open-source-high-content-analysis-utilizing-automated-fluorescence

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Imperial College London

N-myristoylation is the irreversible attachment of a C(14) fatty acid, myristic acid, to the N-terminal glycine of a protein via formation of an amide bond. This modification is catalyzed by myristoyl-coenzyme A (CoA):protein N-myristoyltransferase (NMT), an enzyme ubiquitous in eukaryotes that is up-regulated in several cancers. Here we report a sensitive fluorescence-based assay to study the enzymatic activity of human NMT1 and NMT2 based on detection of CoA by 7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin. We also describe expression and characterization of NMT1 and NMT2 and assay validation with small molecule inhibitors. This assay should be broadly applicable to NMTs from a range of organisms.

A fluorescence-based assay for N-myristoyltransferase activity.

&#x4ECB;&#x7ECD;&#x4E86;&#x9009;&#x62E9;&#x6027;&#x548C;&#x7AD9;&#x70B9;&#x7279;&#x5B9A;&#x9176;&#x6807;&#x8BB0;&#x86CB;&#x767D;&#x7684;&#x4E00;&#x79CD;&#x534F;&#x8BAE;&#x3002;&#x8BE5;&#x65B9;&#x6CD5;&#x5229;&#x7528;&#x86CB;&#x767D;&#x8D28; co-&#x7FFB;&#x8BD1;&#x540E;&#x4FEE;&#x9970;&#x79F0;&#x4E3A; myristoylation&#xFF0C;&#x8089;&#x8C46;&#x853B;&#x9178; (14 &#x78B3;&#x9971;&#x548C;&#x8102;&#x80AA;&#x9178;&#xFF09; &#x79FB;&#x4EA4;&#x50AC;&#x5316;&#x9176; myristoyl N-&#x672B;&#x7AEF;&#x7518;&#x6C28;&#x9178;-CoA:protein N-&#x8089;&#x8C46;&#x853B; &#xFF08;&#x9AD8;&#x8003;&#xFF09;&#x3002;&#x5927;&#x80A0;&#x57C3;&#x5E0C;&#x6C0F;&#x5927;&#x80A0;&#x6746;&#x83CC;&#xFF0C;&#x6709;&#x6CA1;&#x6709;&#x5185;&#x5728;&#x7684;&#x9AD8;&#x8003;&#xFF0C;&#x7528;&#x4E8E;&#x8F6C;&#x79FB;&#x9176;&#x4E0E;&#x76EE;&#x6807;&#x86CB;&#x767D;&#x7684;&#x5171;&#x8868;&#x8FBE;&#x8FDB;&#x884C;&#x6807;&#x8BB0;&#x3001; &#x53C2;&#x4E0E;&#x4F53;&#x5185;&#x7684; N &#x7EC8;&#x7AEF;&#x9644;&#x4EF6;&#x7EFC;&#x5408;&#x6D3E;&#x751F;&#x6807;&#x8BB0;&#x7C7B;&#x4F3C;&#x7269;&#x7684;&#x8089;&#x8C46;&#x853B;&#x9178;&#x8F74;&#x627F; &#39;&#x53EF;&#x70B9;&#x51FB;&#x6807;&#x8BB0;&#x7684;&#x3002;&#x6B64;&#x6807;&#x8BB0;&#x662F;&#x529F;&#x80FD;&#x7EC4;&#xFF0C;&#x53EF;&#x4EE5;&#x63A5;&#x53D7;&#x901A;&#x8FC7;&#x5355;&#x51FB;&#39; &#x5316;&#x5B66;&#xFF0C;&#x4F8B;&#x5982;&#xFF0C;&#x53E0;&#x6C2E;&#x7684;&#x751F;&#x7269;&#x6B63;&#x4EA4;&#x7ED3;&#x624E;&#x5E76;&#x53EF;&#x4EE5;&#x7528;&#x4F5C;&#x8FDB;&#x4E00;&#x6B65;&#x5177;&#x4F53;&#x5730;&#x70B9;&#x8D34;&#x6807;&#x4F53;&#x5916;&#x7684;&#x53E5;&#x67C4;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x4E3A;&#x534F;&#x8BAE;&#x63D0;&#x4F9B;&#x4F53;&#x5185; N &#x7EC8;&#x7AEF;&#x6807;&#x8BB0;&#x7684;&#x91CD;&#x7EC4;&#x86CB;&#x767D;&#x548C;&#x7684;&#x5408;&#x6210;&#x4E0E;&#x5E94;&#x7528;&#x7684;&#x591A;&#x529F;&#x80FD;&#x7684;&#x8BD5;&#x5242;&#xFF0C;&#x4F7F;&#x86CB;&#x767D;&#x6807;&#x8BB0;&#x901A;&#x8FC7;&#x5355;&#x51FB;&#x5316;&#x5B66;&#x4EB2;&#x548C;&#x7EAF;&#x5316;&#x548C;&#x68C0;&#x6D4B;&#x8367;&#x5149;&#x3002;&#x9664;&#x4E86;&#x4E00;&#x822C; N &#x672B;&#x7AEF;&#x86CB;&#x767D;&#x6807;&#x8BB0;&#xFF0C;&#x8BAE;&#x5B9A;&#x4E66; &#x300B; &#x5C06;&#x63D0;&#x4F9B;&#x8BC1;&#x636E;&#x4E3A;&#x672C;&#x673A; myristoylation &#x611F;&#x5174;&#x8DA3;&#xFF0C;&#x8BC1;&#x660E;&#x6D3B;&#x52A8;/&#x9009;&#x62E9;&#x6027;&#x7684; NMTs &#x548C; NMTs &#x800C;&#x4E0D;&#x662F;&#x8BC9;&#x8BF8;&#x5229;&#x7528;&#x653E;&#x5C04;&#x6027;&#x540C;&#x4F4D;&#x7D20;&#x7684;&#x8DE8;&#x7269;&#x79CD;&#x53CD;&#x5E94;&#x6027;&#x86CB;&#x767D;&#x7684;&#x7279;&#x6B8A;&#x7528;&#x9014;&#x3002;

