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-晶状体蛋白是一种复杂的大分子，占成人晶状体蛋白的近 40%。近三十年前发现的α-晶状体蛋白的分子伴侣样活性被认为是一个关键发现通过抑制晶状体蛋白聚集来维持晶状体透明度的成分。 αB-晶状体蛋白 21-28 和 54-61 区域的缺失导致分子伴侣样活性增加（激活、了解激活的晶状体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-晶状体蛋白伴侣样活性的问题，以及人们重新认识到，αB-的增加。缺失突变体中的晶状体蛋白伴侣样活性源于新型寡聚物，其中亚基-亚基研究表明，相互作用导致天然寡聚物中“神秘”伴侣位点的暴露。删除 54-61 或 21-28 和 54-61 序列后表达的重组 αB-晶状体蛋白（导致发现被命名为 αBΔ54-61 和 αBΔ21-28,Δ54-61 的蛋白质形成比野生的更小的寡聚体。型蛋白，但显示出伴侣样活性高达约 25 倍。该提案将揭示驱动 αBΔ21-28,Δ54- 中分子伴侣样活性增加的分子变化 61和αBΔ54-61应用的目的是1)揭示激活的αB-的分子变化。晶状体蛋白，（αBΔ54-61 和 αBΔ21-28，Δ54-61），2）确定增强的生物学意义细胞培养和整个晶状体培养系统中工程蛋白的伴侣样活性。交联剂将用于获得对暴露于其中的“神秘”伴侣位点的新见解。这些研究还将利用定点诱变和质谱分析。分析以揭示活化寡聚体中亚基相互作用水平的分子变化。激活的 αB-晶状体蛋白可用于保护细胞免受氧化损伤、应激诱导的影响将在 HEK293 和 ARPE-19 细胞中研究 H2O2 和碘酸钠等试剂此外，激活的伴侣抑制原纤维聚集和毒性的能力。 β-淀粉样蛋白的形成将在体外和离体进行研究。研究的长期目标是了解激活的 αB-晶状体蛋白的结构-功能关系并开发晶状体蛋白在蛋白质构象疾病和氧化应激条件下具有治疗价值。