Name: preparation and in vivo use of an activity-based probe for n-acylethanolamine acid amidase
Uploaded: 2016-11-23T13:00:00
Description: Watch this Scientific Journal Video about Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase at JoVE.com

The authors thank the Nikon Imaging Center at Istituto Italiano di Tecnologia, Genova, Italy (NIC@IIT).

Cuidado: Todas as reacções de química deve ser levada a cabo numa hotte ventilado e com a utilização de um revestimento do laboratório, luvas, e óculos de protecção. As reacções devem também ser levada a cabo num ambiente de azoto. declaração ética: Nossos procedimentos que envolvem animais são realizados em conformidade com as normas italianas relativas à protecção dos animais utilizados para fins experimentais e outros científicos (DM 116192), e regulamentos Comunidade Económica Europeia (JO de CE L 358/1 1986...

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	<li>Gygi, S. P., Han, D. K., Gingras, A. C., Sonenberg, N., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+analysis+by+mass+spectrometry+and+sequence+database+searching:+tools+for+cancer+research+in+the+post-genomic+era.">Protein analysis by mass spectrometry and sequence database searching: tools for cancer research in the post-genomic era.</a> Electrophoresis. 20, 310-319 (1999).</li><li>Washburn, M. P., Wolters, D., Yates, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+analysis+of+the+yeast+proteome+by+multidimensional+protein+identification+technology.">Large-scale analysis of the yeast proteome by multidimensional protein identification technology.</a> Nat Biotechnol. 19, 242-247 (2001).</li><li>Zhu, H., Bilgin, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics.">Proteomics.</a> Annu Rev Biochem. 72, 783-812 (2003).</li><li>Ito, T., 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=Roles+for+the+two-hybrid+system+in+exploration+of+the+yeast+protein+interactome.">Roles for the two-hybrid system in exploration of the yeast protein interactome.</a> Mol Cell Proteomics. 1, 561-566 (2002).</li><li>Evans, M. J., Cravatt, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism-based+profiling+of+enzyme+families.">Mechanism-based profiling of enzyme families.</a> Chem Rev. 106, 3279-3301 (2006).</li><li>Cravatt, B. F., Wright, A. T., Kozarich, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-based+protein+profiling:+from+enzyme+chemistry+to+proteomic+chemistry.">Activity-based protein profiling: from enzyme chemistry to proteomic chemistry.</a> Annu Rev Biochem. 77, 383-414 (2008).</li><li>Rostovtsev, V. V., Green, L. G., Fokin, V. V., Sharpless, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stepwise+huisgen+cycloaddition+process:+copper(I)-catalyzed+regioselective+"ligation"+of+azides+and+terminal+alkynes.">A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective "ligation" of azides and terminal alkynes.</a> Angew Chem Int Ed Engl. 41, 2596-2599 (2002).</li><li>Meldal, M., Tornoe, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cu-catalyzed+azide-alkyne+cycloaddition.">Cu-catalyzed azide-alkyne cycloaddition.</a> Chem Rev. 108, 2952-3015 (2008).</li><li>Speers, A. E., Adam, G. C., Cravatt, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-based+protein+profiling+in+vivo+using+a+copper(i)-catalyzed+azide-alkyne+[3+++2]+cycloaddition.">Activity-based protein profiling in vivo using a copper(i)-catalyzed azide-alkyne [3 + 2] cycloaddition.</a> J Am Chem Soc. 125, 4686-4687 (2003).</li><li>Saxon, E., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface+engineering+by+a+modified+Staudinger+reaction.">Cell surface engineering by a modified Staudinger reaction.</a> Science. 287, 2007-2010 (2000).</li><li>Kohn, M., Breinbauer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Staudinger+ligation-a+gift+to+chemical+biology.">The Staudinger ligation-a gift to chemical biology.</a> Angew Chem Int Ed Engl. 43, 3106-3116 (2004).</li><li>Romeo, E., 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=Activity-Based+Probe+for+N-Acylethanolamine+Acid+Amidase.">Activity-Based Probe for N-Acylethanolamine Acid Amidase.