&#x591A;&#x529F;&#x80FD;&#x86CB;&#x767D;&#x6807;&#x8BB0;&#x9176; N &#x7AEF;&#x6807;&#x8BB0;&#x548C;&#x9610;&#x8FF0;&#x901A;&#x8FC7;&#x5355;&#x51FB;&#x5316;&#x5B66;&#x3002;

A protocol for selective and site-specific enzymatic labeling of proteins is described. The method exploits the protein co-/post-translational modification known as myristoylation, the transfer of myristic acid (a 14-carbon saturated fatty acid) to an N-terminal glycine catalyzed by the enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT). Escherichia coli, having no endogenous NMT, is used for the coexpression of both the transferase and the target protein to be labeled, which participate in the in vivo N-terminal attachment of synthetically derived tagged analogs of myristic acid bearing a 'clickable' tag. This tag is a functional group that can undergo bio-orthogonal ligation via 'click' chemistry, for example, an azide, and can be used as a handle for further site-specific labeling in vitro. Here we provide protocols for in vivo N-terminal tagging of recombinant protein, and the synthesis and application of multifunctional reagents that enable protein labeling via click chemistry for affinity purification and detection by fluorescence. In addition to general N-terminal protein labeling, the protocol would be of particular use in providing evidence for native myristoylation of proteins of interest, proof of activity/selectivity of NMTs and cross-species reactivity of NMTs without resorting to the use of radioactive isotopes.

Multifunctional protein labeling via enzymatic N-terminal tagging and elaboration by click chemistry.

Un protocole pour le marquage enzymatique s&eacute;lective et sp&eacute;cifique des prot&eacute;ines est d&eacute;crite. La m&eacute;thode exploite la prot&eacute;ine co- / modification post-traductionnelle appel&eacute;e myristoylation, le transfert de l&#39;acide myristique (un acide de gras satur&eacute; de carbone-14) &agrave; une glycine N-terminale, catalys&eacute;e par l&#39;enzyme myristoyl-CoA:protein N-myristoyltransf&eacute;rase (NMT). Escherichia coli, n&#39;ayant aucune NMT endog&egrave;ne, est utilis&eacute; pour la coexpression de la transf&eacute;rase et de la prot&eacute;ine cible doivent &ecirc;tre &eacute;tiquet&eacute;es, qui participent &agrave; l&#39;accessoire N-terminal in vivo de synth&eacute;tiquement d&eacute;riv&eacute;s analogues marqu&eacute;s d&#39;acide myristique, portant une &eacute;tiquette &laquo; cliquable &raquo;. Cette balise est un groupe fonctionnel qui peut subir la ligature bio orthogonale via &laquo; cliquer &raquo; chimie, par exemple, un azoture et peut &ecirc;tre utilis&eacute; comme poign&eacute;e pour encore in situ marquage in vitro. Ici, nous fournissons des protocoles pour marquer in vivo des N-terminale de la prot&eacute;ine recombinante, puis la synth&egrave;se et l&#39;application de r&eacute;actifs multifonctions qui permettent l&#39;&eacute;tiquetage par l&#39;interm&eacute;diaire de prot&eacute;ines sur chimie pour la purification d&#39;affinit&eacute; et la d&eacute;tection par fluorescence. En plus g&eacute;n&eacute;rales marquage de prot&eacute;ines N-terminal, le protocole serait particuli&egrave;rement utile dans la fourniture de preuves pour myristoylation native des prot&eacute;ines d&#39;int&eacute;r&ecirc;t, preuve d&#39;activit&eacute;/s&eacute;lectivit&eacute; des territoires non m&eacute;tropolitains et r&eacute;activit&eacute; h&eacute;t&eacute;rosp&eacute;cifique de territoires non m&eacute;tropolitains sans recourir &agrave; l&#39;utilisation d&#39;isotopes radioactifs.

Prot&eacute;ine multifonctionnelle &eacute;tiquetage via N-terminale de marquage enzymatique et l&#39;&eacute;laboration de chimie de clic.

