Name: purification and aggregation of the amyloid precursor protein intracellular domain
Uploaded: 2012-08-28T12:00:00
Description: Watch this Scientific Journal Video about Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain at JoVE.com

Forfatterne vil gerne takke Dr. Hui Zheng (Baylor College of Medicine) for APP cDNA. Dette arbejde blev finansieret af NIH tilskud R21AG031948 (DB, JMB), F30AG030878 (ESS), R01DK073394 (AFO), John Sealy Memorial Endowment Fund for Biomedical Research (AFO) og Jean C. og William D. Willis Neuroscience Research Endowment (ESS). JMB er en lærd i Translationel Forskning Scholar Program og et medlem fra University of Texas Medical Branch Claude E. Pepper Ældre amerikanere Independence Center (støttet af NIH Tilskud UL1RR029876 og P30-AG-024.832, henholdsvis).

<html> 1. Ekspression af rekombinant APP intracellulært domæne (AICD) <ol><li> Transformere E. coli-stamme BL21 med human AICD (resterne 649-695 af APP, neuronal isoform nummerering) klonet i pGEX-4T-1 (GE Healthcare). Denne vektor vil udtrykke AICD som den C-terminale del af et fusionsprotein med glutathion-S-transferase (GST). Denne vektor koder også en thrombinspaltning sekvens for at lette fjernelse af GST-delen. Detaljer vedrørende kloning AICD til pGEX-4T-1 findes i vores tidligere publikation 4...

