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

Gli autori desiderano ringraziare il Dr. Hui Zheng (Baylor College of Medicine) per la APP cDNA. Questo lavoro è stato finanziato da sovvenzioni NIH R21AG031948 (DB, JMB), F30AG030878 (ESS), R01DK073394 (AFO), il John Sealy Memorial Fondo di dotazione per la ricerca biomedica (AFO), e il C. Jean e William D. Willis Neuroscience Research Endowment (ESS). JMB è uno studioso nel Programma Translational Research Scholar e membro della University of Texas Medical Branch Claude E. Independence Pepper Centro anziani americani (supportato da sovvenzioni NIH UL1RR029876 e P30-AG-024832, rispettivamente).

<html> 1. L&#39;espressione di APP ricombinante dominio intracellulare (AICD) <ol><li> Transform E. coli ceppo BL21 con AICD umana (residui 649-695 di APP, isoforma neuronale numerazione) clonato nel vettore pGEX-4T-1 (GE Healthcare). Questo vettore esprimerà AICD la porzione C-terminale di una proteina di fusione di glutatione-S-transferasi (GST). Questo vettore codifica anche una sequenza trombina clivaggio per facilitare la rimozione della porzione GST. Dettagli della clonazione AICD in pGEX-4T-1 può esse...

<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>

In questo protocollo abbiamo delineato una procedura per ottenere AICD elevata purezza per analisi strutturali, biofisica e biochimica. Questa procedura non richiede attrezzature sofisticate cromatografia ed è quindi accessibile alla maggior parte dei laboratori. Altri gruppi hanno purificato AICD 5-7,16, compreso GST-AICD 17-19, per le analisi biochimiche / strutturale. Svantaggi ai protocolli precedenti includono scarsa solubilità di AICD 16, a meno di ideale purezza 17, e...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> pGEX-4T-1 </td><td> GE Healthcare </td><td> 28-9545-49 </td><td></td></tr><tr><td> Trombina </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 proteine ​​reagenti del dosaggio </td><td> Bio-Rad </td><td> 500-0002 </td><td></td></tr><tr><td> Coomassie blu </td><td> Bio-Rad </td><td> 161-0786 </td><td></td></tr><tr><td> IPTG (isopropil-beta-D tiogalattopiranoside) </td><td> Sigma-Aldrich </td><td> I6758 </td><td></td></tr><tr><td> Glutatione-agarosio </td><td> Sigma-Aldrich </td><td> G4510 </td><td></td></tr><tr&gt; <td> p-aminobenzamidine-agarosio </td><td> Sigma-Aldrich </td><td> A7155 </td><td></td></tr><tr><td> Completa cocktail di inibitori delle proteasi </td><td> Roche </td><td> 11836170001 </td><td></td></tr><tr><td> Slide-A-lizzatore dialisi cassette </td><td> Thermo Scientific </td><td> 66380 </td><td></td></tr><tr><td> Cromatografia colonne </td><td> Evergreen scientifico </td><td> 208-3367-050 </td><td></td></tr><tr><td> Emulsionante </td><td> Avestin, Inc </td><td> Emulsiflex-C3 </td><td> Consigliatissimo </td></tr><tr><td> Eppendorf Thermomixer </td><td> Eppendorf </td><td> 022670107 </td><td></td></tr><tr><td> Mica dischi </td><td> Ted Pella </td><td> 50-12 </td><td></td></tr><tr><td> Cantilever AFM </td><td> Bruker </td><td> MSNL-10 </td><td></td></tr><tr><td> WSxM software </td><td> Nanotec </td><td> N / A </td><td> Pee scaricare </td></tr></tbody></table>

purification and aggregation of the amyloid precursor protein intracellular domain

Proteina precursore dell&#39;amiloide (APP) è un tipo di proteina transmembrana I associato alla patogenesi della malattia di Alzheimer (AD). APP è caratterizzata da un grande dominio extracellulare e un dominio breve citosolico chiamato il dominio intracellulare di APP (AICD). Durante la maturazione attraverso la via secretoria, APP può essere scissa dalla proteasi denominate α, β, e γ-secretasi 1. Sequenziale clivaggio proteolitico di APP con β e γ-secretasi porta alla produzione di un peptide piccolo proteolitico, Ap definito, che è amiloidogena e il componente principale delle placche senili. L&#39;AICD è liberata dalla membrana dopo trasformazione secretasi, e attraverso interazioni con Fe65 e Tip60, può traslocare al nucleo di partecipare nella regolazione della trascrizione di geni bersaglio più 2,3. Proteina-proteina interazioni che coinvolgono il AICD può influenzare il traffico, l&#39;elaborazione, e le funzioni cellulari di olo-APP e il suo C-terminaL frammenti. Abbiamo recentemente dimostrato che AICD può aggregare in vitro, e questo processo è inibito dalla AD-implicato chaperone molecolare ubiquilin-1 4. Coerentemente con questi risultati, il AICD ha esposto domini idrofobici ed è intrinsecamente disordinati in vitro 5,6, tuttavia ottiene stabile struttura secondaria quando si lega al Fe65 7. Abbiamo proposto che ubiquilin-1 impedisce appropriato interazioni inter e intramolecolari di AICD, impedendo l&#39;aggregazione in vitro e in cellule intatte 4. Mentre la maggior parte degli studi si concentrano sul ruolo di APP nella patogenesi di AD, il ruolo di AICD in questo processo non è chiara. Espressione di AICD ha mostrato di indurre apoptosi 8, per modulare i percorsi di segnalazione 9 e di regolare il calcio segnalazione 10. Over-espressione di AICD e Fe65 in un modello di topo transgenico induce Alzheimer come patologia 11, e recentemente AICD è stato rilevato in reggisenonei lisati mediante Western blotting utilizzando appropriate tecniche di recupero di antigene 12. Per facilitare studi strutturali, biochimici e biofisici del AICD, abbiamo sviluppato una procedura per produrre quantità elevate di ricombinante altamente puro proteine ​​AICD. Abbiamo inoltre descrivere un metodo per indurre l&#39;aggregazione in vitro termica AICD e analisi mediante microscopia a forza atomica. I metodi descritti sono utili per la caratterizzazione biochimica, biofisica e strutturale del AICD e gli effetti di chaperone molecolare sull&#39;aggregazione AICD.

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

Un metodo per la grande purificazione del dominio intracellulare APP (AICD) è descritto. Abbiamo anche descrivere la metodologia di indurre<em&gt; In vitro</em&gt; AICD aggregazione e visualizzazione mediante microscopia a forza atomica. I metodi descritti sono utili per la caratterizzazione biochimica / strutturale del AICD e gli effetti di chaperone molecolare sulla sua aggregazione.

Purificazione e aggregazione della proteina precursore dell&#39;amiloide dominio intracellulare

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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