类固醇受体的生化重建•Hsp90的蛋白质复合物和配体激活绑定
Summary
一个<em>在体外</em>准备从纯化的蛋白质和细胞裂解液的功能性糖皮质激素受体（GR）•HSP90蛋白复合物的方法描述。该方法利用免疫吸附的盐剥离和蛋白质复合体重组重组的GR。辅助因子和缓冲条件的重要性进行了讨论，作为潜在的方法应用。
Abstract
HSP90是一个重要和非常丰富，已发现调节超过150个的真核细胞信号蛋白，包括转录因子（如核受体，P53），并参与细胞周期蛋白激酶（如SRC，英国皇家空军，Akt激酶）的分子伴侣蛋白，肿瘤的发生，细胞凋亡和多种真核细胞信号转导通路1,2。许多这些“客户”HSP90蛋白，类固醇受体大会•HSP90复合物是最好的定义（ 图1）。我们在座的适应性强的糖皮质激素受体 （GR）的免疫测定和体外GR•HSP90重组的方法，可随时用来探测HSP90真核细胞的功能活动，HSP90介导的类固醇激素受体 配体的结合，以及分子伴侣辅助因子的要求。例如，此法可用于测试HSP90的辅助因子的要求和加入外源性化合物的重组进程的影响。
遗传资源已经为研究HSP90一个特别有用的系统，必须绑定到HSP90，因为受体有一个开放的配体结合裂访问类固醇 3 。内源性的，unliganded的GR非共价的约束，以HSP90的哺乳动物细胞的细胞质中。如发现内源性GR•HSP90 heterocomplex，GR配体结合裂，是开放的和有约束力的类固醇的。如果从GR HSP90不赞成或如果其功能受到抑制，受体是无法绑定类固醇和需要重组的GR•HSP90 heterocomplex的结合活性类固醇之前恢复 4 。遗传资源，可以从细胞细胞质使用单克隆抗体免疫沉淀，如HSP90蛋白复合物，以遗传资源，可以通过免疫印迹检测。免疫沉淀的GR结合活性的类固醇，可以由孵化与[3H]类固醇immunopellet 。
以前的实验已经证明HSP90介导的GR配体裂约束力的开放需要HSP70，第二个分子伴侣，也为真核细胞的活力至关重要。 HSP90和HSP70的生化活性催化合作伴侣蛋白合，hsp40，和P23 5 。一个多蛋白的伴侣机械HSP90，HSP70，合，和hsp40是在真核细胞的细胞质内源性目前，网织红细胞裂解液提供了一个伴侣，含有丰富的蛋白质来源6。
提出的方法，GR是immunoadsorbed从细胞细胞质和内源性hsp90/hsp70伴侣用温和的盐条件下的机械剥离。盐剥离的GR然后孵育•HSP90 heterocomplex和类固醇的结合活性的激活7，网织红细胞裂解液，三磷酸腺苷，和K +，这在重组的GR结果。可以利用这种方法来测试各种伴侣的辅助因子，新的蛋白质，和实验HSP90或GR抑制剂的影响，以确定其HSP90介导的类固醇具有约束力8-11的功能意义。
Protocol
1。制备细胞细胞质中含有功能的GR 技术说明：所有的缓冲区应冷藏，此协议，包括离心和孵化，每一步应在冰上或在4 ° C，除非另有说明。低温是必不可少的，以防止GR和蛋白质复合物的降解。 使用冷冻离心机，取得了〜1-5毫升颗粒细胞功能的GR表达了高浓度。在遗传资源丰富的细胞来源的例子包括：小鼠成纤维L929细胞，它表达的内源性遗传资源的高浓度，并昆虫重组GR杆状病毒?…
Discussion
上文所述的检测可以适应测试无数影响hsp90/hsp70-based伴侣机械伴侣行动以及GR类固醇具有约束力的条件。这是以前报道的方法 4,8,9,17的修改，旨在充分利用电泳技术的最新进展，并接触到更广泛的研究界。可以添加额外的辅助因子或蛋白质的利益，在GR•HSP90 heterocomplex重组的混合物，在本议定书第4步中所述，以观察对类固醇的约束力，heterocomplex蛋白质相互作用，并共同因素的要求影?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作是由国家健康授予GM086822，希望心脏研究所的基础科学奖，兆焦耳默多克慈善信托基金科学学院研究计划研究院资助。选择电泳试剂和图像分析软件Bio – Rad公司提供的慷慨。
Materials
| Name of the reagent | Company | Catalogue number | Comments |
|---|---|---|---|
| Sf9 insect cells | Invitrogen | for baculovirus expression of recombinant mouse GR | |
| L929 cells | ATCC | CRL-2173 | for endogenous mouse GR cytosol preparation (alternative to baculovirus expression) |
| FiGR hybridoma cells | ATCC | CRL-2173 | for preparing ascites containing anti-GR monoclonal antibody (alternative to purified BuGR2 antibody) |
| Complete-Mini protease inhibitor, EDTA-free | Roche Applied Science | 11836170001 | |
| protein A-Sepharose | Sigma-Aldrich | P3391 | |
| rabbit reticulocyte lysate | Green Hectares (Oregon, WI) | rich source of endogenous hsp90/hsp70 chaperone machinery | |
