Characterization of the cellular function of TBC1D7, independent of- and as the third component of the TSC complex

结节性硬化症复合体（TSC）是由TSC1或TSC2突变引起的，TSC1或TSC2形成功能复合体（TSC复合体）。TSC的主要临床特征是皮肤、心脏、肺和大脑的良性肿瘤生长，并伴有癫痫发作和TSC相关的神经精神疾病（如自闭症谱系障碍）[Henske et al. 2016]。TSC1和TSC2蛋白与TBC1D7蛋白一起在mTORC1上游形成负调控复合体，mTORC1是合成代谢细胞生长和增殖的主要调控因子。TBC1D7是TSC复合物中普遍存在的核心成分，对复合物的稳定性和对Rheb的GAP活性至关重要[Dibble et al. 2012]。到目前为止，尚未在TSC患者中发现TBC1D7基因的种系功能突变缺失，但TBC1D7的缺失与遗传性巨脑畸形综合征相关，并伴有mTORC1信号升高，但与TSC不同[Capo-Chichi et al. 2013]。在过去的10年里，Manning实验室在我们对TSC复合物组分TSC1， TSC2和TBC1D7的理解方面取得了重要的发现。除了作为TSC复合物的组成部分外，TBC1D7还被发现存在于细胞质中的游离蛋白池中。这两种蛋白质的确切功能仍然未知。这项研究的主要目标是确定TBC1D7的分子功能，以及这些功能的改变如何影响TSC不同临床表现的细胞表型。我相信，了解TBC1D7在TSC复合体中的作用，以及作为一个自由实体（在TSC基因缺失时增加）的作用，将为TSC患者异常调节的新途径和过程提供见解。我将利用各种细胞生物学和生物化学方法，在体外和体内研究TBC1D7在大脑中的功能。该研究的三个具体目标是：(1)确定TBC1D7在大脑内的新相互作用伙伴，(2)表征TBC1D7敲除小鼠的全身特征，(3)破译TBC1D7在细胞代谢中的作用。在曼宁实验室的头几个月里，我使用CRISPR/Cas9技术培育了Tbc1d7基因敲除小鼠。这些小鼠是可存活的，没有明显的异常，它们将成为该项目深入了解TBC1D7在生理环境中的作用的重要工具。此外，实验室的初步结果表明，TBC1D7与Rab17和Citron相互作用，以及TBC1D7在调节肌酸代谢中的作用。我将利用这项奖学金进一步探索这些发现，并更详细地分析与其他蛋白质的相互作用。在本提案中，我将专注于更好地理解新通路（包括游离TBC1D7库）与TSC复合物之间的关系，这是在探索TSC治疗意义之前必不可少的基础知识。

Tuberous sclerosis complex (TSC) is caused by mutations in TSC1 or TSC2, which form a functional complex (the TSC complex). Major clinical features of TSC are benign tumor growth of the skin, heart, lung and brain together with seizures and TSC-associated neuropsychiatric disorders (e.g. autism spectrum disorder) [Henske et al. 2016]. Together with the TBC1D7 protein, the TSC1 and TSC2 proteins form a negative regulatory complex upstream of mTORC1, a master regulator of anabolic cell growth and proliferation. TBC1D7 is a ubiquitous core component of the TSC complex, important for the complex´s stability and GAP activity toward Rheb [Dibble et al. 2012]. Germline loss of function mutations in the TBC1D7 gene have not been identified in TSC patients so far, but loss of TBC1D7 has been associated with a hereditary megalencephaly syndrome accompanied with elevated mTORC1 signaling, but distinct from TSC [Capo-Chichi et al. 2013]. Over the last 10 years, the Manning lab has made important discoveries in our understanding of the TSC complex components TSC1, TSC2 and TBC1D7. Besides its role as a component of the TSC complex, TBC1D7 was also found to be present in a pool of free protein in the cell cytoplasm. The exact function of either protein population remains unknown.The main goal of this fellowship is to define the molecular functions of TBC1D7 and how alterations in these functions contribute to cellular phenotypes underlying the diverse clinical manifestations of TSC. I believe that understanding the role of TBC1D7 both within the TSC complex and as a free entity, which increases upon loss of the TSC genes, will provide insights into new pathways and processes that become aberrantly regulated in TSC patients. I will use various cellular biological and biochemical approaches to study the function of TBC1D7 in particular in the brain in vitro and in vivo. The three specific aims of this fellowship are (1) to define new interaction partners of TBC1D7 within the brain, (2) to characterize a whole body Tbc1d7 knock-out mouse and (3) to decipher the role of TBC1D7 in cellular metabolism. In my first months in the Manning lab I generated Tbc1d7 knock-out mice using the CRISPR/Cas9 technique. These mice are viable and show no gross abnormalities, and they will be an essential tool to the project to gain a deeper understanding of the role of TBC1D7 in a physiological context. Furthermore, preliminary results from the lab suggest that TBC1D7 interacts with Rab17 and Citron, as well as a role for TBC1D7 in the regulation of creatine metabolism. I will use this fellowship to further explore these findings and analyze interaction interactions with other proteins in more detail. In this proposal, I will focus on gaining a better comprehension of the relationship between new pathways (involving the free TBC1D7 pool) and the TSC complex, fundamental knowledge that is essential before exploring the therapeutic implications in TSC.