This work was supported by National Institutes of Health (NIH) Grants CA127231 and The Damon Runyon Foundation Clinical Investigator Award (CI 1502) (to S.M). We would like to thank Professor Zdenek Dvorak from Palacky University Olomouc, Czech Republic for his helpful insights into discussing portability of this technique to their institution and standardization of protocol.

1. Construction of PXR and SRC-1 Fusions in Yeast Vectors
<ol>
	<li>PCR amplification of Human PXR LBD (107-434 amino acids) and Human SRC-1 full length (1-1401 amino acids).
	<ol>
		<li>Use pSG5-hPXR plasmid8 as PXR LBD template, use pCMX-SRC1 plasmid as SRC-1 templates.</li>
		<li>Thaw PCR SuperMix, DNA templates and primers (see Materials), keep them on ice.</li>
		<li>Add 0.25 &#956;g/&#956;l DNA template 1 &#956;l, 10 mM primer pairs 2 &#956;l each, PCR SuperMix 45 &#956;l and add H2O to bring total volume to 50 &#956;l.</li>
		<li>Set PCR at one cycle of 94 &#176;C for 2 min, 20 cycles of 94 &#176;C for 30 sec, 55 &#176;C for 30 sec&#160;and 72 &#176;C for 2 min, then one cycle of 72 &#176;C for 10 min&#160;for extension. Check PCR products by running on 1% agarose gel.</li>
	</ol>
	</li>
	<li>PCR products cloning into Y2H vectors.
	<ol>
		<li>Purify PCR products from 1% agarose gel. Digest PXR, LBD, PCR, and Y2H vector pSH2-1 with BamHI and SalI; digest SRC-1 PCR and Y2H vector pGADNOT with Not&#921; and Sal&#921;. To do this, add 1 &#956;g DNA , 3 &#956;l 10x reaction buffer, 1 &#956;l restriction enzymes, and finally H2O to bring total volume to 30 &#956;l.</li>
		<li>Put digestion samples in 37 &#176;C water bath for 1 hr. Check digestion by running on 1% agarose gel.</li>
		<li>Purify digestion samples from 1% agarose gel, ligate digested PCR products and Y2H vectors and transform to DH5&#945; competent cells.</li>
		<li>Pick the colonies and isolate the plasmid DNA.</li>
	</ol>
	</li>
	<li>Identify pSH2-1-PXR plasmid DNA by BamHI/SalI digestion, identify pGADNOT-SRC-1 plasmid DNA by NotI/SalI digestion. Verify all positive plasmid DNA by sequencing.</li>
</ol>
2. Yeast Two-hybrid Assays
<ol>
	<li>Inoculate the yeast strain into 5 ml YPAD and incubate over night by constant agitation (220 rpm at 30 &#176;C). Note: The Saccharomyces cerevisiae strain was CTY10-5d (MATa ade2 trp1-901 leu2-3,112 his3-200 ga l4-gal80- URA3::lexA-lacZ) that contains an integrated GAL1-lacZ gene with lexA operator10. To obtain viable yeast cells resistant to ketoconazole, but those that did not carry transporter alterations as a cause of azole resistance18-20, novel strains of CTY10-5d yeast were obtained by first deleting ERG3 ( erg3&#916;)and then introducing additional deletion in ERG11 ( erg3&#916;/erg11&#916;) genes by homologous recombination. The construction details have been described in a previously published manuscript6 .</li>
	<li>The next day (in the morning), inoculate yeast (1:20) in 5 ml YAPD and incubate to obtain an OD600&#160;~0.2 by constant agitation (220 rpm at 30 &#176;C).</li>
	<li>Pellet the cells at 2,000 rpm in a microcentrifuge for 2 min and discard the supernatant. Then wash the pellet twice with autoclaved water and once with 0.1 M LiAc.</li>