| creatine phosphokinase | Sigma-Aldrich | C3755 | for ATP-regenerating system |
| phosphocreatine | Sigma-Aldrich | P7936 | for ATP-regenerating system |
| anti-GR mouse monoclonal antibody (BuGR2) | Pierce/Thermo Scientific | MA1-510 | used for GR immunoadsorption and as primary antibody in western blotting |
| anti-hsp90 mouse monoclonal antibody (AC88) | Enzo Life Sciences | ADI-SPA-830 | used as primary antibody in western blotting |
| anti-hsp70 mouse monoclonal antibody (N27F3-4) | Enzo Life Sciences | ADI-SPA-820 | used as primary antibody in western blotting |
| IgG from mouse serum | Sigma-Aldrich | 038K7690 | used as nonimmune antibody for negative control of immunoadsorption |
| anti-mouse IgG-peroxidase conjugate | Sigma-Aldrich | A4416 | used as secondary antibody in western blotting |
| Experion microfluidic electrophoresis system | Bio-Rad | 7007001 | alternative to conventional SDS-PAGE |
| [1,2,4,6,7-3H] dexamethasone | PerkinElmer | NET1192001MC | follow safety precautions |
References
- Pratt, W. B., Toft, D. O. Regulation of signaling protein function and trafficking by the hsp90/hsp70-based chaperone machinery. Exp. Biol. Med. 228, 111-133 (2003).
- Murphy, P. J. M. Regulation of glucocorticoid receptor steroid binding and trafficking by the hsp90/hsp70-based chaperone machinery: implications for clinical intervention. Leukemia. 19, 710-712 (2005).
- Pratt, W. B., Morishima, Y., Osawa, Y. The Hsp90 chaperone machinery regulates signaling by modulating ligand binding clefts. J. Biol. Chem. 283, 22885-22889 (2008).
- Dittmar, K. D., Hutchison, K. A., Owens-Grillo, J. K., Pratt, W. B. Reconstitution of the steroid receptor•hsp90 heterocomplex assembly system of rabbit reticulocyte lysate. J. Biol. Chem. 271, 12833-12839 (1996).
- Morishima, Y. The Hsp organizer protein hop enhances the rate of but is not essential for glucocorticoid receptor folding by the multiprotein Hsp90-based chaperone system. J. Biol. Chem. 275, 6894-6900 (2000).
- Murphy, P. J. M., Kanelakis, K. C., Galigniana, M. D., Morishima, Y., Pratt, W. B. Stoichiometry, abundance, and functional significance of the hsp90/hsp70-based multiprotein chaperone machinery in reticulocyte lysate. J. Biol. Chem. 276, 30092-30098 (2001).
- Dittmar, K. D., Pratt, W. B. Folding of the glucocorticoid receptor by the reconstituted Hsp90-based chaperone machinery. The initial hsp90.p60.hsp70-dependent step is sufficient for creating the steroid binding conformation. J. Biol. Chem. 272, 13047-13054 (1997).
