ROLE OF PROLINE DURING OXIDATIVE STRESS AND APOPTOSIS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在植物和真菌中，脯氨酸的积累和耐逆性之间的关系已经得到了很好的证实，但大多数的注意力都集中在脯氨酸作为渗透保护剂的能力上。我们最近对紫花苜蓿的真菌病原菌Collectotrichum trifolii的研究表明，Pro的作用不明显，但可能具有更广泛的功能。三叶草RAS是一种小G蛋白，它的显性活性突变导致小鼠的致癌表型，并在该真菌中表现出营养(饥饿)依赖的表型，导致菌丝异常增殖、极化生长缺陷和无法分化。不同的碳源、氮源、维生素、pH、热量、渗透压以及这些因素的组合都不能逆转表型。值得注意的是，恢复野生型表型只需要脯氨酸。人们注意到，这些被激活的RAS菌株具有异常高的活性氧物种(ROS)水平。当这些菌株被Pro或抗氧化剂处理时，激活的RAS表型逆转，并与ROS水平的显著下降直接相关，表明Pro是ROS的清除剂。此外，激活的RAS诱导的高ROS水平导致三叶锦鸡儿细胞程序性死亡，这与哺乳动物的细胞凋亡有共同的特征，包括DNA凝聚和断裂以及磷脂酰丝氨酸的外化。当真菌补充了脯氨酸时，这些特征就不存在了。 由于植物、真菌和动物体内的ROS水平在各种环境胁迫下都会增加，而ROS是细胞凋亡的媒介，因此，Pro清除ROS的能力可能是这种氨基酸在生物体对细胞胁迫的反应中的一个重要功能。我们研究的中心假设是，在应激过程中，Pro通过清除ROS和维持氧化还原动态平衡而起到抗氧化细胞保护剂的作用。这一建议的主要目的是表征和确定ROS介导的胁迫对Pro细胞保护的程度和范围，并识别调节ROS介导的细胞程序性死亡的植物基因。这两个话题都相对没有被探讨过。 目前还很少研究Pro和ROS之间的联系，对Pro代谢酶的生化表征也不完整。从这项研究中获得的关于脯氨酸在细胞事件中的多种作用的知识将有助于理解脯氨酸的积累和胁迫适应。 这项建议的具体目的是：(1)确定Pro对酵母中氧化还原状态的影响；(2)识别调节ROS诱导的细胞死亡的植物和酵母基因；以及(3)确定Pro的抗氧化作用是否延伸到哺乳动物细胞。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The correlation between proline accumulation and stress tolerance is well established in plants and fungi but most of the attention has been on the ability of proline to function as an osmoprotectant. Evidence for an obscure role for proline and potentially a more broad-based function is from our recent work with the fungal pathogen of alfalfa, Collectotrichum trifolii. Dominant active mutations in Ras from C. trifolii, a small G protein, caused oncogenic phenotypes in mice and displayed a nutrient(starvation)-dependent phenotype in the fungus resulting in abnormal hyphal proliferation, defects in polarized growth and an inability to differentiate. Various carbon sources, nitrogen sources, vitamins, pH, heat, osmoticum and combinations of these factors failed to revert the phenotype. Remarkably, restoration of the wild type phenotype required only proline. It was noted that these activated Ras strains had abnormally high levels of reactive oxygen species (ROS). When these strains were treated with proline or antioxidants, the activated Ras phenotype reverted and was directly coupled to significant decreases in ROS levels, indicating proline is a scavenger for ROS. Furthermore the high ROS levels induced by activated Ras caused a programmed-like cell death in C. trifolii that shares features with mammalian apoptosis including DNA condensation and fragmentation as well as externalization of phosphatidylserine. These features are absent when the fungus is supplemented with proline. Since ROS levels increase in plants, fungi, and animals during a variety of environmental stresses and ROS is a mediator of apoptosis, the ability of proline to scavenge ROS may be an important function of this amino acid in organisms responding to cellular stress. The central hypothesis of our studies is that proline functions as an antioxidant cytoprotectant during stress by scavenging ROS and maintaining redox homeostasis. The major goals of this proposal are to characterize and determine the extent and range of proline cytoportection from ROS mediated stress and to identify plant genes that modulate ROS mediated programmed cell death. Both of these topics are relatively unexplored. The connection between proline and ROS has scarcely been examined and biochemical characterization of proline metabolic enzymes is incomplete. Knowledge gained from this study on the versatile roles of proline in cellular events will contribute to the understanding of proline accumulation and stress adaptation. The specific aims of this proposal are: (1) Determine the effect of proline on the REDOX status in yeast; (2) Identify plant and yeast genes that regulate ROS-induced cell death; and (3) Determine whether the antioxidant effects of proline extend to mammalian cells.