Phosphoinositide-calcium Signaling In Cell Regulation

细胞调节中的磷酸肌醇-钙信号转导

• 批准号: 10676674
• 负责人: TAMAS BALLA
• 金额: $ 193.08万
• 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/10676674
• 关键词: 1-Phosphatidylinositol 4-Kinase; Affect; Apoptosis; Binding; Biochemical Process; Biology; C-terminal; Calcineurin; Calcineurin inhibitor; Calcium Signaling; Canada; Catalytic Domain; Cataract; Cell Differentiation process; Cell division; Cell membrane; Cell physiology; Cells; Cellular Membrane; Cellular Metabolic Process; Communication; Complex; Coupled; Cytokinesis; DNA Sequence Alteration; Data; Defect; Deuterium; Diabetes Mellitus; Disease; Elements; Ensure; Environment; Enzymes; Eukaryotic Cell; Excision; Family; Fishes; Focus Groups; G Protein-Coupled Receptor Signaling; GTP-Binding Proteins; Gene Mutation; Golgi Apparatus; Growth Factor; Hormones; Human; Hydrogen; Immune System Diseases; Immunologic Deficiency Syndromes; Immunologics; Immunosuppressive Agents; Impairment; Individual; Inflammatory Bowel Diseases; Intercept; Interruption; Intestinal Atresia; Intestinal Diseases; Intestines; Ion Channel; Italy; Laboratories; Lead; Life Cycle Stages; Link; Lipids; Malignant Neoplasms; Mammalian Cell; Mediating; Mediator of activation protein; Membrane; Membrane Lipids; Membrane Transport Proteins; Mitosis; Molecular; Multiprotein Complexes; Mus; Names; Neurodegenerative Disorders; Neurologic; Neurotransmitters; Organ; Organelles; Outcome; PPP3CA gene; Pathway interactions; Peptides; Phenotype; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phospholipids; Phosphotransferases; Physiology; Play; Process; Production; Protein Isoforms; Protein Kinase; Protein phosphatase; Proteins; RNA Splicing; Regulation; Reporting; Research; Research Personnel; Reticulum; Role; STIM1 gene; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Signaling Protein; Site; Specificity; Structural Models; Study models; Surface; Tail; Transport Process; Universities; Vacuolar Protein Sorting; Variant; Virus; Work; Yeasts; antimicrobial drug; biochemical model; biophysical properties; cell motility; cell type; cellular targeting; combinatorial; daughter cell; driving force; early onset; fighting; human disease; information processing; inhibitor; insight; lens; leukodystrophy; lipid transport; medical schools; nervous system disorder; oxysterol binding protein; palmitoylation; pathogen; phosphatidylinositol 3,4-diphosphate; phosphatidylinositol 4-phosphate; phosphoinositide-3,4-bisphosphate; premature; prevent; protein complex; receptor; response; senescence; stem; tool; trafficking; transmission process

Every biochemical process that happens in a eukaryotic cell relies upon a molecular information flow that leads from receptors that inform the cell about its environment all the way to the molecular effectors that determine the appropriate cellular response. A proper information transmission requires a high degree of organization where the molecular players are organized into different cellular compartments so that the specificity of the cellular response can be properly maintained. Breakdown of this organization is the ultimate cause of all human