</a> ACS Chem Biol. 10, 2057-2064 (2015).</li><li>Ueda, N., Yamanaka, K., Yamamoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+an+acid+amidase+selective+for+N-palmitoylethanolamine,+a+putative+endogenous+anti-inflammatory+substance.">Purification and characterization of an acid amidase selective for N-palmitoylethanolamine, a putative endogenous anti-inflammatory substance.</a> J Biol Chem. 276, 35552-35557 (2001).</li><li>Tsuboi, K., 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+N-acylethanolamine-hydrolyzing+acid+amidase,+a+novel+member+of+the+choloylglycine+hydrolase+family+with+structural+and+functional+similarity+to+acid+ceramidase.">Molecular characterization of N-acylethanolamine-hydrolyzing acid amidase, a novel member of the choloylglycine hydrolase family with structural and functional similarity to acid ceramidase.</a> J Biol Chem. 280, 11082-11092 (2005).</li><li>Tsuboi, K., Takezaki, N., Ueda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+N-acylethanolamine-hydrolyzing+acid+amidase+(NAAA).">The N-acylethanolamine-hydrolyzing acid amidase (NAAA).</a> Chem Biodivers. 4, 1914-1925 (2007).</li><li>Ribeiro, A., 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=A+Potent+Systemically+Active+N-Acylethanolamine+Acid+Amidase+Inhibitor+that+Suppresses+Inflammation+and+Human+Macrophage+Activation.">A Potent Systemically Active N-Acylethanolamine Acid Amidase Inhibitor that Suppresses Inflammation and Human Macrophage Activation.</a> ACS Chem Biol. 10, 1838-1846 (2015).</li><li>Zhao, L. Y., Tsuboi, K., Okamoto, Y., Nagahata, S., Ueda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolytic+activation+and+glycosylation+of+N-acylethanolamine-hydrolyzing+acid+amidase,+a+lysosomal+enzyme+involved+in+the+endocannabinoid+metabolism.">Proteolytic activation and glycosylation of N-acylethanolamine-hydrolyzing acid amidase, a lysosomal enzyme involved in the endocannabinoid metabolism.</a> Biochim Biophys Acta. 1771, 1397-1405 (2007).</li><li>Wang, J., 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=Amino+acid+residues+crucial+in+pH+regulation+and+proteolytic+activation+of+N-acylethanolamine-hydrolyzing+acid+amidase.">Amino acid residues crucial in pH regulation and proteolytic activation of N-acylethanolamine-hydrolyzing acid amidase.</a> Biochim Biophys Acta. 1781, 710-717 (2008).</li><li>West, J. M., Zvonok, N., Whitten, K. M., Wood, J. T., Makriyannis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometric+characterization+of+human+N-acylethanolamine-hydrolyzing+acid+amidase.">Mass spectrometric characterization of human N-acylethanolamine-hydrolyzing acid amidase.</a> J Proteome Res. 11, 972-981 (2012).</li><li>Bandiera, T., Ponzano, S., Piomelli, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+discovery+of+N-acylethanolamine+acid+amidase+inhibitors.">Advances in the discovery of N-acylethanolamine acid amidase inhibitors.</a> Pharmacol Res. 86, 11-17 (2014).</li><li>Sasso, O., 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=Antinociceptive+effects+of+the+N-acylethanolamine+acid+amidase+inhibitor+ARN077+in+rodent+pain+models.">Antinociceptive effects of the N-acylethanolamine acid amidase inhibitor ARN077 in rodent pain models.</a> Pain. 154, 350-360 (2013).</li><li>Duranti, A., 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=N-(2-oxo-3-oxetanyl)carbamic+acid+esters+as+N-acylethanolamine+acid+amidase+inhibitors:+synthesis+and+structure-activity+and+structure-property+relationships.">N-(2-oxo-3-oxetanyl)carbamic acid esters as N-acylethanolamine acid amidase inhibitors: synthesis and structure-activity and structure-property relationships.</a> J Med Chem. 55, 4824-4836 (2012).</li><li>Ponzano, 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=Synthesis+and+structure-activity+relationship+(SAR)+of+2-methyl-4-oxo-3-oxetanylcarbamic+acid+esters,+a+class+of+potent+N-acylethanolamine+acid+amidase+(NAAA)+inhibitors.">Synthesis and structure-activity relationship (SAR) of 2-methyl-4-oxo-3-oxetanylcarbamic acid esters, a class of potent N-acylethanolamine acid amidase (NAAA) inhibitors.