CATALYSIS OF THIOL/DISULFIDE EXCHANGE

硫醇/二硫化物交换的催化

• 批准号: 2180289
• 负责人: HIRAM F GILBERT
• 金额: $ 18.62万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2180289
• 关键词: active sites; chemical aggregate; chemical binding; chemical kinetics; covalent bond; crosslink; crystallization; cysteine; disulfide bond; enzyme mechanism; enzyme substrate; fluorescent dye /probe; intermolecular interaction; isomerase; light scattering; molecular chaperones; molecular rearrangement; mutant; oxidation reduction reaction; protein folding; site directed mutagenesis; stoichiometry; thermodynamics; thiols; thioredoxin

Recombinant proteins have proven to be medically useful therapeutic agents for the treatment of a wide variety of diseases ranging from bleeding disorders, to diabetes, cystic fibrosis, cancer, and viral infections. The ability to produce biologically active recombinant protein therapeutics in large amounts and at lower cost is very often confounded by improper folding and extensive aggregation of many potentially useful proteins, particularly those that contain disulfide bonds. The long-range goal of this research is to provide more effective strategies that mimic the molecular mechanisms used by cellular folding catalysts and chaperones to promote efficient protein folding. The immediate experimental goals are to investigate the multiple mechanisms for assisting protein folding that are used by protein disulfide isomerase (PDI), a major protein folding catalyst of the eukaryotic endoplasmic reticulum. Functionally, PDI catalyzes slow disulfide bond formation and rearrangement during protein folding, inhibits the aggregation of unfolded proteins at high concentration (chaperone activity), and has the unusual ability to actively increase the aggregation of some unfolded proteins at low concentration (anti- chaperone activity). In addition, PDI is known to bind hydrophobic molecules such as estrogen and thyroid hormone (T3). Experiments using site-directed mutagenesis coupled with the tools of biochemistry are proposed to examine the spatial, structural and functional relationships among the various active sites involved in PDI's interaction with unfolded proteins, thee mechanisms PDI uses to gain access to buried thiols in stabilized folding intermediates, and the contribution of intermolecular reactions to PDI catalysis. The mechanisms of PDI's unusual chaperone/anti-chaperone activities and the mechanisms of protein aggregation in general will be investigated by kinetic and physical studies of aggregation including dynamic light scattering and turbidity measurements, examination of aggregate composition and stability, and an investigation of the specificity involved in the formation of heterogeneous aggregate involving multiple proteins. Catalyzed protein folding and protein aggregation are not well-understood at the molecular level. The proposed studies will provide an important step toward realizing the long-range goals of improved folding and production methods for recombinants proteins of medical importance.

重组蛋白已被证明是医学上有用的治疗 用于治疗多种疾病的药剂， 出血性疾病、糖尿病、囊性纤维化、癌症和病毒性 感染.生产生物活性重组体的能力 大量且成本较低的蛋白质疗法通常 由于折叠不当和许多物质的广泛聚集而感到困惑 潜在有用的蛋白质，特别是那些含有二硫键的蛋白质， 债券这项研究的长期目标是提供更有效的 模拟细胞折叠的分子机制的策略 催化剂和分子伴侣来促进有效的蛋白质折叠。的 当前的实验目标是研究多种机制 用于帮助蛋白质折叠， 异构酶（PDI），真核生物的主要蛋白质折叠催化剂， 内质网在功能上，PDI催化慢二硫键 在蛋白质折叠过程中的形成和重排， 未折叠蛋白质在高浓度下的聚集（分子伴侣 活动），并具有不寻常的能力，积极增加 一些未折叠的蛋白质在低浓度（抗- 伴侣活性）。此外，已知PDI结合疏水性的 雌激素和甲状腺激素（T3）等分子。实验中使用 定点突变与生物化学工具相结合， 建议审查空间、结构和功能关系， 在参与PDI与 未折叠的蛋白质，PDI使用这些机制来获得埋藏的蛋白质。 稳定的折叠中间体中的硫醇，以及 分子间反应到PDI催化。PDI的机制 异常的分子伴侣/抗分子伴侣活性和蛋白质的机制 通常将通过动力学和物理方法研究聚集 包括动态光散射和浊度的聚集研究 测量、骨料组成和稳定性检查，以及 研究参与形成的特异性 涉及多种蛋白质的异质聚集体。催化蛋白 折叠和蛋白质聚集在分子水平上还没有得到很好的理解， 水平拟议的研究将为实现以下目标迈出重要一步： 实现改进折叠和生产方法的长期目标 对于医学上重要的重组蛋白质。