SIGNALING PATHWAYS IN CONTROL OF GROWTH AND DEVELOPMENT

控制生长和发育的信号通路

• 批准号: 10259345
• 负责人: ALAN R KIMMEL
• 金额: $ 149.67万
• 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/10259345
• 关键词: Acute; Adhesions; Adipose tissue; Adrenal Cortex; Adrenal Glands; Adult; Affinity; Age; Agonist; Anabolism; Bacteria; Binding; Binding Proteins; Biochemical; Biological Assay; Biological Models; Brain Hypoxia-Ischemia; Carbon Dioxide; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cell Aggregation; Cell Communication; Cell Density; Cell physiology; Cells; Ceroid; Characteristics; Chemotactic Factors; Chemotaxis; Cholesterol; Cholesterol Esters; Complex; Corticosterone; Corticotropin; Culture Media; Cysteine; Cytoplasmic Tail; Data; Development; Developmental Process; Dictyostelium; Discrimination; Equilibrium; Eukaryota; Excision; Exhibits; Extracellular Protein; FRAP1 gene; Family; Family member; Fatty Acids; Fatty Liver; Food; Gene Expression Profile; Gene Expression Regulation; Generations; Genes; Genetic; Glucose; Glycogen; Growth; Growth and Development function; Hamartoma; Heart; Human; Hydrolysis; Hypoxia; Individual; Infarction; Insulin Resistance; Integral Membrane Protein; Ischemia; Knockout Mice; Length; Ligands; Lipase; Lipids; Lipolysis; Mammalian Cell; Mammals; Mediating; Membrane; Messenger RNA; Metabolic; Metabolic Diseases; Mitochondria; Modeling; Molecular Genetics; Monomeric GTP-Binding Proteins; Multiprotein Complexes; Mus; Muscle Fibers; Mutation; Myocardial; Natural Immunity; Nutrient; Nutrient Depletion; Oleic Acids; Organelles; Organism; Oxides; Pathway interactions; Phagocytes; Phagocytosis; Pharmaceutical Preparations; Pharmacology; Phenocopy; Phenotype; Phosphatidylglycerols; Phospholipids; Physiologic intraventricular pressure; Physiological; Play; Positioning Attribute; Proliferating; Protein Sequence Analysis; Proteins; RNA; RNA-Binding Proteins; Receptor Signaling; Regulation; Regulatory Pathway; Regulon; Risk Factors; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Sirolimus; Source; Specificity; Starvation; Structure; Surface; TIS11 protein; Techniques; Transcript; Transcription Process; Triglycerides; Weight; Withdrawal; Zinc Fingers; alpha-tocopherol transfer protein; amplification detection; cell growth; density; energy balance; experimental study; extracellular; fMet-Leu-Phe receptor; fatty acid oxidation; glycogen metabolism; human disease; improved; loss of function; mRNA Expression; macrophage; mitochondrial membrane; molecular modeling; mutant; non-oncogenic; novel; null mutation; overexpression; oxidation; perilipin; perilipin A; prevent; programs; response; reverse cholesterol transport; sensor; steroid hormone; tumorigenesis; uptake

Multi-protein complexes mTORC1 and mTORC2 are required for growth and development of eukaryotes. mTORC1 is a nutrient sensor that integrates metabolic signals and energy state to regulate cell growth/proliferation; mTORC2 primarily regulates developmental processes. Dictyostelium proliferate in rich growth media, but initiate development upon nutrient depletion. Both mTOR complexes play essential roles in Dictyostelium, where growth and developmental cycles independently require, respectively, mTORC1 or mTORC2. Many protein associations and regulatory pathways for mTORC1 and mTORC2 