Name: photo-induced cross-linking of unmodified proteins (picup) applied to amyloidogenic peptides
Uploaded: 2009-01-12T00:00:00
Description: Watch this Scientific Journal Video about Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides at JoVE.com

Questo lavoro è stato sostenuto dalle concessioni AG027818 e AG030709 dal NIH / NIA, 2005/2E dalla L. Larry Hillblom Foundation, IIRG-07-58334 dall&#39;Associazione Alzheimer, e 07-65798 dalla California Department of Public Health.

1. Peptide preparazione <ol><li> Pesare ~ 100-200 mg di peptide liofilizzato utilizzando una microbilancia e trasferimento in etichetta, siliconato, a basso adsorbente tubi microcentrifuga. Qui, usiamo le sequenze umano di Aβ40, Aβ42, calcitonina, e GRF. </li><li> Qui, usiamo peptidi pre-trattati con 1,1,1,3,3,3-esafluoro-2-propanolo (HFIP) per ottenere omogeneo, aggregato senza preparazioni. Questo passaggio è necessario perché preformato aggregati indurre una rapida aggregazione di proteine ​​amiloidogenica,...

<ol>
	<li>Bitan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+study+of+metastable+amyloidogenic+protein+oligomers+by+photo-induced+cross-linking+of+unmodified+proteins.">Structural study of metastable amyloidogenic protein oligomers by photo-induced cross-linking of unmodified proteins.</a> Methods Enzymol. 413, 217-236 (2006).</li><li>Fancy, D. A., Kodadek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry+for+the+analysis+of+protein-protein+interactions:+rapid+and+efficient+cross-linking+triggered+by+long+wavelength+light.">Chemistry for the analysis of protein-protein interactions: rapid and efficient cross-linking triggered by long wavelength light.</a> Proc. Natl. Acad. Sci. USA. 96, 6020-6024 (1999).</li><li>Kluger, R., Alagic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+cross-linking+and+protein-protein+interactions&#8212;a+review+with+illustrative+protocols.">Chemical cross-linking and protein-protein interactions&#8212;a review with illustrative protocols.</a> Bioorg. Chem. 32, 451-472 (2004).</li><li>Tomohiro, T., Hashimoto, M., Hatanaka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-linking+chemistry+and+biology:+development+of+multifunctional+photoaffinity+probes.">Cross-linking chemistry and biology: development of multifunctional photoaffinity probes.</a> Chem. Rec. 5, 385-395 (2005).</li><li>Bitan, G., Fradinger, E. A., Spring, S. M., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotoxic+protein+oligomers&#8212;what+you+see+is+not+always+what+you+get.">Neurotoxic protein oligomers&#8212;what you see is not always what you get.</a> Amyloid. 12, 88-95 (2005).</li><li>Bitan, G., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+aggregate-free,+low+molecular+weight+amyloid-&#946;+for+assembly+and+toxicity+assays.">Preparation of aggregate-free, low molecular weight amyloid-&#946; for assembly and toxicity assays.</a> Methods Mol. Biol. 299, 3-9 (2005).</li><li>Bitan, G., Lomakin, A., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+&#946;-protein+oligomerization:+prenucleation+interactions+revealed+by+photo-induced+cross-linking+of+unmodified+proteins.">Amyloid &#946;-protein oligomerization: prenucleation interactions revealed by photo-induced cross-linking of unmodified proteins.</a> J. Biol. Chem. 276, 35176-35184 (2001).</li><li>Bitan, G., Kirkitadze, M. D., Lomakin, A., Vollers, S. S., Benedek, G. B., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+&#946;-protein+(A&#946;)+assembly:+A&#946;40+and+A&#946;42+oligomerize+through+distinct+pathways.">Amyloid &#946;-protein (A&#946;) assembly: A&#946;40 and A&#946;42 oligomerize through distinct pathways.</a> Proc. Natl. Acad. Sci. USA. 100, 330-335 (2003).</li><li>Hepler, R. W., Grimm, K. M., Nahas, D. D., Breese, R., Dodson, E. C., Acton, P., Keller, P. M., Yeager, M., Wang, H., Shughrue, P., Kinney, G., Joyce, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution+state+characterization+of+amyloid+&#946;-derived+diffusible+ligands.">Solution state characterization of amyloid &#946;-derived diffusible ligands.</a> Biochemistry (Mosc). 45, 15157-15167 (2006).</li><li>Bitan, G., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+photochemical+cross-linking&#8212;a+new+tool+for+studies+of+metastable,+amyloidogenic+protein+assemblies).">Rapid photochemical cross-linking&#8212;a new tool for studies of metastable, amyloidogenic protein assemblies).</a> Acc. Chem. Res. 37, 357-364 (2004).</li><li>Piening, N., Weber, P., Hogen, T., Beekes, M., Kretzschmar, H., Giese, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photo-induced+crosslinking+of+prion+protein+oligomers+and+prions.">Photo-induced crosslinking of prion protein oligomers and prions.</a> Amyloid. 13, 67-77 (2006).</li><li>Li, H. T., Lin, X. J., Xie, Y. Y., Hu, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+early+events+of+&#945;-synuclein+oligomerization+revealed+by+photo-induced+cross-linking.">The early events of &#945;-synuclein oligomerization revealed by photo-induced cross-linking.</a> Protein Pept. Lett. 13, 385-390 (2006).</li><li>Vollers, S. S., Teplow, D. B., Bitan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Peptide+oligomerization+state+using+rapid+photochemical+crosslinking.">Determination of Peptide oligomerization state using rapid photochemical crosslinking.</a> Methods Mol. Biol. 299, 11-18 (2005).</li></ol>

