HMGB1的聚ADP-核糖基化修饰促进其乙酰化修饰

发布时间：2018-05-08 15:51

本文选题：高迁移率蛋白1 + 聚ADP-核糖聚合酶1　；参考：《华中科技大学》2012年硕士论文

【摘要】：目的：高迁移率族蛋白1(HMGB1)作为重要的晚期炎症介质在炎症(包括SAP)的发生发展中起到重要作用，HMGB1在翻译后修饰包括乙酰化、磷酸化、甲基化和ADP-核糖基化。HMGB1主动分泌过程中，乙酰化修饰扮演了很重要的角色，促进HMGB1向胞浆移位和细胞外分泌。近年的研究发现PARP-1参与了炎症过程的HMGB1核内定位和向细胞外分泌的过程，可能与HMGB1的ADP-核糖基化修饰有关。本研究旨在研究HMGB1的聚ADP-核糖基化修饰及其对乙酰化修饰的影响，探讨PARP-1调控HMGB1细胞内定位和细胞外分泌的可能机制，为PARP-1抑制剂在临床上用于抗HMGB1相关炎症的治疗提供更多的理论依据。方法：以LPS(100ng/ml)、LPS+3-AB(10mmol/l)刺激RAW264.7细胞2h和4h，用RIPA裂解法提取出细胞全蛋白；用HMGB1抗体进行免疫沉淀富集HMGB1；用PAR抗体、乙酰化-赖氨酸抗体进行Western-blot检测HMGB1的聚ADP-核糖基化和乙酰化水平。体外构PARP-1以6-生物素-17-NAD为底物对HMGB1进行聚ADP-核糖基化，用HRP标记的链霉亲和素抗体进行免疫印迹检测生物素化-ADP核糖；体外CBP以乙酰辅酶A为底物对HMGB1进行进一步的的乙酰化，用乙酰化-赖氨酸抗体进行Western-blot检测蛋白的继续乙酰化水平。结果：单用LPS刺激RAW264.7细胞活化PARP-1后，HMGB1的聚ADP-核糖基化水平明显高于对照组（0h），乙酰化水平也明显高于对照组；同时以(PARP-1活性抑制剂)3-AB刺激以后，HMGB1的聚ADP-核糖基化水平明显低于LPS组，乙酰化水平也明显低于LPS组,差异具有统计学意义(P0.05)。体外HMGB1在PARP-1作用下进行聚ADP-核糖基反应后，结果显示PARR-1组HMGB1的聚ADP-核糖基化水平明显高于control组（PARR-1热灭活对照组），，继续乙酰化水平也明显高于control组，差异具有统计学意义(P0.05)。结论：HMGB1的聚ADP-核糖基化修饰促进其乙酰化修饰。
[Abstract]:Aim: high mobility group protein 1 (HMGB1) plays an important role in the occurrence and development of inflammation (including SAP1) as an important late inflammatory mediator. HMGB1 plays an important role in posttranslational modification, including acetylation, phosphorylation, methylation and ADP- ribonylation. HMGB1 is active in the secretion of HMGB1. Acetylation plays an important role in promoting the translocation and exocrine secretion of HMGB1 to the cytoplasm. In recent years, it has been found that PARP-1 is involved in the localization and exocrine of HMGB1 in the process of inflammation, which may be related to the ADP- ribosylation modification of HMGB1. The aim of this study was to investigate the effects of HMGB1 polyADP- ribonylation modification and its effect on acetylation modification, and to explore the possible mechanism of PARP-1 regulating intracellular localization and exocrine secretion of HMGB1 cells. To provide more theoretical basis for the clinical use of PARP-1 inhibitors in the treatment of HMGB1-related inflammation. Methods: RAW264.7 cells were stimulated with LPSN 100ng / ml LPS-3-ABN 10mmol / L for 2h and 4h, the whole cell proteins were extracted by RIPA lysis method, the HMGB1 was enriched by immunoprecipitation with HMGB1 antibody, and the levels of polyADP- ribose glycosylation and acetylation of HMGB1 were detected by Western-blot with PAR antibody and acetyllysine antibody. In vitro PARP-1 was used as the substrate of 6-biotin-17-NAD to carry out polyADPribylation of HMGB1, and HRP labeled streptavidin antibody was used to detect biotinylated ADP ribose by Western blotting, and in vitro CBP used acetyl coenzyme A as substrate for further acetylation of HMGB1. The level of continuous acetylation of protein was detected by Western-blot with acetylated-lysine antibody. Results: the level of polyADP- ribonylation of HMGB1 in RAW264.7 cells activated by LPS alone was significantly higher than that in control group, and the acetylation level was significantly higher than that in control group. At the same time, the level of polyADP- ribonylation of HMGB1 was significantly lower than that of LPS group, and the level of acetylation was significantly lower than that of LPS group after stimulation with PARP-1 activity inhibitor 3-AB. The difference was statistically significant (P 0.05). The results showed that the level of polyADP- ribosylation of HMGB1 in PARR-1 group was significantly higher than that of control group, and the level of continued acetylation was significantly higher than that of control group. The results showed that the level of HMGB1 in PARR-1 group was significantly higher than that in control group, and the level of continued acetylation in control group was significantly higher than that in control group (P 0.05). Conclusion the polyADP- ribonucleylation modification of HMGB1 promotes the acetylation modification of HMGB1.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R3411

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