The mitochondrial calcium uniporter of trypanosomes

描述（由申请人提供）：布鲁氏锥虫属寄生虫是昏睡病或非洲锥虫病的病原体。据世界卫生组织称，撒哈拉以南非洲地区有6000多万人面临感染风险，每年发生数千例病例。自20世纪70年代以来，非洲锥虫病再次出现，化疗仍然不令人满意，特别是对晚期病例。此外，布鲁氏锥虫是最适合进行分子和基因实验的锥虫，并为此开发了强大的遗传工具。在生理条件下，线粒体Ca2+摄取是通过膜内负电位驱动的单口机制进行的，不直接耦合ATP水解或其他离子的运输。早在50多年前（1961- 1962），人们就在啮齿动物线粒体中发现了这种微通道，并对其生物物理特性进行了广泛的研究。在哺乳动物线粒体中发现MCU后的许多年里，人们认为不太复杂的生命形式，如植物、昆虫和其他无脊椎动物，或单细胞生物，如酵母，缺乏特定的线粒体钙摄取途径。直到1989年，我们报道了恰加斯病的病原体克氏锥虫的附生毛线虫具有与哺乳动物线粒体相似的MCU特征：电致运输，对钌红敏感，对阳离子的亲和力低。在锥虫中存在MCU而在酵母中不存在MCU的证据是发现MCU分子身份的关键。哺乳动物线粒体在细胞内Ca2+稳态中起着核心作用，并且已经确定，当细胞质内Ca2+升高几个微摩尔时，线粒体内Ca2+浓度可以达到数十或数百微摩尔值。这是因为线粒体暴露于靠近内质网Ca2+释放位点或质膜Ca2+通道的高Ca2+浓度微结构域。这种Ca2+摄取对于形成细胞质Ca2+增加的幅度和时空模式以及调节导致ATP生成的三种线粒体内脱氢酶的活性以及ATP合成酶的活性是重要的。然而，过量的Ca2+摄取有利于“渗透性过渡孔”的形成，导致细胞质中促凋亡因子的释放和细胞死亡。有趣的是，布鲁氏虾线粒体也暴露于高Ca2+水平附近的酸钙体，酸性钙储存室，我们在1994年发现的布鲁氏虾，含有肌醇1,4,5-三磷酸受体（IP3R） Ca2+释放。我们的假设是，布鲁氏体可以用来研究MCU和线粒体Ca2+摄取的必要性，线粒体和酸钙体之间的关系，以及线粒体Ca2+摄取复合体的其他成分的存在。

DESCRIPTION (provided by applicant): The T. brucei group of parasites is the causative agent of sleeping sickness or African trypanosomiasis. According to the World Health Organization over 60 million people in sub-Saharan Africa are at risk of infection with an incidence of thousands of cases per year. African trypanosomiasis has been reemerging since the 1970s, and chemotherapy remains unsatisfactory especially for advanced cases. T. brucei is, in addition, the trypanosome most amenable to molecular and genetic experimentation, and for which powerful genetic tools have been developed. Under physiological conditions mitochondrial Ca2+ uptake takes place by the uniport mechanism driven electrophoretically by the negative-inside membrane potential without direct coupling to ATP hydrolysis or transport of other ions. This MCU was found more than 50 years ago (1961- 1962) in rodent mitochondria and the biophysical properties of this Ca2+-selective channel were extensively characterized since then. For many years after the discovery of the MCU in mammalian mitochondria, it was thought that less complex life forms such as plants, insects and other invertebrates, or unicellular organisms such as yeast, lacked a specific mitochondrial calcium uptake pathway. This was until we reported, in 1989, that epimastigotes of Trypanosoma cruzi, the etiologic agent of Chagas disease, possesses a MCU with characteristics similar to those described in mammalian mitochondria: electrogenic transport, sensitivity to ruthenium red, and low affinity for the cation. The evidence of the presence of a MCU in trypanosomes but its absence in yeast was the key to the discovery of the molecular identity of MCU. Mammalian mitochondria have a central role in intracellular Ca2+ homeostasis, and it is well established that intramitochondrial Ca2+ concentration can reach tens or hundreds micromolar values upon cytosolic Ca2+ rises of a few micromolar. This is because mitochondria are exposed to microdomains of high Ca2+ concentration in proximity to sites of Ca2+ release at the endoplasmic reticulum, or to Ca2+ channels at the plasma membrane. This Ca2+ uptake is important for shaping the amplitude and spatio-temporal patterns of cytosolic Ca2+ increases and for regulating the activity of three intramitochondrial dehydrogenases that result in ATP generation, as well as the activity of the ATP synthase. Excessive Ca2+ uptake, however, favors the formation of the "permeability transition pore" leading to the release of proapoptotic factors in the cytosol and cell death. Interestingly, T. brucei mitochondrion is also exposed to high Ca2+ levels in proximity to acidocalcisomes, acidic calcium storage compartments that we discovered in T. brucei in 1994, and that contain the inositol 1,4,5- triphosphate receptor (IP3R) for Ca2+ release. Our hypothesis is that T. brucei could be used to investigate the essentiality of the MCU and mitochondrial Ca2+ uptake, the relation between mitochondria and acidocalcisomes, and the presence of other components of the mitochondrial Ca2+ uptake complex that have been postulated to exist.