Ein Protokoll f&uuml;r die selektive und standortspezifische enzymatische Kennzeichnung der Proteine wird beschrieben. Die Methode nutzt die Protein-co- / post-translationale Modifikation bekannt als Myristoylation, die &Uuml;bertragung der Myristins&auml;ure (eine 14-Kohlenstoff ges&auml;ttigte Fetts&auml;ure) an einer N-terminalen Glycin katalysiert durch das Enzym Myristoyl-CoA:protein N-Myristoyltransferase (NMT). Escherichia coli, dass keine endogenen NMT, dient der Coexpression von der Transferase und Zielprotein beschriftet werden, die in der in-vivo N-Terminal-Anlage von synthetisch abgeleiteten markierte Analoga von Myristins&auml;ure tragen einen &quot;anklickbar&quot; Tag teilnehmen. Dieser Tag ist eine funktionelle Gruppe, die Bio-orthogonal Verbindung per &#39;Klick&#39; Chemie, z. B. ein azid, unterziehen k&ouml;nnen und kann f&uuml;r weitere ortsspezifische beschriften in-vitro-als Griff verwendet werden. Hier bieten wir Protokolle f&uuml;r in-vivo-N-Terminal-tagging des rekombinanten Proteins und der Synthese und Anwendung von multifunktionalen Reagenzien, die Beschriftung &uuml;ber Protein aktivieren auf Chemie f&uuml;r Affinit&auml;tsreinigung und Erkennung von Fluoreszenz. Neben allgemeinen N-terminale Protein Etikettieren w&auml;re das Protokoll der besonderen Verwendung in den Nachweis f&uuml;r native Myristoylation Proteine des Interesses, Nachweis der Aktivit&auml;t/Selektivit&auml;t des NMTs und Kreuz-Arten Reaktivit&auml;t des NMTs ohne R&uuml;ckgriff auf die Verwendung radioaktiver Isotope.

Multifunktionale Protein Kennzeichnung &uuml;ber enzymatische N-terminalen tagging und Ausarbeitung von click-Chemie.

Un protocollo per la selettiva e site-specific di etichettatura enzimatica delle proteine &egrave; descritto. Il metodo sfrutta la proteina co- / modifica alberino-di traduzione, conosciuto come myristoylation, il trasferimento di acido miristico (14-carbonio saturato acido grasso) ad una glicina N-terminale catalizzato dall&#39;enzima miristoil-CoA:protein N-miristoiltransferasi (NMT). Escherichia coli, non avendo NMT endogena, viene utilizzato per la coexpression del transferasi e la proteina bersaglio per essere etichettati, che partecipano in vivo allegato N-terminale di analoghi contrassegnati sinteticamente derivati di acido miristico recanti un tag &#39;cliccabile&#39;. Questo tag &egrave; un gruppo funzionale che pu&ograve; subire la legatura bio-ortogonale via chimica &#39;click&#39;, ad esempio, un azoturo e pu&ograve; essere utilizzato come una maniglia per ulteriore site specific etichettatura in vitro. Qui forniamo protocolli in vivo tagging N-terminale della proteina ricombinante e la sintesi e l&#39;applicazione dei reagenti multifunzionali che consentono la proteina etichettatura scegliere chimica per la purificazione di affinit&agrave; e rilevazione di fluorescenza. Oltre al generale N-terminale della proteina etichettatura, il protocollo sarebbe di particolare utilizzo nel fornire prove per myristoylation nativo di proteine di interesse, prova di attivit&agrave;/selettivit&agrave; del NMTs e la reattivit&agrave; del cross-specie di NMTs senza ricorrere all&#39;uso di isotopi radioattivi.

Contrassegno della proteina multifunzionale via enzimatica N-terminale tagging ed elaborazione di scegliere chimica.