in Dictyostelium have context similarity to mammalian cells and specificity to inhibition by the immunosuppressive drug rapamycin. In Dictyostelium, mTORC1 function is inactivated upon starvation-induced development, but development is directly induced through rapamycin-mediated inhibition of mTORC1 activity, even in the absence of nutrient withdrawal. Pharmacologic inhibition of mTORC1, in the absence of nutrient loss, has allowed the identification of a class of essential up-regulated, developmentally-associated signaling genes and down-regulated, growth genes. We showed functional pathway regulations that integrate mTORC1/mTORC2 activities and emphasize complexity of small GTPase regulation of mTORC2 activity. Finally, epistases experiments indicate novel upstream pathway cross-talk in Dictyostelium that requires mTORC1 and mTORC2, but for separate and independent downstream functions. Cellular functions can be regulated by cell-cell interactions and influenced by extra-cellular, density-dependent signaling factors. Dictyostelium grow as individual cells in nutrient-rich sources. As nutrients become depleted, they initiate a multi-cell developmental program dependent upon a cell-density threshold. We hypothesized that novel secreted proteins may serve as density-sensing factors to promote multi-cell developmental fate decisions at a specific cell-density threshold, and use Dictyostelium in the identification of such a factor. We show that multi-cell developmental aggregation in Dictyostelium is lost upon minimal (2-fold) reduction in local cell density. Remarkably, developmental aggregation response at non-permissive cell densities is rescued by addition of conditioned media from high-density, developmentally competent cells. Using rescued aggregation of low-density cells as an assay, we purified a single, 150-kDa extra-cellular protein with density aggregation activity. MS/MS peptide sequence analysis identified the gene sequence, and cells that overexpress the full-length protein accumulate higher levels of a development promoting factor (DPF) activity than parental cells, allowing cells to aggregate at lower cell densities; cells deficient for this DPF gene lack density-dependent developmental aggregation activity and require higher cell density for cell aggregation compared to WT. Density aggregation activity co-purifies with tagged versions of DPF and tag-affinity-purified DPF possesses density aggregation activity. In mixed development with WT, cells that overexpress DPF preferentially localize at centers for multi-cell aggregation and define cell-fate choice during cytodifferentiation. Finally, we show that DPF is synthesized as a larger precursor, single-pass transmembrane protein, with the p150 fragment released by proteolytic cleavage and ectodomain shedding. The TM/cytoplasmic domain of DPF possesses cell-autonomous activity for cell-substratum adhesion and for cellular growth. Post-transcriptional processes mediated by mRNA binding proteins represent important control points in the regulation of gene expression. In mammals, mRNAs containing specific AU-rich motifs are regulated by proteins of the tristetraprolin (TTP) family, which bind to these motifs through a tandem zinc finger (TZF) domain. This binding leads to promotion of subsequent