	<li>After the final wash, discard supernatant and add the following reagents to the cell pellet: 240 &#956;l PEG 3500 50% w/v, 36 &#956;l 1 M LiAc, 50 &#956;l 2 mg/ml boiled salmon sperm ssDNA, H2O 34 &#956;l. Vortex to mix, add 1 &#956;g pSH-PXR and 1 &#956;g pGADNOT-SRC-1 DNA and mix.</li>
	<li>Transfer the mix to polystyrene round-bottom tube, agitate the transformation, mix (220 rpm at 30 &#176;C) for 30 min. Move the tube to 42 &#176;C water bath and incubate for an additional 30 min.</li>
	<li>Centrifuge the tube at 2,000 rpm for 2 min and remove the supernatant. Wash pellet once with autoclaved water.</li>
	<li>Pipette 100 &#956;l sterile water into the tube, re-suspend the cells by gentle finger tapping and pipetting, then plate onto plates of -His/-Leu Dropout Medium (Formulation per One Liter -His/-Leu Dropout Medium: 20 g dextrose, 1.7 g YNB, 0.67 g CSM-His/-Leu, 5 g ammonium sulfate, 1.5% agar) and incubate at 30 &#176;C for 3-4 days to isolate transformants.</li>
</ol>
3. X-gal Filter Assay
<ol>
	<li>Cut nitrocellulose membrane to the size of part of a Petri dish. Label the nitrocellulose membrane to mark the orientation.</li>
	<li>Place premarked nitrocellulose membrane on yeast colonies, make sure that the membrane is in contact with the surface of the plate medium.</li>
	<li>Remove the membrane, place it colony side up on a 3 mm filter paper, and place the membrane and the filter paper at -80 &#176;C for 15 min.</li>
	<li>Make X-gal solution. Add 50 &#956;l 20 mg/ml X-gal and 15 &#956;l &#946;-mercaptoethanol to 5 ml of Z-buffer (1 L buffer contains 16.1 g Na2HPO47H2O, 5.5 g NaH2PO4 H2O, 0.75 g KCl , 0.25 g MgSO4&#8226;7H2O) . If Z Buffer was kept at 4 &#176;C, bring it to room temperature before adding these reagents.</li>
	<li>Place two circles of 3 mm filter paper in a Petri dish and soak them with 5 ml X-gal solution. Drain excess buffer from the Petri dish and place the nitrocellulose membrane with the colony side up.</li>
	<li>Close the lid, ensure that the membrane is making complete contact with the filter paper and it is completely wet. Wrap the Petri dish in foil and incubate at 37 &#176;C for 30 min to overnight.</li>
</ol>
4. Liquid &#946;-gal Assay
<ol>
	<li>Grow Yeast in -His/-Leu Dropout liquid medium to OD600 0.7.</li>
	<li>Transfer 1 ml medium into a 1.5 ml microcentrifuge tube and centrifuge at 3,000 rpm for 2 min. Discard the supernatant.</li>
	<li>Add 1 ml of Z Buffer to resuspend the pellet, centrifuge at 3,000 rpm again for 2 min. Discard the supernatant.</li>
	<li>Add 150 &#956;l of Z Buffer with 2ME to resuspend the cell pellet, mix then add 50 &#956;l chloroform and 20 &#956;l 0.1% SDS. Vigorously vortex the sample for 15 sec.</li>
	<li>Add 700 &#956;l of ONPG Solution (1 mg/ml in Z buffer). Set the timer, incubate at 30 &#176;C long enough to allow the yellow colour to develop and note total reaction time.</li>
	<li>Add 500 &#956;l of 1 M Na2CO3 to stop the reaction and determine the absorbance at 420 nm. The blank is 700 &#956;l ONPG solution plus 500 &#956;l 1 M Na2CO3.</li>
</ol>
Calculate Miller Units as follows: Miller Units = (&#197;420*1,000)/(&#197;600*min*ml)
&#197;420: absorbance units at 420 nanometers; &#197;600: absorbance units at 600 nanometers; min: the reaction time in minutes;&#160;ml: the reaction volume in ml

