Structural and functional studies on molecular evolution of calcium signaling tool kit

钙生物学领域的一个基本且具有挑战性的问题是钙信号机制和遗产在单细胞和多细胞生物的进化中可以追溯到多远？在过去的几年中，我们从寄生原生生物克氏锥虫中鉴定了一个肌醇1，4，5-三磷酸受体（IP 3R）同源物，并证明T。cruziIP 3R（TcIP 3R）是一种新的毒力因子，可调控T.克鲁兹我们未来的研究将集中在钙信号工具包的关键成员，包括那些来自原始多细胞生物，C。优雅在这个提议中，我们将研究人类基质相互作用蛋白（STIM）和奥赖钙通道的原始同源物，这些通道最近被证明在哺乳动物的各种细胞类型中的钙库操纵的钙内流（SOCE）功能中起着至关重要的作用。STIM的两种同种型（STIM 1和STIM 2）作为内质网（ER）腔Ca 2+传感器起作用并激活奥赖Ca 2+通道，其在哺乳动物中具有三种同种型（ORAI 1、2和3）。然而，很少有人知道原始生物中的祖先同源物的STIM和奥赖。在人类和其他哺乳动物中，SOCE是细胞可以维持升高的钙水平的中心机制，这是T细胞活化和其他人类生理学所需的。研究这些蛋白质是如何从原始有机体如C. elegans可能会导致新的见解和更好地理解钙工具包是如何通过进化发展起来的，以及钙信号是如何被用于各种生物体的生理功能的。我们最近对秀丽隐杆线虫STIM（ceSTIM）的结构研究表明，暴露于ER腔的N-末端结构域在N-末端具有先前未鉴定的扭结螺旋，这可以解释在基础ER Ca 2+水平下特异于ceSTIM的在点状细胞外周的组成性定位。我们也在学习C。elegans奥赖（ce奥赖）以及人奥赖同种型，并成功表达和纯化了ce奥赖通道的多聚体功能形式。我们计划对ceSTIM和ceORAI进行进一步的结构和功能研究，以阐明C.和哺乳动物。我们相信，我们的祖先IP 3 R，STIM和奥赖同源物的研究将提供重要的线索，以了解在人类和其他哺乳动物中的个别异构体的异构体特异性的作用，这些作用已经从简单的生物体遗传到人类作为基本的生物学功能，以及这些作用已经在高等生物体中进化，以适应更复杂的生存策略。*****

One of the fundamental and challenging questions in the calcium biology field is how far the calcium signaling mechanisms and legacy go back in the evolution of unicellular and multicellular organisms? In previous years, we identified an inositol 1,4,5-trisphosphate receptor (IP3R) homologue from a parasitic protist, Trypanosoma cruzi and demonstrated that T. cruzi IP3R (TcIP3R) is a novel virulence factor regulating replication, differentiation and infectivity of T. cruzi. Our future studies focus on key members of the calcium signaling toolkit, including those from a primitive multicellular organism, C. elegans. In this proposal we will study the primitive homologues of human stromal interacting proteins (STIMs) and ORAI Ca2+ channels, which were recently shown to play a crucial role in the store-operated calcium entry (SOCE) function in various cell types in mammals. Two isoforms of STIMs (STIM1 and STIM2) function as endoplasmic reticulum (ER) luminal Ca2+ sensors and activate ORAI Ca2+ channels, which have three isoforms (ORAI1, 2 & 3) in mammals. However, very little is known about the ancestral homologues of STIM and ORAI in primitive organisms. In human and other mammals, SOCE is a central mechanism by which cells can maintain an elevated calcium level, which is required for T cell activation and other human physiology. Studying how those proteins evolved from a primitive organism such as C. elegans may lead to new insights and a better understanding of how the calcium toolkit has been developed through evolution and how calcium signals have been utilized for physiological functions of various organisms. Our recent structural studies on C.elegans STIM (ceSTIM) have shown that the N-terminal domain, which is exposed to the ER lumen, possesses a previously unidentified kinked helix at the N-terminus, which may explain the constitutive localization at the cell periphery in puncta under basal ER Ca2+ levels that is specific to ceSTIMs. We have also been studying C. elegans ORAI (ceORAI) as well as human ORAI isoforms and succeeded in expression and purification of the multimeric functional form of the ceORAI channel. We plan to perform further structural and functional studies on ceSTIM and ceORAI in order to elucidate common and diverged mechanisms associated with SOCE between C. elegans and mammals. We believe that our studies of ancestral IP3R, STIM and ORAI homologues will provide important clues to understanding the isoform-specific roles of the individual isoforms in humans and other mammals, which roles have been inherited from simpler organisms to humans as essential biological functions, and which roles have evolved in higher organisms to adapt to more complicated survival strategies. *****