</a> J Med Chem. 56, 6917-6934 (2013).</li><li>Solorzano, C., 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=Synthesis+and+structure-activity+relationships+of+N-(2-oxo-3-oxetanyl)amides+as+N-acylethanolamine-hydrolyzing+acid+amidase+inhibitors.">Synthesis and structure-activity relationships of N-(2-oxo-3-oxetanyl)amides as N-acylethanolamine-hydrolyzing acid amidase inhibitors.</a> J Med Chem. 53, 5770-5781 (2010).</li><li>Vitale, R., 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=Synthesis,+structure-activity,+and+structure-stability+relationships+of+2-substituted-N-(4-oxo-3-oxetanyl)+N-acylethanolamine+acid+amidase+(NAAA)+inhibitors.">Synthesis, structure-activity, and structure-stability relationships of 2-substituted-N-(4-oxo-3-oxetanyl) N-acylethanolamine acid amidase (NAAA) inhibitors.</a> ChemMedChem 9. 9, 323-336 (2014).</li><li>Fiasella, A., 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=3-Aminoazetidin-2-one+derivatives+as+N-acylethanolamine+acid+amidase+(NAAA)+inhibitors+suitable+for+systemic+administration.">3-Aminoazetidin-2-one derivatives as N-acylethanolamine acid amidase (NAAA) inhibitors suitable for systemic administration.</a> ChemMedChem 9. 9, 1602-1614 (2014).</li><li>Armirotti, A., 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=beta-Lactones+Inhibit+N-acylethanolamine+Acid+Amidase+by+S-Acylation+of+the+Catalytic+N-Terminal+Cysteine.">beta-Lactones Inhibit N-acylethanolamine Acid Amidase by S-Acylation of the Catalytic N-Terminal Cysteine.</a> ACS Med Chem Lett. 3, 422-426 (2012).</li><li>Nuzzi, A., 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=Potent+alpha-amino-beta-lactam+carbamic+acid+ester+as+NAAA+inhibitors.+Synthesis+and+structure-activity+relationship+(SAR)+studies.">Potent alpha-amino-beta-lactam carbamic acid ester as NAAA inhibitors. Synthesis and structure-activity relationship (SAR) studies.</a> Eur J Med Chem. 111, 138-159 (2016).</li><li>Bonezzi, F. T., 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=An+Important+Role+for+N-Acylethanolamine+Acid+Amidase+in+the+Complete+Freund's+Adjuvant+Rat+Model+of+Arthritis.">An Important Role for N-Acylethanolamine Acid Amidase in the Complete Freund's Adjuvant Rat Model of Arthritis.</a> J Pharmacol Exp Ther. 356, 656-663 (2016).</li><li>Smith, P. K., 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=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Anal Biochem. 150, 76-85 (1985).</li><li>Speers, A. E., Cravatt, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-Based+Protein+Profiling+(ABPP)+and+Click+Chemistry+(CC)-ABPP+by+MudPIT+Mass+Spectrometry.">Activity-Based Protein Profiling (ABPP) and Click Chemistry (CC)-ABPP by MudPIT Mass Spectrometry.</a> Curr Protoc Chem Biol. 1, 29-41 (2009).</li><li>Rybak, J. N., Scheurer, S. B., Neri, D., Elia, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+biotinylated+proteins+on+streptavidin+resin:+a+protocol+for+quantitative+elution.">Purification of biotinylated proteins on streptavidin resin: a protocol for quantitative elution.</a> Proteomics. 4, 2296-2299 (2004).</li><li>Penna, A., Cahalan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+Blotting+using+the+Invitrogen+NuPage+Novex+Bis+Tris+minigels.">Western Blotting using the Invitrogen NuPage Novex Bis Tris minigels.</a> J Vis Exp. (264), (2007).</li><li>Giuffrida, A., Piomelli, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotope+dilution+GC/MS+determination+of+anandamide+and+other+fatty+acylethanolamides+in+rat+blood+plasma.">Isotope dilution GC/MS determination of anandamide and other fatty acylethanolamides in rat blood plasma.</a> FEBS Lett. 422, 373-376 (1998).</li><li>Buczynski, M. W., Parsons, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+brain+endocannabinoid+levels:+methods,+interpretations+and+pitfalls.">Quantification of brain endocannabinoid levels: methods, interpretations and pitfalls.</a> Br J Pharmacol. 160, 423-442 (2010).</li></ol>

