branched-chain ketoacids (BCKAs) and glutamate. In prior work, our work group has identified BCAT1 as a novel pro-tumorigenic metabolic enzyme in glioblastoma. Most recently, we proposed that BCAT1 supports tumor growth by limiting intracellular α-KG levels and thus the activity of chromatin-modifying DNA and histone demethylases and other α-KG-dependent enzymes. In this doctoral thesis, I aimed to elucidate the mode of action of BCAT1 in glioblastoma cells, using the α-KG depletion model as a working hypothesis. I found that BCAT1 localize to the nucleus as well as the cytoplasm of glioblastoma cells, but immunoprecipitation and mass spectrometry analysis did not identify any α-KG-dependent enzymes as potential BCAT1 interaction partners. Instead, BCAT1 was associated with components of the cytoskeleton and proteins involved in cell cycle regulation, mitosis, and endocytic signaling. Knockout of BCAT1 in glioblastoma cell lines reduced cell proliferation and migration, impeded the cells’ capacity to buffer reactive oxygen species, suppressed peroxide-dependent epidermal growth factor receptor (EGFR) activation and caused extensive mitotic failures. Using a knockout and rescue approach, I showed that re-expression of wildtype and catalytic-mutant BCAT1 rescued the reactive oxygen species-buffering and proliferation phenotypes of BCAT1 knockout cells, whereas re-expression of BCAT1 in which a redox-active CXXC motif had been mutated did not. In summary, I identified a redox-dependent mode of action underlying BCAT1-driven growth of glioblastoma and provided evidence that this new BCAT1 redox mechanism might be more important for clinically relevant glioblastoma phenotypes than the well-established BCAT1 transaminase activity.