耻垢分枝杆菌原核类泛素蛋白对异柠檬酸裂解酶的动态调控

Under the stress condition Pup dynamic regulation of Mycobacterium isocitric acid lyase

目的通过验证异柠檬酸裂解酶（isocitratelyase，Icl）蛋白在缺氧和营养缺乏等应激条件下的原核类泛素蛋白（prokaryotic ubiquitin like protein，Pup）化状态的变化，进而说明Pup一蛋白酶体系统对其进行动态调控。方法构建耻垢分枝杆菌Icl蛋白表达载体，纯化蛋白，制备Icl抗体；构建持续表达His6-Pup的穿梭载体，导人耻垢分枝杆菌中；分别在有氧，缺氧条件（2、4和6d）培养，营养缺乏条件（2、4、6和8d）培养，收集耻垢分枝杆菌菌体，超声破碎后获得总蛋白，通过Ni—NTA柱纯化，获得His6-Pup结合的蛋白；跑SDS聚丙烯酰胺凝胶电泳（SDS-PAGE），转膜后做免疫印迹（Western—blot）实验，分别用抗Pup抗体和抗Icl抗体检测，验证Icl蛋白的表达情况及其被Pup化的状况，所有实验均重复3次。结果发现在缺氧和营养缺乏条件下Icl蛋白的表达呈现增加的趋势，而Pup化的程度有下降的趋势，Pup化和蛋白浓度变化趋势是相反的。说明细胞在应激条件下需要Icl高表达，因而Pup蛋白酶体系统对其降解程度选择性的下降，相应提高了细胞内Icl蛋白的浓度，满足细胞在应激反应中的需要。结论Icl在分枝杆菌细胞的应激过程中发挥重要作用，其浓度随着细胞本身的状态而发生动态变化，这主要是Pup-蛋白酶体系统对它的降解调控发生变化，说明Pup可以动态调控Icl蛋白的浓度水平。

Objective To verify the dynamic regulation of pupylation, we check the pupylation change of isocitrate lyase （Icl） under hypoxia and low nutrient stress conditions. Methods Constructed Icl expression vector, purified protein and make polyclonal antibody of mycobacterium smegmatis Icl protein; Constructed shuttle vector continually expressed His6-Pup, transduced into myeobacterium ; cultured them separately under normal,hypoxia （2 days,4 days and 6 days） and nutrition lack （2 days,4 days, 6 days and 8 days）, collected bacterium pellets, ultrasonic lysis bacterium, collected whole protein component, then purified the binding protein of His6-Pup using Ni-NTA agarose. Ran SDS-PAGE and transfered protein to PVDF membrane, then used anti-pup and anti-Iel anti body to check the expression of Icl protein and pupylation change by western blot. All experiments were been repeated 3 times. Results We found Icl protein expression increasing under hypoxia and lack of nutrition, while at the same time the pupylation situation is the opposite. That is to say Icl high expression was necessary under stress conditions,the pupylation degree of target protein optionally was decreased, so occurred a corresponding increase in the concentration of Icl protein in cytosol, in order to satisfy the need of cells under stress conditions. Conclusions Icl plays an important role in the response to stress in mycobacterium cells,it＇s conceritration depend on the cell circumstance. The process of pupylation recognition manifested for the dynamic change,it proved pupylation can dynamic regulate the concentration level of Icl.