新型肿瘤治疗靶点FACT复合体的结构及其功能研究

FACT heterodimer complex is an important cell transcription factor to assist RNA polymerase II transcription on the DNA template. Recent study revealed that FACT induced apoptosis in tumor cells by activating p53 signal pathway after adding small molecule Curaxin. Xenograft tumor model also had been used to evaluate this anti-tumor activity in animals. Meanwhile, FACT is mainly distributed in the stem cells and tumor cells in human, whereas rarely expressed in adult somatic cells. Therefore, FACT is a desirable anti-cancer target to develop a new generation drug for future clinical treatment. However, the structure of FACT complex and the mechanism how the FACT modulates the p53 signal pathway are unknown. The purpose of this study has two-fold. Firstly, we are to sovle the protein structure of FACT complex. Secondly, we will measure the protein-protein interaction with FACT and other apoptotic proteins both in vivo and in vitro. Base on these information, we are to illustrate the mechanism how FACT consequently activates the cell apoptosis, which is independent of DNA damage. Both aims will lead to the elucidation of the structural and funcational role of FACT in the signal pathway of DNA damage. To achieve this target, we will focus on human FACT complex and its functional domains. We have determined several crystal structures of funtional domains of human FACT. In this study, we are going to solve the other two domains of FACT and put these sturcutral information together piece by piece to construct the structure of full length of FACT complex. EM and SAXS data will be obtained for structural modeling of full length of FACT. Functional studies will be manipulated parallelly to illustrate how FACT works in the signal pathway of apoptosis in vivo. Finally, combination of both in vivo and in vitro data, a DNA damamage independent FACT apoptosis signal pathway will be proposed.

FACT蛋白复合体是一种重要的转录因子，协助RNA聚合酶II在DNA模板上完成转录、延伸。最新研究发现，药物在不损伤细胞DNA前提下，通过FACT可激活p53信号传导通路，诱导肿瘤细胞凋亡，并在移植瘤动物模型上，表现出杀伤、抑制肿瘤的生物活性。同时，FACT主要分布在干细胞和肿瘤细胞中，正常成体细胞含量极少。这为开发特异性、肿瘤靶向治疗新型药物，提供了良好的靶点。但FACT复合体的蛋白结构，及其不依赖DNA损伤，而激活p53传导通路的分子机制尚未阐明。该研究将从蛋白结构与功能研究两个方向，阐明FACT复合体的蛋白生物学功能及其结构基础。为达到这一目的，以人源FACT为对象，在已获得的FACT多个结构功能域的蛋白结构基础上，利用透射电镜及小角散射技术获取FACT整体低分辨率结构信息，构建FACT结构模型。并结合FACT与组蛋白、细胞凋亡蛋白在体内、外的相互作用，阐明FACT诱导凋亡的调控机制

FACT蛋白复合物是RNA聚合酶II的保守辅因子，是组蛋白分子伴侣。在促进核小体形成，DNA复制和转录，DNA损伤修复，核小体的解聚等方面发挥重要作用。FACT主要分布在干细胞和肿瘤细胞中，大部分正常成体细胞含量极少，是一种新型抗肿瘤治疗靶点。人源的FACT蛋白复合物是异元二聚体，由Spt16和SSRP1两个蛋白亚基组成。.本研究成功解析了人源FACT蛋白复合体亚基SSRP1的SSRP1N、SSRP1M和SSRP1 HMG三个相对独立结构域的蛋白晶体结构（分辨率分别为1.65 Å、2.10Å和2.00 Å）以及亚基Spt16的Spt16N结构域的蛋白晶体结构（分辨率为2.1 Å）。利用蛋白诱捕技术及分析型凝胶排阻层析技术，发现 SSRP1N和 SSRP1M不能结合组蛋白，因此推测在SSRP1存在未知的组蛋白结合功能区。HMG是一个典型的DNA结合蛋白，本研究利用凝胶迁移实验及分析型凝胶排阻层析两种实验方法发现SSRP1 HMG的N端loop对结合DNA起着重要作用；利用单分子成像技术进一步探索 SSRP1 HMG 与长链λDNA的相互作用，发现SSRP1 HMG520-616不仅能结合长链 λDNA还能使长链λDNA发生弯折；利用等温量热滴定技术发现SSRPHMG 的 C 端 loop发现与 CK2 的相互作用，初步认为 C 端 loop 这一区域为 CK2β 结合功能区。本研究还运用免疫荧光技术及荧光成像方法首先确认SSRP1的核定位信号序列。本研究利用戊二醛蛋白交联实验、原子吸收光谱、等温量热滴定的等方法对Spt16N蛋白检测，实验结果明确了Spt16N在溶液中的聚合情况，证实Spt16N不结合镁、钴、锌、锰等金属离子。Spt16N蛋白浓度为30-40μM时，Spt16N1-437不结合组蛋白H3/H4，而Spt16N1-510可以结合组蛋白H3/H4，hSpt16N1-437和Spt16N1-510均不结合组蛋白H2A/H2B。.这些研究成果初步阐明了组蛋白分子伴侣FACT蛋白复合体如何激活p53信号通路，引起肿瘤细胞凋亡的信号通路的分子机制，同时为下一步以FACT为靶点的curaxins小分子先导化合物的机制研究及未来的新型药物研发奠定了基础。