A actividade enzimática é finamente regulados em níveis diferentes, incluindo a transcrição de ARN, a síntese de proteínas, a translocação da proteína, a modificação pós-translacional, e interacção proteína-proteína. Muitas vezes, a expressão da enzima sozinha não leva em conta para a sua actividade. ABPP foi desenvolvido para estudar a actividade de proteínas no seu estado nativo. Duas características são necessárias: uma sonda química que covalentemente liga-se ao sítio activo de uma enzima de...

Comumente usado métodos para investigar os padrões de expressão, interações e funções das proteínas, incluindo plataformas de espectrometria de cromatografia de massa de líquido para análise espingarda 1,2, fermento métodos de dois híbridos 3,4 e, em ensaios in vitro, são limitados em que eles são incapaz de avaliar a atividade das proteínas no seu estado nativo. profiling proteína baseado em atividades (ABPP) pode ser usado para preencher esta lacuna. Nesta abordagem, pequena molécula sondas capazes de se ligar covalentemente ao sítio activo de uma enzima de interesse conjugado com um grupo repórter que permite a detecção do alvo. Usando clique química (CC), o repórter pode ser integrado na sonda ou podem ser introduzidos após acoplamento alvo ocorreu 5,6. O último procedimento requer a utilização de sondas que contêm grupos químicos adequados, tais como um alcino ou azida de terminal, que pode ser modificado com uma série de reagentes repórter via reacções bio-ortogonal sucH, tal como o Cu (I) catalisada por Huisgen [3 + 2] cicloadição 7-9 ou Staudinger ligadura 10,11. Recentemente, foi divulgada a ARN14686 composto como o primeiro ABP para o in vitro e in vivo de detecção da hidrolase de cisteína, NAAA 12. NAAA catalisa a desativação hidrolítica FAEs saturados e monoinsaturados, incluindo oleoletanolamina (OEA) e palmitoiletanolamida (PEA), que são agonistas endógenos do anti-inflamatório receptor nuclear PPAR-alfa 13-15. NAAA é predominantemente expresso em macrófagos e outras células derivadas de monócitos, bem como em linfócitos-B 14,16, sugerindo um papel na regulação da resposta imune inata. A enzima é sintetizada no retículo endoplasmático rugoso numa forma inactiva e é activado em compartimentos acídicos da célula através de um mecanismo 17 autoproteolica. A clivagem autoproteolítica gera uma nova cisteína N-terminal (C131 em ratinhos e ratos, C126 em seres humanos), que é o responsável por nucleófilo FAE hidrólise 18,19. A inibição farmacológica da actividade NAAA altera o equilíbrio síntese / degradação FAE em favor do aumento dos níveis celulares de FAEs 16,20,21. Vários derivados β-lactona e β-lactâmicos têm mostrado inibir a actividade de NAAA com elevada potência e selectividade 16,22-26. Estes inibidores actuam através de S-acilação da cisteína catalítica 16,27,28. O ARN14686 composto foi concebido com base na estrutura química do, inibidor β-lactama NAAA-serina derivada sistemicamente activa, ARN726 (N-4 cyclohexylbutyl- - [(S) -2-oxoazetidin-3-il] carbamato de metilo) 16. O grupo 4-butil-ciclo-hexilo de ARN726 foi substituído com uma cadeia alifática em C9 saturado tendo uma marcação alcino terminal para conjugação subsequente com uma tag de CC repórter azida de rolamento. Nós escolhemos para projetar um ABP em duas etapas para minimally alterar a estrutura do andaime original, mantendo, assim, a afinidade da sonda para NAAA. Além do mais, evitando a introdução de etiquetas volumosos, uma tal sonda poderia ser mais adequado para o tratamento in vivo do que um PAF directa. ARN14686 NAAA inibe com elevada potência (hNAAA CI50 = 6 nM, rNAAA IC 50 = 13 nM) através da formação de um aducto covalente com a cisteína catalítica da enzima 12. Experimentos em ratos vivos mostraram que a sonda é seletiva na captura NAAA expressos em pulmões. Ácido ceramidase, outra cisteína-amidase que partilha 33-34% de identidade com NAAA, também foi identificado como um alvo de baixa afinidade ao utilizar concentrações elevadas de sonda (10 uM in vitro, 10 mg / ml por via intravenosa, iv) 12. Temos também utilizado ARN14686 para estudar a presença de NAAA activo em tecidos de rato a seguir à administração inflamadas de adjuvante completo de Freund (CFA) 29. Aqui, descrevemos um protocolo para os preparatina de ARN14686 (Figura 1) e sua aplicação para a investigação de activação NAAA ex vivo. Como exemplo, descreve-se um procedimento experimental para visualizar NAAA nas patas de ratos após a administração CFA. Nesta experiência, as proteínas são extraídas a partir de tecido da pata após a injecção IV da sonda, e o proteoma ABP-marcado é submetido a CC com biotina-azida. amostras biotiniladas são enriquecidas utilizando esferas de estreptavidina, e manchas de proteínas são realizadas. Em outra aplicação, descreve-se a localização de NAAA activo por microscopia de fluorescência em pulmões de ratinho a partir de ratinhos tratados com sonda. Neste caso, o tecido é seccionado e secções são submetidos a CC, por adição de rodamina. Um regime de fluxo de trabalho é ilustrado na Figura 2.

