SIGNALING PATHWAYS IN CONTROL OF GROWTH AND DEVELOPMENT

控制生长和发育的信号通路

• 批准号: 10919481
• 负责人: ALAN R KIMMEL
• 金额: $ 155.05万
• 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/10919481
• 关键词: Adenylate Cyclase; Adhesions; Adipose tissue; Affect; Affinity; Amino Acids; Anabolism; Binding; Binding Proteins; Biochemical; Body Weight; Calcium; Calpain; Cardiovascular Diseases; Catalogs; Cell Communication; Cell Density; Cell Survival; Cell physiology; Cells; Characteristics; Chemotactic Factors; Chemotaxis; Cholesterol; Chromatin; Circulation; Citric Acid Cycle; Communication; Complex; Coupled; Cues; Cyclic AMP; Cytoplasm; Cytoplasmic Tail; Data; Derivation procedure; Development; Dictyostelium; Dioxygenases; Ensure; Epigenetic Process; Epitopes; Eukaryota; Eukaryotic Cell; Family; Fasting; Fatty Liver; Feedback; Folic Acid; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Proteins; Gene Expression; Gene Silencing; Genes; Genetic; Genetic Transcription; Genomics; Genotype; Glucose; Glycolysis; Growth; Growth and Development function; Hepatic; Heterodimerization; High Fat Diet; Homeostasis; Homo; Hydrolysis; Image; Individual; Insulin Resistance; Integral Membrane Protein; Kinetics; Life Cycle Stages; Ligands; Link; Lipid Mobilization; Lipids; Liver; Mammals; Maps; Membrane; Messenger RNA; Metabolic; Metabolic Diseases; Metabolic Pathway; Minor; Modeling; Molecular; Mouse Strains; Movement; Mus; Natural Immunity; Nonesterified Fatty Acids; Nutrient; Nutrient Depletion; Obesity; Organelles; Organism; Pathway interactions; Pattern; Pentosephosphate Pathway; Peptide Hydrolases; Periodicity; Permeability; Phagocytes; Phosphotransferases; Play; Population; Process; Prokaryotic Cells; Property; Protein Family; Protein Secretion; Proteins; RNA Splicing; Receptor Signaling; Regulation; Research; Risk Factors; Role; Signal Pathway; Signal Transduction; Sirolimus; Starvation; Succinates; Surface; System; Territoriality; Testing; Time; Tissues; Totipotent cell; Transcription Process; Transmembrane Domain; United States National Institutes of Health; Variant; Withdrawal; alpha ketoglutarate; cell growth; cell motility; chromatin modification; cofactor; density; desensitization; diet-induced obesity; directional cell; embryonic stem cell; experimental study; glucose tolerance; human disease; interest; mammalian genome; member; metabolic abnormality assessment; metabolic phenotype; metabolomics; molecular modeling; mouse model; novel; perilipin; perilipin A; phosphoric diester hydrolase; pluripotency; posttranscriptional; preference; receptor; recruit; response; self organization; steroid hormone; trafficking; transcriptome; transcriptome sequencing; vector

