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.  

核小体的放置可以直接转录，但是精确的相互作用是复杂的，并且在整个基因组水平上都无法解决。 III型CHD蛋白是SWI2染色质重塑剂的亚家族，仅存在于后生动物和Dictyostelium中，是多细胞发育所需的，并在几种复杂的先天性人体疾病（例如电荷综合征）中突变。 III型CHD在体外介导核小体易位，但未建立对染色质组织和发育调控的体内作用。我们使用野生型（WT）dictyostelium的生长和发育期间对体内的核小体重新定位和体内基因表达进行了深入的序列分析，而缺乏CHDC的细胞（III型CHD）。我们证明了在发育过程中富含差异表达的基因亚群的主要WT染色质重组。相对于WT，CHDC的损失对染色质结构具有非常特异性的影响。尽管CHDC-NULLS中的核小体组织改变的基因组仅限15％，但它发生在WT中重塑的50％的基因中。 CHDC无效中染色质重塑和错误调节的基因表达的缔合表明了多细胞发育过程中主动核小体重位的要求。我们的数据为相关人类疾病提供了新的机械洞察力，显示了III型CHD染色质重塑蛋白的体内功能，用于发育调节的核小体重新定位和基因表达。 趋化性指导癌症转移和慢性炎症的胚胎发生，免疫，细胞更新，伤口愈合以及发病机理。新型小分子探针的鉴定对于剖析机制和发展治疗剂很重要。鉴定抑制趋化性的化合物的当前筛选活动的活性较差，而临床试验则很少。为了鉴定新型的趋化性抑制剂，我们使用快速的激光扫描细胞仪和Dictyostelium开发了一种独特的，公正的测定，用于高通量筛查，这是一种具有与哺乳动物候选细胞相似的趋化机制的系统。我们使用仅在趋化细胞中表达的GFP标记物在微型化系统中定量趋化性。纳入了生存反屏幕以消除有毒化合物。我们以1536孔格式筛选了1,280个小分子化合物，并确定了两种特别抑制GFP表达和趋化性的小分子化合物。两者都被证实在EZ-Taxiscan分析中抑制柱状骨的趋化性和人类嗜中性粒细胞。这种筛查方法可以迅速识别出潜在的药物开发新铅化合物，并与人类和其他系统通用有关的研究工具。 人类两个老年蛋白基因的突变引起家族性阿尔茨海默氏病。 presenilins（PS）既有蛋白水解依赖性和非依赖性。在两个基因删除后，胚胎致死性使分析哺乳动物在哺乳动物中的细胞作用变得复杂。我们先前表明，PS在Dictyostelium的发展中起作用。在这里，我们表明，可以通过人类PS1的表达来挽救PS-NULL的发展。我们还表明，通过对蛋白水解活性必不可少的位点突变的PS蛋白可以改善异常发育。通过底物裂解的损失，证实了PS在DICTyostelium发育过程中PS的蛋白水解独立信号传导。我们的数据表明，PS蛋白在调节细胞内信号传导和发育中的古老非蛋白质作用，并为人类PS功能分析提供了一种新颖的模型系统。 细胞内中性脂质液滴中过量的细胞三酰基甘油三酸酯（TAG）储存是许多代谢疾病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。细胞内脂质液滴（LDS）是储存细胞能量的代谢前体，膜生物合成，类固醇激素合成和信号传导的独特细胞器。 LD表面是由Perilipin（PLIN）家族蛋白具有特异性的Peripin（PLIN）家族蛋白的靶向。 PLIN1是主要的脂肪细胞形式，而PLIN2是肝脏中的主要形式。在诱导的肝脂肪变性期间，我们研究了小鼠脂质存储中的PLIN2功能。将WT和PLIN2 - / - 小鼠放在食用或高脂肪饮食上或为每种饮食禁食过夜。已禁食过夜或放置在高脂饮食上的WT小鼠积累了主要由PLIN2覆盖的肝脏LDS。在所有条件下，与WT相比，PLIN2 - / - 小鼠显示出肝脂质水平的显着降低。从WT和PLIN2 - / - 小鼠的肝脏中分离出LDS，并分析了PLIN蛋白组成；通过在正常的喂养状态下PLIN3和PLIN5的存在以及在禁食状态或高脂肪饮食中PLIN5的增加来补偿PLIN2的不存在。与WT相比，在诱导的肝脂肪变性的条件下，PLIN2 - / - 小鼠的肝LDS显示ATGL（脂肪酶）和ATGL调节蛋白CGI58的募集显着增加。为了更好地理解PLIN2 - / - 肝细胞对脂肪变性的保护，我们研究了TAG存储的主要方面和来自PLIN2 - / - 和WT小鼠的孤立的原代肝细胞中的水解细胞的主要方面。在PLIN2 - / - 原发性肝细胞中，脂肪性速率显着提高，与WT相比，非细胞软骨脂肪酸氧化水平显着升高，对参与脂肪酸氧化涉及的肝基因表达的影响上调。肝细胞中PLIN3和PLIN5的水平升高似乎比主要涂有PLIN2涂层的肝LDS更允许LDS进入脂肪溶酶。 脂质液滴蛋白也与细胞中能量动力学的调节有关。来自氧化组织的细胞具有高且波动的能量需求，需要在脂质液滴中的能量存储与线粒体中的利用率之间有效耦合。 PLIN5在心脏和其他氧化组织中高度表达，可以稳定脂质液滴并促进细胞培养中脂质滴与线粒体之间的关联。由于心肌脂质存储在与线粒体密切相关的液滴中，因此最近对PLIN5的兴趣及其对心脏功能的作用。实际上，PLIN5在小鼠中的表达增加导致心肌脂质积累。在人类中的研究表明，在心肌缺血后心脏功能降低有关，PLIN5的非编码区域的多态性与降低有关。在这里，我们研究了PLIN5及其在心肌缺血后心脏功能障碍和结果中的作用。我们表明，小鼠PLIN5缺乏会导致心脏的代谢柔韧性降低，心肌缺血后心脏功能和预后降低，这表明PLIN5在心脏缺血期间对心脏保护作用起作用，并提供功能性证据表明PLIN5对于心脏物理学是必不可少的。