deadenylation and decay, partly through a conserved carboxyl-terminal CNOT1 binding domain. Four TTP family members are expressed in mice, and possible functional redundancy makes structure-function explorations difficult. We explored the physiological role of the single TTP family member (ttpA) expressed in Dictoystelium discoideum. Two null mutants exhibited slow growth and a multinuclear phenotype. A cysteine point mutant within the TZF domain, which should completely prevent mRNA binding, phenocopied the two null mutations exactly in terms of gene expression patterns. The carboxyl-terminal mutation, which removed the predicted CNOT1 binding domain, exhibited a phenotype indistinguishable from that of the null mutants, raising the possibility that this domain is primarily responsible for interactions with the CCR4/NOT mRNA decay apparatus in this species. Three transcripts which are highly upregulated in the loss-of-function mutants, are co-regulated during differentiation and possess the conserved AU-target motif and. These data suggest that ttpA controls an RNA "regulon", in which a single RNA binding protein co-regulates the expression of a gene group. Most cells store triacylglycerol (TAG) in cytoplasmic lipid droplets (LDs), but steroidogenic cells store primarily cholesteryl esters (CEs). The LD binding protein Plin2 regulates degradation of LDs containing TAG. To determine if Plin2 is also regulates CEs, we characterized adrenals of Plin2 null mice. We observed significantly enlarged adrenal glands, increased size and numbers of CE-LD, elevated cellular free cholesterol levels, and increased expression of macrophage markers and genes facilitating reverse cholesterol transport in adrenals of Plin2-/- mice as compared to Plin2+/+ mice. Mobilization of CE-LDs and secretion of corticosterone induced by ACTH stimulation or starvation were unaffected by the lack of Plin2 in young mice. There was also accumulation of ceroid-like structures containing multilamellar bodies in the adrenal cortex-medulla boundary accompanied with high levels of adrenal phosphatidylglycerols, implying that these lipids are components of the accumulated ceroid-like structures. Our findings indicate that Plin2 is important for regulation of CE-LDs and cellular cholesterol balance in adrenal cortical cells. Perilipin 5 (Plin5) is abundantly expressed in heart where it binds to cardiac lipid droplets (LDs) and facilitates physical interaction between LDs and mitochondria. We isolated cardiomyocytes from adult Plin5+/+ and Plin5-/- mice to study the role of Plin5 for tolerance to hypoxia and ischemia, fatty acid uptake, LD accumulation, and fatty acid oxidation. Cardiomyocytes from Plin5-/- mice stored less LDs than Plin5+/+ following oleic acid stimulation, but comparable levels to Plin5+/+ cardiomyocytes when adipose triglyceride lipase activity was inhibited. The ability to oxidize fatty acids into CO2 was similar between Plin5+/+ and Plin5-/- cardiomyocytes, but Plin5-/- cardiomyocytes had a transient increase in intracellular fatty acid oxidation intermediates. After pre-incubation with oleic acids, Plin5-/- cardiomyocytes retained a higher content of glycogen and showed improved tolerance to hypoxia. Deletion of Plin5 had no important effect on ventricular pressures or infarct