Human gammaD-Crystallin Folding Misfolding and Fibril F*

人类 gammaD-晶状体蛋白折叠错误折叠和原纤维 F*

• 批准号: 7870669
• 负责人: Jonathan Alan King
• 金额: $ 38.66万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7870669
• 关键词: Affect; Age; American; Amino Acid Sequence; Amyloid; Atomic Force Microscopy; Binding; Biological Models; C-terminal; Cataract; Crystalline Lens; Crystallins; Deposition; Disease; Eye; Greek; Human; In Situ; In Vitro; Individual; Lead; Lens Opacities; Maps; Modification; Molecular; Molecular Chaperones; Molecular Weight; N-terminal; Oxidative Stress; Pathology; Pathway interactions; Population; Proteins; Reaction; Recombinants; Recruitment Activity; Site; Solubility; Solutions; System; Therapeutic Agents; Transmission Electron Microscopy; Tryptophan; Vision; Vision research; aggregation pathway; base; conformer; fibrillogenesis; in vitro Model; in vivo; infrared spectroscopy; lens; lens protein; mutant; protein aggregation; research study

Lens cataracts, interfering with normal vision, affect 50% of the American population over age 75. Human yD-crystallin (HyD-Crys) and Human yS-crystallin (HyS-Crys) are monomeric two-domain major proteins of the human eye lens and significant components of mature-onset cataracts.Mature-onset cataracts are thought to be an aggregated state of modified, damaged, or partially unfolded states of lens crystallins. This proposal treats mature-onset cataract as a protein deposition disease, and focuses on in vitro unfolding, refolding and aggregation experiments with purified proteins, to find in vitro models for the precursors to lens protein aggregation and/or chaperone recognition, in situ. For other protein deposition diseases such systems have been key steps toward targeted searches for therapeutic agents inhibiting pathological deposition. Purified recombinant HyD-Crys can be refolded in vitro at 37¿C and neutral pH in the absence of cellular factors. Formation of high molecular weight fibrils competes with productive refolding. A partially unfolded species with the C-terminal domain folded, and the N-terminal domain unfolded, has been identified as an intermediate in the unfolding and refolding pathway, and is a candidate for the precursor in fibril formation. HyS-Crys also refolds in vitro. Though HyS molecules do not self-aggregate during refolding, they are recruited into the HyD fibrils when refolding with both proteins present. These co-aggregation reactions of partially folded intermediates - under conditions of productive refolding - provide in vitro models for lens opacity. In this proposal, we use a set of HyD mutant proteins, including triple-tryptophan replacements, to a) deepen understanding of the molecular basis of the extraordinary stability of the crystallins in their solution state, b) to map the pathway of the partially folded HyDand HyS crystallins polymerizing into fibrillar and other aggregated states, with emphasis on identification of putative nucleating and propagating species, and c) to determine whether partially folded species or their fibrillar state formed in vitro are substrates of the a- rystallins, and whether they are generated in vitro by photo-oxidative stress, thought to be acting on the crystallins within the lens.

晶状体白内障会影响正常视力，影响 50% 的 75 岁以上的美国人口。 人 yD-晶状体蛋白 (HyD-Crys) 和人 yS-晶状体蛋白 (HyS-Crys) 是单体双结构域主要蛋白 人眼晶状体的蛋白质和成熟发病白内障的重要成分。成熟发病 白内障被认为是晶状体改变、损坏或部分展开状态的聚合状态 该提案将成熟发病的白内障视为一种蛋白质沉积疾病，并重点关注于 使用纯化的蛋白质进行体外去折叠、重折叠和聚集实验，以寻找体外模型 晶状体蛋白聚集和/或分子伴侣识别的前体，用于其他蛋白原位沉积。 此类系统已成为有针对性地寻找抑制治疗药物的关键步骤 病理沉积。 纯化的重组 HyD-Crys 可在 37° 体外重折叠C 和中性 pH 条件下不存在 高分子量原纤维的形成与生产性A部分竞争。 已鉴定出 C 端结构域折叠且 N 端结构域展开的未折叠物种 作为展开和重折叠途径的中间体，并且是原纤维前体的候选者 HyS-Crys 也在体外重折叠，尽管 HyS 分子在重折叠过程中不会自我聚集。 当两种蛋白质都存在时，它们会被招募到 HyD 原纤维中。 部分折叠的中间体 - 在有效重折叠的条件下 - 提供晶状体的体外模型 不透明度。 在此提案中，我们使用一组 HyD 突变蛋白，包括三色氨酸替代物，以： 加深对晶体蛋白在溶液中非凡稳定性的分子基础的理解 状态，b) 绘制部分折叠的 HyD 和 HyS 晶体蛋白聚合成纤维状和 其他聚合状态，重点是识别假定的成核和繁殖物种，以及 c) 确定部分折叠的物质或其体外形成的原纤维状态是否是 a-的底物 rystallins，以及它们是否在体外通过光氧化应激产生，被认为作用于 晶状体内的晶状体蛋白。