Targeting the Mitochondrial Calcium Uniporter, Phase I

描述(申请人提供)：本项目的长期目标是在分子水平上了解线粒体钙稳态。在这方面，钙离子单一转运体是一个关键的参与者，因为它是复杂的系统的核心成员，该系统负责将钙离子运入和运出线粒体。因此，单转运蛋白参与维持细胞内钙离子的稳态，调节能量的产生，参与导致坏死性细胞死亡的病理过程，以及通过细胞凋亡发生的细胞死亡。从基础研究的角度来看，单转运体是一种有趣的膜蛋白，也是药物开发的一个有吸引力的靶点。尽管如此，目前还没有关于单转运蛋白分子性质的信息，但很明显，无论从哪种角度来看，都需要这些信息才能取得重大进展。这个项目启动了对单一转运蛋白的分子研究，承诺从多个来源克隆它，并在缺乏内源类似物的酵母线粒体中表达蛋白质。采用了几种新的策略。其中一种是在酵母细胞系中表达cDNA文库，该酵母细胞系被设计为在线粒体中表达光蛋白aequorin。然后，光发射被用来区分也表达单转运蛋白的细胞和不表达单转运蛋白的细胞。在另一种策略中，文库在突变酵母中表达，这些突变酵母在正常钙水平下生长不良，因为细胞内的存储容量无法利用。这些被表达单转运蛋白的细胞中的线粒体基质空间所取代，从而允许通过生长速度来识别它们。第三种策略使用从单一转运体结构的明显部分获得的氨基酸序列信息来设计引物，并通过聚合酶链式反应来克隆它。第四种策略使用生物信息学在基因组数据库中搜索单一转运蛋白候选基因，根据现有数据应该包括的一组基序。在所有情况下，在酵母中鉴定和表达的活性都要经过详细的检查，将它们的生物能量特性与真正的单转运蛋白的特性进行比较。

DESCRIPTION (provided by applicant): The long-range goal of this project is to obtain an understanding of mitochondrial Ca2+ homeostasis at the molecular level. The Ca2+ uniporter is a key player in that regard, in that it is a central member of the elaborate system which transports Ca2+ into and out of mitochondria. The uniporter is therefore involved in maintaining intracellular Ca2+ homeostasis, in regulating energy production, in pathological processes that lead to necrotic cell death, and in cell death occurring via apoptosis. These considerations have established the uniporter as an interesting membrane protein from the perspective of basic research, and as attractive target for drug development. Despite this, there is no information about the molecular properties of the uniporter, while it is clear that such information will be required to make significant progress from either perspective. This program initiates molecular studies of the uniporter by undertaking to clone it from multiple sources and to express the proteins in yeast mitochondria, which lack an endogenous analog. Several novel strategies are employed. In one, cDNA libraries are expressed in a yeast cell line that has been engineered to express the photoprotein aequorin in mitochondria. Light emission is then used to distinguish between cells that also express the uniporter and those that do not. In another strategy, libraries are expressed in mutant yeast that grow poorly at normal Ca2+ levels because intracellular storage volumes cannot be utilized. These are replaced by the mitochondrial matrix space in cells the express the uniporter, allowing their identification by rate of growth. A third strategy uses amino acid sequence information derived from an apparent section of uniporter structure to design primers and to clone it by PCR. A fourth strategy uses bioinformatics to search genomic databases for uniporter candidates, based upon a set of motifs that should be included according to existing data. In all cases, activities identified and expressed in yeast undergo a detailed examination, comparing their bioenergetic characteristics to those of the authentic uniporter.