GADD45β调控MEKK4-p38MAPK信号通路在J亚群禽白血病病毒抑制细胞自噬过程中的作用机制

Subgroup J avian leukosis cause by subgroup J avian leukosis virus (ALV-J) is infectious neoplastic disease which has been seriously plaguing poultry industry. Our team is breeding purebred chicken with high ALV-J resistance in which we filtrate an essential anti-disease gene — GADD45β for study. This gene mainly involves in the courses of repairing DNA damage, and regulating cell proliferation , cell cycle and cell apoptosis. It has been proved by some research results that ALV-J can promote cell proliferation via inhibiting cell autophagy and that GADD45β exerts negative impacts on regulating the process of cell autophagy, which apparently demonstrates the intimate connection between GADD45β and ALV-J proliferation. Based on these research results, our project is to conduct thorough researches to precisely probe into the mechanism of how GADD45 regulates the cell autophagy inhibiting process of ALV-J, elaborately explore what impacts GADD45β could inflict on cell autophagy inhibiting effect of ALV-J through regulating MEKK4-p38MAPK signal path way, and further illuminate the GADD45β’s anti-ALV-J mechanism which functions through promoting cell autophagy. At the same time our group aims to study the relationship between abnormal methylation and expression of GADD45β, focusing on whether the abnormal methylation of this gene is related to its high expression in chicken with high ALV-J resistance. Our research achievements will definitely have an important theoretical and practical meaning in subgroup J avian leukosis prevention and chicken breeding with such disease resistance.

J亚群禽白血病是由ALV-J引起的一种肿瘤性疾病，严重危害养禽业。本团队通过培育对ALV-J具有高抗性的鸡，并从中筛选到一个重要的抗病基因——GADD45β，该基因主要参与调控细胞增生、细胞周期、细胞凋亡和DNA损伤修复。研究发现ALV-J可通过抑制细胞自噬来促进病毒增殖，而GADD45β在细胞自噬过程中发挥负调控作用，且与ALV-J病毒复制密切相关。基于这些研究基础，本项目将深入研究GADD45β对ALV-J抑制细胞自噬的调控机制，探讨GADD45β调控MEKK4-p38MAPK信号通路对ALV-J抑制细胞自噬的影响，阐明GADD45β通过促进细胞自噬实现其抗ALV-J病毒感染的作用机制；同时研究GADD45β异常甲基化与表达量的关系，探讨GADD45β在ALV-J抗性鸡中高表达是否与该基因本身的异常甲基化有关。本项目研究成果对J亚群禽白血病的防控和抗病育种有重要的理论和实践意义。

本项目经过4年的深入研究，圆满完成了研究任务，从分子水平上阐明了GADD45β在ALV-J影响细胞自噬和凋亡过程中的调控作用。研究发现：（1）GADD45β的表达水平在ALV-J感染过程中显著增加：细胞感染ALV-J后，不同时间点的GADD45β的表达水平均呈现上调趋势。（2）ALV-J或过表达GADD45β能显著抑制细胞自噬的发生：细胞感染ALV-J或过表达GADD45β后，细胞内LC3II/LC3I的比值显著下降，自噬体货物SQSTM1显著增加，而ATG5蛋白的表达显著降低，自噬体和溶酶体的融合受到显著抑制。（3）ALV-J通过上调GADD45β的表达激活p38 MAPK/JNK信号通路：ALV-J感染细胞中p38MAPK，JNK磷酸化水平显著上调，而ERK磷酸化水平没有发生变化；沉默GADD45β的表达，能显著抑制p38 MAPK以及JNK的磷酸化水平，并影响ALV-J介导的细胞自噬。（4）GADD45β和MEKK4能发生相互作用，进而激活p38 MAPK信号通路。免疫共沉淀和激光共聚焦显微镜实验表明GADD45β和MEKK4能发生相互作用，共定位在细胞质中，而共转染GADD45β和MEKK4能显著增强p38蛋白的磷酸化水平。（5）p38蛋白的活性与ALV-J感染抑制的细胞自噬：利用抑制剂抑制细胞内p38蛋白的活性，能显著影响自噬关键蛋白LC3II、SQSTM1和ATG5的表达，说明p38磷酸化水平能抑制ALV-J介导的细胞自噬。研究成果系统揭示了GADD45β在ALV-J增殖过程中通过与MEKK4互作，激活p38MAPK和JNK的磷酸化，进而抑制细胞自噬的作用机制。本项目共培养博士研究生1人、硕士研究生3人，在国内外学术会议或学术交流中以学术汇报或壁报形式交流研究成果。相关研究成果在Cell Death and Disease、Journal of Virology, RNA biology, Frontier in Microbiology等国际专业学术期刊发表学术论文9篇。本项目研究为深入了解ALV-J的致病机制提供了理论参考，为研究宿主蛋白参与抗病毒感染的调控开辟了新的视野。