Modeling cAMP Oscilations - Self-organized and excitable signaling activities play important roles in a wide range of cellular functions in eukaryotic and prokaryotic cells. Cells require signaling networks to communicate amongst themselves, but also for response to environmental cues. Such signals involve complex spatial and temporal loops that may propagate as oscillations or waves. When Dictyostelium are starved for nutrients, cells within a territorial space secrete cAMP. Proximal cells move inward toward cAMP and relay the cAMP outward to recruit additional cells. To ensure directed inward movement, cells go through adapted and de-adapted states, for cAMP synthesis/degradation and directional cell movement, that oscillate at 6 min intervals. Developmental cAMP oscillations are characterized by a rise in cAMP synthesis and accumulation, followed by cessation of cAMP synthesis and increased cAMP degradation, with the cycle repeating with a defined temporal periodicity. Although many immediate components that regulate cAMP signaling (including receptors, G proteins, adenyl cyclase, phosphodiesterases, and kinases) are known, others are only inferred. Using biochemical experiments coupled with gene inactivation studies, we have identified new component members and model an integrated large (>25), multi-component kinetic pathway involving activation, inactivation (adaptation), re-activation (re-sensitization), feed-forward, and feed-back controls to generate developmental cAMP oscillations. Metabolomics - Changes in nutrients affect diverse cellular networks, making it challenging to distinguish metabolic paths that regulate growth from a switch to development. The life cycle of Dictyostelium is an excellent model to study metabolic signatures. Dictyostelium grow as single cells in nutrient-rich media, but, with nutrient withdrawal, growth ceases and cells enter multi-cell development. We developed conditions for rapid cell growth in rich-media, but where rapamycin-targeted inactivation of mTORC1 leads to a growth-to-development fate switch. We have shown that nutrient (glucose, amino acids) withdrawal significantly reduces many intermediates within most metabolic pathways, thus, negatively impacting glycolysis, the TCA cycle, pentose-phosphate shunt, etc. Rapamycin-induced development in the absence of nutrient withdrawal is expected to have a more limited influence on metabolic pathways. As part of the trans-NIH Metabolomics Consortium, we have undertaken time-course analyses of metabolomic changes in response to starvation- and rapamycin-induced development, to identify metabolic changes that are associated with a growth-to-development transition, but that are independent of nutrient depletion. We wish to identify metabolic changes that result form development (e.g. autophagic products), but also regulatory metabolites that may promote (e.g. AMP) or inhibit development. Initial results indicate that >5000 metabolite concentration differences are seen between starved-developed and growing cells, whereas <500 differ between growth and rapamycin-induced development in the absence of nutrient withdrawal. We anticipate identifying a defined catalog of metabolites whose concentrations are highly varied during development, independent of nutrient withdrawal. Some may function as epigenetic regulators of cell-fate change. As an example, a-ketoglutarate is a co-factor for dioxygenases that suppress repressive chromatin modifications with impact to transcription and a-ketoglutarate/succinate TCA component ratio differences can promote or suppress ES cell pluripotency. Our preliminary data in Dictyostelium indicate a >4-fold decrease in relative a ketoglutarate to succinate levels as development proceeds. We have further demonstrated that methyl-derivates of a ketoglutarate, for enhanced cell permeability, completely block developmental induction, even under starved conditions, without an effect on cell viability and growth. Data suggest that a ketoglutarate concentration is the primary effector, as developmental inhibition cannot be reversed with increased levels of succinate moieties. We will look for changes in the transcriptome and in chromatin organization that are specific to a-ketoglutarate treatment. Comparison of RNAseq data from cells starved in the presence or absence of exogenous a-ketoglutarate may further discriminate transcriptional changes closely associated with development from those only responsive to nutrient withdrawal. Growth-to-Development Developmental aggregation in Dictyostelium is lost at low cell density, but aggregation at non-permissive cell densities is rescued with secreted factor DPF1. Secreted DPF1 is synthesized as a larger precursor, single-pass transmembrane protein that is released by proteolytic cleavage and ectodomain shedding, leaving a 10 kDa transmembrane (TM) fragment. The TM/cytoplasmic domain of DPF1 possesses independent, cell autonomous activity for cell-substratum adhesion and cellular growth. We have created vectors that solely express the secreted or TM/cytoplasmic forms to understand the different functions. We have also identified a new gene DPF2, which is closely linked to DPF1, that encodes a sequence related protein with similar processing and ectodomain shedding properties. Ectodomain cleavage of both DPF1 and DPF2 is largely dependent upon calcium and calcium-dependent proteases (calpains). Secreted p150 kDa fragments of DPF1 and DPF2 have been purified in mg quantities and are being analyzed by MS/MS to map the specific cleavage sequences. We hypothesize there is pathway interaction between DPF1 and DPF2 and are testing this directly in mixing experiments with differentially epitope-tagged versions of DPF1 and DPF2. We have shown homo- and hetero-dimerization of the transmembrane domains of both DPF1 and DPF2. Lipid Storage during Fasting- Excessive cellular lipid storage can be a risk factor for metabolic disorders, including insulin resistance, cardiovascular disease, and hepatic steatosis. Intracellular lipid droplets are unique organelles that store metabolic precursors of cellular energy, membrane biosynthesis, steroid hormone synthesis, and signaling. The perilipins are a multi-protein family that targets lipid droplet surfaces and regulates lipid storage and hydrolysis. Plin2 binds hepatic LDs with expression levels that correlate with TAG content. We investigated the role of Plin2 in hepatic LD storage in fed and fasted plin2+/+ and plin2-/- mice. Chow-fed plin2-/- mice had comparable body weights, metabolic phenotype, glucose tolerance, and circulating TAG and total cholesterol levels to WT. Overnight fasting stimulates the degradation of stored adipose TAGs, with release of non-esterified fatty acids for circulation. Fasted plin2-/- mice showed substantially reduced accumulation of hepatic TAG compared to fasted WT. RNAseq revealed minor differences in hepatic gene expression between fed plin2+/+ and plin2-/- mice but marked differences in expression between fasted plin2+/+ and plin2-/- mice. Plin2 regulates hepatic lipid droplet size and accumulation of neutral lipid species in the fasted state. Hepatosteatosis - Recent studies describe transcriptome changes associated with hepatosteatosis, but it has been difficult to separate the effects on hepatic gene expression of fatty liver from that of obesity. We studied a plin2-/- mouse model, under conditions that are highly protective to hepatostaetosis, but not diet-induced obesity. We determined the mechanistic functions that protect plin2-/- livers from lipid accumulation and using RNAseq, compared hepatic transcriptomes of chow-fed or high-fat diet plin2+/+ and plin2 /- mice. We show that the Plin2 genotype, and accordingly hepatosteatosis, has a more limited impact on hepatic gene expression than does diet-induced obesity.

