Omvendt gjær to-hybrid system for å identifisere pattedyr Nuclear Receptor Rester som samhandler med Ligander og / eller antagonister

Summary

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.

Abstract

Som en kritisk regulator av legemiddelmetabolisme og inflammasjon, pregnan X Reseptor (PXR), spiller en viktig rolle i sykdoms patofysiologien knytte stoffskiftet og betennelse (f.eks leversteatose) ^1,2. Det har vært mye fremgang i identifisering av agonist ligander for PXR, men det er begrenset beskrivelser av narkotika-lignende antagonister og deres bindingssteder på PXR ^3,4,5. Et kritisk barriere har vært den manglende evne til effektivt å rense full-lengde protein for strukturelle studier med antagonister til tross for at PXR ble klonet og karakterisert i 1998. Vårt laboratorium utviklet en ny høy-kapasitets gjær basert to-hybrid analyse for å definere en antagonist, ketokonazol tallet, bindende rester på PXR ^seks. Vår metode innebærer å skape mutasjons biblioteker som ville redde effekten av enkelt mutasjoner på AF-2 overflaten av PXR forventet å samhandle med ketokonazol. Rescue eller "gain-of-funksjon" second mutasjoner kan gjøres slik at konklusjoner vedrørende den genetiske interaksjon av ketokonazol og residuet flaten (e) på PXR er gjennomførbare. Dermed utviklet vi en høy gjennomstrømming to-hybrid gjær skjermen på PXR mutanter i samspill med sin coactivator, SRC-en. Ved hjelp av denne metode, der gjær ble endret for å få plass til studiet av den soppdrepende stoff, ketokonazol, kunne vi påvise spesifikke mutasjoner på PXR anriket på kloner som ikke er i stand til å binde seg til ketokonazol. Ved omvendt logikk, konkluderer vi med at de opprinnelige rester er direkte interaksjon rester med ketokonazol. Denne analysen representerer en roman, medgjørlig genetiske analysen til skjermen for antagonist bindingssteder på atom reseptor overflater. Denne analysen kan anvendes på en hvilken som helst medikament uavhengig av cytotoksisk potensiale til gjær så vel som til cellulære protein (er) som ikke kan bli undersøkt ved hjelp av standard konstruksjons biologi eller proteomic baserte metoder. Potensielle fallgruver inkluderer tolkning av data (komplementære metoder nyttig), påliteligledelse på single Y2H metode, kompetanse i håndtering av gjær eller utfører gjær to-hybrid-analyser, og analysen optimalisering.

Introduction

The yeast two-hybrid (Y2H) assay is widely used to discover protein-protein interactions and more recently for discovery of novel small molecules that disrupt protein-protein interaction complexes ^{7, 8, 9, 10, 11}. However, the conventional approaches of this assay, used for drug discovery or "hits", do not allow for detection of allosteric interaction residues of chemicals compounds within protein-protein surfaces, that when altered still interact and allow for interrogation of the altered residues¹¹. Indeed, such a method(s), if feasible to develop, would enable a tractable yeast system for high throughput assessment of allosteric interaction residues critical for protein-protein interaction disruption. In the context of drug discovery, the most direct way to establish interaction of compounds with proteins would involve structural determination (e.g. crystalization of protein-inhibitor complex). These methods are cumbersome, use elaborate resources and it is not technically feasible to perform structural studies on every protein.

Tractable genetic drug screening systems have been established in bacteria^{1, 2} and other model systems like mammalian two-hybrid. However, these systems need optimization and alternative systems like Y2H are still the most tested in drug discovery. There are limitations that include poor sensitivity and reliability of interactions using singular methods¹³ , however, a single Y2H assay can be modified to answer specific questions regarding interaction residues. In the field of nuclear receptor research, Y2H has been used to define interacting proteins¹⁴, however, these protein interactions have rarely been used to define the nature in which ligands/antagonists interact with nuclear receptor-protein complexes. Thus, our laboratory focused efforts on defining a method, especially for receptor proteins that are not readily amenable to proteomic based methods, that would unearth novel ligand/antagonist interacting residues using a reverse Y2H based discovery platform.

