Gamma Crystallin Modifications and Mechanisms of Lens Opacity

 描述（由申请方提供）：本提案的长期目标是确定γ-晶状体蛋白修饰（遗传或年龄发作）导致白内障形成的分子机制。我们的方法是使用各种生物物理和生物化学技术来确定将高度可溶的γ-晶体蛋白转化为光散射元素的过程。其中包括对凝聚相中出现的结构的理解，如无定形聚集体，纤维，凝胶或晶体。这样的光散射元件可以是仅蛋白质的聚集体，或与其他细胞组分（例如脂质膜）相关的蛋白质。基于我们目前的知识水平，我们假设：γ-晶体蛋白的修饰改变了分子间的相互作用，并导致（a）自缔合（改变的同源相互作用）或（B）与其他组分的缔合（例如，其它晶体蛋白或膜，即，改变的异源相互作用）。这种变化导致凝聚相或光散射元素的形成。我们将继续研究人γ D-晶状体蛋白（HGD），但也将研究γC-和γ S-晶状体蛋白（HGC和HGS），这是在人透镜中表达的唯一其他γ-晶状体蛋白。我们希望这些研究能够提供更广泛，更全面的知识，γ-晶体蛋白在各种条件下的相互作用，并定义其在光散射元件形成中的作用。为了实现我们的长期目标，我们提出了以下具体目标：（1）确定野生型HGC和选定的HGC和HGD的白内障相关突变体的溶解度和其他热力学和光谱性质，包括它们与模型膜的相互作用。(2)检查特定翻译后修饰（PTM）对HGD和HGS的溶解度和其他热力学和光谱性质的影响。待检查的PTM为：脱酰胺（HGS）、Cys-甲基化（HGD和HGS）和乙酰化（HGD）。(3)检查聚合和形成的组件在HGD展开过程中，介导的特定残基确定的计算研究。拟议的实验将确定由于透镜中的特定γ-晶状体蛋白修饰而导致的光散射和不透明的分子基础，并且应该有助于治疗措施的开发。

 DESCRIPTION (provided by applicant): The long-term objective of this proposal is to determine the molecular mechanisms by which modifications of the γ-crystallins (genetic or age-onset), lead to cataract formation. Our approach is to determine the processes that transform the highly soluble γ-crystallins into light-scattering elements, using a variety of biophysical an biochemical techniques. Included in it is an understanding of the structures appearing in the condensed phase, such as amorphous aggregates, fibers, gels or crystals. Such light scattering elements can be protein-only aggregates, or proteins associated with other cellular components, such as lipid membranes. Based on the current state of our knowledge, we hypothesize that: Modifications of the γ-crystallins alter molecular interactions and lead to either (a) self-association (altered homologous interactions) or (b) association with other components (e.g., other crystallins or membranes, i.e., altered heterologous interactions). Such changes result in the formation of condensed phases or light scattering elements. We will continue to investigate human γD-crystallin (HGD), but will also examine γC- and γS-crystallins (HGC and HGS), which are the only other γ-crystallins expressed in the human lens. We expect these studies to provide a broader, more comprehensive knowledge of γ-crystallin interactions under a variety of conditions and define their role in the formation of light scattering elements. To achieve our long-term goals we propose the following Specific Aims: (1) determine the solubility and other thermodynamic and spectroscopic properties of wild-type HGC and selected, cataract-associated mutants of HGC and HGD, including their interactions with model membranes. (2) Examine the effect of specific post-translational modifications (PTMs) on the solubility and other thermodynamic and spectroscopic properties of HGD and HGS. The PTMs to be examined are: Deamidation (HGS), Cys-methylation (HGD and HGS), and acetylation (HGD). (3) Examine the polymerization and formation of assemblies in HGD during unfolding, mediated by specific residues identified by computational studies. The proposed experiments will identify the molecular bases of light scattering and opacity due to specific γ-crystallin modifications in the lens, and should aid in the development of therapeutic measures.