Phosphoinositide-calcium Signaling In Cell Regulation

细胞调节中的磷酸肌醇-钙信号转导

• 批准号: 7968472
• 负责人: TAMAS BALLA
• 金额: $ 96.37万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7968472
• 关键词: 1-Phosphatidylinositol 3-Kinase; 1-Phosphatidylinositol 4-Kinase; Actins; Acute; Adherence; Agonist; Apoptosis; Appearance; Bacteria; Bacterial Infections; COS-7 Cell; Calcium; Calcium Channel; Calcium Signaling; Calcium ion; Cell Death; Cell membrane; Cell physiology; Cell surface; Cells; Cellular Membrane; Commit; Complex; Dependence; Developed Countries; Developing Countries; Down-Regulation; Enzymes; Escherichia coli; Escherichia coli Infections; Eukaryotic Cell; Event; Family; Fluorescence; Focus Groups; G-Protein-Coupled Receptors; GTP-Binding Proteins; Genetic Transcription; Growth Factor; Homeostasis; Hormones; Human; Immunologic Deficiency Syndromes; Individual; Infantile Diarrhea; Inositol; Invaded; Ion Channel; Label; Laboratories; Life; Link; Lipids; Maintenance; Mammalian Cell; Mediating; Membrane Transport Proteins; Metabolism; Minor; Mitochondria; Molecular; Movement; Muscle Contraction; Mutation; Natural Immunity; Neurotransmitters; Pathogenesis; Pathway interactions; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phospholipase C; Phospholipids; Phosphoric Monoester Hydrolases; Plasma Membrane Lipid Bilayer; Positioning Attribute; Production; Protein Family; Protein Kinase; Proteins; Pump; Receptor Protein-Tyrosine Kinases; Recruitment Activity; Regulation; Research; Reticulum; Role; Route; STIM1 gene; Signal Transduction; Signal Transduction Pathway; Signaling Molecule; Signaling Protein; Site; Surface; T-Cell Activation; Thapsigargin; Tubular formation; Tyrosine; base; cell motility; fighting; inhibitor/antagonist; novel; overexpression; patch clamp; pathogen; phosphatidylinositol 3,4,5-triphosphate; phosphatidylinositol phosphate, PtdIns(4,5)P2; prevent; protein complex; receptor; research study; response; sensor; tool; trafficking; transcription factor

Calcium ions (Ca2+) are one of the most ubiquitously used signaling molecules in eukaryotic cell regulation. They regulate a range of cellular processes such as muscle contraction, hormone secretion or gene transcription. Ca2+ enters the cells via multiple Ca2+ entry routes formed by Ca2+ channels and transporters and is very efficiently eliminated by Ca2+ pumps and exchangers. The tight control of the cells Ca2+ homeostasis is essential for cellular signaling and for the maintenance of cellular integrity. Recent studies identified two protein families (STIM and Orai/CRACM) that mediate a specific form of Ca2+ entry, termed store-operated calcium entry (SOCE). STIM1, is an endoplasmatic reticulum (ER) resident protein that rapidly translocates to the plasma membrane (PM)-adjacent compartment of the ER upon depletion of the ER Ca2+ stores where it activates the calcium channel, Orai1. The importance of this calcium entry pathway is highlighted by the fact that mutations in Orai1 has been linked to severe inborn human immunodeficiencies and that this route of calcium entry is key to the calcium regulated activation of T-cells mediated by the NFAT transcription factors. Studying the molecular details of STIM1 translocation to the PM-adjacent ER compartment and identification of the domains responsible for STIM1/Orai1 interaction will help develop novel molecular approaches for immunosupression. As most ion channels and transporters are regulated by the minor but critically important class of acidic phospholipids, the phosphoinositides, the phosphoinositide dependence of Orai1 channel activation in the PM and of STIM1 movements from the tubular to PM-adjacent ER regions during Ca2+ store depletion were investigated. Phosphatidylinositol 4,5-bisphosphate PtdIns(4,5)P2 levels were changed either with agonist stimulation, or by chemically-induced recruitment of a phosphoinositide 5-phosphatase domain to the PM, while PtdIns4P levels were decreased by inhibition or down regulation of phosphatidylinositol 