diseases even if the affected molecular pathways differ according to the kind of disease, such as cancer, diabetes or neurodegenerative diseases just to name a few. Research described in this report has focused on the question of how cells organize their internal membranes to provide a structural framework on which molecular signaling complexes assemble to ensure proper information processing. Lipid composition of cellular membranes is a major determinant of their biophysical properties and is unique to the different cellular organelles. How cells achieve and maintain the proper lipid composition of their membranes is poorly understood. Cellular processes that affect membrane lipid composition of organelles are often targeted by cellular pathogens such as viruses to force the cells to produce the pathogen instead of performing the cells normal functions. Better understanding of these processes not only can provide new strategies to fight various human diseases but also to intercept the life cycle of cellular pathogens offering an alternative to antimicrobial drugs. Lipid transport and Ca2+ signaling are closely interrelated in plasma membrane (PM) - endoplasmatic reticulum (ER) contact sites Membrane contacts sites (MCS) between various organelles are emerging as key structural elements where important communication between organelles takes place. MCS have been primarily featured in non-vesicular lipid transfer and Ca2+ signal propagation, but their importance is likely to reach beyond these two processes. An important class of molecules that function at MCSs are the ORP (oxysterol-binding protein-related protein) proteins that are the mammalian orthologues of the yeast Osh proteins and mediate the transport of specific lipids between cellular membranes. One of the salient features of Osh/ORP proteins is that they use phosphatidylinositol 4-phosphate (PI4P) gradient as a driving force as they counter-transport PI4P in exchange for the specific lipids they move between membranes. Therefore, lipid transport by Osh/ORP proteins is linked to the activity of PI 4-kinases. In this research period, we investigated the impact of changing PM PI4P levels on the Ca2+ entry process mediated by the STIM1-Orai1 molecular complex that underlies the refilling of the ER luminal Ca2+ stores during receptor stimulation. We found that changing PM PI4P levels through inhibition of the lipid kinase (PI4KA) that produces PI4P in the PM potently inhibits Ca2+ influx through interruption of STIM1 association with the PM. Similarly, manipulation of PM PI4P levels through the expression of ORP5 and ORP8 proteins had major impact on Ca2+ influx. Our studies revealed a tight connection between Ca2+ entry mediated by the STIM1-Orai1 complex and the PI4P-driven lipid transport process at PM-ER contact sites. The critical role of specific phosphoinositide lipids in the late stage of cell division Separation of the two daughter cells at the last stage of mitosis called cytokinetic membrane abscission is a spatially and temporally regulated process that requires membrane remodeling at the midbody, a subcellular organelle that defines the cleavage site. This