共伴侣分子Cdc37从人热休克蛋白90复合体解离中ATP的作用机制  

Identification of the mechanisms related to ATP-driven dissociation of co-chaperone Cdc37 from naturally assembled human HSP90 complex based on IP-FCM

作　　者：曹帆帆[1] 张登海[1] 徐莉敏[2] 池永斌[1] 王莹 李波静[3] CAO Fanfan;ZHANG Denghai;XU Limin;CHI Yongbin;WANG Ying;LI Bojing(Shanghai Health Commission Key Lab of Artificial Intelligence(AI)-Based Management of Inflammation and Chronic Diseases Laboratory,Shanghai Pudong Gongli Hospital,Shanghai 200135,China;Clinical Laboratory,Shanghai Pudong Gongli Hospital,Shanghai 200135,China;Department of Gastroenterology,Shanghai Pudong Gongli Hospital,Shanghai 200135,China)

机构地区：[1]上海市浦东新区公利医院上海市卫健委炎症与慢病管理人工智能重点实验室,上海200135 [2]上海市浦东新区公利医院检验科,上海200135 [3]上海市浦东新区公利医院消化内科,上海200135

出　　处：《中国研究型医院》2022年第1期47-53,共7页Chinese Research Hospitals

基　　金：上海市浦东新区卫生系统学科带头人培养计划资助(PWRd2020-04);上海市浦东新区卫生科技面上项目资助(PW2020A-16);上海市浦东新区卫生系统特色专病项目资助(PWZzb2017-08)。

摘　　要：目的探讨ATP导致共伴侣分子细胞分裂周期蛋白37(Cdc37)从人热休克蛋白90(HSP90)复合体解离的可能机制,为抗肿瘤新药研发提供理论依据。方法用免疫共沉淀法(IP)捕获人组织淋巴瘤细胞(U937细胞)中HSP90复合体后,进行以下实验:(1)用流式细胞术(FCM)检测经过ADP、17-烯丙基胺格尔德霉素(17-AAG)、亚胺二磷酸腺苷(AMPPNP)、ATP处理后HSP90复合体中的Cdc37含量,并分别与经二甲基亚砜(DMSO)处理的对照组进行比较,研究ATP导致Cdc37从HSP90复合体解离是单纯由ATP结合导致还是依赖ATP水解;(2)用FCM检测对照组及ATP处理后复合体中Cdc37和其他共伴侣分子中S期激酶关联蛋白1 G2等位基因抑制因子(Sgt1)、HSP70相互作用蛋白(HIP)、FK-506结合蛋白(FKBP5)、应激诱导蛋白1(STIP1)、热休克蛋白90腺苷三磷酸酶同系物1激活剂(AHSA1)的变化情况,并用IP-westernblot(IP-WB)进行验证,研究ATP导致Cdc37从HSP90复合体解离是否伴随其他共伴侣分子解离;(3)用IP-WB检测对照组及ATP处理组复合体中HSP90、Cdc37及共伴侣分子Sgt1的磷酸化水平;用FCM检测对照组、ATP组及蛋白激酶抑制剂组复合体中的Cdc37及Sgt1的含量。研究ATP导致Cdc37从HSP90复合体解离是否伴随HSP90、Cdc37及其他共伴侣分子磷酸化。结果与对照组相比,ATP处理后HSP90复合体中Cdc37含量显著下降,而ADP、AMPPNP及17-AAG处理对Cdc37无影响;HSP90复合体中加入ATP导致复合体中Cdc37解离,伴随复合体共伴侣分子Sgt1含量降低;HSP90复合体中加入ATP后,复合体中Cdc37、Sgt1磷酸化水平显著增加。而在HSP90复合体中加入蛋白激酶抑制剂后,会阻断ATP导致的Cdc37及Sgt1与复合体解离。结论HSP90复合体中ATP导致Cdc37解离的可能机理是:(1)ATP结合HSP90导致该复合体构象改变;(2)ATP导致Sgt1与该复合体解离,且在这个过程中伴随Cdc37与Sgt1的磷酸化增加。特异性抑制Cdc37和HSP90结合,是抗肿瘤新药研究的方向之一Objective To discuss the molecular basis for Cdc37 dissociation from naturally assembled human HSP90 complex by ATP.This research will provide the basis for the study of new anticancer drugs.Methods HSP90 complex from human monocytic leukemia cell line U937 cell was captured by Immunoprecipitation(IP),then the following experiments were conducted:(1)cytometry(FCM)was used to measure Cdc37 expression in HSP90 complex after adding adenosine diphosphate(ADP),17-N-allylamino-17-demethoxygeldanamycin(17-AAG),Adenosine-5'-[(β,γ)-imido]triphosphate,(AMPPNP),adenosine triphosphate(ATP)into the complex,and these results were compared with that of the group of"con"which was treated by dimethyl sulfoxide(DMSO)to research the Cdc37 dissociation from naturally assembled human HSP90 complex by ATP was depending on ATP binding or ATP hydrolysis.(2)The effects of ATP on the Cdc37 and other co-chaperones,suppressor of G2 allele of skp1(Sgt1),Hsp70-interacting protein(HIP),FK506-binding protein 5(FKBP5),stress-inducible protein 1(STIP1),activator of 90 kDa heat shock protein ATPase homolog 1(AHSA1),expression in HSP90 complex were measured by IP-FCM,and were confirming by western blot,and this will affirm whether or not Cdc37 dissociation from naturally assembled human HSP90 complex by ATP is accompanied by the dissociation of other co-chaperones.(3)Phosphorylation of HSP90,Cdc37 and Sgt1 in HSP90 complex were measured by western blot after DMSO or ATP treatment.Cdc37 and Sgt1 expression in HSP90 complex were measured by IP-FCM after treating by DMSO,ATP or protein Kinase inhibitor.This result will clarify whether Cdc37 dissociation from naturally assembled human HSP90 complex by ATP is accompanied by phosphorylation of HSP90,Cdc37 or other co-chaperones or not.Results Compared with the control group,the expressions of Cdc37 in HSP90 complex was markedly decreased by ATP,thus ADP,AMPPNP and 17-AAG have no effects on Cdc37 expression.Cdc37 dissociation from naturally assembled human HSP90 complex by ATP was accompanied by reducti

关 键 词：HSP90热休克蛋白质类 腺苷三磷酸 药理作用分子作用机制 免疫沉淀法 解离 

分 类 号：R965[医药卫生—药理学]