Name: a high-throughput luciferase assay to evaluate proteolysis of the single-turnover protease pcsk9
Uploaded: 2018-08-28T15:00:00
Description: Watch this Scientific Journal Video about A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9 at JoVE.com

Gli autori ringraziano il generoso sostegno finanziario dal NHLBI/NIH (K08 HL124068 e LRP HMOT1243), NCATS/NIH attraverso la UCSF clinica e traslazionale Science Institute catalizzatore Program (UL1 TR000004), il Senato accademico di UCSF, la Fondazione di Hellman, una Gilead Sciences Research Scholar Award, un premio di cardiovascolare Pfizer ASPIRE (tutte John S. Chorba) e l'Howard Hughes Medical Institute (a Adri M. Galvan e Kevan M. Shokat).

1. mutagenesi di proteasi vettoriale
<ol><li>Progettare e ordinare oligonucleotidi sintetizzati personalizzato per installare una mutazione di interesse usando una modifica di protocolli di mutagenesi sito-diretta standard12. Primer standard dissalate (senza ulteriore purificazione) sono perfettamente accettabili. Nota: Un approccio generale ai disegni di primer comporta la creazione di primer parzialmente sovrapposti come indicato nella tabella 1, utilizzando una calcolatric...

<ol>
	<li>Park, S. W., Moon, Y. A., Horton, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-transcriptional+regulation+of+low+density+lipoprotein+receptor+protein+by+proprotein+convertase+subtilisin/kexin+type+9a+in+mouse+liver.">Post-transcriptional regulation of low density lipoprotein receptor protein by proprotein convertase subtilisin/kexin type 9a in mouse liver.</a> Journal of Biological Chemistry. 279 (48), 50630-50638 (2004).</li><li>Cohen, J. C., Boerwinkle, E., Mosley, T. H., Hobbs, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+variations+in+PCSK9,+low+LDL,+and+protection+against+coronary+heart+disease.">Sequence variations in PCSK9, low LDL, and protection against coronary heart disease.</a> New England Journal of Medicine. 354 (12), 1264-1272 (2006).</li><li>Ridker, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+Efficacy+and+Safety+of+Bococizumab+in+High-Risk+Patients.">Cardiovascular Efficacy and Safety of Bococizumab in High-Risk Patients.</a> New England Journal of Medicine. 376 (16), 1527-1539 (2017).</li><li>Sabatine, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolocumab+and+Clinical+Outcomes+in+Patients+with+Cardiovascular+Disease.">Evolocumab and Clinical Outcomes in Patients with Cardiovascular Disease.</a> New England Journal of Medicine. 376 (18), 1713-1722 (2017).</li><li>Kazi, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cost-effectiveness+of+PCSK9+Inhibitor+Therapy+in+Patients+With+Heterozygous+Familial+Hypercholesterolemia+or+Atherosclerotic+Cardiovascular+Disease.">Cost-effectiveness of PCSK9 Inhibitor Therapy in Patients With Heterozygous Familial Hypercholesterolemia or Atherosclerotic Cardiovascular Disease.</a> Journal of the American Medical Association. 316 (7), 743-753 (2016).</li><li>Kazi, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Updated+Cost-effectiveness+Analysis+of+PCSK9+Inhibitors+Based+on+the+Results+of+the+FOURIER+Trial.">Updated Cost-effectiveness Analysis of PCSK9 Inhibitors Based on the Results of the FOURIER Trial.</a> Journal of the American Medical Association. 318 (8), (2017).</li><li>Pettersen, D., Fjellstr&#246;m, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+modulators+of+PCSK9+-+A+literature+and+patent+overview.">Small molecule modulators of PCSK9 - A literature and patent overview.</a> Bioorganic &amp; Medicinal Chemistry Letters. 28 (7), 1155-1160 (2018).</li><li>Chorba, J. S., Galvan, A. M., Shokat, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stepwise+processing+analyses+of+the+single-turnover+PCSK9+protease+reveal+its+substrate+sequence+specificity+and+link+clinical+genotype+to+lipid+phenotype.">Stepwise processing analyses of the single-turnover PCSK9 protease reveal its substrate sequence specificity and link clinical genotype to lipid phenotype.</a> Journal of Biological Chemistry. 293 (6), 1875-1886 (2018).</li><li>Maxwell, K. N., Breslow, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral-mediated+expression+of+Pcsk9+in+mice+results+in+a+low-density+lipoprotein+receptor+knockout+phenotype.">Adenoviral-mediated expression of Pcsk9 in mice results in a low-density lipoprotein receptor knockout phenotype.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (18), 7100-7105 (2004).</li><li>Benjannet, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NARC-1/PCSK9+and+its+natural+mutants:+zymogen+cleavage+and+effects+on+the+low+density+lipoprotein+(LDL)+receptor+and+LDL+cholesterol.">NARC-1/PCSK9 and its natural mutants: zymogen cleavage and effects on the low density lipoprotein (LDL) receptor and LDL cholesterol.</a> Journal of Biological Chemistry. 279 (47), 48865-48875 (2004).</li><li>Cunningham, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+biophysical+studies+of+PCSK9+and+its+mutants+linked+to+familial+hypercholesterolemia.">Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia.</a> Nature Structural &amp; Molecular Biology. 14 (5), 413-419 (2007).</li><li>Liu, H., Naismith, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+one-step+site-directed+deletion,+insertion,+single+and+multiple-site+plasmid+mutagenesis+protocol.">An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol.</a> BMC Biotechnology. 8, 91 (2008).</li><li>Zhang, J., Chung, T., Oldenburg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Statistical+Parameter+for+Use+in+Evaluation+and+Validation+of+High+Throughput+Screening+Assays.">A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.</a> Journal of Biomolecular Screening. 4 (2), 67-73 (1999).</li><li>Hall, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+luciferase+reporter+from+a+deep+sea+shrimp+utilizing+a+novel+imidazopyrazinone+substrate.">Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate.</a> ACS Chemical Biology. 7 (11), 1848-1857 (2012).</li><li>McNutt, M. C., Lagace, T. A., Horton, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalytic+activity+is+not+required+for+secreted+PCSK9+to+reduce+low+density+lipoprotein+receptors+in+HepG2+cells.">Catalytic activity is not required for secreted PCSK9 to reduce low density lipoprotein receptors in HepG2 cells.</a> Journal of Biological Chemistry. 282 (29), 20799-20803 (2007).</li><li>Chorba, J. S., Shokat, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+proprotein+convertase+subtilisin/kexin+type+9+(PCSK9)+active+site+and+cleavage+sequence+differentially+regulate+protein+secretion+from+proteolysis.">The proprotein convertase subtilisin/kexin type 9 (PCSK9) active site and cleavage sequence differentially regulate protein secretion from proteolysis.</a> Journal of Biological Chemistry. 289 (42), 29030-29043 (2014).</li><li>Benjannet, S., Rhainds, D., Hamelin, J., Nassoury, N., Seidah, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+proprotein+convertase+(PC)+PCSK9+is+inactivated+by+furin+and/or+PC5/6A:+functional+consequences+of+natural+mutations+and+post-translational+modifications.">The proprotein convertase (PC) PCSK9 is inactivated by furin and/or PC5/6A: functional consequences of natural mutations and post-translational modifications.</a> Journal of Biological Chemistry. 281 (41), 30561-30572 (2006).</li><li>Zhao, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+loss-of-function+mutations+in+PCSK9+and+identification+of+a+compound+heterozygote.">Molecular characterization of loss-of-function mutations in PCSK9 and identification of a compound heterozygote.</a> American Journal of Human Genetics. 79 (3), 514-523 (2006).</li></ol>

Le procedure sperimentali descritte sopra presentano un metodo per superare l'attività intrinsecamente bassa della singolo-fatturato proteasi PCSK9 e valutare la sua funzione proteolitica in modo robusto. Il concetto chiave del dosaggio si basa sulla conversione di un evento singolo-fatturato in una lettura enzimaticamente amplificata. I punti di forza del test includono la relativamente breve periodo di tempo e facilità d'uso del reporter di luciferase, come pure la sua scalabilità agli approcci di alto-rendimento. I...

