SIGNALING PATHWAYS IN CONTROL OF GROWTH AND DEVELOPMENT

控制生长和发育的信号通路

• 批准号: 8553636
• 负责人: ALAN R KIMMEL
• 金额: $ 133.64万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 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/8553636
• 关键词: Actins; Adenylate Cyclase; Amino Acids; Bacteria; Biochemical; Biological Models; CHARGE syndrome; Calcium; Cell physiology; Cells; Chemotactic Factors; Chemotaxis; Cholesterol Esters; Chromatin; Complex; Coupled; Cyclic AMP; Cyclic AMP Receptors; Cytoskeleton; DNA Binding; DNA-Binding Proteins; Data; Defect; Development; Dictyostelium; Dictyostelium discoideum; Embryo; Ensure; Equilibrium; Eukaryota; Family; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Proteins; Gene Expression; Genes; Genome; Growth; Growth Factor; Growth and Development function; Haploidy; Homeostasis; Human; Human Genetics; Individual; Laboratories; Leukocytes; Ligand Binding; Ligands; Lipase; Lipids; Liquid substance; MAP Kinase Gene; Maps; Mediating; Messenger RNA; Metabolism; Modeling; Molecular; Molecular Genetics; Movement; Mus; Mutation; Natural Immunity; Neurotransmitters; Nucleosomes; Nutrient; Organism; Pathway interactions; Pattern; Phagocytosis; Phase; Phenotype; Phosphorylation; Physiological Processes; Population; Positioning Attribute; Process; Production; Property; Protein Family; Protein Kinase; Proteins; Proto-Oncogene Proteins c-akt; RIPK3 gene; RNA Splicing; Regulation; Relative (related person); Research; Resources; Role; Second Messenger Systems; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Source; Specificity; Speed; Stimulus; Surface; System; Systems Analysis; Techniques; Transmembrane Domain; Variant; Vision; Yeasts; base; blastocyst; cell growth; cell type; cellular imaging; chemokine; chemokine receptor; chromatin remodeling; detection of nutrient; extracellular; fMet-Leu-Phe receptor; familial Alzheimer disease; fungus; gamma secretase; genetic manipulation; genetic regulatory protein; genome-wide; helicase; human disease; insight; macrophage; mammalian genome; member; morphogens; mutant; neutrophil; notch protein; novel; particle; perilipin; polymerization; preference; presenilin; presenilin-1; protein function; receptor; receptor function; response; scaffold; second messenger; surface coating; uptake

Eukaryotic G protein coupled receptors (GPCRs) control physiological processes, as diverse as, homeostasis, vision, and chemotactic movement. Migratory cells, like mammalian leukocytes and Dictyostelium discoideum, utilize GPCR signaling to regulate MAPK/ERK, PI3K, TORC2/AKT, adenylyl cyclase, and actin polymerization, which collectively direct chemotaxis. Upon ligand binding, mammalian GPCRs are often phosphorylated at cytoplasmic residues, uncoupling G protein pathways, but activating others. In developing Dictyostelium, secreted cAMP serves as a chemoattractant, with extracellular cAMP propagated as oscillating waves to ensure directional migratory signals. The cAMP oscillations derive from a transient excitatory response of adenylyl cyclase, which then rapidly adapts. Most chemotactic responses are similarly transient when cAMP is presented persistently. We have studied chemotactic signaling in Dictyostelium that express non-phosphorylatable cAMP receptors and provide evidence that receptor phosphorylation is required for effective polarization and chemotaxis of Dictyostelium to cAMP. We also show that these cells are unable to regulate adaptation of adenylyl cyclase, which disrupts long-range oscillatory cAMP wave production and cytoskeletal actin response. These data indicate that chemoattractant receptor phosphorylation is required to co-regulate essential pathways for Dictyostelium and perhaps other migratory cells. Mutations in two presenilin proteins cause Familial Alzheimer's disease. These proteins function in both a proteolytic-dependent activity within the gamma-secretase complex and in a scaffold-related function. Analysing the cellular role of these proteins has been complex due to embryonic lethality following deletion of both genes. We had previously shown that in the simple biomedical model, Dictyostelium discoideum, these proteins have a developmental role. Here we show that the developmental role can be rescued by the human presenilin 1 protein, controlling terminal differentiation in multiple cell types. We also show that morphological development can be rescued with the human presenilin protein carrying a single D/A catalytic mutation, suggesting a non-proteolytic role for presenilin proteins in