Relative Roles of HSP70 and HSP25 in the Modulation of Renal Injury

描述(由申请人提供)： 这项拟议的研究项目的长期目标是确定损伤后肾上皮细胞恢复的分子过程。目前的建议集中在两类公认的应激诱导分子伴侣，热休克蛋白70和热休克蛋白25(HSP70和HSP25)及其在调节肾细胞损伤中的功能。这一建议的具体目的是确定a)这两类热休克蛋白作为伤害耐受性介质的相对作用和相互作用，b)它们与一组参与维持细胞完整性和极性的蛋白质(NAK ATPase、ankyrin、Spectrin和actin)的相互作用，以及c)热休克蛋白与这些完整性相关蛋白质相互作用导致细胞结构和极性恢复的分子过程。转染热休克蛋白的产生依赖于载体，不受细胞内源性调节机制的控制。据此，建立了两个过表达HSP70标志(HSP70F)和HSP27(人类HSP25的类似物)的细胞系。在拟议的研究中，内源性热休克蛋白(HSP70和HSP25)在细胞损伤中的作用将与过表达的热休克蛋白所施加的影响区分开来，基因沉默技术(寡核苷酸诱骗和siRNA)将被用于区分内源性热休克蛋白对细胞损伤的影响。拟议的研究将调查这些类别的分子伴侣是否自主、相互或协同作用，以提供保护免受伤害。为了确定参与HSP70和NAK ATPase结合的基序，以及HSP70与每个完整性相关蛋白的结合亲和力，将使用免疫沉淀、蛋白质结合分析和表面等离子体共振。我们将通过免疫组织化学和生物素化研究来探讨调节热休克蛋白丰度对细胞极性恢复动力学的影响，以跟踪损伤后和恢复过程中移位的NAK ATPase的运动。在细胞水平上了解急性肾损伤和恢复的动力学和介体，将有助于加深了解，从而能够制定旨在促进恢复的治疗干预措施，同时避免无意中加重损伤。 拟议的研究将有助于理解急性肾功能衰竭和恢复的分子过程。这些知识将促进和加强对保护肾脏免受损伤和促进恢复的关键分子的理解。

DESCRIPTION (provided by applicant): The long term goal of the proposed research project is to identify the molecular processes involved in the recovery of renal epithelial cells following injury. The current proposal focuses on two well recognized classes of stress inducible molecular chaperones, heat shock protein 70 and heat shock protein 25 (HSP70 and HSP25) and their function in modulating renal cell injury. The specific aims of this proposal are to determine a) the relative role and interaction of these two classes of heat shock protein as mediators of tolerance to injury, b) their interactions with a panel of proteins involved in maintenance of cellular integrity and polarity (NaK ATPase, ankyrin, spectrin and actin) and c) the molecular processes by which HSP interactions with these integrity related proteins result in restitution of cellular structure and polarity. The generation of transfected heat shock protein is dependent on the vector and is not controlled by the endogenous regulatory mechanisms of the cell. Accordingly, two cell lines which have transfected over expression of HSP70 FLAG (HSP70F) and HSP27 (the human analogue of HSP25) have been established. In the proposed studies the effect of endogenous heat shock proteins (HSP70 and HSP25) on cellular injury will be differentiated from the influence exerted by over expressed heat shock proteins using gene silencing techniques (oligonucleotide decoy and siRNA). The proposed studies will investigate whether these classes of molecular chaperone act autonomously, reciprocally or synergistically to provide protection from injury. To determine the motifs involved in the association between HSP70 and NaK ATPase, as well as the binding affinity of HSP70 for each of the integrity related proteins, immunoprecipitation, protein binding assays and surface plasmon resonance will be utilized. The effect of modulating heat shock protein abundance on the kinetics of restitution of cellular polarity will be explored using immunohistochemistry and biotinylation studies to track movement of displaced NaK ATPase following injury and during recovery. Understanding the dynamics and mediators of acute renal injury and recovery at the cellular level will enable an advanced understanding which will allow the development of therapeutic interventions designed to enhance recovery while avoiding inadvertent exacerbation of injury. The proposed studies will help understand the molecular processes involved in acute kidney failure and recovery. This knowledge will advance and strengthen understanding of the key molecules that protect the kidney from injury and enhance recovery.