ANESTHETICS AND STRESS PROTEINS IN BRAIN CELL INJURY

接受神经外科和心脏外科手术的患者脑缺血和中风的风险增加。在寻找更好的脑损伤治疗方法和了解脑损伤机制的过程中，hsp70已被证明是减少缺血性脑损伤基因治疗的良好候选基因。hsp70具有高度的进化保守性，可诱导应激，并在体内和体外对缺血性脑损伤提供保护。目前已知hsp70在细胞内具有多种功能，包括促进蛋白质折叠，协助蛋白质复合物的形成和解离，调节基因表达，防止蛋白质降解的聚集和分拣。到目前为止，还没有关于这些功能中哪一个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。hsp70包含两个主要功能域，ATP结合氨基末端域和羧基末端底物结合域。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是重要的。保护作用将在坏死和凋亡损伤中进行测试。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。复合氧葡萄糖剥夺会损伤培养物。我们还将研究协同伴侣蛋白对hsp70保护作用的影响。在Specific Aim 1中，将测试hsp70的点突变体和缺失突变体，以确定(a) ATPase活性是否对保护至关重要(b) ADP结合的hsp70是否仍然可以结合未折叠的蛋白质，与野生型hsp70提供同等的保护(c)参与ATP结合域和底物结合域相互作用的EEVD四肽是否对保护重要。然后，两个主要结构域的作用将被描述为(d)氨基末端结构域和(e)羧基末端结构域，使用缺失突变体。是否单独任何一个结构域可以提供保护，以及与全长hsp70相比的保护程度将被确定。如果特定突变体在体外具有与全长hsp70相同的保护效果，那么它们将用于大鼠局灶性缺血的保护试验。在绘制出hsp70对缺血保护所需的部分后，我们将在Specific Aim 2中对hsp70相互作用蛋白或共伴侣蛋白的作用进行研究。我们将研究几种hsp70共伴侣蛋白单独过表达和联合过表达对损伤的影响。将测试结合1)atp酶结构域和2)底物结合结构域的共伴侣。增强hsp70 atp酶活性的协同伴侣可能在缺血性损伤的保护中发挥重要作用。对hsp70发挥的各种作用及其与其他蛋白质的相互作用的更深入的了解，目前主要是在试管蛋白质折叠试验中确定的，现在允许对hsp70在脑损伤中的功能进行更详细的分子鉴定。这是一个非常令人兴奋的工作阶段，因为它可能揭示单独hsp70的特定亚域在减少损伤方面是有效的。随着多肽治疗能力的提高，HSP70的一个亚结构域可能被开发成一种有用的药物，以改善中风的预后。

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.