REGULATION OF CELLULAR GROWTH AND ENERGY HOMEOSTASIS

TOR复合物1（TORC 1）是生长所必需的，而TORC 2调节肌动蛋白细胞骨架极化。TORC 1的调节和功能已被充分理解，但TORC 2的调节尚未完全表征;也不清楚TORC 2是否是所有肌动蛋白介导的事件所必需的。我们研究了TORC 2在网骨藻中通过吞噬作用捕获养分的肌动蛋白依赖性过程中的作用，并测试了TORC 2是否参与其中。我们发现，TORC 2组分Rictor/Pia，SIN 1/RIP 3和LST 8的缺失促进了吞噬作用，而TORC 1的失活，通过消耗TORC 1特异性Raptor，对吞噬作用没有影响。我们还表明，吞噬率从体内TORC 1活性解耦。此外，虽然雷帕霉素被认为是TORC 1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TORC 2对吞噬作用的调节，尽管是间接的。因此，尽管TORC 2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TORC 2。最后，我们认为，TORC 2和TORC 1的平衡调节可能是协调和优化增长与能源需求的关键。 我们与C博士合作。Londos，LCDB/NIDDK，以研究与细胞内脂质储存液滴（LSD）特异性相关的蛋白质的功能，所述细胞内脂质储存液滴（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导。我们已经鉴定了新的PAT结构域作为LSD蛋白Perilipin（Peri）、ADRP、TIP 47的定义特征，发现了新的家族成员（PAT 5），并确定了PAT家族成员TIP 47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，我们提出了以下建议：Perilipin现在是Perilipin 1，ADRP是Perilipin 2，TIP 47是Perilipin 3，S3-12是Perilipin 4，PAT 5是Perilipin 5。使用天然和突变形式的Peri 1的功能研究证实了其在PKA介导的三酰甘油脂解中的作用，并显示Peri对于脂解激活期间脂肪敏感性脂肪酶（HSL）的易位是必需的，但是尽管Peri缺失小鼠的脂肪量<WT的30%，但突变小鼠对肝脏中的外周胰岛素抵抗敏感，而不是肌肉。我们还表明，Peri 3（TIP 47）是无法在功能上补偿Peri 1，但可以补充的作用，Peri 2（ADRP）在细胞培养模型系统。为了分析组成哺乳动物蛋白质的Peri家族的5个基因的各种功能，我们启动了一个系统的系列，用于在小鼠ES细胞中产生这些基因的单个和多个靶向突变，并用于它们在小鼠中的种系传递。为此，我们现在有Peri 1（-/-）小鼠，以及ES细胞和小鼠中Peri 2、3和5的突变。其他数据表明，Peri 2（-/-）小鼠仅具有有限的表型，但在这些小鼠中产生的异常Peri 2产物可能具有补偿作用。我们现在通过免疫共沉淀实验显示了Peri 2与独立于Peri 3的通路的联系，并为这些高度相关的蛋白质提出了更复杂和非重叠的调控模式。

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.