Catalysis of Thiol-Disulfide Exchange

说明(由申请人提供)获得正确的构象和结构对于蛋白质的正常功能是必不可少的。在细胞中，蛋白质在加速折叠的酶和抑制聚集的伴侣的帮助下折叠成其固有的结构。蛋白质错误折叠会导致阿尔茨海默氏症、帕金森氏病、Pron介导性感染、肺气肿和囊性纤维化。错误折叠也限制了重组蛋白的有效治疗用途。我们的长期目标是理解和模仿折叠助剂的生物化学和酶学。蛋白质二硫键异构酶(PDI)是内质网(ER)的折叠催化剂和伴侣，对蛋白质二硫键的正确形成和异构化至关重要。提出了以下具体目标。具体目的1.PDI的特异性及其多域结构。PDI的多域结构对于催化异构化是必不可少的，但促进PDI识别和催化的底物相互作用尚未确定。来自底物多肽阵列的数据表明了一种模型，在该模型中，PDI相互作用集中在构成异构化障碍的正电荷底物位置，并且这些位置位于多个PDI结构域上。该模型将通过明确定义的PDI底物中赖氨酸和精氨酸残基的突变以及PDI的结构域缺失突变体与底物序列的多肽阵列的结合来测试。特定目标2.酵母ER中的异构化和还原途径。目前的内质网二硫化物形成模型表明，PDI形成并破坏底物二硫化物，氧化和还原的PDI都是正确折叠所必需的。最近发现的仍支持野生型酵母生长的异构酶缺陷PDI突变体将用于测试维持ER氧化还原平衡的模型。实验还将测试补充PDI活性的补偿途径，包括诱导内质网中非必要的PDI同源物和更一般的未折叠蛋白质反应。具体目标3.异构化反应机理。使PDI成为有效的异构化催化剂的因素尚不清楚。为了确定PDI是否提供了一条涉及还原和再氧化的更快的异构化途径，将向PDI呈现特定的牛胰蛋白酶抑制物(BPTI)折叠中间体，在没有PDI的情况下，这些中间体进行缓慢的分子内异构化。PDI突变体和野生型PDI的单周转实验将检测PDI是否通过改变机制来提供更快的异构化。 改变PDI活性中心的硫醇/二硫键交换动力学和热力学的突变将被用来确定活性中心的哪些化学特征有助于催化。

DESCRIPTION (provided by applicant) Attaining the correct conformation and structure is essential for the proper function of proteins. In the cell, protein folding into its native structure is assisted by enzymes that accelerate folding and by chaperones that inhibit aggregation. Protein misfolding contributes to Alzheimer's disease, Parkinson's disease, prion-mediated infection, emphysema, and cystic fibrosis. Misfolding also limits the effective therapeutic use of recombinant proteins. Our long-term goal is to understand and mimic the biochemistry and enzymology of folding assistants. This proposal focuses on the in vitro and in vivo enzymology of protein disulfide isomerase (PDI), a folding catalyst and chaperone of the endoplasmic reticulum (ER) that is essential for the correct formation and isomerization of protein disulfides. The following specific aims are proposed. Specific Aim 1. PDI specificity and its multi-domain structure. The multidomain structure of PDI is essential for catalysis of isomerization, but substrate interactions that contribute to PDI recognition and catalysis have not been identified. Data from arrays of substrate peptides suggest a model in which PDI interactions are focused on positively charged substrate sites that pose barriers to isomerization and that these sites are located on multiple PDI domains. The model will be tested by mutagenesis of lysine and arginine residues in well-defined PDI substrates and the binding of domain deletion mutants of PDI to peptide arrays of substrate sequences. Specific Aim 2. Isomerization and reductive pathways in the yeast ER. The current model for ER disulfide formation suggests that PDI forms and breaks substrate disulfides and that oxidized and reduced PDI are both needed for proper folding. Recently discovered isomerase-deficient PDI mutants that still support wild-type yeast growth will be used to test models for maintaining ER redox balance. Experiments will also test for compensation pathways to supplement PDI activity, including the induction of non-essential PDI homologues in the ER and the more general unfolded protein response. Specific Aim 3. Mechanisms of isomerization. The factors that make PDI an effective catalyst of isomerization are not understood. To determine if PDI provides a faster pathway of isomerization involving reduction and reoxidation, PDI will be presented with specific folding intermediates of bovine pancreatic trypsin inhibitor (BPTI) which undergo slow, intramolecular isomerization in the absence of PDI. PDI mutants and single turnover experiments with wild-type PDI will detect if PDI provides faster isomerization by changing the mechanism. Mutations that alter the thiol/disulfide exchange kinetics and thermodynamics of the PDI active site will be used to determine which features of the chemistry of the active site contribute to catalysis.