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		<col />
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">1,1&#8217;-sulfonyldiimidazole&#160;</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">367818</td>
			<td style="width:471px;">Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">2-dipyridylcarbonate</td>
			<td style="width:205px;">Fluorochem</td>
			<td style="width:111px;">11331</td>
			<td style="width:471px;">Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">2-Methylbutan</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">M32631</td>
			<td>Flamable, toxic,hazardous to the aquatic environment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">4-(Dimethylamino)pyridine</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">107700</td>
			<td style="width:471px;">Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Acetic acid</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">695092</td>
			<td style="width:471px;">Flammable, Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Acetonitrile&#160;</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">34998</td>
			<td style="width:471px;">Flammable, Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Activated charcoal</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">161551</td>
			<td style="width:471px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Ammonium chloride</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">A9434</td>
			<td style="width:471px;">Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Azide-PEG3-Biotin</td>
			<td style="width:205px;">Jena Biosciences</td>
			<td style="width:111px;">CLK-AZ104P4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Azide-PEG3-Fluor 545</td>
			<td style="width:205px;">Jena Biosciences</td>
			<td style="width:111px;">CLK-AZ109</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">BCA protein assay kit</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td style="width:111px;">23227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Bio-spin columns</td>
			<td style="width:205px;">Biorad</td>
			<td style="width:111px;">732-6204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Biotin</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">B4501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Blocking buffer</td>
			<td style="width:205px;">Li-Cor Biosciences</td>
			<td style="width:111px;">927-40000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">b-mercaptoethanol</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">M6250</td>
			<td>Higly toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Bovin serum albumine (BSA)</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">A7030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Bromophenol blue</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">B0126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Bruker Avance III 400</td>
			<td style="width:205px;">Bruker</td>
			<td style="width:111px;"></td>
			<td style="width:471px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Celite</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">419931</td>
			<td style="width:471px;">Health hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Ceric ammonium nitrate&#160;</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">22249</td>
			<td style="width:471px;">Oxidizing, Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Chloral hydrate</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">C8383</td>
			<td>Higly toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">CuSO4.5H2O&#160;</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">209198</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Cyclohexadiene</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">125415</td>
			<td style="width:471px;">Flammable, Health hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Cyclohexane</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">34855</td>
			<td style="width:471px;">Flammable, Harmful, Health hazard, Environmental hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Dichloromethane</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">34856</td>
			<td style="width:471px;">Harmful, Health hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Diethyl ether</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">296082</td>
			<td style="width:471px;">Flammable, Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:205px;">Acros Organics</td>
			<td style="width:111px;">348441000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Dimethyl sulfoxide d6 (DMSO-d6)</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">175943</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Ethanol</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">2860</td>
			<td style="width:471px;">Flammable, Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Ethyl acetate</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">34858</td>
			<td style="width:471px;">Flammable, Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Glycerol</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">G5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Irdye 680-LT Streptavidin</td>
			<td style="width:205px;">Li-Cor Biosciences</td>
			<td style="width:111px;">925-68031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">IRDye680-LT Streptavidin&#160;</td>
			<td style="width:205px;">Licor</td>
			<td style="width:111px;">925-68031</td>
