乙酰化调控hSSB1的机制，功能及临床意义

Human single-strand DNA binding proteins 1 (hSSB1) has been shown to participate in the DNA damage response. Cells deficient in hSSB1 exhibit defective cell cycle checkpoint activation, increased radiosensitivity and enhanced genomic instability, indicating that hSSB1 may be a therapeutic target. However, the regulaiton of hSSB1 remains elusive. We have provided evidence that hSSB1can interact with both p53 and p21, to protects p53 and p21 from ubiquitin-mediated degradation. Furthermore, hSSB1 associates with the acetyltransferase p300 and is required for efficient transcriptional activation of the p53 target gene p21 by affecting the acetylation of p53 at lysine382. In fact, we recently found that hSSB1 could be acetylated by p300, and this acetylation of hSSB1 were increased in a dose- and time-dependent manner; TSA and NAM, both deacetylation inhibitors, were capable of increasing the acetylation of hSSB1 when they were used alone or combination. This proposal here will continue to investigate following events: To identify the acetylation site of hSSB1 and the deacetyltransferase(s) involved in this acetylation of hSSB1; To investigate the mechanism of interaction between hSSB1 and p300; To determine the biological and clinical significance of this acetylation of hSSB1. At the end of this proposal, we will demonstrate the mechanism and significance of hSSB1 acetylation by p300, and will provide evidence to further support the notion that p300 inhibitors may be used as the sensitizer for chemo- or radiotherapy in patients with cancers.

hSSB1是重要的单链DNA结合蛋白，其缺失导致细胞周期检测点失活，基因组不稳定及放化疗敏感性增加，表明hSSB1是一个潜在的肿瘤治疗靶点。但其被调控的机制尚未明确。我们研究表明，hSSB1既可结合并促进p21及p53蛋白稳定性，又可与p300相互作用，促进p300对p53的382位赖氨酸的乙酰化，增强p53对p21的转录激活作用。最近预实验发现：hSSB1也可被p300乙酰化，且在DNA损伤应激情况下，其乙酰化水平呈时间和剂量依赖性增加；去乙酰化酶抑制剂TSA和NAM单独或联合处理，可促进hSSB1乙酰化表达水平的升高。本项目拟鉴定hSSB1被p300乙酰化的位点，及其去乙酰转移酶；明确p300与hSSB1的相互作用机制；揭示hSSB1乙酰化的生物学功能和临床意义。本项目的完成，将阐明p300对hSSB1乙酰化调控的机制和意义，为p300抑制剂作为放化疗增敏剂在临床上的应用提供实验依据。

肿瘤放化疗如何减毒增效是临床治疗中的重要问题之一，本项目从基础研究出发，找到了一条潜在肿瘤放化疗增敏的干预措施。在本项目中，我们良好的完成了课题既定目标。我们证明了p300乙酰化hSSB1 94位赖氨酸，并鉴定了SIRT4和HDAC10为其去乙酰化酶，K94乙酰化通过拮抗hSSB1的泛素化而使其稳定，并且此位点的乙酰化促进hSSB1与NBS1的结合。这是首次证明hSSB1受到乙酰化修饰调控，并且在DNA损伤应答中发挥重要功能，通过抑制K94乙酰化可以增加放化疗敏感性，具有潜在的临床指导价值。此外，我们进一步深入研究发现，hSSB1 79和94位赖氨酸同样受到SUMO化修饰调控，目前正在深入研究其SUMO化修饰的功能及其生理意义。