TOR Complex 2 and sphingolipid biosynthesis.

描述(申请人提供)：我们的长期目标是了解真核细胞中控制细胞生长的分子机制，特别是雷帕霉素敏感的TOR激酶。包括我自己在内的几个实验室最近的研究表明，TOR是两个不同的蛋白质复合体TORC1和TORC2的一部分，其中TORC1唯一地被雷帕霉素抑制。雷帕霉素被证明具有许多有益的治疗应用，包括作为一种潜在的抗癌治疗，因此人们对更多地了解TOR的细胞作用非常感兴趣。然而，由于它不是雷帕霉素的直接靶点，TORC2的细胞作用仍然没有TORC1那么好。因此，需要其他方法来研究TORC2。为此，我们最近发现，TORC2特异性成分中的突变极大地影响了鞘磷脂途径的最初步骤，特别是在萌芽酵母中神经酰胺的从头形成。最近，我们将这一观察扩展到哺乳动物细胞，我们已经表明，抑制HEK393T细胞中的mTORC2功能也会导致神经酰胺合成酶活性的降低。神经酰胺及其直接前体脂肪酸长链碱基(LCB)是一类越来越被认为在恶性细胞生长、肿瘤发生和衰老中发挥关键作用的脂类。因此，TOR和神经酰胺的生物合成都是生物医学研究的重要领域，我们的发现首次表明它们密切相关。在这里提出的实验中，我们将确定TORC2调控神经酰胺合成的机制，包括对神经酰胺合成酶的详细分析，并将开始描绘参与这一调控的信号通路。为此，我们将使用酵母和哺乳动物细胞作为互补的实验系统。我们还将讨论mTORC2信号在某些癌细胞对化疗药物的反应中所起的作用，众所周知，化疗药物增加神经酰胺的从头合成，并通过诱导神经酰胺介导的细胞凋亡来促进这些药物的细胞杀伤作用。与公共健康相关：拟议的实验解决了由TOR激酶定义的高度保守的信号通路在利用酵母和哺乳动物细胞培养调节合成复杂的鞘磷脂前体，特别是神经酰胺中的作用。众所周知，TOR信号和神经酰胺都在细胞增殖、应激、癌症和衰老中发挥重要作用。因此，我们的工作将这两个调查领域结合在一起，这两个领域对了解人类健康和疾病非常重要，我们希望通过我们的研究对这些过程获得新的见解。

DESCRIPTION (provided by applicant): Our long-term goal is to understand the molecular mechanisms by which cell growth is controlled in eukaryotic cells, in particular by the rapamycin-sensitive TOR kinase. Recent studies in several laboratories, including my own, have demonstrated that TOR functions as part of two distinct protein complexes, TORC1 and TORC2, where TORC1 is uniquely inhibited by rapamycin. Rapamycin is proving to have many beneficial therapeutic applications, including as a potential anti-cancer treatment, and so there is great interest in understanding more about the cellular role of TOR. Because it is not a direct target for rapamycin, however, the cellular role of TORC2 has remained much less well characterized than TORC1. Accordingly, alternative approaches are needed to study TORC2. To this end, we have discovered recently that a mutation within a TORC2-specific component affects dramatically the earliest steps of the sphingolipid pathway, in particular the de novo formation of ceramides, in budding yeast. More recently, we have extended this observation to mammalian cells, where we have shown that inhibiting mTORC2 function in HEK393T cells also leads to a reduction in the activity of the enzyme ceramide synthase. Ceramides, as well as their immediate precursors, the fatty acid long chain bases (LCBs), represent classes of lipids that are increasingly recognized as playing crucial roles in malignant cell growth, tumorigenesis, as well as aging. Thus, both TOR and ceramide biosynthesis represent important areas important for biomedical research, and our findings indicate for the first time that they are intimately linked. In the experiments proposed here, we will identify the mechanism by which ceramide synthesis is regulated by TORC2, including a detailed analysis of the ceramide synthase, and will begin to delineate the signaling pathway involved in this regulation. For this purpose, we will use both yeast and mammalian cells as complementary experimental systems. We will also address the role that mTORC2 signaling plays in the response of certain cancer cells to chemotherapeutic drugs, which are known to increase the de novo synthesis of ceramides and contribute to the cell killing action of these drugs by inducing ceramide-mediated apoptosis. PUBLIC HEALTH RELEVANCE: The proposed experiments address the role of a highly conserved signaling pathway, defined by the TOR kinase, in the regulation of synthesis of precursors to complex sphingolipids, in particular ceramides, using both yeast and mammalian cell culture. Both TOR signaling and ceramides are known to play important roles in cell proliferation, stress, cancer, as well as aging. Thus, our work brings together these two areas of investigation that are very important for understanding human health and disease and we expect to gain novel insights into these processes through our studies.