&#x9078;&#x629E;&#x7684;&#x3068;&#x90E8;&#x4F4D;&#x7279;&#x7570;&#x7684;&#x9175;&#x7D20;&#x5206;&#x985E;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x305F;&#x3081;&#x306E;&#x30D7;&#x30ED;&#x30C8;&#x30B3;&#x30EB;&#x3092;&#x8AAC;&#x660E;&#x3057;&#x307E;&#x3059;&#x3002;&#x30E1;&#x30BD;&#x30C3;&#x30C9; &#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA; co - &#x653B;&#x7565;/&#x7FFB;&#x8A33;&#x5F8C;&#x4FEE;&#x98FE;&#x30DF;&#x30EA;&#x30B9;&#x30C8;&#x30A4;&#x30EB;&#x5316;&#x9175;&#x7D20; myristoyl &#x306B;&#x3088;&#x308B;&#x89E6;&#x5A92;&#x306F; N &#x672B;&#x7AEF;&#x306E;&#x30B0;&#x30EA;&#x30B7;&#x30F3;&#x306B;&#x30DF;&#x30EA;&#x30B9;&#x30C1;&#x30F3;&#x9178; (14 &#x70AD;&#x7D20;&#x98FD;&#x548C;&#x8102;&#x80AA;&#x9178;&#xFF09; &#x306E;&#x8EE2;&#x9001;&#x3068;&#x3057;&#x3066;&#x77E5;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;-CoA:protein N-&#x30DF;&#x30EA;&#x30B9;&#x30C8;&#x30A4;&#x30EB;&#x8EE2;&#x79FB;&#x9175;&#x7D20; &#xFF08;NMT&#xFF09;&#x3002;&#x5185;&#x56E0;&#x6027; NMT &#x3092;&#x6301;&#x305F;&#x306A;&#x3044;&#x5927;&#x8178;&#x83CC;&#x3067;&#x3059;&#x4F7F;&#x7528;&#x3001;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30D5;&#x30A7;&#x30E9;&#x30FC;&#x30BC;&#x3068;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x767A;&#x73FE;&#x3092;&#x30E9;&#x30D9;&#x30EB;&#x4ED8;&#x3051;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#39;&#x30AF;&#x30EA;&#x30C3;&#x30AF;&#39; &#x30BF;&#x30B0; &#x30DF;&#x30EA;&#x30B9;&#x30C1;&#x30F3;&#x9178;&#x306E;&#x5408;&#x6210;&#x7684;&#x6D3E;&#x751F;&#x30BF;&#x30B0;&#x985E;&#x7E01;&#x4F53;&#x306E; in vivo N &#x672B;&#x7AEF;&#x6DFB;&#x4ED8;&#x30D5;&#x30A1;&#x30A4;&#x30EB;&#x306B;&#x53C2;&#x52A0;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x30BF;&#x30B0;&#x306F;&#x3001;&#x6A5F;&#x80FD;&#x30B0;&#x30EB;&#x30FC;&#x30D7; &#39;&#x30AF;&#x30EA;&#x30C3;&#x30AF;&#x3057;&#x3066;&#39; &#x5316;&#x5B66;&#x3001;&#x305F;&#x3068;&#x3048;&#x3070;&#x3001;&#x30A2;&#x30B8;&#x3092;&#x4ECB;&#x3057;&#x3066;&#x30D0;&#x30A4;&#x30AA;&#x76F4;&#x4EA4;&#x7D50;&#x7D2E;&#x3092;&#x53D7;&#x3051;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x3057;&#x3001;&#x3055;&#x3089;&#x306B;&#x30B5;&#x30A4;&#x30C8;&#x56FA;&#x6709; in vitro &#x30E9;&#x30D9;&#x30EA;&#x30F3;&#x30B0;&#x306E;&#x30CF;&#x30F3;&#x30C9;&#x30EB;&#x3068;&#x3057;&#x3066;&#x3092;&#x4F7F;&#x7528;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x3067;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x6211;&#x3005; &#x30D7;&#x30ED;&#x30C8;&#x30B3;&#x30EB;&#x7D44;&#x63DB;&#x3048;&#x86CB;&#x767D;&#x8CEA;&#x306E; N &#x672B;&#x7AEF; in vivo &#x30BF;&#x30AE;&#x30F3;&#x30B0;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x3001;&#x5408;&#x6210;&#x3068;&#x86CB;&#x767D;&#x8CEA;&#x3092;&#x4ECB;&#x3057;&#x3066;&#x30E9;&#x30D9;&#x30EA;&#x30F3;&#x30B0;&#x3092;&#x6709;&#x52B9;&#x306B;&#x3059;&#x308B;&#x591A;&#x6A5F;&#x80FD;&#x306E;&#x8A66;&#x85AC;&#x306E;&#x30A2;&#x30D7;&#x30EA;&#x30B1;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x306B;&#x3088;&#x308B;&#x86CD;&#x5149;&#x5316;&#x5B66;&#x89AA;&#x548C;&#x6027;&#x306E;&#x6D44;&#x5316;&#x3068;&#x691C;&#x51FA;] &#x3092;&#x30AF;&#x30EA;&#x30C3;&#x30AF;&#x3057;&#x307E;&#x3059;&#x3002;&#x4E00;&#x822C;&#x7684;&#x306A; N &#x672B;&#x7AEF;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x30E9;&#x30D9;&#x30EA;&#x30F3;&#x30B0;&#x306B;&#x52A0;&#x3048;&#x3066;&#x3001;&#x30D7;&#x30ED;&#x30C8;&#x30B3;&#x30EB;&#x306E;&#x30CD;&#x30A4;&#x30C6;&#x30A3;&#x30D6; &#x30DF;&#x30EA;&#x30B9;&#x30C8;&#x30A4;&#x30EB;&#x5316;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA; NMTs &#x306E;&#x9078;&#x629E;&#x6027;&#x306E;&#x6D3B;&#x52D5;&#x3068;&#x653E;&#x5C04;&#x6027;&#x540C;&#x4F4D;&#x5143;&#x7D20;&#x306E;&#x4F7F;&#x7528;&#x306B;&#x983C;&#x308B;&#x3053;&#x3068;&#x304C;&#x306A;&#x304F;&#x3001;&#x30AF;&#x30ED;&#x30B9;&#x7A2E;&#x306E;&#x53CD;&#x5FDC;&#x6027;&#x306E; NMTs &#x306E;&#x8A3C;&#x62E0;&#x306E;&#x8208;&#x5473;&#x306E;&#x305F;&#x3081;&#x306E;&#x8A3C;&#x62E0;&#x3092;&#x63D0;&#x4F9B;&#x3059;&#x308B;&#x7279;&#x5B9A;&#x306E;&#x4F7F;&#x7528;&#x306E;&#x3067;&#x3057;&#x3087;&#x3046;&#x3002;

