Unc-51-Like Kinase 4 在神经干细胞增殖和肿瘤发生中的作用

A proper cell proliferation is important for body formation, physiology, and repair. We recently found that ULK4 is a cell cycle regulator and is highly expressed in G2/M phases. Targeted disruption of the Ulk4 in mice dramatically reduced the neural stem cell (NSC) pool at birth and down-regulated expression of the cell cycle-related genes. Emerging evidence showed that ULK4 SNP Ala/542/Thr is associated with multiple myeloma, however the mechanism is unknown. We hypothesize that low ULK4 expression may reduce cell proliferation, while high expression increases proliferation, and ULK4 overexpression and/or over-activity leads to tumorigenesis. We have constructed ULK4 Ala/542 and ULK4 542/Thr expression vectors. In this project, we will transfect the plasmids into human H9NSC line to establish ULK4-overexpression H9NSC lines. These cell lines will be synchronized to G1, S, G2 and M phases of cell cycle, to investigate effects of Ala/542/Thr polymorphism on protein stability/ phosphorylation, cell cycle phase expression, tumor-related gene expression and tumor formation. We also will perform interactive proteomics and bioinformatic analyses to identify ULK4 interactome and 542/Thr-specific binding protein(s). These studies will produce functional data for the Ala/542/Thr polymorphism in the control of cell proliferation and tumnorigenesis. We will also identify NSC-specific ULK4 binding proteins and pathways, which will enhance our understanding how ULK4 influences tumorigenesis and brain functions as well as cardiovascular function, which may lay down biological bases for the development of the therapeutic strategy for the treatment of the three major diseases in the world.

适度细胞增殖和细胞周期调控是个体形成、生理功能和机体修复的基础。我们发现ULK4是一个细胞周期相关因子，其表达在G2/M期最高。Ulk4敲除显著减少新生鼠神经干细胞数目、并下调一系列细胞周期因子。遗传学研究也表明，ULK4 542Ala/Thr多态性与多发性骨髓瘤有关，但是至今尚无机理研究。我们假设：ULK4低表达减少细胞增殖；ULK4高表达增加细胞增殖；ULK4表达/活性过高会引发肿瘤。本课题拟以人的神经干细胞为材料，利用质粒转染实现ULK4过表达，通过细胞同步化，来揭示ULK4 542Thr对蛋白稳定性、磷酸化、细胞周期、肿瘤基因表达、肿瘤形成的影响。同时利用蛋白质组学，找出ULK4在人的神经干细胞中的相互作用蛋白群、以及542特异结合蛋白。预期结果将为ULK4如何影响心血管、肿瘤和大脑功能以及相关的三大疾病，奠定功能性和机理性依据。

细胞的适度增殖是组织、器官和个体形成的基础，是细胞生命活动的重要特征之一。细胞增殖是通过细胞周期来实现的,而细胞周期的正常进行受一系列相关蛋白分子及其信号通路的严格监视和精确调控。其中细周期蛋白依赖性激酶（Cdk）和周期蛋白是调节细胞周期的关键因素。此外，还有细胞周期有关的其他基因及产物也参与细胞周期调控中，如丝氨酸/苏氨酸蛋白激酶是其中重要的一类，在细胞周期级联控制中发挥重要作用。Ulk4是一个推测的丝氨酸/苏氨酸蛋白激酶，前期结果显示Ulk4缺失后影响细胞增殖，但具体作用和分子机制不详。本项目在前期结果的基础上开展了项目计划书的研究内容，具体包括：（1）确证丝氨酸/苏氨酸蛋白激酶Ulk4在细胞细胞增殖和细胞周期调控中的作用；（2）Ulk4调控细胞周期的分子信号通路解析及Ulk4相互作用蛋白的筛选及验证；（3）Ulk4与脑胶质瘤的相关性分析。结果表明蛋白激酶Ulk4调控细胞增殖和细胞周期相关分子的表达，并通过与其他蛋白形成异源二聚体发挥作用。同时通过生物信息学，分析了Ulk4与多种肿瘤的相关性，发现Ulk4与低级别胶质瘤(LGG)的发生密切相关，以上结果将为进一步研究Ulk4在大脑中的功能及脑疾病提供了理论依据。