建模营振荡 - 自组织和激发信号传导活动在真核和原核细胞中广泛的细胞功能中起着重要作用。单元需要信号网络才能彼此交流，也需要对环境提示进行响应。这些信号涉及复杂的空间和颞循环，可能会传播为振荡或波浪。当dictyostelium饿死了营养素时，领土太空中的细胞会分泌营地。近端细胞向内移动向营地，并向外转移营地以募集其他细胞。为了确保向内运动，细胞经过适应和脱位的状态，用于cAMP合成/降解和定向细胞运动，并以6分钟的间隔振荡。发展营地的振荡的特征是cAMP合成和积累的增加，随后停止cAMP合成并增加营地退化，周期重复并定义为时间周期性。尽管已知许多调节CAMP信号传导（包括受体，G蛋白，腺基环酶，磷酸二酯酶和激酶的立即成分），但仅推断出其他。使用与基因失活研究结合的生化实验，我们已经确定了新的成分成员，并建模了涉及激活，灭活（适应），重新激活（重新激活），进料和进料控制的多组分动力学途径的集成（> 25），多组分动力学途径。 代谢组学 - 养分的变化会影响各种细胞网络，从而使区分从转换转向开发的代谢路径区分新陈代谢路径。 Dictyostelium的生命周期是研究代谢特征的出色模型。在富含营养的培养基中，DICTyostelium作为单个细胞生长，但是，随着营养的戒断，生长停止和细胞进入多细胞发育。我们开发了富裕媒介中快速细胞生长的条件，但是在雷帕霉素靶向MTORC1的失活导致生长到开发的命运转换中。我们已经表明，营养（葡萄糖，氨基酸）的戒断显着降低了大多数代谢途径中的许多中间体，因此，对雷蛋白含量促进营养的缺乏，对雷蛋白诱导的雷霉素诱导的发育有望受到更大的影响。作为跨NIH代谢组学联盟的一部分，我们对饥饿和雷帕霉素诱导的发育的响应进行了时间课程分析，以识别与生长到开发过渡相关的代谢变化，但与营养消耗无关。我们希望确定形成形成发展的代谢变化（例如自噬产品），以及可能促进（例如AMP）或抑制发育的调节代谢产物。最初的结果表明，饥饿的细胞和生长细胞之间看到了> 5000个代谢产物浓度差异，而在没有营养的戒断中，生长和雷帕霉素诱导的发育之间的<500差异。我们预计，鉴定出明确的代谢产物目录，其浓度在发育过程中高度变化，而与营养戒断无关。有些可能是细胞效果变化的表观遗传调节剂。例如，A-酮戊二酸是二加氧酶的共同因素，可抑制抑制染色质的修饰，对转录和A-酮酸盐/琥珀酸酯/琥珀酸酯/琥珀酸酯/琥珀酸盐TCA成分比差异差异差异可以促进或抑制ES ES细胞多能力。我们在Dictyostelium中的初步数据表明，随着发育的进行，相对A酮戊二酸到琥珀酸盐水平的相对降低了> 4倍。我们进一步证明，酮谷甲酸酯的甲基衍生物，以增强细胞的渗透性，即使在饥饿的条件下，也完全阻止了发育诱导，对细胞活力和生长没有影响。数据表明，酮戊二酸浓度是主要效应子，因为琥珀酸酯部分水平不断增加发育抑制作用。我们将寻找特定于A-酮戊二酸治疗的转录组和染色质组织中的变化。在存在或不存在外源性A-酮漏斗的情况下饥饿的细胞中的RNASEQ数据比较可能会进一步区分与发育的转录变化与仅响应营养戒断的人的发育密切相关的转录变化。 Dictyostelium中的生长对开发的发育聚集在低细胞密度下丢失，但是通过分泌的因子DPF1挽救了非允许细胞密度的聚集。分泌的DPF1被合成为较大的前体，单次跨膜蛋白，该蛋白是由蛋白水解裂解和外生域脱落而释放的，留下了10 kDa跨膜（TM）片段。 DPF1的TM/细胞质结构域具有独立的细胞自主活性，可用于细胞 - 肌间粘附和细胞生长。我们创建了仅表达分泌或TM/细胞质形式的向量来理解不同的功能。我们还确定了与DPF1紧密相关的新基因DPF2，该基因与序列相关的蛋白质具有相似的加工和外胞状态分裂特性。 DPF1和DPF2的外生域切割在很大程度上取决于钙和钙依赖性蛋白酶（CALPAINS）。 DPF1和DPF2的分泌P150 kDa片段已被纯化，并通过MS/MS分析以绘制特定的切割序列。我们假设DPF1和DPF2之间存在途径相互作用，并在与DPF1和DPF2的差异表位标签版本的混合实验中直接测试了这一点。我们已经显示了DPF1和DPF2的跨膜结构域的同型和异二。 禁食期间的脂质储存 - 过多的细胞脂质储存可能是代谢疾病的危险因素，包括胰岛素抵抗，心血管疾病和肝脂肪变性。细胞内脂质液滴是独特的细胞器，可存储细胞能，膜生物合成，类固醇激素合成和信号传导的代谢前体。围Perilipins是一种靶向脂质液滴表面并调节脂质储存和水解的多蛋白质家族。 PLIN2结合具有与TAG含量相关的表达水平的肝LD。我们研究了PLIN2在FED和禁食的PLIN2+/+和PLIN2 - / - 小鼠中的肝LD存储中的作用。食喂的PLIN2 - / - 小鼠具有可比的体重，代谢表型，葡萄糖耐受性和循环标签，总胆固醇水平为WT。隔夜禁食刺激储存的脂肪标签的降解，并释放了非层化脂肪酸进行循环。与禁食的WT相比，禁食的PLIN2 - / - 小鼠显示出肝标签的积累大幅减少。 RNASEQ揭示了Fed Plin2+/+和Plin2 - / - 小鼠之间肝基因表达的差异，但在禁食的PLIN2+/+和PLIN2 - / - 小鼠之间表达明显差异。 PLIN2调节禁食状态下中性脂质物种的肝脂质液滴的大小和积累。 Hepatosteatosis-最近的研究描述了与肝脏的转录组变化，但是很难将对脂肪肝的肝基因表达的影响与肥胖症分离。我们研究了一个对肝静脉病具有高度保护的条件，但没有饮食引起的肥胖症。我们确定了保护plin2 - / - 肝脏免受脂质积累和使用RNASEQ的机械函数，比较了食物喂养或高脂饮食PLIN2+/+和PLIN2/ - 小鼠的肝转录组。我们表明，与饮食诱导的肥胖相比，PLIN2基因型及其对肝基因表达的影响更有限。

