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Title: Redox binding partners of the hBCAT proteins and their distribution and expression in control and disease human brains : implications in protein folding in neurodegeneration
Author: Lee, Christopher
ISNI:       0000 0004 6351 4287
Awarding Body: University of the West of England
Current Institution: University of the West of England, Bristol
Date of Award: 2017
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INTRODUCTION: Novel binding partners of the branched chain aminotransferase proteins (hBCAT) proteins have been identified and indicate that these metabolic proteins may have alternative functions to their housekeeping role. In particular, the molecular chaperone, protein disulphide isomerase (PDI) was shown to co-localise with mitochondrial hBCAT, where a role for hBCAT in protein folding was demonstrated. Moreover, the cytosolic isoform, hBCATc, showed redox-specific interactions with key regulators of cytoskeletal metabolism, implicating hBCATc in protein trafficking. These associations were influenced by changes in the redox environment, where specific interactions were lost on oxidation. Knowledge of these and other redox-dependent and independent associations are important, in particular as hBCAT are significantly increased in the brains of Alzheimer’s disease. If these cellular associations are dysregulated they could contribute to the pathological changes observed in Alzheimer’s disease brain. We hypothesise that hBCAT is a multifunctional enzyme and its direction of metabolism is directed by its response to changes in the redox environment, dictated through modification of its CXXC redox switch. Moreover, identification of these proteins in neurodegenerative conditions may highlight new markers of disease association. AIMS: The overall aim of this thesis is to identify new binding partners for the hBCAT proteins (both redox-dependent and –independent), which will offer insight into their multifunctional role within the cell. Within this aim, novel indicators of pathology in neurodegenerative conditions, associated with hBCAT function will be identified. These will be examined for their potential role as biomarkers in disease conditions. METHODS: Using nano LC-MS/MS, specific binding partners were identified following affinity hBCAT-tagged chromatography. Under specific redox conditions, proteins extracted from the neuronal cell lines: IMR32 and SH- SY-5Y, and endothelial cells, hCMEC/D3 were shown to differentially bind to affinity columns tagged with either wild-type hBCAT or mutant hBCAT. Key proteins identified in these studies were further mapped in human brain, using immunohistochemistry, to understand their association with hBCAT. Finally, to evaluate their expression in Alzheimer’s disease and Parkinson’s disease, we used immunohistochemistry and Western blot analysis. RESULTS AND DISCUSSION: Here, we have shown for the first time that the hBCAT proteins differentially bind to specific proteins involved in protein folding, cytoskeletal regulation, alternative splicing and metabolic energy metabolism. These interactions were both redox-dependent and redox-independent, indicating that this protein can function in the cell even if oxidised to its inactive metabolic form. Chronic oxidation, as suggested to feature in neurodegenerative conditions, is thought to contribute to protein misfolding,cytoskeletal dysfunction in addition to disrupting other processes such as mitochondrial function, which all contribute to cellular death. Here, oxidation prevented hBCAT interacting with cytoskeleton and key metabolic proteins but did not affect interactions with chaperones or the proteasome, and even evoked new interactions with the enzymes of ubiquitinylation. These and other associations offer exciting new insights into the role of hBCAT in cells. Several of these chaperones that were involved with protein folding, including, the chaperones BiP, BAG1 and TCP1, were mapped to the human brain for the first time. Distribution was ubiquitous throughout the brain in all cell types except for BAG1, which was not present within granular cells of the hippocampus and the cerebellum. Co-expression with hBCATc was evident in several neuronal cell types of the temporal lobe, frontal lobe and the cerebellum, and hBCATm showed co-expression in endothelial cells and some neuronal cells of the same brain regions. Confirmation that these proteins are localised to the same brain cells and regions indicates that these interactions that were identified through these in vitro associations may have relevance in the human brain. To evaluate if these expression patterns are modified in neurodegenerative conditions, expression of hBCAT was examined in the hippocampus of Alzheimer's disease and expression of hBCAT, BiP and TCP1 was examined in the midbrain of Parkinson's disease. Expression of hBCAT was elevated in Alzheimer's disease however, expression of hBCAT, BiP and TCP1 were all decreased in Parkinson's disease and therefore hBCAT may represent a potential biomarker with good differentiation between diseases. Overall, these studies indicate that proteins involved in protein folding/chaperone activity are modified differentially in certain brain areas of Alzheimer's disease and Parkinson's disease. CONCLUSION: Overall, data generated in this thesis supports the role of hBCAT as a multi-functional cellular protein. Future studies of their functional role with these partners will offer exciting insight into these moonlighting roles. Moreover, their co-localisation in the human brain and differential expression may point to new areas of biomarker development and therapeutic targeting.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: hBCAT proteins ; brains