Identification of molecular determinants that govern distinct STIM2 activation dynamics.
Identification of molecular determinants that govern distinct STIM2 activation dynamics.
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识别控制不同 STIM2 激活动力学的分子决定因素
DOI：
10.1371/journal.pbio.2006898
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发表时间：
2018-11
期刊：
影响因子：
9.8
通讯作者：
Wang Y
中科院分区：
文献类型：
--
作者：
Zheng S;Ma G;He L;Zhang T;Li J;Yuan X;Nguyen NT;Huang Y;Zhang X;Gao P;Nwokonko R;Gill DL;Dong H;Zhou Y;Wang Y
内质网（ER） Ca2+传感器基质相互作用分子1 （STIM1）和STIM2，连接ER Ca2+消耗和细胞外Ca2+内流，是维持哺乳动物细胞Ca2+稳态的关键。尽管最近在解开STIM2在Ca2+信号传导中的作用方面取得了进展，但其激活的机制基础仍未得到充分探索。我们使用工程方法将ER-resident STIM引导到质膜（PM），同时保持其正确的膜拓扑结构，以及Förster共振能量转移（FRET）传感器，可以在蜂窝中实时监测STIM的活动。这使我们能够确定STIM1和STIM2在细胞内和原位的钙亲和力，解释了目前文献中的差异。我们还确定了关键的结构决定因素，特别是STIM1中相应的G残基，它们定义了STIM2独特的激活动力学。嵌合的E470G突变可以将STIM2从一个缓慢而微弱的Orai通道激活剂转变为一个像STIM1一样快速而有效的激活剂，反之亦然。通过蛋白质工程对STIM2激活的系统解剖，为阐明哺乳动物中STIM2介导的信号传导的调控和功能奠定了基础。钙离子在细胞的生理生化过程中起着重要的调节作用，因此钙离子的水平和活性应受到严格的调控。基质相互作用分子（STIMs）是内质网（ER）内钙水平的传感器，内质网是主要的细胞内钙储存，介导内质网和质膜之间的通信，被认为是哺乳动物细胞中普遍存在的钙信号传导的中心角色。STIM2作为质膜上Orai1钙通道的缓慢弱激活剂，直接结合；然而，stim对钙的亲和力或如何激活Orai1通道仍不清楚。在这项研究中，我们系统地分析了控制STIM蛋白激活的分子决定因素。采用蛋白工程的方法，使内质膜上的STIM蛋白能够重新定位，我们测定了哺乳动物细胞生理条件下STIM蛋白的钙亲和力。我们确定了stim中的一个关键位置，它定义了它们不同的静息状态和激活动力学，以及激活Orai1通道的功效。这些发现揭示了STIM2如何有效地响应内质网腔内的微小变化，以调节哺乳动物细胞中的钙稳态和信号传导。
The endoplasmic reticulum (ER) Ca2+ sensors stromal interaction molecule 1 (STIM1) and STIM2, which connect ER Ca2+ depletion with extracellular Ca2+ influx, are crucial for the maintenance of Ca2+ homeostasis in mammalian cells. Despite the recent progress in unraveling the role of STIM2 in Ca2+ signaling, the mechanistic underpinnings of its activation remain underexplored. We use an engineering approach to direct ER-resident STIMs to the plasma membrane (PM) while maintaining their correct membrane topology, as well as Förster resonance energy transfer (FRET) sensors that enabled in cellulo real-time monitoring of STIM activities. This allowed us to determine the calcium affinities of STIM1 and STIM2 both in cellulo and in situ, explaining the current discrepancies in the literature. We also identified the key structural determinants, especially the corresponding G residue in STIM1, which define the distinct activation dynamics of STIM2. The chimeric E470G mutation could switch STIM2 from a slow and weak Orai channel activator into a fast and potent one like STIM1 and vice versa. The systemic dissection of STIM2 activation by protein engineering sets the stage for the elucidation of the regulation and function of STIM2-mediated signaling in mammals. Calcium ions play a major regulatory role in the physiology and biochemistry of the cell, and thus their levels and activities should be tightly regulated. The stromal interaction molecules (STIMs) are sensors of the calcium levels within the endoplasmic reticulum (ER)—which serves as a major intracellular calcium store—to mediate communication between the ER and the plasma membrane and are regarded as ubiquitous central players of calcium signaling in mammalian cells. STIM2 acts as a slow and weak activator of Orai1 calcium channels on the plasma membrane by direct binding; however, the affinity of STIMs for calcium or how Orai1 channels are activated remain unclear. In this study, we systematically analyzed the molecular determinants that govern the activation of STIM proteins. Adopting protein engineering approaches that enable the relocation of ER-resident STIM proteins at the plasma membrane, we determined the calcium affinities of STIMs under physiological conditions in mammalian cells. We identified a critical position within STIMs, which defines their distinct resting states and activation kinetics, as well as the efficacy to activate Orai1 channels. These findings shed new light on how STIM2 can efficiently respond to small changes within the ER lumen to regulate calcium homeostasis and signaling in mammalian cells.
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影响因子：
3.7
作者：
Henderson MJ;Baldwin HA;Werley CA;Boccardo S;Whitaker LR;Yan X;Holt GT;Schreiter ER;Looger LL;Cohen AE;Kim DS;Harvey BK
通讯作者：
Harvey BK
DOI：
10.1016/j.bbrc.2015.02.110
发表时间：
2015-04-24
影响因子：
3.1
作者：
Hogan, Patrick G.;Rao, Anjana
通讯作者：
Rao, Anjana
影响因子：
4.8
作者：
Cai, Xiangyu;Zhou, Yandong;Gill, Donald L.
通讯作者：
Gill, Donald L.
影响因子：
5.5
作者：
Best, Robert B.;Zhu, Xiao;Shim, Jihyun;Lopes, Pedro E. M.;Mittal, Jeetain;Feig, Michael;MacKerell, Alexander D., Jr.
通讯作者：
MacKerell, Alexander D., Jr.
影响因子：
4.4
作者：
FELLER, SE;ZHANG, YH;BROOKS, BR
通讯作者：
BROOKS, BR