process mediated by a multi-protein molecular complex, called ESCRT and its defective function can lead to cataract. It is not known how ESCRT defects can lead to cataract and whether it is related to cytokinesis defects. In a collaborative study led by the Hirsch laboratory in Italy, it was found that a lens-specific cytokinetic process required the lipid kinase, PI3K-C2 (phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2),and its lipid product PI(3,4)P2 (phosphatidylinositol 3,4-bisphosphate). These studies showed that the ESCRT-II subunit VPS36 (vacuolar protein-sorting-associated protein 36)requires PI(3,4)P2 binding and loss of each of the ESCRT-II components led to impaired cytokinesis, triggering premature senescence in the lens of fish, mice, and humans. Importantly, the PI4P substrate for the PI3K-C2 enzyme to support this process was provided by the PI4KA enzyme. This evolutionarily conserved pathway underlies the cell type specific control of cytokinesis that helps to prevent early onset cataract by protecting from senescence. Specific calcineurin splice form targets the multi-protein complex of PI4KA Calcineurin, the conserved protein phosphatase and target of immunosuppressants, is a critical mediator of Ca2+ signaling. In a collaborative study led by the Cyert laboratory at Stanford University that focused on the understudied calcineurin isoform, CNA1 it was discovered that unlike canonical cytosolic calcineurin, CNA1 localizes to the plasma membrane and Golgi due to reversible palmitoylation of its divergent C-terminal tail. Palmitoylation targets CNA1 to a distinct set of membrane-associated interactors including the multi-protein PI4KA complex containing EFR3B, PI4KA, TTC7B and FAM126A. Hydrogen-deuterium exchange studies performed in the Burke laboratory at the University of Victoria, Canada, found multiple contacts in the calcineurin-PI4KA complex, including a calcineurin-binding peptide motif in the disordered tail of FAM126A, which was establish as a calcineurin substrate. In cellular studies, Calcineurin inhibitors decreased PI4P production during Gq-coupled GPCR signaling, suggesting that calcineurin dephosphorylates and promotes PI4KA complex activity. This work revealed a calcineurin-regulated signaling pathway and identified the PI4KA complex as a regulatory target. It also showed that dynamic palmitoylation provides the CNA1 enzyme a unique localization to increase its access its substrates providing unique specificity and regulation to the protein. PI4KA variants in human cause neurological, intestinal and immunological disease The lipid kinase PI4KA generates PI4P in the PM playing critical roles in the physiology of multiple cell types. PI4KA requires its assembly into a heterotetrameric complex with EFR3, TTC7 and FAM126. Sequence alterations in two of these molecular partners, TTC7 (encoded by TTC7A or TCC7B) and FAM126, in humans have been associated with a heterogeneous group of either neurological (FAM126A) or intestinal and immunological (TTC7A) conditions. In this multi-center collaborative study led by researchers of the University of Exeter Medical School in the UK, biallelic