Human gammaD-Crystallin Folding, Misfolding and Fibril Forms

描述：晶状体白内障干扰正常视力，影响美国75岁以上人口的50%。人类GammaD-晶体蛋白(HGammaD-Crys)和人类GammaS-Crys(HGammaS-Crys)是人眼晶状体中的两个单体主要蛋白质，是成熟性白内障的重要组成部分。成熟性白内障被认为是晶状体晶体蛋白修饰、受损或部分未折叠状态的聚集状态。这项建议将成熟性白内障视为一种蛋白质沉积性疾病，重点是用纯化的蛋白质进行体外去折叠、复性和聚集实验，以寻找晶状体蛋白质聚集和/或伴侣识别的前体的体外模型。对于其他蛋白质沉积疾病，这样的系统一直是有针对性地寻找抑制病理沉积的治疗剂的关键步骤。 纯化的重组HGammaD-Crys可在37℃、中性pH条件下进行体外复性。高分子量纤维的形成与高效复性竞争。一种部分未折叠的物种，具有折叠的C-末端结构域和展开的N-末端结构域，已被确定为去折叠和再折叠途径的中间产物，是纤维形成的候选前体。HGammaS-Crys在体外也可以折叠。虽然HGammaS分子在复性过程中不会自我聚集，但在两种蛋白质都存在的情况下，它们会被招募到HGammaD纤维中。这些部分折叠的中间体在高效复性条件下的共聚反应为晶状体混浊提供了体外模型。 在这个建议中，我们使用一组HGammaD突变蛋白，包括三重色氨酸替代物，以a)加深对晶体蛋白在其溶液状态下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路径，重点是鉴定可能的成核和繁殖物种，以及c)确定部分折叠的物种或它们在体外形成的纤维状状态是否是α晶体蛋白的底物，以及它们是否是通过光氧化应激在体外产生的，被认为作用于晶状体内的晶体蛋白。

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.