&#x591A;&#x6A5F;&#x80FD;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x9175;&#x7D20;&#x306E; N &#x672B;&#x7AEF;&#x306E;&#x30BF;&#x30B0;&#x4ED8;&#x3051;&#x3068;&#x7CBE;&#x7DFB;&#x5316;&#x306B;&#x3088;&#x3063;&#x3066;&#x3092;&#x4ECB;&#x3057;&#x3066;&#x30E9;&#x30D9;&#x30EA;&#x30F3;&#x30B0;&#x5316;&#x5B66;] &#x3092;&#x30AF;&#x30EA;&#x30C3;&#x30AF;&#x3057;&#x307E;&#x3059;&#x3002;

&#xC120;&#xD0DD;&#xC801;&#xC774; &#xACE0; &#xC0AC;&#xC774;&#xD2B8; &#xAD00;&#xB828; &#xD6A8;&#xC18C; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xB77C;&#xBCA8;&#xC5D0; &#xB300; &#xD55C; &#xD504;&#xB85C;&#xD1A0;&#xCF5C;&#xC744; &#xC124;&#xBA85; &#xD569;&#xB2C8;&#xB2E4;. &#xBA54;&#xC11C;&#xB4DC;&#xB97C; &#xC774;&#xC6A9;&#xD55C; &#xACF5;&#xACA9; &#xB2E8;&#xBC31;&#xC9C8; &#xACF5;&#xB3D9;-/ &#xD3EC;&#xC2A4;&#xD2B8; &#xBC88;&#xC5ED; &#xC0C1; &#xC218;&#xC815; myristoylation, &#xCD09;&#xB9E4; &#xD6A8;&#xC18C; myristoyl&#xC5D0; &#xC758;&#xD574; N-&#xD130;&#xBBF8;&#xB110; &#xAE00;&#xB9AC;&#xC2E0; myristic &#xC0B0; (14-&#xD0C4;&#xC18C; &#xD3EC;&#xD654; &#xC9C0;&#xBC29;&#xC0B0;)&#xC758; &#xC804;&#xC1A1;&#xC73C;&#xB85C; &#xC54C;&#xB824;&#xC9C4;-CoA:protein N myristoyltransferase (NMT). &#xB300;&#xC7A5;&#xADE0;, &#xC544;&#xB2C8; &#xB0B4; &#xC0DD; NMT&#xB294; &#xB370; &#xC0AC;&#xC6A9; &#xB429;&#xB2C8;&#xB2E4; &#xC5EC;&#xACFC; &#xBC0F; &#xB300;&#xC0C1; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; coexpression &#xD45C;&#xC2DC; &#xB420; myristic &#xC0B0; &#39;&#xD074;&#xB9AD;&#xD560;&#39; &#xD0DC;&#xADF8; &#xBCA0;&#xC5B4;&#xB9C1;&#xC758; &#xC885;&#xD569;&#xC801; &#xD30C;&#xC0DD;&#xB41C; &#xD0DC;&#xADF8; &#xC720;&#xC0AC; &#xCCB4;&#xC758; in vivo N &#xB2E8;&#xC790; &#xCCA8;&#xBD80; &#xD30C;&#xC77C;&#xC5D0; &#xCC38;&#xC5EC; &#xD558;&#xB294;. &#xC774; &#xD0DC;&#xADF8;&#xB294; &#39;&#xD074;&#xB9AD;&#39; &#xD654;&#xD559;, &#xC608;&#xB97C; &#xB4E4;&#xC5B4;&#xB294; &#xC544; &#xC9C0; &#xB4DC;&#xB97C; &#xD1B5;&#xD574; &#xBC14;&#xC774;&#xC624; &#xC9C1;&#xAD50; &#xACB0; &#xCC30;&#xC744; &#xBC1B;&#xC744; &#xC218; &#xBC0F; &#xCD94;&#xAC00; &#xC0AC;&#xC774;&#xD2B8;&#xBCC4; &#xB77C;&#xBCA8; &#xCCB4; &#xC678;&#xC5D0; &#xB300; &#xD55C; &#xD578;&#xB4E4;&#xC744; &#xC0AC;&#xC6A9;&#xD560; &#xC218; &#xC788;&#xB294; &#xAE30;&#xB2A5; &#xADF8;&#xB8F9;&#xC785;&#xB2C8;&#xB2E4;. &#xC7AC;&#xC870;&#xD569; &#xD615; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; in vivo N &#xD130;&#xBBF8;&#xB110; &#xD0DC;&#xADF8;&#xC5D0; &#xB300; &#xD55C; &#xD504;&#xB85C;&#xD1A0;&#xCF5C;&#xC744; &#xC81C;&#xACF5; &#xD558;&#xB294; &#xC5EC;&#xAE30;&#xB97C; &#xD569;&#xC131; &#xBC0F; &#xB2E8;&#xBC31;&#xC9C8;&#xC744; &#xD1B5;&#xD574; &#xB77C;&#xBCA8;&#xC744; &#xC0AC;&#xC6A9;&#xD560; &#xC218; &#xC788;&#xB294; &#xB2E4;&#xAE30;&#xB2A5; &#xC2DC; &#xC57D;&#xC758; &#xC751;&#xC6A9; &#xD654;&#xD559; &#xCE5C; &#xD654;&#xB825; &#xC815;&#xD654; &#xBC0F; &#xAC80;&#xCD9C;&#xC744; &#xC704;&#xD55C; &#xD615;&#xAD11;&#xC744; &#xD074;&#xB9AD;. &#xC77C;&#xBC18;&#xC801;&#xC778; N &#xB9E8;&#xB05D; &#xB2E8;&#xBC31;&#xC9C8; &#xB77C;&#xBCA8;, &#xBFD0;&#xB9CC; &#xC544;&#xB2C8;&#xB77C; &#xD504;&#xB85C;&#xD1A0;&#xCF5C; &#xAD00;&#xC2EC;, &#xD65C;&#xB3D9;/&#xC120;&#xD0DD;&#xB3C4; NMTs &#xBC0F; Nmts&#xC758; &#xBC29;&#xC0AC;&#xC131; &#xB3D9;&#xC704; &#xC6D0;&#xC18C; &#xC0AC;&#xC6A9;&#xC5D0;&#xC758; &#xC9C0; &#xD558;&#xC9C0; &#xC54A;&#xACE0; &#xAC04; &#xC885; &#xBC18;&#xC751;&#xC131;&#xC758; &#xC99D;&#xAC70;&#xC758; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xB124;&#xC774;&#xD2F0;&#xBE0C; myristoylation&#xC5D0; &#xB300; &#xD55C; &#xC99D;&#xAC70;&#xB97C; &#xC81C;&#xACF5; &#xD558; &#xACE0; &#xD2B9;&#xC815; &#xC0AC;&#xC6A9; &#xB420; &#xAC83; &#xC774;&#xB2E4;.

