Elucidating a mechanism of mTORC1 activation independent of amino acids signaling

描述（由申请人提供）：mTOR活性促进衰老的机制尚不清楚。mTOR活性的失调导致衰老加速，而mTORC1活性的上调发生在衰老的生物体中。我们最近描述了Rag蛋白家族是氨基酸激活mTORC1的关键，通过一种需要mTORC1穿梭到溶酶体表面的机制。我们已经从缺乏RagA基因的基因工程小鼠中获得了一系列永生细胞系。尽管在raga缺乏的胚胎中几乎检测不到mTORC1活性，但从它们衍生的细胞系已经重新激活了mTORC1信号，该信号现在对氨基酸退出不敏感，但对生长因子退出敏感。有趣的是，在这些细胞系中，mTORC1虽然有活性，但并不定位于溶酶体，这表明mTORC1激活的另一种未知机制在起作用。基于转录谱分析和基于蛋白质组学的方法的结合，我们计划找到负责mTORC1溶酶体非依赖性激活的基因，并了解这是如何发生的。然后，我们将利用RagA-null肝脏在体内研究改变候选基因表达的后果。进一步阐明导致mTORC1对氨基酸退出脱敏的分子机制，以及该途径中可药物靶点的鉴定，可能为衰老和年龄相关疾病的新治疗方法铺平道路。

DESCRIPTION (provided by applicant): The mechanisms underlying the pro-aging consequences of mTOR activity are obscure. Deregulated mTOR activity leads to accelerated aging and upregulated mTORC1 activity occurs in aging organisms. We recently described the Rag family of proteins as key for mTORC1 activation by amino acids, through a mechanism that requires mTORC1 shuttling to the lysosomal surface. We have generated a series of immortalized cell lines derived from genetically-engineered mice that lack the RagA gene. Although mTORC1 activity is barely detectable in RagA-deficient embryos, cell lines derived from them have reactivated mTORC1 signaling, which is now insensitive to amino acids withdrawal, but sensitive to growth factors withdrawal. Intriguingly, in these lines mTORC1, although active, does not localize to lysosomes, indicating that an additional unknown mechanism of mTORC1 activation is at work. Based on a combination of transcriptional profiling and proteomic-based approaches, we plan to find the genes responsible for the lysosomal-independent activation of mTORC1 and to understand how this occurs. We will then take advantage of RagA-null livers to study in vivo the consequences of altering the expression of the candidates found. Further elucidation of the molecular mechanism leading to mTORC1 desensitization to amino acid withdrawal, together with the identification of druggable targets within this pathway, may pave the way for novel therapeutic approaches to aging and age-related diseases. PUBLIC HEALTH RELEVANCE: Deregulated cell growth signals arising from the mechanistic target of rapamycin (mTOR) protein kinase occur in diabetes, cancer and aging. Thus, understanding mTOR signaling pathway will enable the pursuit of improved therapies against these diseases. Taking advantage of novel mouse models of deregulated mTOR activity we have generated cell lines with a unique and intriguing regulation of mTOR activation, resistant to nutrient deprivation, which normally inactivates this signaling pathway. The identification of the responsible genes and the molecular mechanisms governing this particular signaling alteration that we plan to investigate in the present proposal will help understand deregulated cell growth states. The series of novel mouse models will also allow us to explore the consequences of this deregulated growth state in mammalian physiology, helping us to develop novel therapeutic avenues to manipulate the mTORC1 pathway in human disease.