Human gammaD-Crystallin Folding Misfolding & Fibril Form

描述：透镜白内障，干扰正常视力，影响50%的美国人口超过75岁。人γ D-晶状体蛋白（HgammaD-Crys）和人γ S-晶状体蛋白（HgammaS-Crys）是人眼透镜的单体双结构域主要蛋白，是成熟型白内障的重要组成部分。成熟型白内障被认为是透镜晶体蛋白的修饰、受损或部分未折叠状态的聚集状态。该建议将成熟发作的白内障视为蛋白质沉积疾病，并专注于用纯化的蛋白质进行体外解折叠、重折叠和聚集实验，以在原位找到透镜蛋白聚集和/或伴侣识别的前体的体外模型。对于其他蛋白质沉积疾病，这样的系统已经成为有针对性地寻找抑制病理性沉积的治疗剂的关键步骤。 纯化的重组HgammaD-Crys可以在37 ℃和中性pH下在不存在细胞因子的情况下体外重折叠。高分子量原纤维的形成与生产性重折叠竞争。具有折叠的C-末端结构域和未折叠的N-末端结构域的部分未折叠的种类已被鉴定为未折叠和再折叠途径中的中间体，并且是原纤维形成中的前体的候选者。HgammaS-Crys也在体外重折叠。虽然HgammaS分子在重折叠过程中不会自我聚集，但当重折叠时，它们会被招募到HgammaD原纤维中，同时存在两种蛋白质。这些部分折叠的中间体的共聚集反应-在生产性再折叠的条件下-提供了透镜混浊的体外模型。 在该提议中，我们使用一组HgammaD突变蛋白，包括三重色氨酸置换，以a）加深对晶体蛋白在其溶液状态下的非凡稳定性的分子基础的理解，B）绘制部分折叠的HgammaD和HgammaS晶体蛋白聚合成纤维状和其他聚集状态的途径，重点是鉴定推定的成核和繁殖物种，和c）确定体外形成的部分折叠的种类或它们的纤维状状态是否是α-晶状体蛋白的底物，以及它们是否是通过被认为作用于透镜内的晶状体蛋白的光氧化应激在体外产生的。

DESCRIPTION: Lens cataracts, interfering with normal vision, affect 50% of the American population over age 75. Human gammaD-crystallin (HgammaD-Crys) and Human gammaS-crystallin (HgammaS-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 HgammaD-Crys can be refolded in vitro at 37 degrees 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. HgammaS-Crys also refolds in vitro. Though HgammaS molecules do not self-aggregate during refolding, they are recruited into the HgammaD 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 HgammaD 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 HgammaD and HgammaS 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 alpha-crystallins, and whether they are generated in vitro by photo-oxidative stress, thought to be acting on the crystallins within the lens.