			<td>Briefly centrifuge before use to precipitate protein complexes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Methanol</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">34966</td>
			<td>Highly toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Methanol</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">34860</td>
			<td style="width:471px;">Flammable, Toxic, Health hazard</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">N-(3-Dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">E7750</td>
			<td style="width:471px;">Harmful, Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">N,N-diisopropylethylamine</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">D125806</td>
			<td style="width:471px;">Flammable, Corrosive, Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">N,N-dimethylformamide</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">227056</td>
			<td style="width:471px;">Flammable, Harmful, Health hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">N-Cbz-L-Serine</td>
			<td style="width:205px;">Fluorochem&#160;</td>
			<td style="width:111px;">M03053</td>
			<td style="width:471px;">Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Nikon A1 confocal microscopy</td>
			<td style="width:205px;">Nikon</td>
			<td style="width:111px;"></td>
			<td>Read&#160; the user manual</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">NuPAGE 4-12% Bis-Tris gel</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td style="width:111px;">NP0335BOX</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Palladium on carbon</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">330108</td>
			<td style="width:471px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">p-anisidine</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">A88255</td>
			<td style="width:471px;">Toxic, Health hazard, Environmental hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Paraformaldehyde</td>
			<td style="width:205px;">sigma Aldrich</td>
			<td style="width:111px;">441244</td>
			<td>Toxic, respiratory harmful, corrosive, falmable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly(ethylene glycol)&#160;</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">P3265</td>
			<td style="width:471px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">ProLong Gold antifade mountant with DAPI&#160;</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td style="width:111px;">P36931</td>
			<td>Avoid bubbles formation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Protease inhibitor cocktail</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">P8340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Sodium bicarbonate</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">S6014</td>
			<td style="width:471px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Sodium dodecyl sulfate (SDS)&#160;</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">L3771</td>
			<td>Toxic, corrosive, falmmable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Sodium hydride&#160;</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">452912</td>
			<td style="width:471px;">Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Sodium sulfate</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">239313</td>
			<td style="width:471px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Starion FLA-9000 immage scanner</td>
			<td style="width:205px;">FUJIFILM</td>
			<td style="width:111px;"></td>
			<td>Read&#160; the user manual</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Streptavidin agarose</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td>20349</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Sucrose</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">S7903</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Tert-butanol</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">360538</td>
			<td>Toxic, flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Tetrahydrofuran</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">186562</td>
			<td style="width:471px;">Flammable, Harmful, Health hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Thiourea</td>
			<td style="width:205px;">Acros Organics</td>
			<td style="width:111px;">424542500</td>
			<td>Toxic, warm at 50 &#176;C to dissolve</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Tris</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">RDD008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris(2-carboxyethyl)phosphine (TCEP)</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">C4706</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA)</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">678937</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Triton-x100</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">X100</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Tween-20</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">P9416</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Tween-80</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">P1754</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Ultra turrax IKA T18 basic tissue homogenizer</td>
			<td style="width:205px;">IKA</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Undec-10-yn-1-ol</td>
			<td style="width:205px;">Fluorochem</td>
			<td style="width:111px;">13739</td>
			<td style="width:471px;">Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Urea</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">U5378</td>
			<td>Toxic, warm at 50 &#176;C to dissolve</td>
		</tr>
	</tbody>
</table>

