CATALYSIS OF THIOL DISULFIDE EXCHANGE

蛋白质必须折叠成正确的构象才能发挥生物学功能。 在细胞中，蛋白质折叠是由催化剂，加速折叠和分子伴侣，抑制聚集。 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的用途。 我们解决这些问题的长期方法是理解和模拟细胞折叠助手在促进正确折叠方面的行为。 该建议的重点是蛋白质二硫键异构酶（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。作为分子伴侣，它抑制底物聚集，但在某些条件下，PDI可以促进聚集。 直接的目标是定义催化的机制，并确定PDI如何抑制或刺激聚集。 具体目标1。 待检验的假设是PDI通过多个还原和氧化循环催化二硫键异构化。 诱变作用将被用于在体外和在S.酿酒酵母，以检查特定途径的贡献。 具体目标2。该假设是PDI通过其稳定性来区分错误折叠的蛋白质和天然蛋白质。 如果正确，该假设表明PDI展开其底物的能力应与其稳定性相关。 具体目标3。PDI的催化有效性可归因于活性位点的中间氧化还原电位和/或PDI到结构域中的排列。 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。 缺失突变体的催化性质将决定辅助结构域的贡献。 具体目标4。工作模型表明，PDI通过共价和非共价相互作用交联较小的底物聚集体来促进聚集。 沉降速度实验将确定聚集的物种。 将研究缺失一个或多个结构域的PDI突变体以定位底物相互作用的位点。 这些目标的完成将建议采取新的战略，鼓励适当的合并，不鼓励聚合。

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.