Metastable Crystallins: Structure and Stabilization

亚稳态晶体蛋白：结构和稳定性

基本信息

批准号：
10657220
负责人：
KRISHNA K SHARMA
金额：
$ 46.29万
依托单位：
UNIVERSITY OF MISSOURI-COLUMBIA
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-05-01 至 2027-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10657220
关键词：
3-Dimensional Adult Affect Amyloid beta-Protein Apoptosis Apoptotic Arginine Biological Biological Response Modifier Therapy Cataract Cell Culture Techniques Cells Chemicals Complex Crosslinker Cryoelectron Microscopy Crystalline Lens Crystallins Cytoprotection Development Disease Engineering Exposure to Family suidae Fluorescence Resonance Energy Transfer Future Goals Grant Hydrogen Peroxide In Vitro Investigation Knowledge Lead Maintenance Measures Methods Mitochondria Modality Modeling Modification Molecular Molecular Chaperones Mutation Oxidative Stress Pathway interactions Phosphorylation Phosphorylation Site Preparation Prevention Property Protein Conformation Protein Engineering Proteins Recombinants Reporting Research Personnel Resistance Role Site Site-Directed Mutagenesis Sodium Selenite Stress Structure Structure-Activity Relationship System Techniques Testing Therapeutic Toxic effect age related alpha-Crystallins experimental study fascinate gain of function image processing in vivo insight lens lens transparency macromolecule mutant novel oxidative damage particle preservation prevent reconstruction replication stress small molecule sodium iodate stem three dimensional structure tool

项目摘要

Abstract α-Crystallin is a complex macromolecule that accounts for nearly 40% of the adult lens proteins. The chaperone-like activity of α-crystallin, which was discovered nearly three decades ago, is implicated as a key component in the maintenance of lens transparency by suppression of crystallin aggregation. It was found that the deletion of 21-28 and 54-61 regions of αB-crystallin leads to increased chaperone-like activity (activation, gain of function). Understanding the molecular organization and properties of crystallin subunits in activated chaperones would help answer questions on how α-crystallin chaperone-like activity might be harnessed and manipulated for the development of protein-based therapeutics. It is hypothesized that the increased αB- crystallin chaperone-like activity in deletion mutants stems from new type of oligomers where subunit–subunit interactions lead to the exposure of “cryptic” chaperone sites in the native oligomers. Studies show a recombinant αB-crystallin expressed after deleting either 54-61 or 21-28 and 54-61 sequences (resulting in a protein designated as αBΔ54-61 and αBΔ21-28,Δ54-61) was found to form smaller oligomers than the wild- type protein but to show up to ~25-fold increase in chaperone-like activity. The experiments proposed in this proposal will uncover the molecular changes that drive the increased chaperone-like activity in αBΔ21-28,Δ54- 61 and αBΔ54-61. The aims of the application are 1) Uncover the molecular changes in the activated αB- crystallins, (αBΔ54-61 and αBΔ21-28,Δ54-61), 2) determine the biological implications of enhanced chaperone-like activity of engineered proteins in the cell culture and whole lens culture system. Novel crosslinker(s) will be used to gain fresh insights into the “cryptic” chaperone sites getting exposed in the activated crystallin. The studies will also make use of site-directed mutagenesis and mass spectrometric analysis to uncover the molecular changes at subunit interaction level in activated oligomers. To see whether the activated αB-crystallin can be exploited to protect cells from oxidative injury, the effects of stress-inducing agents such as H2O2 and sodium iodate will be investigated in HEK293 and ARPE-19 cells in presence of activated crystallins. Further, the ability of activated chaperones to suppress aggregation and toxicity of fibril- forming β-amyloid will be investigated both in vitro and ex-vivo. The long-term goals of the studies are to understand the structure–function relationship of activated αB-crystallins and develop crystallin proteins that have therapeutic value in protein conformational diseases and oxidative stress conditions.

抽象的 α-晶状体是一种复杂的大分子，占成年晶状体蛋白的几乎40％。这大约三十年前发现的α-晶状体的伴侣样活性被视为钥匙通过抑制结晶蛋白聚集来维持晶状体透明度的成分。发现 αB-晶状体蛋白的21-28和54-61区域的缺失导致伴侣样活性增加（激活，功能获得）。了解激活的结晶蛋白亚基的分子组织和特性伴侣将有助于回答有关如何利用α-晶状体蛋白伴侣的活动的问题，并且操纵基于蛋白质的治疗的发展。假设增加的αB- 删除突变体中的结晶蛋白伴侣样活性，从新型的低聚物中逐步进行亚基 - subunit 相互作用导致在天然低聚物中暴露“隐性”伴侣位点。研究表明重组αB-晶状蛋白在删除54-61或21-28和54-61序列后表达（导致A 发现被指定为αBδ54-61和αBδ21-28，δ54-61）的蛋白质比野生 - 类型蛋白质，但显示出伴侣样活性的增加约25倍。提出的实验提案将发现分子变化，这些变化驱动αBΔ21-28，Δ54-的伴侣样活性增加的分子变化。 61和αBδ54-61。应用的目的是1）发现活化αB-的分子变化 Crystallins，（αBδ54-61和αBδ21-28，δ54-61），2）确定增强的生物学意义工程蛋白在细胞培养和整个晶状体培养系统中类似伴侣的活性。小说交叉链接器（S）将用于获得对“隐秘”伴侣站点的新见解。活化的结晶蛋白。这些研究还将利用位置定向的诱变和质谱法分析以发现活性寡聚中亚基相互作用水平的分子变化。看看是否可以探索活化的αB-晶状体蛋白以保护细胞免受氧化损伤，这是应力诱导的影响在存在的情况下活化的结晶蛋白。此外，活化的伴侣抑制原纤维的聚集和毒性的能力形成β-淀粉样蛋白的体外和前体都将研究。研究的长期目标是了解活化的αB-晶状蛋白的结构 - 功能关系，并发展出结晶蛋白蛋白在蛋白质构象疾病和氧化应激状况中具有治疗价值。