स्टेरॉयड रिसेप्टर की बायोकेमिकल पुनर्गठन • HSP90 प्रोटीन ligand के परिसर और पुनर्सक्रियण बंधन
Summary
एक<em> इन विट्रो में</em> • HSP90 प्रोटीन परिसरों शुद्ध प्रोटीन और सेलुलर lysates से कार्यात्मक glucocorticoid रिसेप्टर (जीआर) की तैयारी के लिए विधि वर्णित है. विधि पुनः संयोजक नमक के विपठ्ठन और प्रोटीन जटिल पुनर्गठन द्वारा बाद जीआर के immunoadsorption का इस्तेमाल करता. cofactors और बफर स्थितियों के महत्व पर चर्चा कर रहे हैं के रूप में संभावित विधि आवेदन कर रहे हैं.
Abstract
Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, including transcription factors (e.g. nuclear receptors, p53) and protein kinases (e.g. Src, Raf, Akt kinase) involved in cell cycling, tumorigenesis, apoptosis, and multiple eukaryotic signaling pathways 1,2. Of these many ‘client’ proteins for hsp90, the assembly of steroid receptor•hsp90 complexes is the best defined (Figure 1). We present here an adaptable glucocorticoid receptor (GR) immunoprecipitation assay and in vitro GR•hsp90 reconstitution method that may be readily used to probe eukaryotic hsp90 functional activity, hsp90-mediated steroid receptor ligand binding, and molecular chaperone cofactor requirements. For example, this assay can be used to test hsp90 cofactor requirements and the effects of adding exogenous compounds to the reconstitution process.
The GR has been a particularly useful system for studying hsp90 because the receptor must be bound to hsp90 to have an open ligand binding cleft that is accessible to steroid 3. Endogenous, unliganded GR is present in the cytoplasm of mammalian cells noncovalently bound to hsp90. As found in the endogenous GR•hsp90 heterocomplex, the GR ligand binding cleft is open and capable of binding steroid. If hsp90 dissociates from the GR or if its function is inhibited, the receptor is unable to bind steroid and requires reconstitution of the GR•hsp90 heterocomplex before steroid binding activity is restored 4 . GR can be immunoprecipitated from cell cytosol using a monoclonal antibody, and proteins such as hsp90 complexed to the GR can be assayed by western blot. Steroid binding activity of the immunoprecipitated GR can be determined by incubating the immunopellet with [3H]steroid.
Previous experiments have shown hsp90-mediated opening of the GR ligand binding cleft requires hsp70, a second molecular chaperone also essential for eukaryotic cell viability. Biochemical activity of hsp90 and hsp70 are catalyzed by co-chaperone proteins Hop, hsp40, and p23 5. A multiprotein chaperone machinery containing hsp90, hsp70, Hop, and hsp40 are endogenously present in eukaryotic cell cytoplasm, and reticulocyte lysate provides a chaperone-rich protein source 6.
In the method presented, GR is immunoadsorbed from cell cytosol and stripped of the endogenous hsp90/hsp70 chaperone machinery using mild salt conditions. The salt-stripped GR is then incubated with reticulocyte lysate, ATP, and K+, which results in the reconstitution of the GR•hsp90 heterocomplex and reactivation of steroid binding activity 7. This method can be utilized to test the effects of various chaperone cofactors, novel proteins, and experimental hsp90 or GR inhibitors in order to determine their functional significance on hsp90-mediated steroid binding 8-11.
Protocol
Discussion
ऊपर वर्णित परख hsp90/hsp70-based निगरानी मशीनरी की निगरानी कार्रवाई के रूप में अच्छी तरह के रूप में जीआर स्टेरॉयड बंधन को प्रभावित करने की स्थिति की असंख्य परीक्षण करने के लिए अनुकूलित किया जा सकता है. यह पहले की …
Declarações
The authors have nothing to disclose.
Acknowledgements
यह काम राष्ट्रीय स्वास्थ्य अनुदान GM086822, आशा है कि हार्ट इंस्टीट्यूट बेसिक साइंसेज पुरस्कार, और एम.जे. Murdock चैरिटेबल ट्रस्ट कॉलेज साइंस रिसर्च प्रोग्राम के संस्थान के द्वारा वित्त पोषित किया गया था. वैद्युतकणसंचलन अभिकर्मकों का चयन करें और छवि विश्लेषण सॉफ्टवेयर उदारता जैव रेड द्वारा प्रदान किया गया.
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 |
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