OGT及O-GlcNAc蛋白糖基化在DNA损伤应答及肿瘤治疗中的作用研究

O-linked N-acetylglucosamine linkage (O-GlcNAcylation) to Serine or threonine residues regulates numerous biological processes. Recently, we found that OGT and O-GlcNAcylation participate in gene transcription regulation and DNA damage response. OGT binds with TET2, which can recruit OGT to specific chromatin positions to O-GlcNAcylate H2B on Ser112. O-GlcNAcylated H2B could regulate the transcription of a specific group of genes. We also found that, in response to DNA damage, OGT reclocates to the sites of DNA damage to O-GlcNAcylate multiple DNA damage repair factors including H2AX and MDC1. Because aberrant OGT and O-GlcNAcylation have been found in multiple tumors including prostate cancer, breast cancer and leukemia etc, OGT might be a promising target for tumor treatment. In this proposal, we plan to explore the detailed mechanism, by which OGT and O-GlcNAcyltion participate in DNA damage response, and finally study whether we can combine OGT inhibitor and DNA damage inducing chemicals for tumor treatment. As protein ubiqutination happened in DNA damage response, we will first explore what is the role of O-GlcNAcylation in protein ubiquitination processes after DNA damage. Next, we will determine the epigenetic modification changes after DNA damage and elucidate the role of O-GlcNAcylation in this process. We will try to identify key factors in DNA damage response that are modified and regulated by OGT. As we have found that, following DNA damage, depletion of OGT prolongs the G2/M checkpoint and reduces cell viability, we finally want to study the effects of treatment with OGT inhibitor and DNA damage inducing chemicals on the viability of different type of tumor cells. We will analyze the functions and protein levels of the key factor regulated by OGT, and find the correlation between the sensitivity of tumor cells to the combined treatment and functions of these key factors. We believe that our study could identify key factors regulated by OGT following DNA damage and we could use these factors as predictive markers for precise treatment of tumors by OGT inhibitors and DNA damage inducing chemicals.

DNA损伤修复机制的研究，对于研究肿瘤的发生机制以及肿瘤治疗有实际意义。DNA损伤发生后，多种蛋白修饰参与DNA损伤应修复过程，比如蛋白泛素化、磷酸化等，我们前期发现OGT及O-GlcNAc蛋白糖基化修饰能被募集到DNA损伤位点并调控DNA损伤应答过程。有意思的是，OGT及O-GlcNAc糖基化修饰在多种肿瘤中水平异常，而且OGT能够O-GlcNAc糖基化修饰多种肿瘤相关蛋白，并影响蛋白稳定性或调控基因表达，参与肿瘤发生，暗示O-GlcNAc糖基化修饰在肿瘤发生中有作用。因此本项研究将以OGT及蛋白糖基化在DNA损伤应答中的作用为切入点，通过研究DNA损伤发生后OGT与蛋白泛素化的关系及OGT对表观遗传修饰的调控，发现OGT调控DNA损伤发生的关键分子，为使用OGT抑制剂治疗肿瘤提供分子依据，并探索OGT抑制剂和DNA损伤诱导类化疗药物联合用药治疗肿瘤的可行性。

DNA损伤修复机制的研究，对于研究肿瘤的发生机制以及肿瘤治疗有实际意义。DNA损伤发生后，多种蛋白修饰参与DNA损伤应修复过程，比如蛋白泛素化、磷酸化和OGT介导的糖基化修饰等，同时最近的研究发现DNA损伤修复过程也受到细胞质组分的调控，比如微管骨架。本项研究的成果主要分为两方面的内容，第一方面，紧扣项目申请计划书，以OGT介导的糖基化修饰在DNA损伤修复中的作用为切入点，筛选比发现了多个OGT参与DNA损伤修复的可能的新机制，比如，我们发现Zyxin是OGT的新底物，研究发现糖基化修饰能促进Zyxin和14-3-3蛋白的结合，最终促进Zyxin的入核，在功能上，OGT通过糖基化Zyxin调控DNA损伤发生后p53蛋白活性，从而调控UV引起的细胞死亡；同时，我们还发现OGT的另一个新底物SCML2，研究发现SCML2的糖基化修饰能够促进其蛋白稳定性，在功能上，SCML2的蛋白量升高能够促进肿瘤细胞对DNA损伤的抵抗性，在分子机制上，SCML2分别通过调控p53活性或CHK1的蛋白稳定性参与DNA损伤修复过程。第二方面，在第一方面的研究中，我们发现DNA损伤修复过程和微管动态变化之间存在联系，并通过深入研究发现DNA损伤能主动调控微管的动态变化并促进DNA双链断裂修复。我们将这种新的DNA双链断裂诱导的微管应激机制命名为DMSR。在DMSR发生时，DNA双链断裂能诱导间期细胞中心体成熟以及微管成核和延伸能力增强，这一过程伴随着中心体对中心粒外周蛋白(PCM)的募集，特别是PCNT(Pericentrin)，γ-tubulin，NEDD1等；随着PCM的募集，中心体成熟，中心体成核能力增强，从而促进DSBs运动性的增加以及c-NHEJ修复过程的发生。此外，DNA双链断裂促进的中心体成熟主要发生在细胞静止期和间期，并且依赖于DNA-PK-AKT 信号通路，而不是传统的有丝分裂细胞时期中促进中心体成熟的PLK1激酶。当敲除中心体蛋白之后，DMSR和DSBs运动性会极大的减弱，进而导致c-NHEJ修复进程延迟。综上所述，本项目进行过程中，我们一方面紧扣申请计划书开展探索性研究，取得了多项进展，同时，针对研究中发现的新现象进行了拓展研究，并取得了不错的成果。