SIGNALING PATHWAYS IN CONTROL OF GROWTH AND DEVELOPMENT

控制生长和发育的信号通路

• 批准号: 8939697
• 负责人: ALAN R KIMMEL
• 金额: $ 140.24万
• 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/8939697
• 关键词: Actins; Adipocytes; Alzheimer' s Disease; Amino Acids; Bacteria; Biochemical; Biological Assay; Biological Models; CHARGE syndrome; Cardiac; Cardiovascular Diseases; Cell Culture Techniques; Cells; Chemotactic Factors; Chemotaxis; Chromatin; Chromatin Structure; Chronic; Clinical Trials; Complex; Congenital Disorders; Coupling; Cyclic AMP; Cyclic AMP Receptors; Cytoskeleton; Data; Dementia; Development; Dictyostelium; Diet; Disease; Embryo; Embryonic Development; Enzymes; Eukaryota; Family; Fasting; Fatty Liver; Fatty acid glycerol esters; Functional disorder; G-Protein-Coupled Receptors; GTP-Binding Proteins; Gene Expression; Genes; Genetic Polymorphism; Genetic Transcription; Genome; Growth; Growth Factor; Growth and Development function; Heart; Hepatic; Hepatocyte; Human; Hydrolysis; Immunity; In Vitro; Individual; Inflammation; Insulin Resistance; Ischemia; Laboratories; Laser Scanning Cytometry; Lead; Ligands; Lipase; Lipids; Liver; Mammals; Mediating; Membrane; Metabolic; Metabolic Diseases; Mitochondria; Modeling; Molecular; Molecular Genetics; Mus; Mutate; Mutation; Myocardial; Myocardial Ischemia; Natural Immunity; Neoplasm Metastasis; Neurotransmitters; Nucleosomes; Nutrient; Obesity; Organelles; Organism; Orthologous Gene; Outcome; Pathogenesis; Pathology; Phagocytosis; Physiological; Physiological Processes; Physiology; Play; Poison; Population; Positioning Attribute; Process; Protein Family; Proteins; Regulation; Relative (related person); Resources; Risk Factors; Role; SMARCA1 gene; Sequence Analysis; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Site; Source; Specificity; Stimulus; Surface; System; Systems Analysis; Techniques; Therapeutic; Tissues; Variant; Wild Type Mouse; Wound Healing; cell growth; chemokine; chemokine receptor; chromatin remodeling; deep sequencing; drug development; familial Alzheimer disease; fatty acid oxidation; feeding; flexibility; fungus; genetic regulatory protein; genome-wide; heart function; high throughput screening; human disease; improved; in vivo; inhibitor/antagonist; insight; interest; loss of function mutation; macrophage; miniaturize; morphogens; neutrophil; novel; perilipin; presenilin; receptor function; research and development; response; screening; small molecule; steroid hormone biosynthesis; tool

Nucleosome placement can direct transcription, but precise interactions are complex and largely unresolved at the whole genome level. Type III CHD proteins, a subfamily of SWI2 chromatin remodelers, are only present in metazoa and Dictyostelium, required for multicellular development, and mutated in several complex congenital human disorders (eg CHARGE syndrome). Type III CHDs mediate nucleosome translocation in vitro, but in vivo actions on chromatin organization and developmental regulation are not established. We investigated nucleosome re-positioning and gene expression in vivo, using deep sequence analyses of genome-wide chromatin structure and transcription profiles during growth and development of wild-type (WT) Dictyostelium and cells lacking ChdC, a Type III CHD. We demonstrate major WT chromatin reorganization of a gene subset enriched for differential expression during development. Loss of chdC has a very specific effect on chromatin structure relative to WT. Although altered nucleosomal organization in chdC-nulls is restricted to only 15% of the genome, it occurs in 50% of genes that are remodeled in WT. Association of altered chromatin remodeling and mis-regulated gene expression in chdC-nulls demonstrates the requirement for active nucleosomal re-positioning during multicellular development. Our data provide new mechanistic insight for related human diseases, showing in vivo function of Type III CHD chromatin remodeling proteins, for developmentally regulated nucleosome re-positioning, and gene expression. Chemotaxis directs embryogenesis, immunity, cell renewal, wound healing, and pathogenesis of cancer metastasis and chronic inflammation. Identification of novel small molecule probes of chemotaxis is important to dissect mechanisms and develop therapeutics. Current screens that identified compounds that inhibit chemotaxis have had poor activity, with few advanced to clinical trials. To identify novel chemotactic inhibitors, we developed a unique, unbiased assay for high-throughput screening using rapid, laser-scanning cytometry and Dictyostelium, a system with similar chemotactic mechanisms as mammalian migratory cells. We quantify chemotaxis in a miniaturized system using a GFP-marker only expressed