size after ischemia in isolated, perfused hearts. Old Plin5-/- mice had reduced levels of cardiac triacylglycerides, increased heart weight and elevated Myh7 and Cd36 mRNA expression compared to Plin5+/+. Still, most other genes involved in fatty acid oxidation, glycogen metabolism and glucose utilization were essentially unchanged by age or removal of Plin5. Plin5 seems to facilitate cardiac LD storage primarily by repressing adipose triglyceride lipase activity without altering cardiac fatty acid oxidation capacity. Expression of Plin5 and cardiac LD content does not appear to be of vital importance for tolerance to acute hypoxia and ischemia.

多蛋白质复合物mTORC1和MTORC2是真核生物的生长和发展所必需的。 MTORC1是一种营养传感器，它整合了代谢信号和能量状态以调节细胞生长/增殖。 MTORC2主要调节发展过程。在丰富的生长培养基中，dictyostelium繁殖，但会在营养耗尽时启动发展。这两种MTOR络合物在dictyostelium中都起着重要作用，在Dictyostelium中，生长和发育周期分别独立地要求MTORC1或MTORC2。 MTORC1和MTORC2在Dictyostelium中的MTORC1和MTORC2的许多蛋白质关联和调节途径与哺乳动物细胞具有相似性，并且具有免疫抑制药物雷帕霉素抑制的特异性。在Dictyostelium中，MTORC1的功能在饥饿诱导的发育后被灭活，但是即使没有营养戒断，雷帕霉素介导的MTORC1活性的抑制也会直接诱导发育。在没有养分损失的情况下，MTORC1的药理抑制已允许鉴定一类必需的上调，发育相关的信号基因和下调的生长基因。我们展示了整合MTORC1/MTORC2活性并强调MTORC2活性的小GTPase调节的复杂性的功能途径法规。最后，Epistases实验表明需要MTORC1和MTORC2的dictyostelium中的新型上游途径串扰，但用于独立且独立的下游函数。 细胞功能可以受细胞 - 细胞相互作用的调节，并受细胞外密度依赖的信号传导因子的影响。 dictyostelium在富含营养的来源中随着单个细胞而生长。随着养分的耗尽，它们启动了一个多细胞发育程序，取决于细胞密度阈值。我们假设新型分泌的蛋白质可以用作密度感应因子，以在特定的细胞密度阈值下促进多细胞发育命运决策，并在鉴定这种因素时使用dictyostelium。我们表明，在局部细胞密度最小（2倍）降低时，DIDYOSTELIUM中的多细胞发育聚集损失。值得注意的是，通过添加高密度，发育能力的细胞来挽救非腐败细胞密度的发育聚集反应。我们使用低密度细胞作为测定的聚集，我们纯化了具有密度聚集活性的单个150 kDa细胞外蛋白。 MS/MS肽序列分析确定了基因序列，而过表达全长蛋白的细胞比亲本细胞积累了更高水平的发育促进因子（DPF）活性，从而使细胞可以在较低的细胞密度下聚集。与WT相比，缺乏该DPF基因缺乏该DPF基因的细胞缺乏密度依赖性的发育聚集活性，并且需要更高的细胞聚集。密度聚集活性与标记版本的DPF和TAG亲和力纯化的DPF共同绘制具有密度聚集活性。在与WT的混合发育中，过表达DPF的细胞优先定位在中心，以用于多细胞聚集并定义细胞分化过程中的细胞污染选择。最后，我们表明DPF被合成为较大的前体单跨跨膜蛋白，其中P150片段由蛋白水解裂解和胞外域脱落释放。 DPF的TM/细胞质结构域具有细胞自主活性，可用于细胞肌间粘附和细胞生长。 由mRNA结合蛋白介导的转录后过程代表基因表达调节中的重要控制点。在哺乳动物中，含有特定AU依赖基序的mRNA受三翼蛋白（TTP）家族的蛋白质调节，该蛋白通过串联锌指（TZF）结构域与这些基序结合。这种结合导致促进随后的去苯和衰减，部分地通过保守的羧基末端CNOT1结合结构域。在小鼠中表达了四个TTP家族成员，可能的功能冗余使结构功能探索变得困难。我们探讨了在迪斯托型迪斯特型中表达的单个TTP家族成员（TTPA）的生理作用。两个无效突变体表现出缓慢的生长和多核表型。 TZF结构域内的半胱氨酸点突变体应完全防止mRNA结合，这是根据基因表达模式精确的两个无效突变。去除预测的CNOT1结合结构域的羧基末端突变表现出与零突变体的表型没有区别，这增加了该域主要导致该物种中该域与CCR4/NOT NOT MRNA衰减相互作用的可能性。在功能丧失突变体中高度上调的三个转录本在分化过程中是共同调节的，并具有保守的Au-Target基序。这些数据表明TTPA控制了RNA“调节量”，其中单个RNA结合蛋白共同调节基因组的表达。 大多数细胞将三酰基甘油（TAG）储存在细胞质脂质液滴（LDS）中，但类固醇生成细胞主要存储主要是胆固醇酯（CES）。 LD结合蛋白PLIN2调节包含TAG的LD的降解。为了确定PLIN2是否也是调节的CE，我们表征了PLIN2 NULL小鼠的肾上腺。我们观察到与Plin2+/+小鼠相比，我们观察到显着肿大的肾上腺，CE-LELD的增加和数量增加，CE-LED升高，细胞不含胆固醇水平升高，巨噬细胞标记物和基因的表达增加，促进PLIN2-/ - 小鼠肾上腺中反向胆固醇的转运。 CE-LDS的动员和由ACTH刺激或饥饿诱导的皮质酮的分泌不受年轻小鼠缺乏PLIN2的影响。在肾上腺皮质 - 梅德拉边界中还积累了含有多层体的类凝胶状结构，并伴有高水平的肾上腺磷脂酰甘油，这意味着这些脂质是累积的类似硅烷类似结构的成分。我们的发现表明，PLIN2对于调节肾上腺皮质细胞中CE-LDS和细胞胆固醇平衡很重要。 Perilipin 5（PLIN5）在心脏中大量表达，它与心脏脂质液滴（LDS）结合，并促进LDS与线粒体之间的物理相互作用。我们从成年PLIN5+/+和PLIN5 - / - 小鼠中分离出心肌细胞，以研究PLIN5对低氧和缺血，脂肪酸摄取，LD的积累和脂肪酸氧化的耐受性的作用。在油酸刺激后，来自PLIN5-/ - 小鼠的心肌细胞比PLIN5+/+少的LDS较少，但是当抑制脂肪甘油三酸酯脂肪酶活性时，与PLIN5+/+心肌细胞相当的水平与PLIN5+/+心肌细胞相当。在PLIN5+/+和PLIN5 - / - 心肌细胞之间，将脂肪酸氧化为CO2的能力相似，但是PLIN5 - / - 心肌细胞的细胞内脂肪酸氧化中间体的瞬时增加。在与油酸预孵育后，PLIN5 - / - 心肌细胞保留了更高的糖原含量，并显示出对缺氧的耐受性的提高。 PLIN5的缺失对孤立的，灌注心脏的缺血后对心室压力或梗死大小没有重要影响。与PLIN5+/+相比，旧的PLIN5 - / - 小鼠的心脏三酰基甘油三酸酯水平降低，心脏重量增加，MYH7和CD36 mRNA表达升高。尽管如此，大多数其他参与脂肪酸氧化，糖原代谢和葡萄糖利用的基因基本上是通过年龄或去除PLIN5的变化。 PLIN5似乎主要是通过抑制脂肪甘油三酸酯脂肪酶活性而不改变心脏脂肪酸氧化能力来促进心脏LD的储存。 PLIN5和心脏LD含量的表达对于对急性低氧和缺血的耐受性似乎并不重要。