项目成果

Oscillatory signaling and network responses during the development of Dictyostelium discoideum.

盘基网柄菌发育过程中的振荡信号和网络反应。

• DOI: 10.1016/j.arr.2008.04.003
• 发表时间: 2008
• 期刊: Ageing research reviews
• 影响因子: 13.1
• 作者: McMains,VanessaC;Liao,Xin-Hua;Kimmel,AlanR
• 通讯作者: Kimmel,AlanR

Perilipin family members preferentially sequester to either triacylglycerol-specific or cholesteryl-ester-specific intracellular lipid storage droplets.

• DOI: 10.1242/jcs.104943
• 发表时间: 2012-09-01
• 期刊: Journal of cell science
• 影响因子: 4
• 作者: Hsieh K;Lee YK;Londos C;Raaka BM;Dalen KT;Kimmel AR
• 通讯作者: Kimmel AR

Isolated Plin5-deficient cardiomyocytes store less lipid droplets than normal, but without increased sensitivity to hypoxia.

• DOI: 10.1016/j.bbalip.2020.158873
• 发表时间: 2020-12
• 期刊: Biochimica et biophysica acta. Molecular and cell biology of lipids
• 作者: Yuchuan Li;M. Torp;F. Norheim;P. Khanal;A. Kimmel;K. Stensløkken;J. Vaage;K. Dalen

Regulation of nucleosome positioning by a CHD Type III chromatin remodeler and its relationship to developmental gene expression in Dictyostelium.

• DOI: 10.1101/gr.216309.116
• 发表时间: 2017-04
• 期刊: Genome research
• 影响因子: 7
• 作者: Platt JL;Kent NA;Kimmel AR;Harwood AJ
• 通讯作者: Harwood AJ

DPF is a cell-density sensing factor, with cell-autonomous and non-autonomous functions during Dictyostelium growth and development.

DPF是一种细胞密度传感因子，在盘基网柄菌生长发育过程中具有细胞自主和非自主功能。

• DOI: 10.1186/s12915-019-0714-9
• 发表时间: 2019
• 期刊: BMC biology
• 影响因子: 5.4
• 作者: Meena,NetraPal;Jaiswal,Pundrik;Chang,Fu-Sheng;Brzostowski,Joseph;Kimmel,AlanR
• 通讯作者: Kimmel,AlanR