in chemotaxing cells; a viability counter-screen is incorporated to eliminate toxic compounds. We screened 1,280 small molecule compounds in 1536-well formats and identified two that specifically inhibited GFP expression and chemotaxis. Both were confirmed to inhibit chemotaxis of Dictyostelium and human neutrophils in EZ-TAXIscan assays. This screening approach can rapidly identify potential new lead compounds for drug development and research tools universal to humans and other systems. Mutations in two presenilin genes in humans cause familial Alzheimers disease. Presenilins (PS) have both proteolytic-dependent and -independent. Analyzing the cellular role of presenilins in mammals has been complicated by embryonic lethality following deletion of both genes. We previously showed that PS has a role in the development of Dictyostelium. Here we show that development of PS-nulls can be rescued by expression of human PS1. We also show that aberrant development can be improved with PS proteins mutated at sites essential for proteolytic activity. Proteolytic- independent signaling by PS during Dictyostelium development is confirmed, as assayed by loss of substrate cleavage. Our data suggest an ancient non-proteolytic role for PS proteins in regulating intracellular signaling and development, and provide a novel model system for the analysis of human PS function. Excessive cellular triacylglyceride (TAG) storage within intracellular neutral lipid droplets is a well-known risk factor for many metabolic disorders, including insulin resistance, cardiovascular disease, and hepatic steatosis. Intracellular lipid droplets (LDs) are unique organelles that store metabolic precursors of cellular energy, membrane biosynthesis, steroid hormone synthesis, and signaling. LD surfaces are targeted by Perilipin (Plin) family proteins with specificity to different cells; Plin1 is the major adipocyte form, while Plin2 is the predominant form in liver. We studied Plin2 function in lipid storage in mice during induced hepatic steatosis. WT and plin2-/- mice were placed on chow or high fat diets or fasted overnight for each diet. WT mice that had been fasted overnight or placed on a high-fat diet accumulate abnormal levels of liver LDs that are primarily coated by Plin2. plin2-/- mice showed very significantly reduced hepatic lipid levels compared to WT under all conditions. LDs were isolated from livers of WT and plin2-/- mice and Plin protein compositions analyzed; the absence of Plin2 was compensated by the presence of Plin3 and Plin5 in the normal fed state, and by more dramatic increases in Plin5 during the fasted states or the high fat diet. Hepatic LDs of plin2-/- mice showed significantly increased recruitment of ATGL (lipase) and the ATGL regulatory protein CGI58 during conditions of induced hepatic steatosis as compared to WT. To better understand the protection of plin2-/- hepatocytes to steatosis, we studied primary aspects of TAG storage and hydrolyses in isolated primary hepatocytes from plin2-/- and WT mice. Lipolytic rates are significantly enhanced in plin2-/- primary hepatocytes, with significantly elevated levels of non-mitochondrial fatty acid oxidation and upregulated effects on hepatic gene expressions involved in fatty acid oxidation compared to WT. The increased levels of Plin3 and Plin5 in hepatocytes appear more permissive for access of LDs to lipolytic enzymes than are hepatic LDs primarily coated with Plin2. Lipid droplet proteins are also implicated in regulation of energy dynamics in the cell. Cells from oxidative tissue have high and fluctuating energy demands that require efficient coupling between energy storage in lipid droplets and utilization in mitochondria. Plin5 is highly expressed in heart and other oxidative tissues, and can stabilize lipid droplets and facilitate association between lipid droplets and mitochondria in cell culture. As myocardial lipids are stored in droplets that are closely associated with mitochondria, there is recent interest in Plin5 and its role for cardiac function. Indeed, increased expression of Plin5 in mouse leads to myocardial lipid accumulation. Studies in humans showed that a polymorphism in a noncoding region of PLIN5 is associated with decreased heart function after myocardial ischemia. Here, we investigated Plin5 and its role for cardiac dysfunction and outcome after myocardial ischemia. We show that deficiency in Plin5 in mice results in diminished metabolic flexibility of the heart and reduced heart function and outcome after myocardial ischemia, suggesting that Plin5 plays a role for cardioprotection during ischemia in the heart, and providing functional evidence that Plin5 is essential for cardiac physiology.  

