Metastable Crystallins: Structure and Stabilization

亚稳态晶体蛋白：结构和稳定性

• 批准号: 10200048
• 负责人: KRISHNA K SHARMA
• 金额: $ 37.59万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10200048
• 关键词: Adult; Amyloid beta-Protein; Arginine; Back; Biological; Biological Response Modifier Therapy; Cataract; Cell Culture System; Cell Culture Techniques; Cells; Chemicals; Complex; Crosslinker; Crystalline Lens; Crystallins; Development; Disease; Etoposide; Event; Excision; Future; Goals; Hela Cells; High temperature of physical object; Human; Hydrogen Peroxide; Hydrophobicity; In Vitro; Investigation; Knowledge; Maintenance; Modeling; Modification; Molecular; Molecular Chaperones; Onset of illness; Oxidative Stress; Preparation; Property; Protein Conformation; Proteins; Recombinants; Role; S-crystallin; Site; Site-Directed Mutagenesis; Staurosporine; Stress; Structure; Structure-Activity Relationship; Testing; Therapeutic; Urea; alpha-Crystallins; base; biophysical techniques; fascinate; improved; in vitro activity; insight; lens; lens transparency; macromolecule; mutant; novel; oxidative damage; preservation; prevent; protein oligomer; replication stress; response; small molecule; stem; thermal stress; tool

ABSTRACT -Crystallin is a complex macromolecule that accounts for nearly 40% of the adult lens proteins. The chaperone-like activity of -crystallin, is implicated as a key component in the maintenance of lens transparency by suppression of crystallin aggregation. In vitro studies of α-crystallin have shown that chaperone activity is increased when α-crystallin is subjected to heat (48-60oC) and then brought back to 25- 37oC. Similarly, α-crystallin subjected to urea-induced unfolding and refolding also displays increased chaperone activity. Understanding the molecular organization and properties of crystallin subunits in activated chaperones would help answer questions on how -crystallin chaperone-like activity might be harnassed and manipulated for the development of protein-based therapeutics. In our studies of the role of subunit interactions in chaperone activity, a recombinant αB-crystallin expressed after deleting the 54-61 sequence (resulting in a protein designated as αB∆54-61) was found to form ~ 40 % smaller oligomer than the wild-type protein oligomer but to show a 10-fold increase in chaperone activity. The Specific Aims of this project will uncover the molecular changes that account for the increased chaperone activity in heat- and urea-treated α-crystallin and deletion mutant of αB-crystallin. Specific Aim 1: a) Determine the mechanism of αB-crystallin chaperone activation after deletion of the 54-61 sequence and b) determine the biological implications of enhanced chaperone activity in cell culture system. Specific Aim 2: a) Determine the functional units and mechanism of activation in α-crystallin chaperone after thermal stress and urea-induced unfolding and refolding and b) investigate the cytoprotective effect of heat- and urea-activated crystallins. Novel cross-linker(s) will be used to gain fresh insights into the “cryptic” chaperone sites getting exposed in the activated crystallins. The studies will also make use of site-directed mutagenesis, mass spectrometric analysis and biophysical techniques to uncover the molecular changes at the secondary and tertiary structure levels and to delineate the quaternary organization of the subunits in the oligomers showing increased chaperone activity. To see whether the activated α-crystallins can be exploited to protect cells from oxidative injury, the effects of stress-inducing agents such as H2O2, staurosporine or etoposide will be investigated in Cos-7, HeLa, HEK293 and ARPE-19 cells. Further, the ability of activated chaperones to suppress aggregation of mutant proteins (αAG98R) as well as 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 α-crystallin and develop crystallin proteins that have therapeutic value in protein conformational diseases.

抽象的 -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 -晶状体的类似伴侣的活性是维护镜头的关键组成部分 通过抑制结晶蛋白聚集的透明度。 α-晶状体蛋白的体外研究表明 当α-晶链蛋白受到热量（48-60oC），然后将伴侣活性增加，然后将其带回25-- 37oc。同样，受尿素引起的展开和重折的α-晶状体也增加了 伴侣活动。了解激活的结晶蛋白亚基的分子组织和特性 伴侣将帮助回答有关-crystallin伴侣伴侣的活动的问题 操纵基于蛋白质的治疗的发展。在我们对亚基相互作用的作用的研究中 在伴侣活性中，删除54-61序列后表达的重组αB-晶状蛋白（导致A 发现被指定为αBΔ54-61）的蛋白质比野生型蛋白质形成约40％的低聚物 低聚物，但显示伴侣活性增加了10倍。该项目的具体目的将发现 分子变化是造成热和尿素处理的α-晶体蛋白和伴侣活性增加的分子变化 αB-晶状体蛋白的缺失突变体。特定目的1：a）确定αB-晶状体链蛋白的机制 缺失54-61序列和b）确定增强的生物学意义 细胞培养系统中的伴侣活性。特定目标2：a）确定功能单元和机制 热应力和尿素引起的展开和重折叠后，α-晶链蛋白链蛋白的激活以及b） 研究热和尿素激活的晶体的细胞保护作用。新颖的跨链接器将用于 在激活的结晶蛋白中暴露于“隐秘”伴侣的位点，获得新的见解。研究 还将利用位置定向的诱变，质谱分析和生物物理技术 揭示次级和三级结构水平的分子变化，并描述第四纪 低聚物中亚基的组织显示伴侣活性增加。看看是否 可以探索活化的α-晶状蛋白以保护细胞免受氧化损伤的影响，这是应激诱导的影响 H2O2，Staurosporine或Etoposide等药物将在COS-7，HELA，HEK293和ARPE-19中进行研究 细胞。此外，活化的伴侣抑制突变蛋白聚集（αAG98R）的能力 由于形成原纤维的β-淀粉样蛋白将在体外和前体内进行研究。研究的长期目标是 了解活化的α-晶链蛋白的结构 - 功能关系，并发展出结晶蛋白蛋白 在蛋白质构象中具有治疗价值。