Metastable Crystallins: Structure and Stabilization

摘要 - 晶状体蛋白是一种复杂的大分子，占成人透镜蛋白质的近40%。的 β-晶状体蛋白的伴侣样活性是维持透镜的关键成分 通过抑制晶体蛋白聚集而实现透明。α-晶状体蛋白的体外研究表明， 当α-晶状体蛋白受热（48- 60 ℃），然后恢复到25- 60 ℃时， 摄氏37度。同样，α-晶状体蛋白在尿素诱导的去折叠和重折叠过程中， 伴侣活性。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晶状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。在我们对亚基相互作用的研究中， 在分子伴侣活性方面，缺失54-61序列后表达的重组α B-晶状体蛋白（导致 被命名为αB 54-61的蛋白质）被发现形成比野生型蛋白质小~ 40 寡聚体，但显示伴侣活性增加10倍。该项目的具体目标将揭示 分子变化，解释了加热和尿素处理的α-晶状体蛋白中伴侣活性增加， α B-晶状体蛋白缺失突变体。具体目标1：a）确定α B-晶状体蛋白伴侣的作用机制 和B）确定在54-61序列缺失后增强的激活的生物学意义， 细胞培养系统中的伴侣活性。具体目标2：a）确定以下方面的功能单位和机制： 在热应激和尿素诱导的解折叠和重折叠后α-晶状体蛋白伴侣蛋白中的活化和B） 研究热激活和尿素激活的晶状体蛋白的细胞保护作用。新型交联剂将用于 获得新的见解“神秘的”伴侣网站越来越暴露在激活晶体蛋白。研究 还将利用定点诱变、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， 在低聚物中的亚基的组织显示增加的伴侣活性。看是否 活化的α-晶体蛋白可用于保护细胞免受氧化损伤， 将在Cos-7、HeLa、HEK 293和ARPE-19中研究H2 O2、staurosporine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（α AG 98 R）聚集的能力也被证实是有效的。 因为将在体外和离体研究形成纤维的β-淀粉样蛋白。研究的长期目标是 了解激活的α-晶状体蛋白的结构-功能关系，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.