&#xB2E4;&#xAE30;&#xB2A5; &#xB2E8;&#xBC31;&#xC9C8; &#xD6A8;&#xC18C; N &#xB9E8;&#xB05D;&#xC5D0; &#xD0DC;&#xADF8;&#xB97C; &#xCD94;&#xAC00; &#xD558; &#xACE0; &#xC815;&#xAD50; &#xD558; &#xC5EC; &#xD1B5;&#xD574; &#xB77C;&#xBCA8; &#xD654;&#xD559;&#xC744; &#xD074;&#xB9AD; &#xD569;&#xB2C8;&#xB2E4;.

Se describe un protocolo para el etiquetado enzim&aacute;tica espec&iacute;fica y selectiva de las prote&iacute;nas. El m&eacute;todo explota la prote&iacute;na co- / modificaci&oacute;n post-translacional conocido como myristoylation, la transferencia de &aacute;cido mir&iacute;stico (un &aacute;cido graso saturado de carbono 14) a una glicina N-terminal, catalizada por la enzima myristoyl-CoA:protein N-myristoyltransferase (NMT). Escherichia coli, no tener NMT end&oacute;geno, se utiliza para la coexpresi&oacute;n de la transferasa y la prote&iacute;na diana a etiquetarse, que participan en la fijaci&oacute;n de N-terminal in vivo de an&aacute;logos etiquetados sint&eacute;tico derivados del &aacute;cido mir&iacute;stico teniendo una etiqueta &#39;clic&#39;. Esta etiqueta es un grupo funcional que puede someterse a la ligadura bio-ortogonal v&iacute;a qu&iacute;mica &quot;clic&quot;, por ejemplo, una azida y puede utilizarse como un mango para m&aacute;s espec&iacute;fica etiquetado in vitro. Aqu&iacute; ofrecemos protocolos para in vivo el etiquetado N-terminal de la prote&iacute;na recombinante y la s&iacute;ntesis y la aplicaci&oacute;n de reactivos multifuncionales que permiten prote&iacute;na etiquetado v&iacute;a en qu&iacute;mica para la purificaci&oacute;n de la afinidad y detecci&oacute;n por fluorescencia. Adem&aacute;s de general N-terminal prote&iacute;na etiquetado, el protocolo ser&iacute;a de uso particular en proporcionar evidencia para myristoylation nativa de prote&iacute;nas de inter&eacute;s, prueba de actividad/selectividad de territorios no metropolitanos y la reactividad de la especie de los territorios no metropolitanos sin recurrir al uso de is&oacute;topos radiactivos.

Prote&iacute;na multifuncional etiquetado v&iacute;a enzim&aacute;tica N-terminal marcado y elaboraci&oacute;n por haga clic en qu&iacute;mica.

Fluorescence lifetime measurements can provide quantitative readouts of local fluorophore environment and can be applied to biomolecular interactions via Förster resonant energy transfer (FRET). Fluorescence lifetime imaging (FLIM) can therefore provide a high content analysis (HCA) modality to map protein-protein interactions (PPIs) with applications in drug discovery, systems biology and basic research. We present here an automated multiwell plate reader able to perform rapid unsupervised optically sectioned FLIM of fixed and live biological samples and illustrate its potential to assay PPIs through application to Gag protein aggregation during the HIV life cycle. We demonstrate both hetero-FRET and homo-FRET readouts of protein aggregation and report the first quantitative evaluation of a FLIM HCA assay by generating dose response curves through addition of an inhibitor of Gag myristoylation. Z' factors exceeding 0.6 are realised for this FLIM FRET assay.

