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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.

摘要 α-晶体蛋白是一种复杂的大分子，占成人透镜蛋白质的近40%。的近三十年前发现的α-晶体蛋白的伴侣样活性被认为是一个关键因素，通过抑制晶状体蛋白聚集来维持透镜透明度的组分。结果发现 α B-晶状体蛋白21-28和54-61区的缺失导致增加的分子伴侣样活性（活化，功能的增益）。了解激活的晶状体蛋白亚基的分子结构和性质分子伴侣将有助于回答关于如何利用α-晶体蛋白分子伴侣样活性的问题，用于开发基于蛋白质的疗法。假设增加的αB- 缺失突变体中晶体蛋白伴侣样活性源于新型寡聚体，其中亚基-亚基相互作用导致天然寡聚体中“隐蔽”分子伴侣位点的暴露。研究显示在缺失54-61或21-28和54-61序列后表达的重组α B-晶状体蛋白（导致被命名为αBΔ54-61和αBΔ21-28，Δ54-61的蛋白）被发现形成比野生型更小的寡聚体。型蛋白质，但显示高达约25倍的伴侣样活性增加。在此提出的实验该提案将揭示驱动αBΔ21-28，Δ54- 28中分子伴侣样活性增加的分子变化。 61和αBΔ54-61。本研究的目的是：1）揭示活化的αB- 晶体蛋白（αBΔ54-61和αBΔ21-28，Δ54-61），2）确定增强的在细胞培养物和整个透镜培养系统中工程化蛋白质的伴侣样活性。小说交联剂将用于获得对暴露在细胞中的“神秘”分子伴侣位点的新见解。激活晶体蛋白这些研究还将利用定点突变和质谱分析技术，分析以揭示活化寡聚体中亚基相互作用水平的分子变化。以查看是否活化的α B-晶状体蛋白可用于保护细胞免受氧化损伤，将在HEK 293和ARPE-19细胞中研究H2 O2和碘酸钠等试剂，激活晶体蛋白此外，活化的伴侣蛋白抑制原纤维的聚集和毒性的能力也被证实。将在体外和离体研究β-淀粉样蛋白的形成。研究的长期目标是了解激活的α B-晶体蛋白的结构-功能关系，并开发晶体蛋白蛋白，在蛋白质构象疾病和氧化应激病症中具有治疗价值。