REGULATION OF CELLULAR GROWTH AND ENERGY HOMEOSTASIS

TOR复合体1(TORC1)是细胞生长所必需的，而TORC2调节肌动蛋白细胞骨架极化。TORC2的调控和功能已经被很好地理解了，但TORC2的调控还没有完全的特征；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在依赖肌动蛋白通过吞噬获取营养的过程中的作用，并测试了TORC2是否参与其中。我们发现TORC2组分Rictor/Pia、SIN1/RIP3和LST8的丢失促进了吞噬作用，而TORC1的失活，通过耗尽TORC1特异性Raptor，对吞噬作用没有影响。我们还发现吞噬细胞的速率与体内的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吞噬功能的调节，尽管是间接的。因此，尽管TORC2是细胞极化所必需的，但并不是所有肌动蛋白依赖的过程都需要TORC2。最后，我们认为，TORC2和TORC1的平衡监管可能是协调和优化增长与能源需求的关键。 我们与LCDB/NIDDK的C.Londos博士合作，研究与细胞内脂肪储存液滴(LSD)特定相关的蛋白质的功能，LSD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递。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家族成员的第一结构，TIP47。这些不同蛋白质之间的结构关系需要一个新的命名法。因此，我们提出了如下建议：Perilipin现在是Perilipin 1，AdRP是Perilipin 2，TIP47是Perilipin 3，S3-12是Perilipin 4，PAT5是Perilipin 5。使用天然和突变形式的Peri1的功能研究证实了它在PKA介导的三酰甘油脂解中的作用，并表明Peri在脂解激活过程中对于激素敏感脂肪酶(HSL)的移位是必不可少的，但是尽管Peri缺失的小鼠的脂肪质量是WT的30%，但突变的小鼠对肝脏的外周胰岛素抵抗敏感，但对肌肉不敏感。我们还表明，Peri3(TIP47)不能在功能上补偿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个基因的不同功能，我们启动了一系列系统的研究，以在小鼠ES细胞中产生这些基因的单靶向突变和多靶向突变，并用于它们在小鼠中的种系传播。为此，我们现在有了Peri1(-/-)小鼠，以及ES细胞和Peri2、3和5的突变小鼠。其他数据表明，Peri2(-/-)小鼠只有有限的表型，但这些小鼠产生的异常Peri2产物可能具有代偿作用。我们现在通过免疫共沉淀实验证明了Peri2与一条独立于Peri3的途径的联系，并为这些高度相关的蛋白质提出了一种更复杂和不重叠的调控模式。

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.