PCSK9 è destinato il ricevitore di LDL (LDL-R) per la degradazione, innalzamento del colesterolo LDL (LDL-C) e guida la malattia cardiaca aterosclerotica1,2. Therapeutics targeting PCSK9 robusto abbassare LDL-C e per migliorare i risultati cardiovascolari per i pazienti, anche quando aggiunto ad una terapia ipolipemizzante aggressiva con le statine3,4. Tuttavia, terapie attualmente approvate sono limitate ad approcci basati su anticorpi e soffrono di una mancanza di redditività5,6. Per risolvere questo problema, le alternative terapeutiche meno costose, un mezzo per identificare i pazienti probabilmente ottenere maggiori benefici, o entrambi, sono necessari.
Approcci della piccolo-molecola bersaglio intracellulare PCSK9, fornirebbe una migliore via di somministrazione e ridurre i costi, che li rende il "Santo Graal" in questa zona7. Tuttavia, PCSK9 è dimostrato difficile da droga di piccole molecole. Come una proteasi, targeting funzione proteolitica di PCSK9 è un'attraente strategia, come self-proteolisi sono la tappa limitante di PCSK9 maturazione8 ed sono necessaria per il suo massimo effetto su LDL-R9. Fin qui, tuttavia, questa strategia non ha avuto successo, probabilmente a causa di biochimica unica di PCSK9: PCSK9 fende solo sé10, eseguendo una reazione di singolo-fatturato, e dopo auto-fenditura, la PCSK9 propeptide rimane legato nel sito attivo come un auto-inibitore11, impedendo la lettura di qualsiasi ulteriore attività di proteasi.
Questo articolo presenta un metodo per valutare la funzione proteolitica PCSK9 in moda di alto-rendimento8. Attraverso mutagenesi sito-diretta, gli investigatori possono utilizzare questo test per sondare gli effetti di codifica SNPs trovati nella clinica per valutarli per effetti sulla proteolisi, la tappa limitante di PCSK9 maturazione. Inoltre, questo metodo sarà utile nella progettazione di schermi ad alta produttività per identificare i modulatori della proteolisi PCSK9, che si prevedono, infine, interferire con la presentazione di PCSK9 al LDL-R (e modulare l'effetto ipercolesterolemico di PCSK9) . Infine, questo protocollo può essere adattato alle altre proteasi con attività intrinsecamente bassa, purché i) una coppia di substrato-proteasi specifiche possa essere trovata, e ii) un adeguato ancoraggio intracellulare può essere stabilita per il substrato.