4-kinases (PI4Ks). Agonist-induced phospholipase C activation and PI4K inhibition but not isolated PtdIns(4,5)P2 depletion substantially reduced endogenous or STIM1/Orai1 mediated SOCE without preventing STIM1 movements toward the PM upon Ca2+ store depletion. Patch clamp analysis of cells overexpressing STIM1 and Orai1 proteins confirmed that phospholipase C activation or PI4K inhibition greatly reduced ICRAC currents (the electrophysiological correlate of STIM1/Orai1 mediated SOCE). These results suggest an inositide requirement of Orai1 activation but not STIM1 movements and indicate that PtdIns4P rather than PtdIns(4,5)P2 is a likely determinant of the Orai1 channel activity. In another set of studies it was examined whether subplasmalemmal mitochondria are located in close proximity to the ER-PM regions close to the activated Orai1 channels. This question was important as such a group of mitochondria could preferentially respond to Ca2+ influx occuring via the STIM1/Orai1 mechanism. For this purpose COS-7 cells were cotransfected with Orai1, STIM1 labeled with YFP or mRFP and a mitochondrially targeted Ca2+ sensitive fluorescent protein, inverse Pericam. Depletion of ER Ca2+ with ATP + thapsigargin (Tg) (in Ca2+ -free medium) induced the appearance of STIM1 puncta in the ≤ 100 nm wide subplasmalemmal space, as examined with total internal reflection fluorescence (TIRF) microspcopy. In such cells, mitochondria were located either in the gaps between STIM1-tagged puncta or in remote, STIM1-free regions. After addition of Ca2+ to initiate Ca2+ influx via the activated Orai1 channles, mitochondrial (Ca2+m) increased similarly regardless of the mitochondrion-STIM1 distance. These observations indicated that specially positioned mitochondria are not likely to serve as uniquely sensitive sensors of Ca2+ influx occuring at the PM and the subplasmalemmal ER. Another research focus of the group was the analysis of the phosphoinositide changes upon invasion of mammalian cells by the enteropathogenic bacteria, E. coli (EPEC). EPEC is a major cause of severe infantile diarrhea in developing countries. Many bacterial pathogens use the cells own trafficking machinery to invade and move around within the cells and help spred the bacteria from one cell to another. Understanding the underlying molecular events will help us find new strategies to better fight bacterial infections. Phosphatidylinositol 4,5-bisphosphate PtdIns(4,5)P2 and phosphatidylinositol 3,4,5-trisphosphate PtdIns(3,4,5)P3 are phosphoinositides (PIs) present in small amounts in the inner leaflet of the plasma membrane (PM) lipid bilayer of host target cells. They modulate the activity of proteins involved in EPEC infection. However, the role of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in EPEC pathogenesis remains obscure. In a set of experiments utilizing the fluorescent phosphoinositide probes developed in our laboratory, we collaborated with Dr. Benjamin Aroetis group to show that EPEC induces a transient PtdIns(4,5)P2 accumulation at bacterial infection sites. Simultaneous actin accumulation, likely involved in the construction of the actin-rich pedestal, was also observed at these sites. Acute PtdIns(4,5)P2 depletion partially diminished EPEC adherence to the cell surface and actin pedestal formation. These findings were consistent with a bimodal role, whereby PtdIns(4,5)P2 contributes to EPEC association with the cell surface and to the maximal induction of actin pedestals. Finally, it was shown that EPEC induces PtdIns(3,4,5)P3 clustering at bacterial infection sites, in a translocated intimin receptor (Tir)-dependent manner. Tir phosphorylated on tyrosine 454, but not on tyrosine 474, formed complexes with an active phosphatidylinositol 3-kinase (PI3K), suggesting that PI3K recruited by Tir prompts the production of PtdIns(3,4,5)P3 beneath EPEC attachment sites. The functional significance of this event may be related to the ability of EPEC to modulate cell death and innate immunity.