PICUP è stato sviluppato originariamente per studiare complessi proteici stabili 2. Il metodo è stato applicato in seguito allo studio quantitativo delle assemblee metastabili proteina amiloide, tra cui Aβ 10, prioni e le malattie associate PrP Sc 11 e α-sinucleina 12. I fattori più importanti che devono essere considerati quando si progetta un esperimento PICUP sono la stechiometria dei reagenti, tempo di irradiazione, e la preparazione procedura di esempio. I primi due problemi può richied...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Dolan-Jenner 200-W incandescent lamp</td>
<td>Other</td>
<td>Dolan-Jenner Industries</td>
<td>Model 170-D</td>
<td>The heat generated by the lamp does not affect samples for short incubation periods.</td>
</tr>
<tr>
<td>35-mm SLR camera body</td>
<td>Tool</td>
<td>Pentax</td>
<td>SP500 model</td>
<td>In our settings, a bellows is attached to the body of the camera to provide a convenient chamber for irradiation of the sample placed 10 cm away from the light source.</td>
</tr>
<tr>
<td>Clear, thin walled PCR tubes</td>
<td>Other</td>
<td>Eppendorf</td>
<td>951010006 supplied by Fisher L22-003-24</td>
<td>&nbsp;</td>
<tr>
<td>Glass vials (1.8 mL)</td>
<td>Other</td>
<td>Kimble Chromatography</td>
<td>60940A 2, supplied by Fisher 03-340-60</td>
<td>&nbsp;</td>
<tr>
<td>GRF</td>
<td>Reagent</td>
<td>Bachem</td>
<td>H-3695</td>
<td>&nbsp;</td>
<tr>
<td>HFIP</td>
<td>Reagent</td>
<td>TCI America</td>
<td>H0424</td>
<td>Use in a fume hood.</td>
</tr>
<tr>
<td>A&beta;40 and A&beta;42</td>
<td>Reagent</td>
<td>UCLA Biopolymers Laboratory</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Calcitonin</td>
<td>Reagent</td>
<td>American Peptide</td>
<td>22-1-10</td>
<td>&nbsp;</td>
<tr>
<td>Tris(2,2-bipridyl)dichlororuthenium(II) hexahydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>224758-1G</td>
<td>Vortex until the solution is clear. Cover the RuBpy tube with foil to protect the reagent from ambient light. RuBpy is prepared freshly each time and should be used within 48 h.</td>
</tr>
<tr>
<td>Ammonium persulfate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A-7460</td>
<td>Vortex until the solution is clear. APS is prepared freshly each time and should be used within 48 h. </td>
</tr>
<tr>
<td>&beta;-mercaptoethanol</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M7154-25 ML</td>
<td>Can be used when SDS-PAGE analysis is performed.</td>
</tr>
<tr>
<td>Novex Tricine SDS Sample Buffer (2x)</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>LC1676</td>
<td>&nbsp;</td>
<tr>
<td>XCell SureLock Mini-Cell</td>
<td>Tool</td>
<td>Invitrogen</td>
<td>EI0001</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine Gels (10-20%)</td>
<td>Other</td>
<td>Invitrogen</td>
<td>EC6625B0X</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine SDS Running Buffer (10x )</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC1675</td>
<td>&nbsp;</td>
<tr>
<td>Silver Express Staining Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC6100</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

photo-induced cross-linking of unmodified proteins (picup) applied to amyloidogenic peptides