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No conflicts of interest declared.

In our modified Y2H assay, we have identified important residues for ketoconazole interactions on PXR6. Since SRC-1 is a coactivator (and was cloned into the pGADNot vector), we also tested whether SRC-1 could activate lacZ expression when cloned into the pSH vector system and whether this would change the activation profile and/or affect the leakiness of the yeast two-hybrid assay. Using our redesigned plasmids we performed two-hybrid assays in erg3&#916;/erg11&#916; yeast. As before, we sho...

A levedura de dois híbridos (Y2H) ensaio é utilizado para descobrir as interacções proteína-proteína e, mais recentemente, para a descoberta de novas moléculas pequenas que perturbem complexos proteína-proteína de interacção 7, 8, 9, 10, 11. No entanto, as abordagens convencionais deste ensaio, usado para a descoberta de medicamentos ou &quot;hits&quot;, não permitem a detecção de resíduos de interação de compostos químicos alostéricos dentro superfícies proteína-proteína, que, quando alterados ainda interagir e permitir o interrogatório dos resíduos alterados 11 . Com efeito, um tal método (s), se possível desenvolver, permitiria um sistema de levedura tratável para avaliação de alto rendimento de resíduos de interacção alostéricos críticos para a interacção proteína-proteína ruptura. No contexto da descoberta da droga, a maneira mais direta de estabelecer a interação de compostos com proteínas envolveria determinação estrutural (por exemplo, cristalização do complexo proteína-inibidor). Estes métodos são cumbersome, utilizar os recursos elaborados e não é tecnicamente viável a realização de estudos estruturais em cada proteína. Sistemas de rastreio de drogas genética tratável foram estabelecidas em bactérias 1, 2 e outros sistemas modelo como mamíferos de dois híbridos. No entanto, esses sistemas precisam de otimização e sistemas alternativos como Y2H ainda são os mais testados na descoberta de medicamentos. Há limitações que incluem pouca sensibilidade e confiabilidade das interações usando métodos singulares 13, no entanto, um ensaio Y2H único pode ser modificada para responder a perguntas específicas sobre resíduos de interação. No campo da investigação de receptores nucleares, Y2H foi usado para definir as proteínas que interagem 14, no entanto, estas interacções proteína raramente foram utilizados para definir a natureza, em que os ligandos / antagonistas do receptor interajam com complexos proteicos nucleares. Assim, o nosso laboratório concentrado esforços na definição de um método, especialmente para as proteínas receptoras que não sejamprontamente passíveis de proteômica métodos baseados, que desenterrar novela ligante / antagonista interagindo resíduos utilizando uma plataforma baseada Y2H descoberta inversa. Com base em nossa constatação anterior que o cetoconazol interrompe PXR e seu ativador SRC-1, foi desenvolvido um novo sistema de reverter Y2H que nos permitirá definir e interrogar cetoconazol interagir resíduos em PXR 6. O nosso método baseia-se nas propriedades da proteína GAL4 de levedura, que consiste em domínios separáveis ​​responsáveis ​​pela activação de ligação a ADN e transcricional. A proteína PXR LBD é expressa como uma fusão com o domínio de ligação de LexA de ADN (DNA-BD), enquanto que as de comprimento total de co-activadores de SRC-1 (coactivator do receptor de esteróides 1) as proteínas são expressas como fusões com o domínio de activação de GAL4 (AD ). Interacção entre PXR e SRC-1 As proteínas de fusão conduz à activação da transcrição de GAL4, sítios de ligação que contêm genes repórter β-Lac Z, que é integrado no genom levedurae. Cetoconazol, um antagonista de PXR, interrompe PXR e SRC-1 interacção 15, 16, 17 e que pode detectar a interacção de PXR e SRC-1 na presença ou ausência de cetoconazol após coloração colónias em filtros para a actividade de X-gal. O princípio de Y2H é ilustrada na Figura 1 e o procedimento experimental é resumido na Figura 2.

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Yeast Strain CTY10-5d erg3&#916;/erg11&#916;</td>
			<td>Our lab</td>
			<td></td>
			<td>CTY10-5d yeast was double knocked out ERG3 and ERG11 (erg3&#916;/erg11&#916;) genes6 .</td>
		</tr>
		<tr>
			<td>YPD Growth Medium</td>
			<td>BD Biosciences</td>
			<td>630409</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Yeast Nitrogen Base (YNB) w/o Amino Acids and Ammonium Sulfate</td>
			<td>BD Biosciences</td>
			<td>233520</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>BD Biosciences</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM-His/-Leu Complete Supplement Mixture</td>
			<td>MP Biomedicals</td>
			<td>4250-412</td>
			<td></td>
		</tr>
		<tr>
			<td>ONPG (o-Nitrophenyl &#914;-D- Galactopyranoside).</td>
			<td>Sigma-Aldrich</td>
			<td>N1127</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>Luria Broth (LB)</td>
			<td>Sigma-Aldrich</td>
			<td>L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>X-Gal</td>
			<td>Fisher</td>
			<td>BP-1615</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicated Salmon Sperm DNA boiled (10 mg/ml)</td>
			<td>Life Technology</td>
			<td>156-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Acros Organics</td>
			<td>61177</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketoconazole</td>
			<td>Sigma-Aldrich</td>
			<td>K1003</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Dimethylformamide</td>
			<td>Acros Organics</td>
			<td>326871000</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>L4158</td>
			<td></td>
		</tr>
		<tr>
			<td>50% PEG-3350 solution, filter-sterilized</td>
			<td>Sigma-Aldrich</td>
			<td>P-3640</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose Membrane</td>
			<td>Whatman</td>
			<td>10402091</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm Petri Dish</td>
			<td>Fisher</td>
			<td>875712</td>
			<td></td>
		</tr>
		<tr>
		</tr>
	</tbody>
</table>

