ANESTHETICS AND STRESS PROTEINS IN BRAIN CELL INJURY

接受神经外科手术和心脏手术的患者患脑缺血和中风的风险增加。在寻求更好的治疗和了解脑损伤机制的过程中，HSP7O已被证明是减少缺血性脑损伤的基因治疗的良好候选基因。HSP7O在进化上高度保守，是应激诱导的，并在体内和体外提供脑缺血损伤的保护。目前已知HSP7O在细胞内执行多种功能，包括促进蛋白质折叠，协助蛋白质复合体的形成和解离，调节基因表达，防止蛋白质降解的聚集和分类。到目前为止，还没有关于这些功能中哪些与缺血保护最相关的信息，或者是否所有这些功能都是必需的。HSP7O含有两个主要功能结构域，即ATP结合氨基末端结构域和羧基末端底物结合结构域。将采用遗传方法来确定HSP70的哪些区域和功能在缺血保护中起重要作用。将对坏死性和凋亡性损伤进行保护测试。逆转录病毒载体将用于在原代小鼠脑细胞培养中表达突变体。这些培养物会受到联合氧糖剥夺的伤害。还将研究辅助伴侣对HSP7O提供的保护的影响能力。在具体目标1中，将测试HSP70的点突变和缺失突变，以确定(A)ATPase活性是否对保护至关重要(B)ADP结合的HSP7O是否具有与野生型HSP70相同的保护作用(C)参与ATP结合结构域和底物结合结构域相互作用的EEVD四肽是否对保护起重要作用。然后，将使用缺失突变体来表征两个主要结构域中的每一个的作用：(D)氨基末端结构域，和(E)羧基末端结构域。这两个结构域是否可以单独提供保护，以及与全长HSP70相比保护的大小将被确定。如果特定突变体在体外的保护作用与全长HSP70一样有效，则将测试它们对大鼠局灶性缺血的保护作用。在绘制了HSP70的缺血保护所需的部分图谱后，将针对特定的目的研究HSP70相互作用蛋白或辅助伴侣的作用。2.单独过度表达几个HSP70辅助伴侣以及联合过表达HSP70对损伤的影响。将测试结合1)ATPase结构域和2)底物结合结构域的辅助伴侣。增强HSP70-ATPase活性的辅伴蛋白可能在脑缺血损伤保护中发挥重要作用。对HSP70的各种作用及其与其他蛋白质相互作用的更多了解，目前主要是在试管蛋白折叠分析中确定的，现在可以开发更详细的分子评价HSP70在脑损伤中的功能。这是这项工作的一个非常令人兴奋的阶段，因为它可能揭示HSP70的特定亚域单独在减少伤害方面是有效的。随着递送多肽治疗能力的提高，HSP70的一个亚结构域有可能被开发成一种有用的药物来改善中风的预后。

Patients undergoing neurosurgery and cardiac surgery are at increased risk of cerebral ischemia and stroke. In the search for better treatments for and understanding of mechanisms of brain injury, HSP7O has proven to be a good candidate gene for gene therapy to reduce ischemic brain injury. HSP7O is highly evolutionarily conserved, stress inducible, and provides in vivo and in vitro brain protection from ischemic injury. HSP7O is now known to carry out diverse functions within the cell including facilitating protein folding, assisting in the formation and dissociation of protein complexes, modulating gene expression, preventing aggregation and sorting for protein degradation. To date, there is no information on which of these functions is most relevant to ischemic protection, or if all of them are required. HSP7O contains two main functional domains, the ATP binding amino terminal domain and the carboxy terminal substrate binding domain. A genetic approach will be taken to determine which regions and functions of hsp70 are important in ischemic protection. Protection will be tested in both necrotic and apoptotic injuries. Retroviral vectors will be used to express mutants in primary mouse brain cell culture. The cultures will be injured by combined oxygen glucose deprivation. The ability of co- chaperones to affect the protection afforded by HSP7O will also be studied. In Specific Aim 1, point and deletion mutants of hsp70 will be tested to determine (a) whether ATPase activity is critical to protection (b) whether ADP bound HSP7O which can still bind unfolded proteins provides equivalent protection to wild type hsp70 (c) whether the EEVD tetrapeptide involved in the interaction between the ATP binding and substrate binding domains is important to protection. The roles of each of the two major domains will then be characterized (d) the amino terminal domain, and (e) the carboxyl terminal domain, using deletion mutants. Whether either domain alone can provide protection, and the magnitude of protection compared to full length hsp70 will be determined. If specific mutants are as effective as full length hsp70 at protection in vitro, they will be tested for protection against focal ischemia in rats. After mapping the parts of hsp70 required for ischemic protection, a study of the role of hsp70 interacting proteins or co-chaperones will be performed in Specific Aim 2. The effect on injury of overexpressing several hsp70 co-chaperones alone and in combination with hsp70 overexpression will be studied. Co-chaperones that bind 1) the ATPase domain and 2) the substrate binding domain will be tested. Co-chaperones that augment hsp70 ATPase activity may play an important role in protection from ischemic injury. The greater understanding of the variety of roles played by hsp70 and of its interactions with other proteins, determined to date primarily in test tube protein folding assays, now permits the development of a more detailed molecular appreciation of hsp70 function in brain injury. This is a very exciting phase of the work as it may reveal that specific subdomains of hsp70 alone are effective at reducing injury. With improving ability to deliver peptide therapeutics it is possible that a subdomain of HSP70 could be developed into a useful pharmaceutical to improve outcome from stroke.