L&#39;assemblaggio delle proteine ​​amiloidogenica in oligomeri tossici è un evento fondamentale nella patogenesi delle malattie misfolding, tra cui l&#39;Alzheimer, il Parkinson, la corea di Huntington e le malattie, ereditarie sclerosi laterale amiotrofica, e diabete di tipo 2. A causa della natura metastabile di queste assemblee di proteine, è difficile valutare la distribuzione delle dimensioni oligomero quantitativamente con metodi classici, come l&#39;elettroforesi, cromatografia luce, fluorescenza, o dinamico dispersione. Oligomeri di proteine ​​amiloidogenica esistono come miscele metastabile, in cui gli oligomeri dissociano in monomeri e associare in grandi gruppi contemporaneamente. PICUP stabilizza popolazioni oligomero da covalenti cross-linking e in combinazione con metodi di frazionamento, come il sodio dodecil solfato elettroforesi su gel di poliacrilammide (SDS-PAGE) o la dimensione-cromatografia di esclusione (SEC), PICUP fornisce istantanee delle distribuzioni di dimensioni oligomero che esisteva prima croce -linking. Quindi, PICUP permette la visualizzazione e l&#39;analisi quantitativa delle popolazioni di proteine ​​metastabili e possono essere utilizzati per monitorare le relazioni di assemblaggio e decifrare tra le modifiche sequenza e oligomerizzazione<sup&gt; 1</sup&gt;. Meccanicisticamente, PICUP coinvolge foto-ossidazione del Ru<sup&gt; 2 +</sup&gt; In un (bipyridyl) tris Ru (II), complessi (RuBpy) alla Ru<sup&gt; 3 +</sup&gt; Per irraggiamento con luce visibile in presenza di un accettore di elettroni. Ru<sup&gt; 3 +</sup&gt; È un forte ossidante di un elettrone in grado di astrarre un elettrone da una molecola di proteina vicini, generando una proteina radicale<sup&gt; 1,2</sup&gt;. I radicali sono instabili, specie altamente reattiva e quindi scomparire rapidamente attraverso una serie di reazioni intra ed intermolecolari. Un radicale può utilizzare l&#39;energia di un elettrone spaiato a reagire con un altro monomero proteina dimerica formando un radicale, che perde quindi un atomo di idrogeno e forma un stabile, covalentemente legato dimero. Il dimero può quindi reagire ulteriormente attraverso un meccanismo simile con monomeri e dimeri di altri per formare oligomeri di ordine superiore. Vantaggi del PICUP rispetto ad altri foto-chimiche o cross-linking metodi<sup&gt; 3,4</sup&gt; Sono brevi (≤ 1 s) l&#39;esposizione a non distruttiva la luce visibile, senza bisogno di<i&gt; Pre facto</iModifica&gt; la stessa sequenza, e di lunghezza zero covalente cross-linking. Inoltre, consente PICUP cross-linking delle proteine ​​all&#39;interno di pH e temperatura, inclusi i parametri fisiologici. In questo studio dimostriamo applicazione di PICUP di cross-linking di tre proteine ​​amiloidogenica il 40 - e 42-β-amiloide residui di proteine ​​varianti (Aβ40 e Aβ42) e la calcitonina, e una proteina di controllo, l&#39;ormone della crescita-releasing factor (GRF).

Watch this Scientific Journal Video about Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides at JoVE.com

Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides

Foto-indotta cross-linking delle proteine ​​non modificato (PICUP) permette la caratterizzazione della distribuzione dimensionale oligomeri in miscele proteiche metastabili. Dimostriamo applicazione di PICUP a tre peptidi rappresentante amiloidogenico il 40 - e 42-residuo forme di β-amiloide, proteina, e la calcitonina, e un peptide di controllo della crescita-fattore di rilascio dell&#39;ormone.

Photo-Induced Cross-Linking delle proteine ​​non modificati (PICUP) applicata a peptidi amiloidogenico

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, ...

Research

JoVE Journal

Biology

Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides

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Photo-Induced Cross-Linking delle proteine ​​non modificati (PICUP) applicata a peptidi amiloidogenico

Photo-Induced Cross-Linking delle proteine non modificati (PICUP) applicata a peptidi amiloidogenico