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 则调节肌动蛋白细胞骨架极化。 TORC1的调控和功能已被充分了解，但TORC2的调控尚未完全表征；也不清楚 TORC2 是否是所有肌动蛋白介导的事件所必需的。我们研究了TORC2在盘基网柄菌通过吞噬作用捕获营养物的肌动蛋白依赖性过程中的作用，并测试了TORC2是否参与其中。我们发现TORC2成分Rictor/Pia、SIN1/RIP3和LST8的丢失会促进吞噬作用，而TORC1的失活会促进吞噬作用，通过消耗 TORC1 特异性 Raptor，对吞噬作用没有影响。我们还表明吞噬速率与体内 TORC1 活性无关。此外，虽然雷帕霉素被认为是 TORC1 的特异性抑制剂，但我们的额外数据表明雷帕霉素也能够抑制 TORC2 吞噬作用的调节，尽管是间接的。因此，虽然 TORC2 是细胞极化所必需的，但并非所有肌动蛋白依赖性过程都需要它。最后，我们建议 TORC2 和 TORC1 的平衡监管对于协调和优化增长与能源需求可能至关重要。 我们与 LCDB/NIDDK 的 C. Londos 博士合作，研究与细胞内脂质存储液滴 (LSD) 特异性相关的蛋白质的功能，这些脂质存储液滴组装三酰甘油和胆固醇酯，用于能量代谢、类固醇激素合成、膜生物合成和细胞信号传导。我们已经确定新的 PAT 结构域是 LSD 蛋白 Perilipin (Peri)、ADRP、TIP47 的定义特征，发现了新的家族成员 (PAT5)，并确定了 PAT 家族成员 TIP47 的第一个结构。这些不同蛋白质之间的结构关系需要新的命名法。因此，我们提出以下建议：Perilipin 现在为 Perilipin 1，ADRP 为 Perilipin 2，TIP47 为 Perilipin 3，S3-12 为 Perilipin 4，PAT5 为 Perilipin 5。使用 Peri1 的天然和突变形式的功能研究证实了其在 PKA 中的作用- 介导三酰甘油的脂肪分解，并表明 Peri 对于激素敏感脂肪酶 (HSL) 的易位至关重要脂肪分解激活，但虽然peri-null小鼠的脂肪量<WT的30%，但突变小鼠的肝脏对外周胰岛素抵抗敏感，但对肌肉则不然。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。为了分析组成哺乳动物蛋白 Peri 家族的 5 个基因的各种功能，我们启动了一系列系统性研究，用于在小鼠 ES 细胞中产生这些基因的单靶点和多靶点突变，并在小鼠中进行种系传递。为此，我们现在拥有 Peri1(-/-) 小鼠，以及 ES 细胞和 Peri2、3 和 5 小鼠的突变。其他数据断言 Peri2 (-/-) 小鼠仅具有有限的表型，但具有异常的表型。这些小鼠体内产生的 Peri2 产品可能具有补偿作用。我们现在通过免疫共沉淀实验展示了 Peri2 与独立于 Peri3 的途径的联系，并提出了这些高度相关的蛋白质的更复杂和非重叠的调控模式。