development. The conserved function of presenilin proteins in these distantly related species is also shown in that the D. discoideum proteins show proteolytic (Notch cleavage) activity in mammalian (mouse blastocyst)-derived cells. We further show that elevated cyclic AMP (cAMP) levels and enhanced stimulation-induced calcium release result from presenilin loss in D. discoideum, suggesting dysregulation of intracellular signalling pathways involving these second messengers in this model. Our data therefore suggests ancient roles for presenilin proteins in regulating intracellular signalling and development and provides a novel, model system for the analysis of human presenilin function. Perilipin family proteins (Plins) coat the surface of intracellular neutral lipid storage droplets in various cell types. Studies across diverse species demonstrate that Plins regulate lipid storage metabolism through recruitment of lipases and other regulatory proteins to lipid droplet surfaces. Mammalian genomes encode 5 distinct Plin gene members and additional protein forms derive from specific mRNA splice variants. However, it is not known if the different Plins have distinct functional properties. Using biochemical, cellular imaging, and flow cytometric analyses, we now show that within individual cells of various types, the different Plin proteins preferentially sequester to separate pools of lipid storage droplets. By examining ectopically expressed GFP fusions and all endogenous Plin protein forms, we demonstrate that different Plins sequester to lipid droplets, comprised distinctly of either triacylcerides or of cholesterol esters. Further, Plins with strong association preferences to TAG (or CE) droplets can re-direct the relative intracellular TAG/CE balance toward the targeted lipid. Our data suggest diversity of Plin function, alter previous assumptions about shared collective actions of the Plins, and indicate that each Plin can have separate and unique functions. The TOR protein kinase functions in two distinct complexes, TOR Complexes 1 (TORC1) and 2 (TORC2). TORC1 is required for growth in response to growth factors, nutrients, and cellular energy state; TORC2 is documented to regulate AKT signaling, which can modulate cytoskeletal polarization. In its ecological niche, Dictyostelium engulf bacteria and yeast for nutrient capture. Despite the essential role of TORC1 in control of cellular growth, we show that nutrient particle capture, phagocytosis, in Dictyostelium is independent of TORC1 mediated nutrient sensing and growth regulation. However, loss of Dictyostelium TORC2 components Rictor/Pia, SIN1/RIP3, and Lst8 promotes nutrient particle uptake; inactivation of TORC2 leads to increased efficiency and speed of phagocytosis. In contrast to phagocytosis, we show that macropinocytosis, an AKT-dependent process for cellular uptake of fluid phase nutrients, is not regulated by either TOR complex. The integrated and balanced regulations of TORC1 and TORC2 may be critical in Dictyostelium to coordinate growth and energy needs with other essential TOR-regulated processes. The CHD (Chromodomain-Helicase-DNA binding) family is one of the major ATP-dependent, chromatin remodeling proteins that regulate nucleosome positioning and gene expression in eukaryotes. Mammalian CHD proteins group into three subfamilies and several human diseases are associated with impaired CHD function. Here, we identify three CHDs (ChdA, ChdB, and ChdC) in Dictyostelium discoideum that are expressed with unique developmental patterns. Null mutants for each have distinct, non-redundant phenotypes, indicating functional specificity. ChdC clusters with members of CHD subfamily 3 that includes mammalian CHD7. Mutations in CHD7 are associated with the human CHARGE syndrome, characterized by multiple congenetical developmental defects, and in Dictyostelium, chdC-nulls similarly have severe and diverse developmental phenotypes. To understand the mechanistic function of ChdC and related CHDs, we compared genome-wide nucleosomal maps of wild-type (WT) cells during growth and development and those of chdC-nulls. We show that nucleosome-spacing is altered in a subset of genes in chdC-nulls and that gene-specific transcriptional profiling changes correlate statistically with these unique nucleosomal patterning differences and phenotypic defects. This study provides novel mechanistic insight into the action of a class of CHDs in chromatin organization and may serve as a basis to better understand certain human genetic defects.

