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:9769023
- 负责人:
- 金额:$ 38.75万
- 依托单位:
- 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 2022-06-30
- 项目状态:已结题
- 来源:
- 关键词:AdultAmyloid beta-ProteinArginineBackBiologicalBiological Response Modifier TherapyCataractCell Culture SystemCell Culture TechniquesCellsChemicalsComplexCrosslinkerCrystalline LensCrystallinsDevelopmentDiseaseEtoposideEventExcisionFutureGoalsHela CellsHigh temperature of physical objectHumanHydrogen PeroxideHydrophobicityIn VitroInjuryInvestigationKnowledgeMaintenanceModelingModificationMolecularMolecular ChaperonesOnset of illnessOxidative StressPreparationPropertyProtein ConformationProteinsRecombinantsRoleS-crystallinSiteSite-Directed MutagenesisStaurosporineStressStructureStructure-Activity RelationshipTestingTherapeuticUreaalpha-Crystallinsbasebiophysical techniquesfascinateimprovedin vitro activityinsightlenslens transparencymacromoleculemutantnovelpreservationpreventprotein oligomerreplication stressresponsesmall moleculestemthermal stresstool
项目摘要
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
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%。这
α-晶状体蛋白的分子伴侣样活性,是维持晶状体的关键成分
通过抑制晶状体蛋白聚集来提高透明度。 α-晶状体蛋白的体外研究表明
当 α-晶状体蛋白受热 (48-60oC) 然后恢复到 25-
37℃。类似地,α-晶状体蛋白在尿素诱导的解折叠和重折叠作用下也表现出增加
陪伴活动。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动的问题,以及
用于开发基于蛋白质的疗法。在我们对亚基相互作用作用的研究中
在分子伴侣活性中,删除 54-61 序列后表达的重组 αB-晶状体蛋白(导致
发现指定为 αBΔ54-61 的蛋白质形成的寡聚体比野生型蛋白质小约 40%
寡聚体,但伴侣活性增加了 10 倍。该项目的具体目标将揭示
分子变化导致经过热处理和尿素处理的 α-晶状体蛋白中分子伴侣活性增加,
αB-晶状体蛋白的缺失突变体。具体目标 1:a) 确定 αB-晶状体蛋白伴侣的机制
删除 54-61 序列后激活,b) 确定增强的生物学意义
细胞培养系统中的伴侣活性。具体目标 2: a) 确定功能单元和机制
热应激和尿素诱导的去折叠和重折叠后α-晶状体蛋白伴侣的激活和b)
研究热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
获得对活性晶状体蛋白中暴露的“神秘”伴侣位点的新见解。研究
还将利用定点诱变、质谱分析和生物物理技术来
揭示二级和三级结构水平的分子变化并描绘四级结构
寡聚物中亚基的组织显示出伴侣活性增加。看看是否
活化的 α-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
H2O2、十字孢菌素或依托泊苷等药物将在 Cos-7、HeLa、HEK293 和 ARPE-19 中进行研究
细胞。此外,激活的伴侣还具有抑制突变蛋白(αAG98R)聚集的能力
因为纤维形成的β-淀粉样蛋白将在体外和离体进行研究。研究的长期目标是
了解激活的 α-晶状体蛋白的结构-功能关系并开发晶状体蛋白
对蛋白质构象疾病具有治疗价值。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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KRISHNA K SHARMA其他文献
KRISHNA K SHARMA的其他文献
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{{ truncateString('KRISHNA K SHARMA', 18)}}的其他基金
Metastable Crystallins: Structure and Stabilization
亚稳态晶体蛋白:结构和稳定性
- 批准号:
8470982 - 财政年份:2013
- 资助金额:
$ 38.75万 - 项目类别:
Metastable Crystallins: Structure and Stabilization
亚稳态晶体蛋白:结构和稳定性
- 批准号:
9132472 - 财政年份:2013
- 资助金额:
$ 38.75万 - 项目类别:
Metastable Crystallins: Structure and Stabilization
亚稳态晶体蛋白:结构和稳定性
- 批准号:
10200048 - 财政年份:2013
- 资助金额:
$ 38.75万 - 项目类别:
Metastable Crystallins: Structure and Stabilization
亚稳态晶体蛋白:结构和稳定性
- 批准号:
8841373 - 财政年份:2013
- 资助金额:
$ 38.75万 - 项目类别:
Metastable Crystallins: Structure and Stabilization
亚稳态晶体蛋白:结构和稳定性
- 批准号:
10657220 - 财政年份:2013
- 资助金额:
$ 38.75万 - 项目类别:
Metastable Crystallins: Structure and Stabilization
亚稳态晶体蛋白:结构和稳定性
- 批准号:
9265858 - 财政年份:2013
- 资助金额:
$ 38.75万 - 项目类别:
Metastable Crystallins: Structure and Stabilization
亚稳态晶体蛋白:结构和稳定性
- 批准号:
8657443 - 财政年份:2013
- 资助金额:
$ 38.75万 - 项目类别:
Crystallin-Derived Anti-Chaperones in the Lens
晶状体中的晶状体蛋白衍生的反伴侣分子
- 批准号:
8306862 - 财政年份:2010
- 资助金额:
$ 38.75万 - 项目类别:
Crystallin-Derived Mini-Chaperones as Protein Aggregation Inhibitors
晶状体蛋白衍生的微型伴侣作为蛋白质聚集抑制剂
- 批准号:
7976444 - 财政年份:2010
- 资助金额:
$ 38.75万 - 项目类别:
Crystallin-Derived Anti-Chaperones in the Lens
晶状体中的晶状体蛋白衍生的反伴侣分子
- 批准号:
7992774 - 财政年份:2010
- 资助金额:
$ 38.75万 - 项目类别:
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