Spectral revelations of mitochondrial Ca2+ flux interactome

描述(申请人提供)：线粒体是细胞质[Ca~(2+)]通量的多元信号处理器。线粒体[Ca~(2+)]通过激活线粒体酶复合体参与细胞能量的产生，但也可促进不同方式的细胞死亡。线粒体通过多种途径巧妙地维持细胞内钙梯度。线粒体钙摄取的失调与许多细胞功能障碍有关，包括慢性氧化负担、自噬和细胞死亡的敏化。高胞浆[Ca~(2+)]通过一种未知的单转运蛋白促进线粒体Ca~(2+)的积累，直到2011年。最近，我们发现线粒体钙摄取1(MICU1)和线粒体钙单一转运体调节器1(MCUR1)在静息和活动状态下对线粒体钙单转运体孔洞提交(MCU)活性具有负性和正性调控作用。MCU分别与MICU1和MCUR1相互作用，在线粒体内膜形成不同的MCU复合体。鉴于线粒体Ca~(2+)对MCU相互作用组的关键作用，我们假设MCU是MCU复合体的主要“枢纽”，并与蛋白质网络相互作用形成MCU复合体。此外，MCU相互作用的组件对于MCU复合体的组装是至关重要的，并将依赖于细胞内钙离子水平。该提案的目标是通过应用最先进的蛋白质组技术和超分辨率成像来识别和验证哺乳动物MCU结合伙伴。为了完成对线粒体钙通量相互作用组功能的鉴定，招募了四名高素质的研究人员。Muniswamy和Merali实验室将确定MCU相互作用的伙伴，并重点研究与MCU分子相互作用的身份和功能。此外，将在重建系统(曼西亚实验室)中测试MCU交互组的物理相互作用。最后，卡普兰实验室将用超分辨率成像可视化MCU交互组复合体。线粒体生物学的关键下一步需要这四个研究人员和技术的合作，从而为未来大分子相互作用的合作提供了一个理想的战略。这些结果将极大地加深我们对MCU复合体及其在细胞正常和活动状态下线粒体钙内流中的作用的理解。

DESCRIPTION (provided by applicant): Mitochondria are multivariate signal processors of cytoplasmic [Ca2+] flux. Mitochondrial [Ca2+] participates in cellular energy production via activation of mitochondrial enzymatic complexes yet can also promote different modes of cell death. Mitochondria exquisitely maintain cytosolic Ca2+ gradient via numerous pathways. Dysregulation of mitochondrial Ca2+ uptake has been linked to numerous cellular dysfunctions including chronic oxidative burden, autophagy and sensitization for cell death. High cytoplasmic [Ca2+] facilitates mitochondrial Ca2+ accumulation via an unknown until 2011 uniporter. Recently, we demonstrated that mitochondrial Ca2+ uptake 1 (MICU1) and mitochondrial Ca2+ uniporter regulator 1 (MCUR1) negatively and positively control the mitochondrial Ca2+ uniporter pore submit (MCU) activity under resting and active state. MCU interacts with MICU1 and MCUR1 separately and forms different MCU complexes at the mitochondrial inner membrane. Given the critical importance of mitochondrial Ca2+ for the MCU interactome, we hypothesize that MCU is a major "hub" of the MCU complex and interacts with a network of proteins to form the MCU complex. Further, MCU interacting components are crucial for MCU complex assembly and will be dependent on cytosolic Ca2+ levels. The goal of the proposal is to identify and validate mammalian MCU binding partners by the application of state-of-the-art proteomic technologies and super-resolution imaging. To accomplish the identification of the functional mitochondrial Ca2+ flux interactome, four highly qualified investigators are recruited. Muniswamy and Merali laboratories will identify the MCU interacting partners and focus on the identity and function of molecular interactions with MCU. Further, the physical interaction of the MCU interactome will be tested in a reconstituted system (Mancia lab). Finally, the Caplan lab will visualize the MCU interactome complex with super-resolution imaging. The critical next step in mitochondrial biology requires the collaboration of these four investigators and technologies thus providing an ideal strategy for future collaborations of macromolecular interactions. The results will greatly enhance our understanding of the MCU complex and its role in mitochondrial Ca2+ influx during the normal and active states of the cell.