PI4KA sequence alterations in humans were shown to be associated with neurological disease, in particular hypomyelinating leukodystrophy. In addition, some affected individuals may also present with inflammatory bowel disease, multiple intestinal atresia and combined immunodeficiency. Biochemical and structural modelling studies indicated that PI4KA-associated phenotypic outcomes probably stem from impairment of PI4KIII-TTC7-FAM126's organ-specific functions, due to defective catalytic activity or altered intra-complex functional interactions. Together, these data define PI4KA gene alteration as a cause of a variable phenotypical spectrum and provide fundamental new insight into the combinatorial biology of the PI4KIII-FAM126-TTC7-EFR3 molecular complex.

在真核细胞中发生的每一个生化过程都依赖于分子信息流，该分子信息流来自受体，该信息一直通知细胞的环境，一直到确定适当细胞反应的分子效应子。适当的信息传输需要高度的组织，其中分子玩家被组织到不同的细胞室中，以便可以正确维护细胞反应的特异性。即使受影响的分子途径因癌症，糖尿病或神经退行性疾病等疾病的类型而异，该组织的崩溃是所有人类疾病的最终原因。 本报告中描述的研究集中在细胞如何组织其内部膜的问题上，以提供一个结构框架，分子信号传导复合物组装以确保适当的信息处理。细胞膜的脂质组成是其生物物理特性的主要决定因素，并且在不同的细胞细胞器中是独有的。细胞如何实现和维持其膜的适当脂质组成的理解很少。影响细胞器的膜脂质组成的细胞过程通常是由病毒等细胞病原体靶向的，迫使细胞产生病原体，而不是执行细胞正常功能。更好地了解这些过程不仅可以提供与各种人类疾病作斗争的新策略，而且还可以拦截提供抗菌药物替代品的细胞病原体生命周期。 脂质转运和Ca2+信号在质膜（PM） - 内质网（ER）接触位点紧密相互关联 各种细胞器之间的膜接触位点（MCS）正在出现，随着细胞器之间重要通信的关键结构元素。 MC主要在非二维脂质转移和Ca2+信号传播中出现，但其重要性可能超出了这两个过程。在MCSS中起作用的一类重要分子是ORP（氧蛋白酶结合蛋白相关的蛋白）蛋白，它们是酵母OSH蛋白的哺乳动物直系同源物，并介导细胞膜之间特定脂质的转运。 OSH/ORP蛋白的显着特征之一是它们使用磷脂酰肌醇4-磷酸（PI4P）梯度作为驱动力，因为它们会反向传输PI4P来换取它们在膜之间移动的特定脂质。因此，OSH/ORP蛋白通过OSH/ORP蛋白的脂质转运与PI 4-激酶的活性有关。在这一研究期间，我们研究了PM PI4P水平对由STIM1-ORAI1分子复合物介导的Ca2+进入过程的影响，该过程是受体刺激过程中ER腔Ca2+储存的补充的基础。我们发现，通过抑制脂质激酶（PI4KA）而改变PM PI4P水平，该脂质激酶（PI4KA）在PM中产生PI4P，通过中断STIM1与PM的关联而有效抑制Ca2+流入。同样，通过ORP5和ORP8蛋白的表达对PM PI4P水平的操纵对Ca2+流入产生了重大影响。我们的研究表明，由STIM1-ORAI1复合物介导的Ca2+进入与PM-ER接触位点的PI4P驱动的脂质传输过程之间存在紧密的联系。 特定磷酸肌醇脂质在细胞分裂的后期的关键作用 在有丝分裂的最后阶段将两个子细胞的分离称为细胞力学膜脱落是在空间和时间调节的过程中，需要在中体中进行膜重塑，这是一个定义裂解位点的亚细胞细胞器。由多蛋白分子复合物（称为ESCRT及其缺陷功能）介导的该过程可导致白内障。尚不清楚ESCRT缺陷如何导致白内障以及它是否与细胞因子缺陷有关。在意大利的赫希实验室领导的一项合作研究中，发现透镜特异性细胞动力学过程需要脂质激酶PI3K-C2（磷脂酰肌醇-4-磷酸3-磷酸3-激酶催化亚基2）及其脂质产物Pi（3,4,4）p2（phostater）p2（phossiTylIn）。这些研究表明，ESCRT-II亚基VPS36（液泡蛋白与蛋白质相关的蛋白36）需要PI（3,4）P2 P2结合和每个ESCRT-II成分的损失，导致细胞因子受损，从而触发了鱼类，小鼠，小鼠和猫的镜头中的早产。重要的是，PI4KA酶提供了PI3K-C2酶支持此过程的PI4P底物。该进化保守的途径是细胞类型特异性控制的细胞因子的控制，这有助于通过保护衰老来防止早期发作性白内障。 特定的钙调神经酶剪接形式靶向PI4KA的多蛋白质复合物 钙调蛋白是保守的蛋白质磷酸酶和免疫抑制剂的靶标，是Ca2+信号传导的关键介体。在斯坦福大学的Cyert实验室的一项合作研究中，该研究重点是研究的钙调蛋白同工型，CNA1发现，与规范的胞质钙调蛋白不同，CNA1本地化于质膜和高尔基，这是由于其相差C-末端尾部可逆的棕榈酰化而引起的。棕榈酰化将CNA1靶向一组不同的膜相关相互作用者，包括含有EFR3B，PI4KA，TTC7B和FAM126A的多蛋白PI4KA复合物。在加拿大维多利亚大学的伯克实验室进行的氢 - 居民交换研究发现了Clacineurin-PI4KA综合体的多个接触，其中包括FAM126A无序尾巴中的钙调神经蛋白结合肽基序，该基序是钙调蛋白底物的建立。在细胞研究中，钙调蛋白抑制剂在GQ耦合的GPCR信号传导过程中降低了PI4P的产生，这表明钙调神经磷酸酶脱磷酸化并促进PI4KA复合活性。这项工作揭示了钙调蛋白调节的信号通路，并将PI4KA复合物确定为调节靶标。它还表明，动态棕榈酰化为CNA1酶提供了独特的定位，以增加其底物的访问，从而为蛋白质提供了独特的特异性和调节。 人类原因的PI4KA变体神经，肠道和免疫疾病 脂质激酶PI4KA在PM中生成PI4P，在多种细胞类型的生理学中发挥关键作用。 PI4KA需要将其组装到EFR3，TTC7和FAM126中的异常综合体中。这些分子伴侣的两个序列改变TTC7（由TTC7A或TCC7B编码）和人类FAM126与神经（FAM126A）或肠道和免疫学（TTC7A）条件的异质组有关。在这项由英国埃克塞特大学医学院的研究人员领导的这项多中心协作研究中，人类的双重PI4KA序列改变与神经系统疾病有关，尤其是白细胞营养不良的人性疾病。此外，一些受影响的个体也可能出现炎症性肠病，多个肠闭锁和联合免疫缺陷。生化和结构建模研究表明，与PI4KA相关的表型结局可能是由于PI4KIII-TTC7-FAM126的器官特异性功能损害，这是由于缺陷催化活性或改变了内部功能的功能。这些数据共同将PI4KA基因的改变定义为可变的表型谱的原因，并为PI4KIII-FAM126-TTC7-EFR3分子复合物的组合生物学提供了基本的新见解。