Proteins must fold into a correct conformation to attain biological function. In the cell, protein folding is assisted by catalysts that accelerate folding and by chaperones that inhibit aggregation. Protein misfolding and aggregation is a primary contributor to Alzheimer's disease, prion-mediated infection, emphysema, and cystic fibrosis. Misfolding also limits the use of recombinant proteins for therapeutic needs. Our long-term approach to these problems is to understand and mimic the behavior of cellular folding assistants in promoting correct folding. This proposal focuses on protein disulfide isomerase (PDI), a folding catalyst and a chaperone. PDI accelerates folding by catalyzing disulfide bond formation and rearrangement. As a chaperone, it inhibits substrate aggregation, but under certain conditions, PDI can facilitate aggregation. The immediate goals are to define the mechanisms for catalysis and to determine how PDI inhibits or stimulates aggregation.
Specific Aim 1. The hypothesis to be tested is that PDI catalyzes disulfide isomerization by multiple cycles of reduction and oxidation. Mutagenesis will be used to inactivate alternative pathways for isomerization in vitro and in S. cerevisiae to examine the contributions of specific pathways.
Specific Aim 2. The hypothesis is that PDI distinguishes between misfolded and native proteins by their stability. If correct, the hypothesis suggests that the ability of PDI to unfold its substrates should correlate with their stability.
Specific Aim 3. The catalytic effectiveness of PDI can be attributed to an intermediate redox potential of the active site and/or the arrangement of PDI into structural domains. Mutagenesis will be used to alter the redox potential of PDI active sites to test its contribution to catalysis. The catalytic properties of deletion mutants will define the contribution of accessory domains.
Specific Aim 4. A working model suggests that PDI facilitates aggregation by cross- linking smaller substrate aggregates through covalent and non- covalent interactions. Sedimentation velocity experiments will identify the species that aggregate. PDI mutants missing one or more structural domains will be studied to localize the sites of substrate interaction. The completion of these goals will suggest new strategies to encourage proper folding and to discourage aggregation.
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