- Kanelakis, K. C. Differential effects of the hsp70-binding protein BAG-1 on glucocorticoid receptor folding by the hsp90-based chaperone machinery. J. Biol. Chem. 274, 34134-34140 (1999).
- Morishima, Y., Kanelakis, K. C., Murphy, P. J. M., Shewach, D. S., Pratt, W. B. Evidence for iterative ratcheting of receptor-bound hsp70 between its ATP and ADP conformations during assembly of glucocorticoid receptor.hsp90 heterocomplexes. Biochimie. 40, 1109-1116 (2001).
- Murphy, P. J. M. Pifithrin-alpha inhibits p53 signaling after interaction of the tumor suppressor protein with hsp90 and its nuclear translocation. J. Biol. Chem. 279, 30195-30201 (2004).
- Murphy, P. J. M., Morishima, Y., Kovacs, J. J., Yao, T. P., Pratt, W. B. Regulation of the dynamics of hsp90 action on the glucocorticoid receptor by acetylation/deacetylation of the chaperone. J. Biol. Chem. 280, 33792-33799 (2005).
- Morishima, Y., Murphy, P. J. M., Li, D. P., Sanchez, E. R., Pratt, W. B. Stepwise assembly of a glucocorticoid receptor•hsp90 heterocomplex resolves two sequential ATP-dependent events involving first hsp70 and then hsp90 in opening of the steroid binding pocket. J. Biol. Chem. 275, 18054-18060 (2000).
- Pratt, W. B., Toft, D. O. Steroid receptor interactions with heat shock protein and immunophilin chaperones. Endocr. Rev. 18, 306-360 (1997).
- Bodwell, J. E. Identification of phosphorylated sites in the mouse glucocorticoid receptor. J. Biol. Chem. 266, 7549-7555 (1991).
- Penna, A., Cahalan, M. W. e. s. t. e. r. n. Blotting using the Invitrogen NuPage Novex Bis Tris minigels. J. Vis. Exp. (7), e264-e264 (2007).
- Choo, Y. S., Zhang, Z. Detection of Protein Ubiquitination. J. Vis. Exp. (30), e1293-e1293 (2009).
- Murphy, P. J. M. Visualization and mechanism of assembly of a glucocorticoid receptor•Hsp70 complex that is primed for subsequent Hsp90-dependent opening of the steroid binding cleft. J. Biol. Chem. 278, 34764-34773 (2003).
- Schrum, A. G. High-sensitivity detection and quantitative analysis of native protein-protein interactions and multiprotein complexes by flow cytometry. Sci STKE. 389, pl2-pl2 (2007).
- Davis, T. R., Schrum, A. G. IP-FCM: Immunoprecipitation Detected by Flow Cytometry. J. Vis. Exp. (46), e2066-e2066 (2010).
- Felts, S. J., Karnitz, L. M., Toft, D. O. Functioning of the Hsp90 machine in chaperoning checkpoint kinase I (Chk1) and the progesterone receptor (PR). Cell Stress Chaperones. 12, 353-363 (2007).
- Cintron, N. S., Toft, D. Defining the requirements for Hsp40 and Hsp70 in the Hsp90 chaperone pathway. J. Biol. Chem. 281, 26235-26244 (2006).
Tags
Biochemical ReconstitutionSteroid ReceptorHsp90 Protein ComplexesLigand BindingMolecular ChaperoneEukaryotic Signaling ProteinsTranscription FactorsProtein KinasesCell CyclingTumorigenesisApoptosisSignaling PathwaysGlucocorticoid Receptor (GR)Immunoprecipitation AssayIn Vitro Reconstitution MethodHsp90 Functional ActivitySteroid Receptor Ligand BindingMolecular Chaperone Cofactor Requirements
Citer Cet Article
Murphy, P. J. M., Franklin, H. R., Furukawa, N. W. Biochemical Reconstitution of Steroid Receptor•Hsp90 Protein Complexes and Reactivation of Ligand Binding. J. Vis. Exp. (55), e3059, doi:10.3791/3059 (2011).