Lipids Interactions of Small Heat Shock Proteins

描述（由申请人提供） 拟议的研究是NIH资助#RO 1-GM 42762 -11的延伸，并将支持主要在匈牙利塞格德生物化学研究所Laszlo Vigh博士实验室进行的工作。这项研究涉及一个普遍存在的应激蛋白家族，即小热休克蛋白（sHsps），它被认为是应激和病变细胞中的分子伴侣。父母资助的目的是了解sHsp在体内的功能，以确定哪些细胞成分/过程中，他们保护或相互作用的压力，并进一步定义sHsp活动的机制。要求FIRCA补助金将探索一种替代，但不一定相互排斥的假设，即sHsps的功能，以调节膜的特性，既改善膜流动性的快速变化引起的热应激，并控制热休克蛋白的表达。研究将利用P.I.的工作建立了集胞藻热休克蛋白16.6的遗传学和生物化学，以及合作者在脂质生物化学和膜生物物理学方面的专业知识。所提出的研究的具体目的是：目的1：为了确定如何sHsps影响的相态，动态参数，和微区（“筏”）组织的类囊体（和细胞质）膜在体内的野生型集胞藻和细胞缺乏的sHsp或携带突变sHsp的这些属性的比较。目标二：为了测试是否sHsp脂质相互作用与膜功能的热保护相关，通过全链光合电子传递速率和PSII活性（类囊体）和离子泄漏（细胞质膜）来测量。目标3：确定热休克蛋白16.6的脂质相互作用特性，包括与脂质结合的可逆性，与天然集胞藻和合成脂质相互作用的特异性，以及脂质相互作用所需蛋白的结构特征。目标4：为了测试由于sHsp缺失或突变引起的特定膜重塑是否改变了Hsp合成的诱导温度和持续时间，与应激的“膜传感理论”一致。研究结果将为建立更完整的sHsps细胞功能模型提供数据。资金将为外国实验室的两名博士生提供支持，实验的跨学科性质将大大扩展他们的理论和实践培训，以确保他们在未来的研究中取得成功。

DESCRIPTION (provided by applicant) The proposed research is an extension of NIH grant # RO1-GM42762-11 and will support work to be carried out primarily in the laboratory of Dr. Laszlo Vigh, Institute of Biochemistry, Szeged, Hungary. The parent grant concerns a ubiquitous family of stress proteins, the small heat shock proteins (sHsps), that are proposed to act as molecular chaperones in stressed and diseased cells. The parent grant aims to understand sHsp function in vivo, to identify what cellular components/processes they protect or interact with during stress, and to define further the mechanism of sHsp activity. The requested FIRCA grant will explore an alternative, though not necessarily mutually exclusive hypothesis, that sHsps function to modulate membrane properties, both to ameliorate the rapid changes in membrane fluidity induced by heat stress, and to control Hsp expression. Studies will take advantage of the P.I.'s work establishing both the genetics and biochemistry of Synechocystis Hsp 16.6 and the collaborator's expertise in lipid biochemistry and membrane biophysics. The specific aims of the proposed research are: Aim 1: To determine how sHsps affect the phase state, dynamic parameters, and microdomain ("raft") organization of the thylakoid (and cytoplasmic) membrane in vivo by comparison of these properties in wild type Synechocystis and cells lacking the sHsp or carrying a mutant sHsp. Aim 2: To test if sHsp lipid interactions are correlated with thermoprotection of membrane functions as measured by the rate of whole chain photosynthetic electron transport and of PSII activity (thylakoid) and leakage of ions (cytoplasmic membrane). Aim 3: To define lipid interaction properties of Hsp 16.6, including reversibility of association with lipids, the specificity of interactions with native Synechocystis and synthetic lipids, and the structural features of the protein required for lipid interactions. Aim 4: To test if specific membrane remodelling due to sHsp deletion or mutation alters the temperature of induction and duration of Hsp synthesis, consistent with the "membrane sensing theory" of stress. Results will provide data on which to build a more complete model of the cellular function of sHsps. Funds will provide support for two PhD students in the foreign laboratory, and the interdisciplinary nature of the experiments will greatly expand their theoretical and practical training to ensure their success in future research.