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

钙生物学领域的一个基本和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？在过去的几年里，我们从寄生原生生物克氏锥虫中鉴定出了肌醇1,4,5-三磷酸受体（IP3R）同源物，并证明克氏锥虫IP3R （TcIP3R）是一种调节克氏锥虫复制、分化和传染性的新型毒力因子。我们未来的研究重点是钙信号工具箱的关键成员，包括来自原始多细胞生物秀丽隐杆线虫的成员。在本研究中，我们将研究人类基质相互作用蛋白（STIMs）和ORAI Ca2+通道的原始同源物，它们最近被证明在哺乳动物各种细胞类型的储存操作钙进入（SOCE）功能中起着至关重要的作用。stim的两种异构体（STIM1和STIM2）作为内质网（ER）腔内Ca2+传感器并激活ORAI Ca2+通道，其中三种异构体（orai1,2）钙信号已被用于多种生物的生理功能。我们最近对秀丽隐杆线虫STIM （ceSTIM）的结构研究表明，暴露于内质网腔的n端结构域在n端具有先前未被识别的扭曲螺旋，这可能解释了ceSTIM在基础内质网Ca2+水平下在细胞外围点的组成定位。我们也一直在研究秀丽隐杆线虫的ORAI （ceORAI）和人类的ORAI同工型，并成功地表达和纯化了ceORAI通道的多聚功能形式。我们计划对ceSTIM和ceORAI进行进一步的结构和功能研究，以阐明秀丽隐杆线虫和哺乳动物之间SOCE的共同和不同机制。我们相信，我们对祖先IP3R、STIM和ORAI同系物的研究将为理解个体异构体在人类和其他哺乳动物中的特定作用提供重要线索，哪些作用是作为基本生物功能从简单生物遗传给人类的，哪些作用是在高等生物中进化以适应更复杂的生存策略的。

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 lcium 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.