Metastable Crystallins: Structure and Stabilization

摘要 -晶体蛋白是一种复杂的大分子物质，占成人晶状体蛋白的近40%。这个 -晶状体蛋白的伴侣样活性是维持晶状体的关键成分 通过抑制晶体蛋白聚集而实现的透明性。α-晶体蛋白的体外研究表明， 当α-晶体蛋白受热(48-60oC)并随后恢复到25oC时，伴侣活性增加。 摄氏37度。同样，α-晶体蛋白在尿素诱导下的去折叠和复性也表现出增加 监护人活动。了解激活状态下晶体蛋白亚基的分子结构和性质 伴侣将有助于回答有关-晶体蛋白伴侣样活性如何被利用和 以蛋白质为基础的疗法的发展。在我们对亚单位相互作用作用的研究中 在伴侣活性中，在删除54-61序列后表达的重组αB-晶体蛋白(导致 被命名为αB∆54-61的蛋白质形成的寡聚体比野生型蛋白质小约40% 齐聚物，但显示伴侣活性增加10倍。该项目的具体目标将揭示 加热和尿素处理的α晶体蛋白中伴侣活性增加的分子变化 αB-晶状体蛋白缺失突变体。具体目标1：A)确定αB-晶体蛋白伴侣的作用机制 54-61序列缺失后的激活和b)决定了增强的生物学意义 细胞培养体系中的伴侣活性。具体目标2：a)确定 热应激和尿素诱导的去折叠和复性后α-晶体蛋白伴侣蛋白的激活 研究热激活和尿素激活的晶状体蛋白的细胞保护作用。新型交联剂(S)将用于 获得新的洞察力，了解在激活的晶体蛋白中暴露的“神秘”伴侣部位。这些研究 还将利用定点突变、质谱分析和生物物理技术 揭示二级和三级结构水平上的分子变化，并圈定第四系 寡聚体中亚单位的组织显示出伴侣活性的增加。以查看是否有 激活的α-晶体蛋白可被用来保护细胞免受氧化损伤、应激诱导的作用 将在CoS-7、HeLa、HEK293和ARPE-19中研究过氧化氢、星形孢子素或依托泊苷等药物 细胞。此外，激活的伴侣蛋白抑制突变蛋白聚集的能力(αAG98R)也是如此 作为形成纤维的β-淀粉样蛋白，将在体外和体外进行研究。这些研究的长期目标是 了解激活的α-晶状体蛋白的结构与功能关系，并开发 在蛋白质构象疾病中具有治疗价值。

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.