<table>
	<tbody>
		<tr>
			<td>PCR Tubes</td>
			<td>USA Scientific</td>
			<td>1402-2900</td>
			<td>For PCR</td>
		</tr>
		<tr>
			<td>Q5 Hot Start</td>
			<td>New England Biolabs</td>
			<td>M0493L</td>
			<td>High-fidelity DNA Polymerase</td>
		</tr>
		<tr>
			<td>Deoxynucleotide Solution Mix</td>
			<td>New England Biolabs</td>
			<td>N0447L</td>
			<td>dNTPs (for PCR)</td>
		</tr>
		<tr>
			<td>pPCSK9-NLucProteaseAssay-WT</td>
			<td>Authors</td>
			<td>n/a</td>
			<td>Available from authors</td>
		</tr>
		<tr>
			<td>pPCSK9-NLucProteaseAssay-S386A</td>
			<td>Authors</td>
			<td>n/a</td>
			<td>Available from authors</td>
		</tr>
		<tr>
			<td>Agarose LE</td>
			<td>Gold Biotechnology</td>
			<td>A-201-100</td>
			<td>For DNA gels</td>
		</tr>
		<tr>
			<td>E-Gel Imager System with Blue Light Base</td>
			<td>ThermoFisher Scientific</td>
			<td>4466612</td>
			<td>For imaging DNA gels</td>
		</tr>
		<tr>
			<td>SYBR Safe DNA Gel Stain</td>
			<td>ThermoFisher Scientific</td>
			<td>S33102</td>
			<td>For DNA gels</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>ThermoFisher Scientific</td>
			<td>BP152-1</td>
			<td>For DNA gel running buffer</td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>ThermoFisher Scientific</td>
			<td>A38-500</td>
			<td>For DNA gel running buffer</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid solution</td>
			<td>Millipore Sigma</td>
			<td>3690</td>
			<td>EDTA, for DNA gel running buffer</td>
		</tr>
		<tr>
			<td>1 kb DNA ladder</td>
			<td>Gold Biotechnology</td>
			<td>D010</td>
			<td>DNA ladder</td>
		</tr>
		<tr>
			<td>DpnI</td>
			<td>New England Biolabs</td>
			<td>R0176S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>LB Agar plates with 100 &#181;g/mL carbenicillin</td>
			<td>Teknova</td>
			<td>L1010</td>
			<td>LB-Carb plates</td>
		</tr>
		<tr>
			<td>One Shot Mach1 T Phage-Resistent Chemically Competent E. coli</td>
			<td>ThermoFisher Scientific</td>
			<td>C862003</td>
			<td>Chemically competent cells</td>
		</tr>
		<tr>
			<td>LB Broth, Miller</td>
			<td>ThermoFisher Scientific</td>
			<td>BP1426-2</td>
			<td>LB</td>
		</tr>
		<tr>
			<td>Carbenicillin</td>
			<td>Gold Biotechnology</td>
			<td>C-103-5</td>
			<td>Selective antibiotic</td>
		</tr>
		<tr>
			<td>E.Z.N.A. Plasmid Mini Kit I</td>
			<td>Omega BioTek</td>
			<td>D6942-02</td>
			<td>DNA Purification Miniprep kit</td>
		</tr>
		<tr>
			<td>NanoDrop 2000 Spectrophotomer</td>
			<td>ThermoFisher Scientific</td>
			<td>ND-2000C</td>
			<td>Spectrophotometer</td>
		</tr>
		<tr>
			<td>293T Cells</td>
			<td>American Tissue Culture Collection (ATCC)</td>
			<td>CRL-3216</td>
			<td>HEK 293T cells</td>
		</tr>
		<tr>
			<td>DMEM, high glucose, pyruvate</td>
			<td>ThermoFisher Scientific</td>
			<td>11995065</td>
			<td>DMEM, mammalian cell media</td>
		</tr>
		<tr>
			<td>Fetal Bovine Sera</td>
			<td>Axenia Biologix</td>
			<td>F001</td>
			<td>FBS</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>ThermoFisher Scientific</td>
			<td>25300062</td>
			<td>Trypsin, for cell dissociation</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>ThermoFisher Scientific</td>
			<td>10010023</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Countess automated cell counter</td>