真核G蛋白耦合受体（GPCR）控制生理过程，如稳态，视力和趋化运动。迁移细胞，例如哺乳动物白细胞和dictyostelium discoideum，利用GPCR信号来调节MAPK/ERK，PI3K，TORC2/AKT，腺苷酸环化酶和肌动蛋白聚合，从而集体直接趋化性。配体结合后，哺乳动物GPCR通常在细胞质残基上被磷酸化，从而解开G蛋白途径，但会激活其他途径。在开发dictyostelium的过程中，分泌的营地用作趋化剂，细胞外营地传播为振荡波，以确保方向性的迁移信号。 cAMP振荡源自腺苷酸环化酶的短暂兴奋反应，然后迅速适应。当camp持续出现时，大多数趋化反应类似地是短暂的。我们已经研究了表达非磷酸化cAMP受体的Dictyostelium中的趋化信号传导，并提供了证据表明，受体磷酸化是有效的极化和dictyostelium趋化性所需要的。我们还表明，这些细胞无法调节腺苷酸环化酶的适应性，这破坏了远距离振荡性cAMP的产生和细胞骨架肌动蛋白反应。这些数据表明，趋化剂受体磷酸化需要共同调节DICETYOSTELIUM和其他迁移细胞的基本途径。 两种老年蛋白蛋白的突变引起家族性阿尔茨海默氏病。这些蛋白质在γ-分泌酶复合物中的蛋白水解依赖性活性以及与脚手架相关的蛋白水解依赖性活性中起作用 功能。分析这些蛋白质的细胞作用是由于两个基因缺失后的胚胎致死性而变得复杂的。我们先前曾表明，在简单的生物医学模型，迪斯特尔迪斯特尔，这些蛋白质具有发育作用​​。在这里，我们表明，人类Presenilin 1蛋白可以挽救发育作用，从而控制多种细胞类型的末端分化。我们还表明，可以通过携带单个D/A催化突变的人类presenilin蛋白来挽救形态学发育，这表明质子蛋白在发育中的非蛋白质作用。在这些遥远相关的物种中，老年蛋白蛋白的保守功能也显示在D. discoideum蛋白中 在哺乳动物（小鼠胚泡）衍生的细胞中显示蛋白水解（缺口裂解）活性。我们进一步表明，环状AMP（CAMP）水平升高和刺激诱导的钙释放 D. discoideum中的presenilin损失导致，表明在该模型中涉及这些第二个使者的细胞内信号传导途径失调。因此，我们的数据表明古老 presenilin蛋白在调节细胞内信号传导和发育中的作用，并提供了一种新型的模型系统，用于分析人类presenilin功能。 Perilipin家族蛋白（PLIN）在各种细胞类型的细胞内中性脂质储存液滴的表面上覆盖。跨不同物种的研究表明，PLIN通过募集脂肪酶和其他调节蛋白来调节脂质储存代谢，从而调节脂质液滴表面。哺乳动物基因组编码5种不同的plin基因成员和其他蛋白质形式，源自特定的mRNA剪接变体。但是，尚不清楚不同的PLIN是否具有不同的功能特性。使用生化，细胞成像和流式细胞仪分析，我们现在表明，在各种类型的单个细胞中，不同的PLIN蛋白优先隔离了脂质存储液滴的池。通过检查异位表达的GFP融合和所有内源性plin蛋白形式，我们证明了不同的plins隔离为脂质液滴，这些液滴明显由三酰基酸酯或胆固醇酯组成。此外，对标签（或CE）液滴具有较强关联偏好的PLIN可以将相对细胞内标签/CE平衡重新指向靶向脂质。我们的数据表明，PLIN功能的多样性，更改有关PLIN共享集体行动的先前假设，并表明每个PLIN都可以具有独特的独特功能。 Tor蛋白激酶在两个不同的复合物中发挥作用，Tor复合物1（Torc1）和2（Torc2）。对于生长因子，养分和细胞能状态，TORC1是生长所必需的。记录了TORC2以调节AKT信号，该信号传导可以调节细胞骨架极化。在其生态利基市场中，dictyostelium吞噬细菌和酵母可捕获营养。尽管TORC1在控制细胞生长方面的重要作用，但我们表明，在Dictyostelium中，营养颗粒捕获，吞噬作用与Torc1介导的营养感应和生长调节无关。然而，dictyostelium torc2组件的损失rictor/pia，sin1/rip3和lst8促进了营养颗粒的吸收。 Torc2的失活导致吞噬作用的效率和速度提高。与吞噬作用相反，我们表明巨型细胞增多症是Akt依赖性液相养分的AKT依赖性过程，不受任何TOR复合物的调节。 TORC1和TORC2的综合和平衡法规在Dictyostelium中可能至关重要，以与其他必要的TOR调节过程协调生长和能量需求。 CHD（染色体 - 羟基酶-DNA结合）家族是调节真核生物中核小体定位和基因表达的主要ATP染色质重塑蛋白之一。哺乳动物冠心病蛋白分为三个亚科，几种人类疾病与CHD功能受损有关。在这里，我们在Dictyostelium Discoideum中识别出三个CHD（CHDA，CHDB和CHDC），它们以独特的发展模式表达。每个的空突变体具有不同的非冗余表型，表明功能特异性。 CHDC群集与包括哺乳动物CHD7在内的CHD亚家族成员。 CHD7中的突变与人类电荷综合征有关，其特征是多种相互性发育缺陷，而在Dictyostelium中，CHDC-Null类似地具有严重和多样化的发育表型。为了了解CHDC和相关CHD的机械功能，我们比较了生长和发育过程中野生型（WT）细胞的全基因组核小体图和CHDC无效的细胞。我们表明，在CHDC无效的基因子集中有核小体间距发生了变化，并且基因特异性转录分析的变化与这些独特的核小体模式差异和表型缺陷在统计上相关。这项研究提供了对一类CHD在染色质组织中的作用的新型机械洞察力，并可以作为更好地理解某些人类遗传缺陷的基础。