<ol>
	<li>De Strooper, B., Vassar, R., Golde, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+secretases:+enzymes+with+therapeutic+potential+in+Alzheimer+disease.">The secretases: enzymes with therapeutic potential in Alzheimer disease.</a> Nature reviews. Neurology. 6, 99-107 (2010).</li><li>Chang, K. A., Suh, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possible+roles+of+amyloid+intracellular+domain+of+amyloid+precursor+protein.">Possible roles of amyloid intracellular domain of amyloid precursor protein.</a> BMB reports. 43, 656-663 (2010).</li><li>McLoughlin, D. M., Miller, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+FE65+proteins+and+Alzheimer's+disease.">The FE65 proteins and Alzheimer's disease.</a> J. Neurosci. Res. 86, 744-754 (2008).</li><li>Stieren, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquilin-1+is+a+molecular+chaperone+for+the+amyloid+precursor+protein.">Ubiquilin-1 is a molecular chaperone for the amyloid precursor protein.</a> J. Biol. Chem. 286, 35689-35698 (2011).</li><li>Ramelot, T. A., Nicholson, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation-induced+structural+changes+in+the+amyloid+precursor+protein+cytoplasmic+tail+detected+by+NMR.">Phosphorylation-induced structural changes in the amyloid precursor protein cytoplasmic tail detected by NMR.</a> J. Mol. Biol. 307, 871-884 (2001).</li><li>Ramelot, T. A., Gentile, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+structure+of+the+amyloid+precursor+protein+cytoplasmic+tail+indicates+preordering+of+structure+for+binding+to+cytosolic+factors.">Transient structure of the amyloid precursor protein cytoplasmic tail indicates preordering of structure for binding to cytosolic factors.</a> Biochem. 39, 2714-2725 (2000).</li><li>Radzimanowski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+intracellular+domain+of+the+amyloid+precursor+protein+in+complex+with+Fe65-PTB2.">Structure of the intracellular domain of the amyloid precursor protein in complex with Fe65-PTB2.</a> EMBO Rep. 9, 1134-1140 (2008).</li><li>Ohkawara, T., Nagase, H., Koh, C. S., Nakayama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amyloid+precursor+protein+intracellular+domain+alters+gene+expression+and+induces+neuron-specific+apoptosis.">The amyloid precursor protein intracellular domain alters gene expression and induces neuron-specific apoptosis.</a> Gene. 475, 1-9 (2011).</li><li>von Rotz, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+APP+intracellular+domain+forms+nuclear+multiprotein+complexes+and+regulates+the+transcription+of+its+own+precursor.">The APP intracellular domain forms nuclear multiprotein complexes and regulates the transcription of its own precursor.</a> J. Cell Sci. 117, 4435-4448 (2004).</li><li>Hamid, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+precursor+protein+intracellular+domain+modulates+cellular+calcium+homeostasis+and+ATP+content.">Amyloid precursor protein intracellular domain modulates cellular calcium homeostasis and ATP content.</a> J. Neurochem. 102, 1264-1275 (2007).</li><li>Ghosal, K., Stathopoulos, A., Pimplikar, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=APP+intracellular+domain+impairs+adult+neurogenesis+in+transgenic+mice+by+inducing+neuroinflammation.">APP intracellular domain impairs adult neurogenesis in transgenic mice by inducing neuroinflammation.</a> PLoS ONE. 5, e11866 (2010).</li><li>Pimplikar, S. W., Suryanarayana, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+APP+intracellular+domain+in+brain+tissue.">Detection of APP intracellular domain in brain tissue.</a> Met. Molecul. Biol. 670, 85-91 (2011).</li><li>Buchner, J., Grallert, H., Jakob, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+chaperone+function+using+citrate+synthase+as+nonnative+substrate+protein.">Analysis of chaperone function using citrate synthase as nonnative substrate protein.</a> Met. Enzymol. 290, 323-338 (1998).</li><li>Hansma, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+atomic+force+microscopy+of+DNA.">Recent advances in atomic force microscopy of DNA.</a> Scanning. 15, 296-299 (1993).</li><li>Valbuena, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quasi-simultaneous+imaging/pulling+analysis+of+single+polyprotein+molecules+by+atomic+force+microscopy.">Quasi-simultaneous imaging/pulling analysis of single polyprotein molecules by atomic force microscopy.</a> Rev. Sci. Instrum. 78, 113707 (2007).</li><li>Radzimanowski, J., Beyreuther, K., Sinning, I., Wild, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overproduction,+purification,+crystallization+and+preliminary+X-ray+analysis+of+human+Fe65-PTB2+in+complex+with+the+amyloid+precursor+protein+intracellular+domain.">Overproduction, purification, crystallization and preliminary X-ray analysis of human Fe65-PTB2 in complex with the amyloid precursor protein intracellular domain.</a> Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 64, 409-412 (2008).</li><li>Chen, T. Y., Liu, P. H., Ruan, C. T., Chiu, L., Kung, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intracellular+domain+of+amyloid+precursor+protein+interacts+with+flotillin-1,+a+lipid+raft+protein.">The intracellular domain of amyloid precursor protein interacts with flotillin-1, a lipid raft protein.</a> Biochem. Biophys. Res. Commun. 342, 266-272 (2006).</li><li>Kim, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Notch1+signaling+by+the+APP+intracellular+domain+facilitates+degradation+of+the+Notch1+intracellular+domain+and+RBP-Jk.">Regulation of Notch1 signaling by the APP intracellular domain facilitates degradation of the Notch1 intracellular domain and RBP-Jk.</a> J. Cell Sci. 124, 1831-1843 (2011).</li><li>Lazarov, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+transport,+amyloid+precursor+protein,+kinesin-1,+and+the+processing+apparatus:+revisited.">Axonal transport, amyloid precursor protein, kinesin-1, and the processing apparatus: revisited.</a> J. Neurosci. 25, 2386-2395 (2005).</li><li>Kakuda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equimolar+production+of+amyloid+beta-protein+and+amyloid+precursor+protein+intracellular+domain+from+beta-carboxyl-terminal+fragment+by+gamma-secretase.">Equimolar production of amyloid beta-protein and amyloid precursor protein intracellular domain from beta-carboxyl-terminal fragment by gamma-secretase.</a> J Biol. Chem. 281, 14776-14786 (2006).</li><li>Gosal, W. S., Myers, S. L., Radford, S. E., Thomson, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+under+the+atomic+force+microscope.">Amyloid under the atomic force microscope.</a> Protein Pept. Lett. 13, 261-270 (2006).</li></ol>