			<td>ThermoFisher Scientific</td>
			<td>C10227</td>
			<td>Automated cell counting</td>
		</tr>
		<tr>
			<td>Countess cell counting chamber slides</td>
			<td>ThermoFisher Scientific</td>
			<td>C10228</td>
			<td>Slides for cell counting</td>
		</tr>
		<tr>
			<td>CELLSTAR Tissue Culture Plates, White, White-Bottom, with Lid</td>
			<td>Grenier Bio-One</td>
			<td>655083</td>
			<td>White, white-bottom 96 well plate</td>
		</tr>
		<tr>
			<td>TempPlate non-skirted 96-well PCR plate, natural</td>
			<td>USA Scientific</td>
			<td>1402-9596</td>
			<td>96 well plate for master plasmid plate</td>
		</tr>
		<tr>
			<td>Nunc 2.0mL DeepWell Plates</td>
			<td>ThermoFisher Scientific</td>
			<td>278743</td>
			<td>96 well deep well plate</td>
		</tr>
		<tr>
			<td>Lipofectamine 3000</td>
			<td>ThermoFisher Scientific</td>
			<td>L3000008</td>
			<td>Lipid transfection reagent, Lf3K</td>
		</tr>
		<tr>
			<td>P3000 Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>L3000008</td>
			<td>DNA pre-complexation reagent, provided with Lf3K</td>
		</tr>
		<tr>
			<td>OptiMEM I Reduced Serum Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>31985062</td>
			<td>Reduced serum medium for transfection</td>
		</tr>
		<tr>
			<td>(+)-Sodium L-ascorbate</td>
			<td>Millipore Sigma</td>
			<td>A4034</td>
			<td>Sodium ascorbate</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Millipore Sigma</td>
			<td>S9888</td>
			<td>NaCl</td>
		</tr>
		<tr>
			<td>Albumin, Bovine Serum, Fraction V, Low Heavy Metals</td>
			<td>Millipore Sigma</td>
			<td>12659</td>
			<td>BSA</td>
		</tr>
		<tr>
			<td>Methanol (HPLC)</td>
			<td>ThermoFisher Scientific</td>
			<td>A4524</td>
			<td>MeOH</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>VWR</td>
			<td>JT9535-2</td>
			<td>Concentrated HCl</td>
		</tr>
		<tr>
			<td>Coelenterazine</td>
			<td>Gold Biotechnology</td>
			<td>CZ2.5</td>
			<td>Luciferase substrate</td>
		</tr>
		<tr>
			<td>Syringe Filter, Sterile</td>
			<td>ThermoFisher Scientific</td>
			<td>09-720-3</td>
			<td>Sterile filter, PVDF, 0.22 &#181;m pore</td>
		</tr>
		<tr>
			<td>Pipet-Lite Multi Pipette L12-200XLS+</td>
			<td>Rainin</td>
			<td>17013810</td>
			<td>Multichannel pipette</td>
		</tr>
		<tr>
			<td>Pipet-Lite Multi Pipette L12-20XLS+</td>
			<td>Rainin</td>
			<td>17013808</td>
			<td>Multichannel pipette</td>
		</tr>
		<tr>
			<td>Pipet-Lite Multi Pipette L12-10XLS+</td>
			<td>Rainin</td>
			<td>17013807</td>
			<td>Multichannel pipette</td>
		</tr>
		<tr>
			<td>Reagent reservoir</td>
			<td>Corning</td>
			<td>4870</td>
			<td>Trough for reagents</td>
		</tr>
		<tr>
			<td>Centrifuge tubes, 15 mL</td>
			<td>ThermoFisher Scientific</td>
			<td>05-539-12</td>
			<td>15 mL tubes</td>
		</tr>
		<tr>
			<td>Centrifuge tubes, 50 mL</td>
			<td>Corning</td>
			<td>430829</td>
			<td>50 mL tubes</td>
		</tr>
		<tr>
			<td>Spark Microplate Reader</td>
			<td>Tecan</td>
			<td>N/a</td>
			<td>Plate Reader</td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td>2016 for Mac</td>
			<td>Spreadsheet software</td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad Software</td>
			<td>v7</td>
			<td>Scientific data analysis software</td>
		</tr>
	</tbody>
</table>