钙离子（Ca2+）是真核细胞调节中普遍使用的信号分子之一。它们调节一系列细胞过程，例如肌肉收缩，激素分泌或基因转录。 Ca2+通过由Ca2+通道和转运蛋白形成的多个CA2+进入途径进入细胞，并通过Ca2+泵和交换器非常有效地消除了细胞。 细胞Ca2+稳态的严格控制对于细胞信号传导和维持细胞完整性至关重要。最近的研究确定了两个蛋白质家族（Stim和Orai/cracm）介导特定形式的Ca2+进入，称为商店经营的钙进入（SOCE）。 STIM1是一种内质网（ER）驻留蛋白，在ER Ca2+储存量耗尽后，它迅速转移到ER的质膜（PM） - Adjacent室，在该eR激活钙通道，Orai1。 这一钙进入途径的重要性是由ORAI1中的突变与严重的先天人免疫缺陷有关的事实，并且这种钙进入途径是由NFAT转录因子介导的钙调节T细胞的钙调节激活的关键。 研究STIM1转移到PMADJACACENT ER室的分子细节以及负责STIM1/ORAI1相互作用的域的鉴定将有助于开发新的分子方法以进行免疫选择。 由于大多数离子通道和转运蛋白受到次要但至关重要的酸性磷脂的调节，因此在PM中，ORAI1通道激活的磷酸肌醇依赖性以及从管状运动到PM-ADJACACENT of Stim1移动到PM-储存量的储存过程中的磷酸sim1运动。磷脂酰肌醇4,5-双磷酸PTDIN（4,5）P2水平通过激动剂刺激或化学诱导的磷酸肌醇5-磷酸酶结构域募集到PM PM，而PTDINS4P水平降低了PTDINS4P的抑制作用或下降降低了pTDINS4P的水平，或者通过抑制降低了磷酸化糖酶4-磷酸4-磷酸4-磷酸4-磷酸4-- pPI。 激动剂诱导的磷脂酶C活化和PI4K抑制作用，但没有分离的PTDINS（4,5）P2耗竭可大大降低内源性或STIM1/ORAI1介导的SOCE，而无需在CA2+储存量deptetiT耗尽时阻止刺激速度向PM朝向PM。 对过表达STIM1和ORAI1蛋白的细胞的斑块夹分析证实，磷脂酶C的激活或PI4K抑制大大降低了ICRAC电流（STIM1/ORAI1介导的SOCE的电生理相关性）。这些结果表明ORAI1激活的唯一识别要求，而不是STIM1运动，并表明PTDINS4P而不是PTDINS（4,5）P2可能是Orai1通道活性的决定因素。 在另一项研究中，检查了下型线粒体是否位于接近激活的Orai1通道附近的ER-PM区域。这个问题很重要，因为这样的线粒体可以优先响应通过STIM1/ORAI1机制发生的Ca2+涌入。 为此，用ORAI1共转染了cos-7细胞，用YFP或MRFP标记的stim1以及线粒体靶向的Ca2+敏感荧光蛋白逆佩里卡姆逆pericam。用ATP+ Thapsigargin（Tg）（在Ca2+的培养基中）耗尽ER Ca2+会诱导≤100nm宽的亚质量空间中的STIM1点的出现，并使用总内反射荧光（TIRF）微观进行了检查。 在这样的细胞中，线粒体位于刺激标记的点的差距或无刺激区域之间的间隙中。在添加Ca2+以通过激活的Orai1通道启动Ca2+涌入后，无论线粒体stim1距离如何，线粒体（Ca2+ M）也同样增加。这些观察结果表明，特殊位置的线粒体不可能用作PM和亚质量ER处发生的Ca2+涌入的独特敏感传感器。 该小组的另一个研究重点是分析肠病细菌大肠杆菌（EPEC）对哺乳动物细胞侵袭的磷酸肌醇变化。 EPEC是发展中国家严重婴儿腹泻的主要原因。 许多细菌病原体都使用细胞自己的运输机制来侵入并在细胞内移动，并帮助将细菌从一个细胞转移到另一个细胞。了解潜在的分子事件将有助于我们找到新的策略，以更好地抵抗细菌感染。 Phosphatidylinositol 4,5-bisphosphate PtdIns(4,5)P2 and phosphatidylinositol 3,4,5-trisphosphate PtdIns(3,4,5)P3 are phosphoinositides (PIs) present in small amounts in the inner leaflet of the plasma membrane (PM) lipid bilayer of host target cells.它们调节参与EPEC感染的蛋白质的活性。然而，PTDINS（4,5）P2和PTDINS（3,4,5）P3在EPEC发病机理中的作用仍然晦涩。在利用实验室中开发的荧光磷酸肌醇探针的一系列实验中，我们与本杰明·阿罗泰斯（Benjamin Aroetis）组合作，表明EPEC诱导了瞬时PTDINS（4,5）P2在细菌感染部位积累。在这些部位也观察到了可能参与富含肌动蛋白的基座的同时肌动蛋白积累。急性PTDINS（4,5）P2耗竭部分减少了EPEC依从性对细胞表面和肌动蛋白基座形成。这些发现与双峰作用一致，在这种情况下，PTDINS（4,5）P2促进了EPEC与细胞表面的关联和肌动蛋白基座的最大诱导。最后，结果表明，EPEC诱导细菌感染部位的PTDINS（3,4,5）P3聚类，以易位的内膜素受体（TIR）依赖性方式诱导PTDINS（3,4,5）。在酪氨酸454上磷酸化的TIR磷酸化，而不是在酪氨酸474上磷酸化，该磷酸化形成了与活性磷脂酰肌醇3-激酶（PI3K）形成的复合物，这表明TIR募集的PI3K促使PTDINS（3,4,5）P3 P3 P3 P3在Epecec Eccectect附着位置下产生。该事件的功能意义可能与EPEC调节细胞死亡和先天免疫的能力有关。