शोधन और amyloid प्रोटीन अग्रदूत intracellular डोमेन के एकत्रीकरण
Summary
एपीपी intracellular डोमेन (AICD) बड़े पैमाने पर की शुद्धि के लिए एक विधि का वर्णन है. हम भी पद्धति का वर्णन करने के लिए प्रेरित<em> इन विट्रो में</em> AICD और परमाणु शक्ति माइक्रोस्कोपी द्वारा एकत्रीकरण दृश्य. वर्णित विधियों का जैव रासायनिक संरचनात्मक / AICD की विशेषताओं और अपने एकत्रीकरण पर आणविक संरक्षिकाओं के प्रभाव के लिए उपयोगी होते हैं.
Abstract
Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer’s disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed α, β, and γ-secretases1. Sequential proteolytic cleavage of APP with β and γ-secretases leads to the production of a small proteolytic peptide, termed Aβ, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer’s like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.
Protocol
Discussion
इस प्रोटोकॉल में हम संरचनात्मक, biophysical और जैव रासायनिक विश्लेषण के लिए अत्यधिक शुद्ध AICD प्राप्त करने के लिए एक प्रक्रिया को रेखांकित किया है. इस प्रक्रिया परिष्कृत क्रोमैटोग्राफी उपकरण की आवश्यकता नही?…
Declarações
The authors have nothing to disclose.
Acknowledgements
लेखकों के लिए सीडीएनए एपीपी के लिए डॉ. हुई Zheng (मेडिसिन के Baylor कॉलेज) का शुक्रिया अदा करना चाहते हैं. इस काम NIH R21AG031948 अनुदान (DB, JMB), F30AG030878 (ईएसएस), R01DK073394 (AFO), जॉन बायोमेडिकल रिसर्च के लिए Sealy मेमोरियल बंदोबस्ती कोष (AFO), और जीन सी. और विलियम डी. विलिस तंत्रिका विज्ञान अनुसंधान बंदोबस्ती द्वारा वित्त पोषित किया गया था (ईएसएस). JMB translational अनुसंधान स्कॉलर प्रोग्राम में एक विद्वान और टेक्सास चिकित्सा शाखा क्लाउड ई. काली मिर्च पुराने अमेरिकियों स्वतंत्रता केंद्र विश्वविद्यालय (NIH UL1RR029876 और P30 एजी-०,२४,८३२, क्रमशः अनुदान द्वारा समर्थित) के एक सदस्य है.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| pGEX-4T-1 | GE Healthcare | 28-9545-49 | |
| Thrombin | GE Healthcare | 27-0846-01 | |
| Ampicillin | Fisher Scientific | BP1760 | |
| Bradford protein assay reagent | Bio-Rad | 500-0002 | |
| Coomassie blue | Bio-Rad | 161-0786 | |
| IPTG ( isopropyl-beta-D thiogalactopyranoside) | Sigma-Aldrich | I6758 | |
| Glutathione-agarose | Sigma-Aldrich | G4510 | |
| p-aminobenzamidine-agarose | Sigma-Aldrich | A7155 | |
| Complete protease inhibitor cocktail | Roche | 11836170001 | |
| Slide-A-Lyzer dialysis cassettes | Thermo Scientific | 66380 | |
| Chromatography columns | Evergreen Scientific | 208-3367-050 | |
| Emulsifier | Avestin, Inc | EmulsiFlex-C3 | Highly recommended |
| Eppendorf Thermomixer | Eppendorf | 022670107 | |
| Mica Disks | Ted Pella | 50-12 | |
| AFM cantilevers | Bruker | MSNL-10 | |
| WSxM software | Nanotec | N/A | Free download |
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