a high-throughput luciferase assay to evaluate proteolysis of the single-turnover protease pcsk9

Proteina convertasi subtilisin/kexin tipo 9 (PCSK9) è una proteasi di singolo-fatturato che regola i livelli di lipoproteina a bassa densità (LDL) del siero e, di conseguenza, la malattia cardiovascolare. Anche se PCSK9 proteolisi sono richiesto per il suo effetto completo ipercolesterolemico, la valutazione della sua funzione proteolitica è impegnativa: PCSK9 è noto solo per fendere se stesso, subisce solo un singolo fatturato e dopo proteolisi, conserva il suo substrato in suo sito attivo come inibitore di auto. I metodi presentati qui descrivono un'analisi che supera queste sfide. L'analisi si concentra sulla proteolisi intermolecolari in un contesto di basati su cellule e avvenuta scissione collegamenti per l'attività luciferasica secrete, che possono essere facilmente lette nel medium condizionato. Via passaggi sequenziali di mutagenesi, trasfezione transiente e una lettura di luciferasi, il dosaggio può sondare PCSK9 proteolisi in condizioni di perturbazione o genetici o molecolari in un modo ad alta velocità. Questo sistema è adatto per entrambi la valutazione biochimica di missenso clinicamente scoperto polimorfismi a singolo nucleotide (SNPs), anche per quanto riguarda lo screening di piccole molecole inibitori di PCSK9 proteolisi.

Il saggio di proteolisi di alto-rendimento si basa su superare tre grandi sfide. In primo luogo, per superare l'uscita intrinsecamente bassa di una proteasi PCSK9 singolo-fatturato, una proteasi di PCSK9 manca l'inibitoria propeptide è utilizzato, con la sequenza di clivaggio Tail il propeptide collegata a una luciferasi che possono essere secrete14. In secondo luogo, per soddisfare la necessità per la proteasi piegare complesso con suoi inibitori propeptide, che...

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A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

Un'analisi di Luciferase High-Throughput per valutare la proteolisi di PCSK9 la proteasi singolo-fatturato

Questo protocollo presenta un metodo per valutare l'attività proteolitica di una proteasi fatturato intrinsecamente bassa attività, unico in un contesto cellulare. In particolare, questo metodo viene applicato per valutare l'attività proteolitica di PCSK9, un driver chiave del metabolismo lipidico cui attività proteolitica è richiesto per la sua funzione ipercolesterolemici ultimate.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, ...

Research

JoVE Journal

Biochemistry

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

High-throughput screening degli enzimi carboidrati di degradazione Utilizzando Novel insolubile Chromogenic Substrate Assay Kit

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

Ricerca

Biochimica

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We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, ...

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Un metodo semplice per High Throughput Screening chimico in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

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Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, ...

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

Riconoscimento della sequenza del DNA da parte del primase del DNA utilizzando la profilazione primasise ad alto valore a più velocità

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

Adattamento semi-alto/throughput del saggio ATPase accoppiato NADH per lo screening degli inibitori delle piccole molecole

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

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Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced ...

Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening

Saggio fluorometrico basato sulla chimica dei clic per l'apolipoproteina N-aciltransferasi dalla caratterizzazione enzimatica allo screening ad alta produttività

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

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