CATALYSIS OF THIOL DISULFIDE EXCHANGE

蛋白质必须折叠成正确的构象才能获得生物学功能。在细胞中，蛋白质折叠由加速折叠的催化剂和抑制聚集的伴侣蛋白辅助。蛋白质错误折叠和聚集是阿尔茨海默病、朊病毒介导的感染、肺气肿和囊性纤维化的主要原因。错误折叠也限制了重组蛋白用于治疗需要。我们解决这些问题的长期方法是理解和模仿细胞折叠助手在促进正确折叠方面的行为。这一建议的重点是蛋白质二硫异构酶（PDI），折叠催化剂和伴侣。PDI通过催化二硫键的形成和重排来加速折叠。作为伴侣，它抑制底物聚集，但在一定条件下，PDI可以促进聚集。当前的目标是确定催化机制，并确定PDI如何抑制或刺激聚集。具体目标要验证的假设是PDI通过多次还原和氧化循环催化二硫异构化。诱变将用于灭活体外和酿酒酵母中异构化的替代途径，以检查特定途径的贡献。具体目标2。假设是PDI通过稳定性来区分错误折叠蛋白和天然蛋白。如果正确，该假设表明PDI展开底物的能力应该与其稳定性相关。具体目标3。PDI的催化效能可归因于活性位点的中间氧化还原电位和/或PDI在结构域中的排列。诱变将用于改变PDI活性位点的氧化还原电位，以测试其对催化的贡献。缺失突变体的催化特性将决定辅助结构域的贡献。具体目标一个工作模型表明，PDI通过共价和非共价相互作用使较小的底物聚集体交联，从而促进聚集。沉降速度实验将确定聚集的种类。缺少一个或多个结构域的PDI突变体将被研究以定位底物相互作用的位点。完成这些目标将提出新的策略，以鼓励适当的折叠和阻止聚集。

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.