Introduction and aims: The branched-chain aminotransferase (BCAT) enzymes are important in the regulation of brain L-glutamate. A unique function of these aminotransferases is their regulation by the redox environment, where our group have shown their function as oxidoreductases, and their ability to refold misfolded proteins in particular when S-glutathionylated. Our group recently showed a significant increase in the level of these proteins in Alzheimer’s disease brain. An increase in BCAT metabolism could generate excess glutamate, contributing to the excitotoxic environment observed under pathogenic conditions. Alternatively, we hypothesize that if this protein is modified in response to cellular stress, it may play a more prominent role in regulating redox status or protein folding. To address these questions this project focussed on the design of chemical inhibitors and knock-down models together with a co-culture model of the human cerebrovasculature and specifically targeted key metabolic and redox pathways.
Methods: Several chemical inhibitors based on 4-Benzyloxyphenylacetic acid were identified using the DockBlaster and Schrödinger software suites, synthesized and structurally verified using proton nuclear magnetic resonance (NMR) spectroscopy. The functional impact and specificity of these inhibitors was assessed using the BCAT radiolabelled assay and a coupled-enzyme assay. In tandem, siRNA was used to knock-down both isoforms in SH-SY5Y cells, and validated using Western blot analysis and RT-PCR. The impact of BCAT inhibition on the expression of redox proteins, in addition to selected metabolic proteins, was assessed by Western blot analysis. Functional redox assays, including glutathione concentration and metabolic activity, were also used to investigate the impact of human BCAT (hBCAT) expression in neuronal cells. Finally, a model of the blood-brain barrier (BBB) was developed and validated for studies into the role of mitochondrial hBCAT (hBCATm) in brain microvasculature.
Results: For the first time we have identified a family of chemical inhibitors of hBCATm. In particular, benzofenac has a two-fold greater enzyme affinity for hBCATm (Ki=43 μM) than cytosolic hBCAT (hBCATc) (Ki=93 μM) and a four-fold greater inhibition relative to alanine transaminase (ALT; Ki=167 μM). These inhibitors will require further optimisation but have potential as tools to assess the cellular function of hBCAT. In separate studies, knock-down of hBCATm in SH-SY5Y neuronal cells demonstrated that hBCATm expression has an impact on the metabolic and redox status of the cell. In particular, knock-down caused a >70% decrease in glutaredoxin (GRx), thioredoxin (TRx), branched-chain α-keto acid dehydrogenase α-subunit (BCKDHA), and AU-rich binding homolog of enoyl-CoA hydratase (AUH) expression. Interestingly, this effect was attenuated when cells were treated with L-leucine, indicating that these mechanisms may be regulated by a metabolic signal. L-glutamate treatment was also found to significantly increase hBCATm expression, but decreased glutamate dehydrogenase (GDH) expression, except in cells overexpressing hBCATm, suggesting a metabolic synergy between the two enzymes. Finally, total glutathione concentration was significantly decreased on hBCATm knock-down, while sensitivity to L-glutamate toxicity was significantly increased.
Discussion: Results from this work has significantly contributed to the design of cellular models, which can be used to further investigate the role of BCAT in metabolic and redox metabolism. A model of the human blood-brain barrier, developed in this thesis, will also contribute to evaluating the endothelial role of hBCATm, particular to humans. The initial impact of limiting BCAT expression is both a reduction in the expression of key metabolic proteins and also the cellular reductants of the cell. Together this had an impact on cell viability and survival. Knowledge of these pathways and their regulation will be important to our understanding, of not only the regulation of brain glutamate, but also the role of BCAT in protein folding and as a contributor to cellular redox status. These factors are fundamentally important to the development of neurodegenerative conditions but also to tumour development such as gliomas.
The role of increased hBCATm in the endothelial cells of patients with Alzheimer’s disease. (Thesis). University of the West of England