Spatio-temporal regulation of mTORC1 signaling in normal and disease states

项目摘要 细胞感知营养的分子机制在很大程度上仍然未知，但它们的 阐明是我们理解正常和疾病状态下代谢调节的关键。中心 营养传感和生长调节的关键是一种古老的蛋白激酶，被称为营养传感和生长调节的机制靶点。 雷帕霉素复合物1（mTORC 1）。为了响应营养素等代谢输入的联合作用， 生长因子、能量和氧，mTORC 1从细胞质易位到溶酶体表面， 在那里它被激活。越来越多的证据表明，异常的mTORC 1激活， 溶酶体可能是从癌症到2型糖尿病再到神经退行性疾病的驱动力。 mTORC 1的溶酶体易位和激活需要异二聚体Rag鸟苷 三磷酸酶（GTP酶），其与五聚体Ragulator复合物一起，形成营养调节的 支架复合物，其将mTORC 1物理锚定到溶酶体表面。将动态成像与 细胞与生化重建和结构的方法，我们最近发现，Ragulator-Rag 复合体不是静态的，而是被营养物质积极地重塑，导致Rag的空间循环 溶酶体表面和细胞质之间的GTP酶。反过来，Rag自行车赛限制了 mTORC 1捕获的效率，并可能促进其失活时，营养水平下降。重要的是，Rag 循环被影响mTORC 1信号传导的癌症特异性突变改变。基于这些发现，我们 假设mTORC 1支架时空调节是一种新的未被认识的机制 调节mTORC 1信号传导反应的效力和选择性，并且其破坏可能会驱动 mTORC 1驱动的癌症的异常生长，包括肾细胞癌和淋巴瘤。 我们将通过两个高度互补和创新的研究目标来验证这一假设。一是 采用结构引导的诱变来剖析控制mTORC 1- 脚手架复杂的响应不断变化的营养输入。第二，我们将描述 我们最近发现的新一代化合物的作用，它阻止了溶酶体的组装， mTORC 1支架复合物，并确定其抑制mTORC 1 - 100代谢和生长的能力。 驱动癌症。 总的来说，拟议的研究将产生新的知识的时空调控， mTORC 1信号，并指出新的策略来操纵mTORC 1信号在正常和 疾病状态。

PROJECT SUMMARY The molecular mechanisms through which cells sense nutrients remain largely unknown, but their elucidation is key to our understanding of metabolic regulation both in normal and disease states. At the center of nutrient sensing and growth regulation is an ancient protein kinase known as the mechanistic Target of Rapamycin Complex 1 (mTORC1). In response to the combined action of metabolic inputs such as nutrients, growth factors, energy and oxygen, mTORC1 translocates from the cytoplasm to the surface of lysosomes, where it becomes activated. Accumulating evidence indicates that aberrant mTORC1 activation at the lysosome could be a driving force in diseases ranging from cancer to type-2 diabetes to neurodegeneration. Lysosomal translocation and activation of mTORC1 requires the heterodimeric Rag guanosine triphosphatases (GTPases), which together with the pentameric Ragulator complex, form a nutrient-regulated scaffolding complex that physically anchors mTORC1 to the lysosomal surface. Combining dynamic imaging in cells with biochemical reconstitution and structural approaches, we recently discovered that the Ragulator-Rag complex is not static but is rather actively remodeled by nutrients, leading to spatial cycling of the Rag GTPases between the lysosomal surface and the cytoplasm. In turn, Rag cycling places a limit on the efficiency of mTORC1 capture and may facilitate its inactivation when nutrient levels fall. Importantly, Rag cycling is altered by cancer-specific mutations that affect mTORC1 signaling. Based on these findings, we hypothesize that spatial-temporal regulation of mTORC1 scaffolding is a novel and unrecognized mechanism to modulate the potency and selectivity of mTORC1 signaling responses, and that its disruption may drive the aberrant growth of mTORC1-driven cancers, including renal cell carcinoma and lymphoma. We will test this hypothesis via two highly complementary and innovative research aims. First, we will employ structure-guided mutagenesis to dissect the mechanisms that govern the assembly of the mTORC1- scaffolding complex in response to changing nutrient inputs. Second, we will characterize the mechanism of action of new-generation compounds we recently discovered, which block the assembly of the lysosomal mTORC1 scaffolding complex, and determine their ability to inhibit the metabolism and growth of mTORC1- driven cancers. Collectively, the proposed studies will generate new knowledge on the spatial-temporal regulation of mTORC1 signaling, and point the way to novel strategies to manipulate mTORC1 signaling in both normal and disease states.