Automated fluorescence lifetime imaging plate reader and its application to Förster resonant energy transfer readout of Gag protein aggregation.

Protein N-myristoylation is a ubiquitous co- and post-translational modification that has been implicated in the development and progression of a range of human diseases. Here, we report the global N-myristoylated proteome in human cells determined using quantitative chemical proteomics combined with potent and specific human N-myristoyltransferase (NMT) inhibition. Global quantification of N-myristoylation during normal growth or apoptosis allowed the identification of >100 N-myristoylated proteins, >95% of which are identified for the first time at endogenous levels. Furthermore, quantitative dose response for inhibition of N-myristoylation is determined for >70 substrates simultaneously across the proteome. Small-molecule inhibition through a conserved substrate-binding pocket is also demonstrated by solving the crystal structures of inhibitor-bound NMT1 and NMT2. The presented data substantially expand the known repertoire of co- and post-translational N-myristoylation in addition to validating tools for the pharmacological inhibition of NMT in living cells.

Global profiling of co- and post-translationally N-myristoylated proteomes in human cells.

Protein lipidation is unique amongst post-translational modifications (PTMs) in enabling direct interaction with cell membranes, and is found in every form of life. Lipidation is important in normal function and in disease, but its intricate interplay with disease context presents a challenging for drug development. Global whole-proteome profiling of protein lipidation lies beyond the range of standard methods, but is well-suited to metabolic tagging with small 'clickable' chemical reporters that do not disrupt metabolism and function; chemoselective reactions are then used to add multifunctional labels exclusively to tagged-lipidated proteins. This chemical proteomic technology has opened up the first quantitative whole-proteome studies of the known major classes of protein lipidation, and the first insights into their full scope in vivo.

Global profiling of protein lipidation using chemical proteomic technologies.

Nearly isosteric oxo to thioxo substitution was employed to interrogate the structure of foldamers with a urea backbone and explore the relationship between helical folding and hydrogen-bonding interactions. A series of oligomers with urea bonds substituted by thiourea bonds at discrete or all positions in the sequence have been prepared and their folding propensity was studied by using a combination of spectroscopic methods and X-ray diffraction. The outcome of oxo to thioxo replacements on the helical folding was found to depend on whether central or terminal ureas were modified. The canonical helix geometry was not affected upon insertion of thioureas close to the negative end of the helix dipole, whereas thioureas close to the positive pole were found to increase the terminal flexibility and cause helix fraying. Perturbation was amplified when a selenourea was incorporated instead, leading to a structure that is only partly folded.

Isosteric substitutions of urea to thiourea and selenourea in aliphatic oligourea foldamers: site-specific perturbation of the helix geometry.

Proteins are acylated by a variety of metabolites that regulates many important cellular pathways in all kingdoms of life. Acyl groups in cells can vary in structure from the smallest unit, acetate, to modified long-chain fatty acids, all of which can be activated and covalently attached to diverse amino acid side chains and consequently modulate protein function. For example, acetylation of Lys residues can alter the charge state of proteins and generate new recognition elements for protein-protein interactions. Alternatively, long-chain fatty-acylation targets proteins to membranes and enables spatial control of cell signalling. To facilitate the analysis of protein acylation in biology, acyl analogues bearing alkyne or azide tags have been developed that enable fluorescent imaging and proteomic profiling of modified proteins using bioorthogonal ligation methods. Herein, we summarize the currently available acylation chemical reporters and highlight their utility to discover and quantify the roles of protein acylation in biology.

Chemical reporters for exploring protein acylation.

Hedgehog signaling is critical for correct embryogenesis and tissue development. However, on maturation, signaling is also found to be aberrantly activated in many cancers. Palmitoylation of the secreted signaling protein sonic hedgehog (Shh) by the enzyme hedgehog acyltransferase (Hhat) is required for functional signaling. To quantify this important posttranslational modification, many in vitro Shh palmitoylation assays employ radiolabeled fatty acids, which have limitations in terms of cost and safety. Here we present a click chemistry armed enzyme-linked immunosorbent assay (click-ELISA) for assessment of Hhat activity through acylation of biotinylated Shh peptide with an alkyne-tagged palmitoyl-CoA (coenzyme A) analogue. Click chemistry functionalization of the alkyne tag with azido-FLAG peptide allows analysis through an ELISA protocol and colorimetric readout. This assay format identified the detergent n-dodecyl β-d-maltopyranoside as an improved solubilizing agent for Hhat activity. Quantification of the potency of RU-SKI small molecule Hhat inhibitors by click-ELISA indicated IC50 values in the low- or sub-micromolar range. A stopped assay format was also employed that allows measurement of Hhat kinetic parameters where saturating substrate concentrations exceed the binding capacity of the streptavidin-coated plate. Therefore, click-ELISA represents a nonradioactive method for assessing protein palmitoylation in vitro that is readily expandable to other classes of protein lipidation.

Click chemistry armed enzyme-linked immunosorbent assay to measure palmitoylation by hedgehog acyltransferase.

