REGULATION OF CELLULAR GROWTH AND ENERGY HOMEOSTASIS

细胞生长和能量稳态的调节

• 批准号: 7733970
• 负责人: ALAN R KIMMEL
• 金额: $ 41.24万
• 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/7733970
• 关键词: Actins; Adipose tissue; Amino Acids; Anabolism; Bacteria; Biological Models; Cells; Cholesterol Esters; Classification; Co-Immunoprecipitations; Complement; Complex; Cultured Cells; Data; Development; Dictyostelium; Energy Metabolism; Equilibrium; Event; Family; Family member; Gene Mutation; Genes; Growth; Growth Factor; Homeostasis; Insulin Resistance; Knockout Mice; Link; Lipids; Lipolysis; Liver; Mammals; Mediating; Molecular; Mus; Muscle; Mutant Strains Mice; Mutate; Mutation; Natural Immunity; Nomenclature; Nutrient; Organism; Pathway interactions; Peripheral; Personal Satisfaction; Phagocytosis; Phenotype; Process; Production; Proteins; Raptors; Rate; Regulation; Relative (related person); Resources; Role; Series; Signal Transduction; Sirolimus; Source; Stimulus; Structure; System; Testing; Triglycerides; cell growth; detection of nutrient; embryonic stem cell; fungus; in vivo; inhibitor/antagonist; macrophage; membrane synthesis; mouse model; novel; perilipin; protein function; research study; response; steroid hormone; sterol esterase; transmission process

TOR Complex 1 (TORC1) is required for growth, while TORC2 regulates actin cytoskeletal polarization. The regulation and function of TORC1 is well understood, but TORC2 regulation has not been fully characterized; nor, is it clear if TORC2 is required for all actin-mediated events. We examined the role of TORC2 during the actin-dependent process of nutrient capture by phagocytosis in Dictyostelium and tested if TORC2 were involved.We show that loss of TORC2 components Rictor/Pia, SIN1/RIP3, and LST8 promotes phagocytosis, while inactivation of TORC1, by depletion of TORC1-specific Raptor, has no effect on phagocytosis. We also show that rates of phagocytosis are uncoupled from in vivo TORC1 activity. Also, while rapamycin is suggested to be a specific inhibitor of TORC1, our additional data indicate that rapamycin is also able to inhibit TORC2 regulation of phagocytosis, albeit indirectly. Thus, although TORC2 is required for cell polarization, it is not required for all actin-dependent processes. Finally, we suggest that the balanced regulations of TORC2 and TORC1 may be critical to coordinate and optimize growth with energy needs. We have collaborated with Dr. C. Londos, LCDB/NIDDK, to study the function of proteins that associate specifically with the intracellular lipid storage droplets (LSDs) that assemble triacylglycerols and cholesteryl esters for energy metabolism, steroid hormone synthesis, membrane biosynthesis, and cell signaling. We had identified the novel PAT domain as a defining feature for LSD proteins Perilipin (Peri), ADRP, TIP47, discovered new family members (PAT5), and determined the first structure of a PAT family member, TIP47. The structural relationships among these various proteins had necessitated a new nomenclature. Accordingly we have proposed the following: Perilipin is now Perilipin 1, ADRP is Perilipin 2, TIP47 is Perilipin 3, S3-12 is Perilipin 4, and PAT5 is Perilipin 5. Functional studies using native and mutated forms of Peri1 confirmed its role in PKA-mediated lipolysis of triacylglycerols and showed that Peri is essential for the translocation of hormone-sensitive lipase (HSL) during lipolytic activation, but that while the adipose mass of peri-null mice is <30% that of WT, the mutant mice are susceptible to peripheral insulin resistance in liver, but not muscle. We also show that Peri3 (TIP47) is unable to functionally compensate for Peri1 but can complement the role of Peri2 (ADRP) in a cell culture model system. To analyze the various functions of the 5 genes that comprise the Peri family of mammalian proteins, we initiated a systematic series for production of singly and multiply targeted mutations of these genes in mouse ES cells and for their germline transmission in mice. To this end we now have Peri1(-/-) mice, and mutations in ES cells and mice for Peri2, 3, and 5. Others data had asserted that Peri2 (-/-) mice only had a limited phenotype, but an aberrant Peri2 product produced in those mice may have had a compensating effect. We now show by co-immunoprecipitation experiments a link of Peri2 to a pathway that is independent of Peri3 and suggest a more complex and non-overlapping regulatory mode for these highly related proteins.

TOR复合物1（TORC1）是生长所必需的，而TORC2调节肌动蛋白细胞骨架极化。 TORC1的调节和功能已被充分理解，但是TORC2调节尚未完全表征。也不清楚所有肌动蛋白介导的事件是否需要TORC2。我们检查了TORC2在肌动蛋白依赖性的营养捕获过程中的作用，在dictyostelium中吞噬作用捕获，并测试了是否涉及TORC2。我们表明，Torc2成分RICTOR/PIA，SIN1/RIP3和LST8的损失通过DETORC1的作用，同时de cap1-Sperion of torc1-spec1-spec1-spec1-吞噬作用。我们还表明，吞噬作用的发生率与体内Torc1活性未耦合。同样，虽然雷帕霉素被认为是TORC1的特定抑制剂，但我们的其他数据表明雷帕霉素也能够抑制吞噬作用的TORC2调节，尽管是间接的。因此，尽管TORC2是细胞极化所必需的，但所有依赖肌动蛋白的过程并不需要。最后，我们建议TORC2和TORC1的平衡法规对于与能源需求进行协调和优化生长至关重要。 我们已经与LCDB/NIDDK的C. Londos博士合作研究了与细胞内脂质储物液滴（LSD）专门关联的蛋白质的功能，这些液滴（LSD）组装了三酰基甘油和胆固醇酯的能量代谢，用于能量代谢，用于代谢，类固醇激素的综合，膜生物元素和细胞信号。我们已经将新颖的PAT领域确定为LSD蛋白peripin（Peri），ADRP，TIP47的定义特征，发现了新的家庭成员（PAT5），并确定了PAT家庭成员TIP47的第一个结构。这些各种蛋白质之间的结构关系需要新的命名法。 Accordingly we have proposed the following: Perilipin is now Perilipin 1, ADRP is Perilipin 2, TIP47 is Perilipin 3, S3-12 is Perilipin 4, and PAT5 is Perilipin 5. Functional studies using native and mutated forms of Peri1 confirmed its role in PKA-mediated lipolysis of triacylglycerols and showed that Peri is essential for the translocation of hormone-sensitive lipase （HSL）在脂解激活过程中，但是虽然尿中小鼠的脂肪质量小于WT的30％，但突变小鼠容易受到肝脏外周胰岛素抵抗的影响，但不容易受到肌肉的抗性。我们还表明，Peri3（TIP47）无法在功能上补偿PERI1，但可以补充Peri2（ADRP）在细胞培养模型系统中的作用。为了分析构成哺乳动物蛋白质家族的5个基因的各种功能，我们启动了一个系统的序列，用于在小鼠ES细胞中单独生产这些基因的突变，并在小鼠中生产其生殖线传播。为此，我们现在有PERI1（ - / - ）小鼠，以及在PERI2、3和5的ES细胞和小鼠中的突变。其他数据已经断言，Peri2（ - / - ）小鼠只有有限的表型，但是在这些小鼠中产生的异常PERI2产物可能具有补偿效应。现在，我们通过共免疫沉淀实验显示了Peri2与独立于Peri3的途径的联系，并建议针对这些高度相关的蛋白质一种更复杂和非重叠的调节模式。