This phenylalanine located near to the catalytic site of E2 has been found to be responsible for substrate specificity and its substitution could be directly responsible for decreased enzymatic activity in this case. No major structural changes were observed in this mutant core. In summary, our recombinant PDC model has proved to be of considerable benefit in enabling us to gain a more informed insight into the molecular mechanisms of pathogenesis underlying these rare E2-linked mutations, particularly in the case of the E35-E2 mutant. In the absence of the recombinant model, such detailed investigations would have proved impossible owing to the lack of access to human tissue from individual patients. As a corollary to the main aim of the thesis, a preliminary attempt was made to create an equivalent recombinant OGDC model. OGDC is also a mitochondrial assembly that is involved in the TCA cycle and is increasingly implicated in the aetiology of various neurodegenerative diseases linked to oxidative stress including Alzheimer’s and Parkinson’s disease. The basic organisation of the OGDC is directed by the self-assembly of 24 copies of dihydrolipoyl succinyltransferase (E2o) to form a cubic core, to which multiple copies of 2-oxoglutarate dehydrogenase (E1o) and dihydrolipoamide dehydrogenase (E3) bind non-covalently. The mammalian E2o is unusual in lacking any obvious E3 or E1o binding domain. In this study, E2o and E3 were successfully overexpressed and purified. Initially it was confirmed that E2o and E3 do not interact with each other on gel filtration although stable association of all 3 constituent enzymes occurs in the native complex. Full-length E1o was also cloned successfully although it proved impossible to achieve detectable expression in our E. coli BL21 host system. Previous studies employing subunit-specific proteolysis have identified the extreme N-terminal segment of E1o as a key region involved in the maintenance of complex stability and integrity and is required for E2 and E3 binding. To investigate this region in more detail, three N-terminal E1o fragments of decreasing size were overexpressed, one in His-tag form (193 amino acids) and two as E1o-GST fusion proteins (166 and 83 amino acids). In co-expression, purification and gel filtration studies, it was found that all these N-terminal truncates of E1o appeared capable of interacting with E2o although problems were encountered with rapid degradation and unambiguous identification in some cases. However, Western blotting revealed conclusively that even the shortest N-terminal E1o fragment (83 amino acids) was able to enter into a stable association with E2o. Owing to time constraints and difficulties with rapid degradation and/or solubility of the E1o truncates, it remains to be determined whether this N-terminal region of E1o can also interact with E2o in a post-translational fashion and whether it is directly involved in mediating E3 binding. However, this type of approach should continue to provide additional insights in the unique subunit organisation of OGDC and is an important step towards creating a recombinant model of OGDC. This will be invaluable for future studies on an important metabolic assembly that has been increasingly implicated in disorders linked to oxidative stress and neurodegeneration.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:499428 |
| Date | January 2008 |
| Creators | Singh, Geetanjali |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/214/ |
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