Based on our previous finding that ketoconazole disrupts PXR and its activator SRC-1, we developed a novel reverse Y2H system that enable us to define and interrogate ketoconazole interacting residues on PXR⁶. Our method is based on the properties of the yeast GAL4 protein that consists of separable domains responsible for DNA-binding and transcriptional activation. The PXR LBD protein is expressed as a fusion to the LexA DNA-binding domain (DNA-BD), while the full length co-activators SRC-1 (steroid receptor coactivator 1) proteins are expressed as fusions to the GAL4 activation domain (AD). Interaction between PXR and SRC-1 fusion proteins leads to the transcriptional activation of GAL4-binding sites containing reporter gene β-LacZ that is integrated into the yeast genome. Ketoconazole, a PXR antagonist, disrupts PXR and SRC-1 interaction ^{15, 16, 17} and we can detect the interaction of PXR and SRC-1 in the presence or absence of ketoconazole after staining colonies on filters for X-gal activity. The principle of Y2H is illustrated in Figure 1 and the experimental procedure is summarized in Figure 2.

Protocol

1. Construction of PXR and SRC-1 Fusions in Yeast Vectors PCR amplification of Human PXR LBD (107-434 amino acids) and Human SRC-1 full length (1-1401 amino acids). Use pSG5-hPXR plasmid8 as PXR LBD template, use pCMX-SRC1 plasmid as SRC-1 templates. Thaw PCR SuperMix, DNA templates and primers (see Materials), keep them on ice. Add 0.25 μg/μl DNA template 1 μl, 10 mM primer pairs 2 μl each, PCR SuperMix 45 μl and add H2O to brin…

Representative Results

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…

Discussion

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 erg3Δ/erg11Δ yeast. As before, we sho…

Materials

Name	Company	Catalog Number	Comments
Yeast Strain CTY10-5d erg3Δ/erg11Δ	Our lab		CTY10-5d yeast was double knocked out ERG3 and ERG11 (erg3Δ/erg11Δ) genes6 .
YPD Growth Medium	BD Biosciences	630409
Difco Yeast Nitrogen Base (YNB) w/o Amino Acids and Ammonium Sulfate	BD Biosciences	233520
Bacto Agar	BD Biosciences	214010
CSM-His/-Leu Complete Supplement Mixture	MP Biomedicals	4250-412
ONPG (o-Nitrophenyl Β-D- Galactopyranoside).	Sigma-Aldrich	N1127
2-Mercaptoethanol	Sigma-Aldrich	M6250
Luria Broth (LB)	Sigma-Aldrich	L3022
X-Gal	Fisher	BP-1615
Sonicated Salmon Sperm DNA boiled (10 mg/ml)	Life Technology	156-017
Ampicillin	Acros Organics	61177
Ketoconazole	Sigma-Aldrich	K1003
N,N-Dimethylformamide	Acros Organics	326871000
Lithium Acetate	Sigma-Aldrich	L4158
50% PEG-3350 solution, filter-sterilized	Sigma-Aldrich	P-3640
Nitrocellulose Membrane	Whatman	10402091
10 cm Petri Dish	Fisher	875712

5'-ACCGGATCCCGATGAAGA AGGAGATGATCATGTCC-3'	our lab		PXR LBD forward primer for pSH2-1
5'-AGAGTCGACTCAGCTA CCTGTGATGCC -3'	our lab		PXR LBD reverse primer for pSH2-1
5'-TATAGC GGCCGCATGAGTG GCCTCGGGGACAGTTCATCC -3'	our lab		SRC-1 forward primer for pGADNOT
5'-GCGGTCGACTTATTCAGTCA GTAGCTG -3'	our lab		SRC-1 reverse primer for pGADNOT
Platinum PCR Supermix	Invitrogen	11306-016
BamHI	our lab	R0136
SalI	our lab	R0138
NotI	our lab	R0189