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 蛋白偶联受体 (GPCR) 控制多种生理过程，如体内平衡、视觉和趋化运动。迁移细胞，如哺乳动物白细胞和盘基网柄菌，利用 GPCR 信号传导来调节 MAPK/ERK、PI3K、TORC2/AKT、腺苷酸环化酶和肌动蛋白聚合，这些共同指导趋化性。配体结合后，哺乳动物 GPCR 通常在细胞质残基处磷酸化，从而解偶联 G 蛋白途径，但激活其他途径。在盘基网柄菌的发育过程中，分泌的 cAMP 作为化学引诱剂，细胞外 cAMP 以振荡波的形式传播，以确保定向迁移信号。 cAMP 振荡源自腺苷酸环化酶的瞬时兴奋反应，然后迅速适应。当 cAMP 持续存在时，大多数趋化反应同样是短暂的。我们研究了盘基网柄菌中表达非磷酸化 cAMP 受体的趋化信号传导，并提供了证据表明，盘基网柄菌对 cAMP 的有效极化和趋化性需要受体磷酸化。我们还表明，这些细胞无法调节腺苷酸环化酶的适应，从而破坏长程振荡 cAMP 波的产生和细胞骨架肌动蛋白反应。这些数据表明，趋化剂受体磷酸化是共同调节盘基网柄菌和其他迁移细胞的重要途径所必需的。 两种早老素蛋白的突变会导致家族性阿尔茨海默病。这些蛋白质在γ-分泌酶复合物内的蛋白水解依赖性活性和支架相关的活性中发挥作用。 功能。由于删除这两个基因后胚胎会致死，分析这些蛋白质的细胞作用变得很复杂。我们之前已经证明，在简单的生物医学模型盘基网柄菌中，这些蛋白质具有发育作用​​。在这里，我们表明，人类早老素 1 蛋白可以挽救发育作用，控制多种细胞类型的终末分化。我们还表明，携带单个 D/A 催化突变的人早老素蛋白可以挽救形态发育，这表明早老素蛋白在发育中具有非蛋白水解作用。这些远缘相关物种中早老素蛋白的保守功能还表现在 D. discoideum 蛋白 显示哺乳动物（小鼠囊胚）来源的细胞中的蛋白水解（Notch 切割）活性。我们进一步表明，环磷酸腺苷 (cAMP) 水平升高和刺激诱导的钙释放增强 这是由于 D. discoideum 中早老素的丢失造成的，表明该模型中涉及这些第二信使的细胞内信号传导途径失调。因此，我们的数据表明古代 早老素蛋白在调节细胞内信号传导和发育中的作用，并为分析人类早老素功能提供了一种新颖的模型系统。 Perilipin 家族蛋白 (Plins) 覆盖在各种细胞类型的细胞内中性脂质储存液滴的表面。跨不同物种的研究表明，Plins 通过向脂滴表面募集脂肪酶和其他调节蛋白来调节脂质储存代谢。哺乳动物基因组编码 5 个不同的 Plin 基因成员和源自特定 mRNA 剪接变体的其他蛋白质形式。然而，尚不清楚不同的 Plin 是否具有不同的功能特性。利用生化、细胞成像和流式细胞术分析，我们现在表明，在各种类型的单个细胞内，不同的 Plin 蛋白优先隔离到单独的脂质存储液滴池中。通过检查异位表达的 GFP 融合体和所有内源性 Plin 蛋白形式，我们证明不同的 Plins 隔离于脂滴，脂滴明显由三酰酯或胆固醇酯组成。此外，与 TAG（或 CE）液滴具有强烈关联偏好的 Plins 可以将相对的细胞内 TAG/CE 平衡重新导向目标脂质。我们的数据表明 Plin 功能的多样性，改变了之前关于 Plin 共享集体行动的假设，并表明每个 Plin 可以具有单独且独特的功能。 TOR 蛋白激酶在两种不同的复合物中发挥作用：TOR 复合物 1 (TORC1) 和 2 (TORC2)。 TORC1 是生长所必需的，以响应生长因子、营养物质和细胞能量状态；据记录，TORC2 可以调节 AKT 信号传导，从而调节细胞骨架极化。在其生态位中，盘基网柄菌吞噬细菌和酵母以捕获营养。尽管TORC1在控制细胞生长中发挥着重要作用，但我们发现盘基网柄菌中营养颗粒的捕获和吞噬作用独立于TORC1介导的营养感应和生长调节。然而，盘基网柄菌 TORC2 成分 Rictor/Pia、SIN1/RIP3 和 Lst8 的损失会促进营养颗粒的吸收； TORC2 失活可提高吞噬作用的效率和速度。与吞噬作用相反，我们发现巨胞饮作用是细胞摄取液相营养物质的 AKT 依赖性过程，不受任一 TOR 复合物的调节。 TORC1 和 TORC2 的综合和平衡调节对于盘基网柄菌来说可能至关重要，以协调生长和能量需求与其他重要的 TOR 调节过程。 CHD（染色质结构域-解旋酶-DNA 结合）家族是主要的 ATP 依赖性染色质重塑蛋白之一，可调节真核生物中的核小体定位和基因表达。哺乳动物 CHD 蛋白分为三个亚家族，几种人类疾病与 CHD 功能受损有关。在这里，我们在盘基网柄菌中鉴定出三种以独特的发育模式表达的 CHD（ChdA、ChdB 和 ChdC）。每个无效突变体都具有不同的、非冗余的表型，表明功能特异性。 ChdC 与 CHD 亚家族 3 的成员形成簇，其中包括哺乳动物 CHD7。 CHD7 中的突变与人类 CHARGE 综合征相关，其特征是多种先天性发育缺陷，而盘基网柄菌中的 chdC 缺失同样具有严重且多样化的发育表型。为了了解 ChdC 和相关 CHD 的机制功能，我们比较了生长和发育过程中野生型 (WT) 细胞与 chdC-null 细胞的全基因组核小体图谱。我们发现，chdC-null 中的一部分基因的核小体间距发生了改变，并且基因特异性转录谱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。这项研究为一类先心病在染色质组织中的作用提供了新的机制见解，并可作为更好地了解某些人类遗传缺陷的基础。