<table border="1">
	<tbody>
		<tr>
			<td>5&#39;-ACCGGATCCCGATGAAGA AGGAGATGATCATGTCC-3&#39;</td>
			<td>our lab</td>
			<td></td>
			<td>PXR LBD forward primer for pSH2-1</td>
		</tr>
		<tr>
			<td>5&#39;-AGAGTCGACTCAGCTA CCTGTGATGCC -3&#39;</td>
			<td>our lab</td>
			<td></td>
			<td>PXR LBD reverse primer for pSH2-1</td>
		</tr>
		<tr>
			<td>5&#39;-TATAGC GGCCGCATGAGTG GCCTCGGGGACAGTTCATCC -3&#39;</td>
			<td>our lab</td>
			<td></td>
			<td>SRC-1 forward primer for pGADNOT</td>
		</tr>
		<tr>
			<td>5&#39;-GCGGTCGACTTATTCAGTCA GTAGCTG -3&#39;</td>
			<td>our lab</td>
			<td></td>
			<td>SRC-1 reverse primer for pGADNOT</td>
		</tr>
		<tr>
			<td>Platinum PCR Supermix</td>
			<td>Invitrogen</td>
			<td>11306-016</td>
			<td></td>
		</tr>
		<tr>
			<td>BamHI</td>
			<td>our lab</td>
			<td>R0136</td>
			<td></td>
		</tr>
		<tr>
			<td>SalI</td>
			<td>our lab</td>
			<td>R0138</td>
			<td></td>
		</tr>
		<tr>
			<td>NotI</td>
			<td>our lab</td>
			<td>R0189</td>
			<td></td>
		</tr>
	</tbody>
</table>

reverse yeast two-hybrid system to identify mammalian nuclear receptor residues that interact with ligands and/or antagonists

Como um regulador fundamental no metabolismo de drogas e inflamação, Pregnane X Receptor (PXR), desempenha um papel importante na patofisiologia da doença ligando metabolismo e inflamação (por exemplo, a esteatose hepática) 1,2. Tem havido muito progresso na identificação de ligantes agonistas para PXR, no entanto, há descrições limitadas de antagonistas droga-como e seus sítios de ligação PXR 3,4,5. Uma barreira crítica tem sido a incapacidade de purificar de forma eficiente a proteína de comprimento completo para estudos estruturais com antagonistas, apesar do facto de PXR foi clonado e caracterizado em 1998. O nosso laboratório desenvolveu uma nova levedura de alto rendimento com base ensaio de dois híbridos para definir um antagonista, de cetoconazol, os resíduos de ligação em PXR 6. O nosso método envolve a criação de bibliotecas de mutações que seriam resgatar o efeito das mutações individuais em FA-2 superfície de PXR o esperado para interagir com cetoconazol. Resgate ou &quot;ganho de função&quot; secomutações nd pode ser feita de tal forma que conclusões a respeito da interação genética de cetoconazol e do resíduo (s) de superfície em PXR são viáveis. Assim, foi desenvolvido um alto rendimento de dois híbridos tela levedura de mutantes PXR interagindo com sua coactivator, SRC-1. Usando esta abordagem, em que a levedura foi modificado para acomodar o estudo da droga antifúngica, cetoconazol, conseguimos demonstrar mutações específicas em PXR enriquecidas em clones incapazes de se ligar ao cetoconazol. Pela lógica inversa, podemos concluir que os resíduos originais são resíduos diretos de interação com cetoconazol. Este ensaio representa um romance, ensaio genético tratável para a tela por sítios de ligação do antagonista do receptor nuclear em superfícies. Este ensaio pode ser aplicado a qualquer medicamento, independentemente do seu potencial citotóxico de levedura, bem como a proteína (s) celular, que não pode ser estudado usando biologia estrutural padrão ou métodos proteomic base. Armadilhas potenciais incluem interpretação de dados (métodos complementares útil), reliance em método único Y2H, experiência em lidar com fermento ou levedura realizando ensaios de dois híbridos e otimização do ensaio.

We performed an assay to see if we could detect a colorimetric readout of the association of PXR and steroid receptor coactivator-1 (SRC-1). Since yeast has significant sterol production, it has previously been shown that lacZ expression in yeast can be induced without the need for additional exogenous ligand. We found that lacZ expression (blue colonies) is also induced in the yeast strain transformed with PXR and SRC-1; however, there is no induction of LacZ expression (white colonies) in yeast transformed with empty v...

Watch this Scientific Journal Video about Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists at JoVE.com

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

Reverter levedura Sistema de dois híbridos para identificar mamíferos Receptor Nuclear Resíduos que interagem com ligantes e / ou antagonistas

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

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12.7K visualizações.  Albert Einstein College of Medicine. Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.