核小体的放置可以指导转录，但精确的相互作用很复杂，并且在整个基因组水平上很大程度上尚未解决。 III 型 CHD 蛋白是 SWI2 染色质重塑蛋白的一个亚家族，仅存在于多细胞发育所需的后生动物和盘基网柄菌中，并在几种复杂的先天性人类疾病（例如 CHARGE 综合征）中发生突变。 III 型 CHD 在体外介导核小体易位，但在体内对染色质组织和发育调节的作用尚未确定。我们对野生型 (WT) 盘基网柄菌和缺乏 ChdC（一种 III 型 CHD）的细胞生长和发育过程中的全基因组染色质结构和转录谱进行深度序列分析，研究了体内核小体重新定位和基因表达。我们证明了在发育过程中富集差异表达的基因子集的主要 WT 染色质重组。相对于 WT，chdC 的丢失对染色质结构具有非常特殊的影响。尽管 chdC-null 中核小体组织的改变仅限于基因组的 15%，但它发生在 WT 中重构的 50% 的基因中。 chdC-null 中染色质重塑改变和基因表达失调的关联表明多细胞发育过程中需要主动核小体重新定位。我们的数据为相关人类疾病提供了新的机制见解，显示了 III 型 CHD 染色质重塑蛋白的体内功能，用于发育调节核小体重新定位和基因表达。 趋化性指导胚胎发生、免疫、细胞更新、伤口愈合以及癌症转移和慢性炎症的发病机制。鉴定新型趋化性小分子探针对于剖析机制和开发治疗方法非常重要。目前筛选出的抑制趋化性化合物的活性较差，很少进入临床试验。为了鉴定新型趋化抑制剂，我们开发了一种独特的、无偏倚的高通量筛选方法，使用快速激光扫描细胞术和盘基网柄菌（Dictyostelium），这是一种与哺乳动物迁移细胞具有相似趋化机制的系统。我们使用仅在趋化细胞中表达的 GFP 标记来量化小型化系统中的趋化性；结合了活性计数器屏幕以消除有毒化合物。我们在 1536 孔板中筛选了 1,280 种小分子化合物，并鉴定了两种特异性抑制 GFP 表达和趋化性的小分子化合物。在 EZ-TAXIscan 检测中，两者均被证实可抑制盘基网柄菌和人中性粒细胞的趋化性。这种筛选方法可以快速识别潜在的新先导化合物，用于人类和其他系统通用的药物开发和研究工具。 人类两个早老素基因的突变会导致家族性阿尔茨海默病。早老素（PS）具有蛋白水解依赖性和非蛋白水解依赖性。分析早老素在哺乳动物中的细胞作用因删除这两个基因后的胚胎致死性而变得复杂。我们之前表明 PS 在盘基网柄菌的发育中发挥作用。在这里，我们证明 PS-null 的发展可以通过表达人类 PS1 来挽救。我们还表明，通过在蛋白水解活性必需的位点突变 PS 蛋白可以改善异常发育。通过底物裂解损失的测定，证实了盘基网柄菌发育过程中 PS 的不依赖于蛋白水解的信号传导。我们的数据表明 PS 蛋白在调节细胞内信号传导和发育中具有古老的非蛋白水解作用，并为分析人类 PS 功能提供了一种新的模型系统。 细胞内中性脂滴内过多的细胞三酰甘油（TAG）储存是许多代谢紊乱的众所周知的危险因素，包括胰岛素抵抗、心血管疾病和肝脂肪变性。细胞内脂滴 (LD) 是一种独特的细胞器，可储存细胞能量、膜生物合成、类固醇激素合成和信号传导的代谢前体。 LD 表面被 Perilipin (Plin) 家族蛋白靶向，对不同细胞具有特异性； Plin1 是主要的脂肪细胞形式，而 Plin2 是肝脏中的主要形式。我们研究了诱导性肝脂肪变性期间小鼠脂质储存中 Plin2 的功能。 WT和plin2-/-小鼠被置于食物或高脂肪饮食中，或者每种饮食禁食过夜。禁食过夜或接受高脂肪饮食的 WT 小鼠会积累异常水平的肝脏 LD，这些 LD 主要由 Plin2 包被。在所有条件下，与 WT 小鼠相比，plin2-/- 小鼠的肝脂质水平均显着降低。从 WT 和 plin2-/- 小鼠的肝脏中分离 LD，并分析 Plin 蛋白组成；在正常进食状态下，Plin2 的缺失被 Plin3 和 Plin5 的存在所补偿，而在禁食状态或高脂肪饮食期间，Plin5 的增加则更加显着。与 WT 相比，plin2-/- 小鼠的肝脏 LD 在诱导性肝脂肪变性条件下显示 ATGL（脂肪酶）和 ATGL 调节蛋白 CGI58 的募集显着增加。为了更好地了解 plin2-/- 肝细胞对脂肪变性的保护作用，我们研究了来自 plin2-/- 和 WT 小鼠的分离原代肝细胞中 TAG 储存和水解的主要方面。与WT相比，plin2-/-原代肝细胞中的脂解率显着增强，非线粒体脂肪酸氧化水平显着升高，并且对涉及脂肪酸氧化的肝基因表达的影响上调。与主要包被有 Plin2 的肝 LD 相比，肝细胞中 Plin3 和 Plin5 水平的增加似乎更容易允许 LD 接触脂肪分解酶。 脂滴蛋白还参与细胞能量动态的调节。来自氧化组织的细胞具有高且波动的能量需求，需要脂滴中的能量存储和线粒体中的利用之间的有效耦合。 Plin5 在心脏和其他氧化组织中高表达，可以稳定脂滴并促进细胞培养中脂滴与线粒体之间的结合。由于心肌脂质储存在与线粒体密切相关的液滴中，最近人们对 Plin5 及其对心脏功能的作用产生了兴趣。事实上，小鼠中 Plin5 表达的增加会导致心肌脂质积累。对人类的研究表明，PLIN5 非编码区的多态性与心肌缺血后心脏功能下降有关。在这里，我们研究了 Plin5 及其对心肌缺血后心功能障碍和结局的作用。我们发现小鼠 Plin5 缺陷会导致心脏代谢灵活性降低，心肌缺血后心脏功能和结局降低，这表明 Plin5 在心脏缺血期间发挥心脏保护作用，并提供功能证据证明 Plin5 对于心肌缺血至关重要。生理。