Targeting the Mitochondrial Calcium Uniporter, Phase I

描述（由申请人提供）：本项目的长期目标是在分子水平上了解线粒体Ca2+稳态。Ca2+单向转运蛋白是这方面的关键参与者，因为它是将Ca2+转运进出线粒体的复杂系统的中心成员。因此，单向转运蛋白参与维持细胞内Ca 2+稳态、调节能量产生、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，这些考虑使单向转运蛋白成为一种有趣的膜蛋白，并成为药物开发的有吸引力的靶点。尽管如此，目前还没有关于单向转运蛋白的分子特性的信息，但很明显，无论从哪一个角度来看，都需要这些信息来取得重大进展。该计划通过从多个来源克隆单向转运蛋白并在缺乏内源性类似物的酵母线粒体中表达蛋白质来启动单向转运蛋白的分子研究。采用了几种新的策略。在一种方法中，cDNA文库在酵母细胞系中表达，所述酵母细胞系已被工程化以在线粒体中表达发光蛋白水母发光蛋白。然后使用光发射来区分也表达单向转运体的细胞和不表达单向转运体的细胞。在另一种策略中，库在突变酵母中表达，这些酵母在正常Ca 2+水平下生长不良，因为无法利用细胞内储存体积。这些被表达单向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率对其进行鉴定。第三种策略使用来自单向转运蛋白结构的表观部分的氨基酸序列信息来设计引物并通过PCR克隆它。第四种策略使用生物信息学根据现有数据应包含的一组基序来搜索基因组数据库中的单向转运候选者。在所有情况下，在酵母中鉴定和表达的活动都要经过详细的检查，将其生物能量特征与真正的单向转运体进行比较。

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.