Protein fatty-acylation in eukaryotes has been associated with many fundamental biological processes. However, the diversity, abundance and regulatory mechanisms of protein fatty-acylation in vivo remain to be explored. Herein, we review the proteomic analysis of fatty-acylated proteins, with a focus on N-myristoylation and S-palmitoylation. We then highlight major challenges and emerging methods for direct site identification, quantitation, and lipid structure characterization to understand the functions and regulatory mechanisms of fatty-acylated proteins in physiology and disease.

Proteomic analysis of fatty-acylated proteins.

Fatty acylation of cysteine residues provides spatial and temporal control of protein function in cells and regulates important biological pathways in eukaryotes. Although recent methods have improved the detection and proteomic analysis of cysteine fatty (S-fatty) acylated proteins, understanding how specific sites and quantitative levels of this posttranslational modification modulate cellular pathways are still challenging. To analyze the endogenous levels of protein S-fatty acylation in cells, we developed a mass-tag labeling method based on hydroxylamine-sensitivity of thioesters and selective maleimide-modification of cysteines, termed acyl-PEG exchange (APE). We demonstrate that APE enables sensitive detection of protein S-acylation levels and is broadly applicable to different classes of S-palmitoylated membrane proteins. Using APE, we show that endogenous interferon-induced transmembrane protein 3 is S-fatty acylated on three cysteine residues and site-specific modification of highly conserved cysteines are crucial for the antiviral activity of this IFN-stimulated immune effector. APE therefore provides a general and sensitive method for analyzing the endogenous levels of protein S-fatty acylation and should facilitate quantitative studies of this regulated and dynamic lipid modification in biological systems.

Mass-tag labeling reveals site-specific and endogenous levels of protein S-fatty acylation.

In this data article we describe synthetic and characterisation data for four members of the 5-acyl-6,7-dihydrothieno[3,2-c]pyridine (termed "RU-SKI") class of inhibitors of Hedgehog acyltransferase, including associated NMR spectra for final compounds. RU-SKI compounds were selected for synthesis based on their published high potencies against the enzyme target. RU-SKI 41 (9a), RU-SKI 43 (9b), RU-SKI 101 (9c), and RU-SKI 201 (9d) were profiled for activity in the related article "Click chemistry armed enzyme linked immunosorbent assay to measure palmitoylation by Hedgehog acyltransferase" (Lanyon-Hogg et al., 2015) [1]. (1)H NMR spectral data indicate different amide conformational ratios between the RU-SKI inhibitors, as has been observed in other 5-acyl-6,7-dihydrothieno[3,2-c]pyridines. The synthetic and characterisation data supplied in the current article provide validated access to the class of RU-SKI inhibitors.

Synthesis and characterisation of 5-acyl-6,7-dihydrothieno[3,2-c]pyridine inhibitors of Hedgehog acyltransferase.

N-Myristoyltransferase (NMT) covalently attaches a C14 fatty acid to the N-terminal glycine of proteins and has been proposed as a therapeutic target in cancer. We have recently shown that selective NMT inhibition leads to dose-responsive loss of N-myristoylation on more than 100 protein targets in cells, and cytotoxicity in cancer cells. N-myristoylation lies upstream of multiple pro-proliferative and oncogenic pathways, but to date the complex substrate specificity of NMT has limited determination of which diseases are most likely to respond to a selective NMT inhibitor. We describe here the phenotype of NMT inhibition in HeLa cells and show that cells die through apoptosis following or concurrent with accumulation in the G1 phase. We used quantitative proteomics to map protein expression changes for more than 2700 proteins in response to treatment with an NMT inhibitor in HeLa cells and observed down-regulation of proteins involved in cell cycle regulation and up-regulation of proteins involved in the endoplasmic reticulum stress and unfolded protein response, with similar results in breast (MCF-7, MDA-MB-231) and colon (HCT116) cancer cell lines. This study describes the cellular response to NMT inhibition at the proteome level and provides a starting point for selective targeting of specific diseases with NMT inhibitors, potentially in combination with other targeted agents.

N-Myristoyltransferase Inhibition Induces ER-Stress, Cell Cycle Arrest, and Apoptosis in Cancer Cells.

Dietary unsaturated fatty acids, such as oleic acid, have been shown to be covalently incorporated into a small subset of proteins, but the generality and diversity of this protein modification has not been studied. We synthesized unsaturated fatty-acid chemical reporters and determined their protein targets in mammalian cells. The reporters can induce the formation of lipid droplets and be incorporated site-specifically onto known fatty-acylated proteins and label many proteins in mammalian cells. Quantitative proteomics analysis revealed that unsaturated fatty acids modify similar protein targets to saturated fatty acids, including several immunity-associated proteins. This demonstrates that unsaturated fatty acids can directly modify many proteins to exert their unique and often beneficial physiological effects in vivo.

Emmanuelle Thinon

Chemical Biology Section,
Department of Chemistry,
Chemical Biology Section, Department of Chemistry

Imperial College London

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

Emmanuelle Thinon

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Chemical Biology Section,Department of Chemistry,Chemical Biology Section, Department of Chemistry

Imperial College London

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

Chemical Biology Section,
Department of Chemistry,
Chemical Biology Section, Department of Chemistry