preparation and in vivo use of an activity-based probe for n-acylethanolamine acid amidase

perfis de proteína à base de Actividade (ABPP) é um método para a identificação de uma enzima de interesse em um proteoma do complexo através da utilização de uma sonda que tem como alvo química sítios activos da enzima. Uma etiqueta repórter introduzidos na sonda permite a detecção da enzima marcada por análise em gel de fluorescência, mancha de proteína, microscopia de fluorescência, ou espectrometria de massa de cromatografia de líquido. Aqui, descrevemos a preparação e utilização do ARN14686 composto, uma sonda baseada em atividades de cliques química (CC-ABP) que reconhece seletivamente a enzima N amidase ácido -acylethanolamine (NAAA). NAAA é uma hidrolase cisteína que promove a inflamação desactivando -alfa agonistas endógenos do receptor proliferador de peroxissoma activado (PPAR), tais como palmitoiletanolamida (PEA) e oleoletanolamina (OEA). NAAA é sintetizada como uma proenzima de comprimento completo inactiva, que é activada pelo autoproteolysis no pH ácido do lisossoma. estudos de localização have mostrado que NAAA é predominantemente expresso em macrófagos e outras células derivadas de monócitos, bem como em linfócitos-B. Nós fornecem exemplos de como ARN14686 podem ser usadas para detectar e quantificar NAAA activo ex vivo em tecidos de roedores por mancha de proteína e microscopia de fluorescência.

ARN14686 foi projetado com base no cadafalso da ARN726 inibidor NAAA. O grupo 4-butil-ciclo-hexilo de ARN726 foi substituído com uma cadeia alifática em C9 saturado tendo uma marcação alcino terminal (Figura 1). A tag alcino foi introduzida, a fim de permitir a utilização de um procedimento de marcação de dois passos para adicionar um fluoróforo ou uma molécula de biotina através de CC. Esta característica torna ARN14686 uma ferramenta muito versátil para so...

Watch this Scientific Journal Video about Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase at JoVE.com

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Aqui, nós descrevemos a preparação e utilização de uma sonda com base na actividade (ARN14686, undec-10-ynyl- N - [(3 S) -2-oxoazetidin-3-il] carbamato de metilo), que permite a detecção e quantificação da forma activa da enzima pró-inflamatória N-amidase ácido -acylethanolamine (NAAA), tanto in vitro como ex vivo.

preparação e<em&gt; In Vivo</em&gt; O uso de uma sonda baseado em atividades para<em&gt; N</em&gt; Amidase Ácido -acylethanolamine

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical ...

Research

JoVE Journal

Biochemistry

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

Um método eficiente para o isolamento de ARN altamente purificada a partir de sementes para Utilização em Análise Quantitativa do transcriptoma

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

Bioquímica

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

Preparação de gigantes Vesículas Encapsulamento microesferas por centrifugação de uma emulsão de água-em-óleo

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Preparação da amostra para identificação de espectrometria de massa-baseada das regiões de ligação de RNA

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

Preparação de hidrolisado de queratina de penas de galinha e sua aplicação em cosméticos

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

An In Vivo Estrogen Deficiency Mouse Model for Screening Exogenous Estrogen Treatments of Cardiovascular Dysfunction After Menopause

Um modelo in vivo de deficiência de estrogênio mouse para triagem tratamentos de estrogênio exógeno de disfunção cardiovascular após a menopausa

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning ...

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

Uso do elemento Alu contendo Minigenes para analisar RNAs circulares

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Preparação de amostras para espectrometria de ionização de eletrospray de sonda

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

Preparation of High-Quality Fermented Fish Product

Preparação de produtos de peixe fermentado de alta qualidade

This protocol provides a method for preparation of industrialized fermented fish product with sturgeon (Aquilaria sinensis) meat product. The procedures ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

Uso de Termoforese microescala para medir interações proteína-lipídicas

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

Plant Sample Preparation for Nucleoside/Nucleotide Content Measurement with An HPLC-MS/MS

Preparação da amostra de plantas para medição de conteúdo nucleoídeo/nucleotídeo com hplc-MS/MS

Nucleosides/nucleotides are building blocks of nucleic acids, parts of cosubstrates and coenzymes, cell signaling molecules, and energy carriers, which ...