Lipids Interactions of Small Heat Shock Proteins

描述(由申请人提供) 这项拟议的研究是美国国立卫生研究院#RO1-GM42762-11号拨款的延伸，将支持主要在匈牙利塞格德生物化学研究所Laszlo Vigh博士的实验室开展的工作。父母的资助涉及一个普遍存在的应激蛋白家族，小热休克蛋白(SHsps)，被认为在应激和疾病细胞中扮演分子伴侣的角色。父母资助的目的是了解SHSP在体内的功能，确定它们在应激过程中保护或与哪些细胞成分/过程相互作用，并进一步定义SHSP的活性机制。申请的FIRCA拨款将探索另一种尽管不一定是互斥的假设，即sHSPs功能调节膜特性，既改善由热应激引起的膜流动性的快速变化，又控制HSP的表达。研究将利用P.I.S的工作，建立聚球藻HSP16.6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方面的专业知识。本研究的具体目的是：目的1：通过比较野生型集胞藻和缺失SHSP或携带突变型SHSP的细胞的这些特性，确定sHSP对体内类囊体膜(和细胞质)的相态、动力学参数和微域(RAFT)组织的影响。目的：用全链光合作用电子传递速率、PSII活性(类囊体膜)和离子渗漏(细胞膜)来检测SHSP脂相互作用是否与膜功能的热保护有关。目的：确定HSP16.6的脂类相互作用特性，包括与脂类结合的可逆性，与天然集胞藻和合成脂类相互作用的特异性，以及脂类相互作用所需蛋白质的结构特征。目的4：检测SHSP缺失或突变引起的特异性膜重塑是否改变了HSP合成的诱导温度和持续时间，这与应激的“膜感觉理论”相一致。这些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极大地扩大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.