家蚕生长抑制蛋白ING5乙酰化修饰在杆状病毒侵染过程中的调控作用

Regulatory Role of Acetylation Modification of Silkworm Growth Inhibitor Protein ING5 in the Process of Baculovirus Infection

生长抑制蛋白5(inhibitor of growth protein 5,ING5)是生长抑制蛋白家族的成员之一,参与调节细胞周期、细胞增殖和凋亡等多种生命活动。家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)侵染家蚕卵巢细胞BmN前后的蛋白质乙酰化修饰差异组学分析结果显示,BmN细胞中的ING5蛋白有3个赖氨酸残基位点(K136、K137和K154)的乙酰化修饰水平在BmNPV感染后显著下调。为了探究ING5乙酰化修饰对其功能的影响以及在BmNPV侵染过程中的调控作用机制,首先克隆了家蚕ING5基因,并将BmNPV感染后乙酰化修饰水平显著下调的赖氨酸(K)定点突变为谷氨酰胺(Q)以模拟乙酰化修饰,突变为精氨酸(R)以模拟去乙酰化修饰。然后构建瞬时表达载体并转染BmN细胞,结果显示ING5蛋白过表达具有显著抑制细胞活力的作用,而ING5乙酰化修饰则可以显著提高细胞活力。进一步研究发现,过表达ING5蛋白具有显著促细胞凋亡作用,而ING5乙酰化修饰则显著抑制细胞凋亡。酵母双杂交试验结果显示,野生型ING5与凋亡相关蛋白P53可以互作,但ING5的乙酰化修饰影响了该互作关系;同时还发现ING5的乙酰化可显著降低P53蛋白稳定性。上述结果表明,家蚕ING5可能通过P53依赖的方式参与细胞凋亡调控,但K136、K137和K154位点的乙酰化修饰改变了ING5与P53的相互作用,进而影响细胞凋亡。研究结果将为深入解析家蚕ING5家族蛋白调控BmNPV侵染的作用机制提供试验依据,同时也可为家蚕的抗病毒育种提供新的理论依据。

Inhibitor of growth protein 5(ING5),a member of the growth inhibitory protein family,is involved in regulating various life activities such as cell cycle,cell proliferation,and apoptosis.The differential omics analysis of protein acetylation modification of Bombyx mori ovarian cell BmN before and after infection with Bombyx mori nucleopolyhedrovirus(BmNPV)revealed that the acetylation modification levels of ING5 at three lysine residues(K136,K137,and K154)were significantly down-regulated after BmNPV infection.In order to explore the impact of acetylation modification on the function of ING5 and its regulatory mechanism during the process of BmNPV infection,the ING 5 gene of silkworm was cloned.The certain lysine(K),whose acety-lation modification level was significantly reduced after BmNPV infection,was site-mutated into glutamine(Q)to simulate acetylation modification and into arginine(R)to simulate deacetylation modification.Subsequently,instantaneous expression vectors were constructed and transfected into BmN cells.The results indicated that the ING5 protein overexpression significantly inhibited cell viability,while ING5 acetylation modification significantly improved cell viability.Meanwhile,it was found that the overexpression of ING5 protein significantly promoted apoptosis,whereas ING5 acetylation significantly inhibited apoptosis.Moreover,the analysis of yeast two-hybrid demonstrated an interaction between wild-type ING5 and apoptosis-related protein P53,which could be affected by the acetylation of ING5.Furthermore,it was also proved that the acetylation of ING5 could significantly reduce the stability of P53 protein.The above results indicate that silkworm ING5 may regulate host cell apoptosis in a P53-dependent pathway,but acetylation modifications at K136,K137,and K154 sites alter the interaction between ING5 and P53,thereby affecting cell apoptosis.The research results will provide an experimental basis for further understanding the mechanism of ING5 family proteins regulating BmNPV infection and provide new theoretical basis for antiviral breeding of silkworm.