I denne protokol har vi skitseret en procedure for opnåelse af yderst ren AICD for strukturelle, biofysik, og biokemiske analyser. Denne procedure kræver ikke sofistikeret chromatografi udstyr og er derfor tilgængelig for de fleste laboratorier. Andre grupper har oprenset AICD 5-7,16, herunder GST-AICD 17-19, for biokemiske / strukturelle analyser. Ulemper til tidligere protokoller omfatter dårlig opløselighed af AICD 16, mindre end ideel renhed 17, og kravet om størrels...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> pGEX-4T-1 </td><td> GE Healthcare </td><td> 28-9545-49 </td><td></td></tr><tr><td> Thrombin </td><td> GE Healthcare </td><td> 27-0846-01 </td><td></td></tr><tr><td> Ampicillin </td><td> Fisher Scientific </td><td> BP1760 </td><td></td></tr><tr><td> Bradford Protein Assay Reagent </td><td> Bio-Rad </td><td> 500-0002 </td><td></td></tr><tr><td> Coomassie blue </td><td> Bio-Rad </td><td> 161-0786 </td><td></td></tr><tr><td> IPTG (isopropyl-beta-D-thiogalactopyranosid) </td><td> Sigma-Aldrich </td><td> I6758 </td><td></td></tr><tr><td> Glutathion-agarose </td><td> Sigma-Aldrich </td><td> G4510 </td><td></td></tr><tr&gt; <td> p-aminobenzamidin-agarose </td><td> Sigma-Aldrich </td><td> A7155 </td><td></td></tr><tr><td> Complete protease inhibitor cocktail </td><td> Roche </td><td> 11836170001 </td><td></td></tr><tr><td> Slide-A-Lyzer dialyse kassetter </td><td> Thermo Scientific </td><td> 66.380 </td><td></td></tr><tr><td> Kromatografisøjler </td><td> Evergreen Scientific </td><td> 208-3367-050 </td><td></td></tr><tr><td> Emulgator </td><td> Avestin, Inc. </td><td> EmulsiFlex-C3 </td><td> Stærkt anbefales </td></tr><tr><td> Eppendorf Thermomixer </td><td> Eppendorf </td><td> 022670107 </td><td></td></tr><tr><td> Mica Disks </td><td> Ted Pella </td><td> 50-12 </td><td></td></tr><tr><td> AFM køreledningsophæng </td><td> Bruker </td><td> MSNL-10 </td><td></td></tr><tr><td> WSxM software </td><td> Nanotec </td><td> N / A </td><td> Free downloades </td></tr></tbody></table>

purification and aggregation of the amyloid precursor protein intracellular domain

Amyloidprecursorprotein (APP) er et type I transmembrant protein associeret med patogenesen af ​​Alzheimers sygdom (AD). APP er kendetegnet ved et stort ekstracellulært domæne og en kort cytosolisk domæne kaldet APP intracellulære domæne (AICD). Under modning gennem den sekretoriske sti, kan APP spaltes af proteaser kaldet α, β, og γ-sekretaser 1. Sekventiel proteolytisk spaltning af APP med β-og γ-sekretaser fører til produktion af en lille proteolytisk peptid, betegnet Ap, som er amyloidogene og kernen bestanddel af senile plaques. The AICD er også frigøres fra membranen efter sekretasebearbejdning, og gennem interaktioner med Fe65 og Tip60, kan translokere til kernen for at deltage i transskriptionsregulering af flere målgener 2,3. Protein-protein interaktioner, der involverer AICD kan påvirke handelen, forarbejdning og cellulære funktioner i holo-APP og dets C-terminal-fragmenter. Vi har for nylig vist, at AICD kan aggregere in vitro, og denne fremgangsmåde inhiberes af AD-impliceret molekylchaperone ubiquilin-1 4. I overensstemmelse med disse resultater har AICD eksponerede hydrofobe domæner og er uløseligt uorden in vitro 5,6, men det opnår en stabil sekundær struktur, når det er bundet til Fe65 7. Vi har foreslået at ubiquilin-1 forhindrer uhensigtsmæssige inter-og intramolekylære interaktioner af AICD, forhindrer aggregering in vitro og i intakte celler 4. Mens de fleste undersøgelser fokuserer på den rolle APP i patogenesen af ​​AD, hvilken rolle AICD i denne proces er ikke klar. Ekspression af AICD er blevet vist at inducere apoptose 8, til at modulere signaleringsveje 9, og at regulere calcium signalering 10. Over-ekspression af AICD og Fe65 i en transgen musemodel inducerer Alzheimers som patologi 11, og for nylig AICD er blevet påvist i brai lysater ved Western blotting, når du bruger passende antigen hentning teknikker 12. For at lette strukturelle, biokemiske og biofysiske undersøgelser af AICD, har vi udviklet en fremgangsmåde til fremstilling af rekombinant store mængder yderst rent AICD protein. Vi beskriver yderligere en fremgangsmåde til induktion af in vitro termiske aggregering af AICD og analyse ved atomic force mikroskopi. De beskrevne fremgangsmåder er nyttige til biokemiske, biofysiske og strukturelle karakterisering af AICD og virkningerne af molekylære chaperoner til AICD aggregering.

Watch this Scientific Journal Video about Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain at JoVE.com

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

En fremgangsmåde til stor skala oprensning af APP intracellulære domæne (AICD) er beskrevet. Vi beskriver også metoder til at fremkalde<em&gt; In vitro</em&gt; AICD sammenlægning og visualisering ved atomic force mikroskopi. De beskrevne fremgangsmåder er nyttige til biokemiske / strukturel karakterisering af AICD og virkningerne af molekylære chaperoner på dens aggregering.

Oprensning og Aggregering af amyloidprecursorprotein intracellulært domæne

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

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