This work was funded by NIH research grants K01AR053210, R01AR061484 and a grant from National Psoriasis Foundation.

1. Background information
The yeast two-hybrid system is a powerful genetic technique used to discover protein-protein interactions 16, 17. Several kinds of 2-hybrid systems, such as lexA-based systems, the Sos Recruitment System, and bacteria- or mammalian cell-based 2-hybrids, are commercial available, this paper specifically focuses on the modifications of the most commonly used GAL4 based yeast 2-hybrid system. Briefly, the method is based on the properties of the yeast GAL4 protein that consists of separable domains responsible for DNA-binding and transcriptional activation. The bait protein is expressed as a fusion to the GAL4 DNA-binding domain (DNA-BD), while the prey proteins are expressed as fusions to the GAL4 activation domain (AD). Interaction between bait and prey fusion proteins leads to the transcriptional activations of GAL4-binding sites containing reporter genes that are integrated into the yeast genome. The principle of Y2H is illustrated in Fig. 1 and the experimental procedure is summarized in Fig. 2.
2. Required materials and solutions
<ol>
	<li>YPD Growth Medium (a blend of peptone, yeast extract, and dextrose in optimal proportions for growing most Saccharomyces cerevisiae strains).</li>
	<li>Minimal SD Bases (Minimal synthetic defined (SD) bases include a yeast nitrogen base, ammonium sulfate, and a carbon source, dextrose. Dropout (DO) supplements can be added to the Minimal SD Base to make a synthetic, defined medium lacking the specified nutrients).</li>
	<li>Leu/-Trp Dropout (DO) supplement (containing every essential amino acid except for leucine and tryptophan)</li>
	<li>-His/-Leu/-Trp/-Ura Dropout (DO) Supplement (containing every essential amino acid except for leucine, tryptophan, histidine, and uracil)</li>
	<li>Luria broth (LB) (Tryptone 10 g/L, Yeast extract 5 g/L, NaCl 5 g/L)</li>
	<li>X-Gal (5-bromo-4-chloro-3-indolyl-&beta;-D-galactopyranoside) solution: prepared as a 20 mg/ml stock solution in N, N-Dimethylformamide</li>
	<li>10X TE (100 mM Tris-HCl (pH 7.5), 10 mM EDTA, autoclaved)</li>
	<li>Sonicated Herring or Salmon Sperm DNA, boiled (10 mg/ml)</li>
	<li>10X LiAc (1 M lithium acetate, autoclaved)</li>
	<li>50% PEG-3350 solution, filter-sterilized</li>
	<li>3-Amino-1, 2, 4-Triazole (3AT)</li>
	<li>Kanamycin</li>
	<li>Ampicillin</li>
	<li>ELECTROMAX DH5&alpha; cells</li>
	<li>Z buffer: 16.1 g Na2HPO4 7H2O (or 8.52 g anhydrous), 5.5 g NaH2PO4 H2O (or 4.8 ganhydrous), 0.75 g KCl, 0.246 g MgSO4 7H2O (or 0.12 g anhydrous), dissolved in 1 L autoclaved, distilled water and adjusted to pH 7.0.</li>
</ol>
3. Bait generation (pDBLeu-PGRN)
A cDNA fragment encoding PGRN lacking signal peptide (a.a.21-588) was directionally cloned into the Sal I-Not I sites of the pDBLeu vector (the ProQuest two-hybrid system, Invitrogen), keeping the same translation reading frame as the GAL4 DNA Binding Domain to generate pDBLeu-PGRN.
<ol>
	<li>Amplify the cDNA fragment of PGRN described above by PCR using oligonucleotide primers designed to contain restriction sites (Sal I at the 5&prime;end and Not I at the 3&prime;end) to allow in-frame fusion.</li>
	<li>Gel purify the PCR product, digest with restriction endonucleases Sal I and Not I. 
	 Prepare the DB vector pDBLeu by double restriction digestion with the same restriction endonucleases.</li>
	<li>Ligate the restriction PGRN fragment into the linearized pDBLeu vector and transform into DH5&alpha; with selection for LB+kanamycin at 25 &mu;g/ml.</li>
	<li>Verify the correction of the construct by DNA sequencing.</li>
</ol>
4. Small scale transformation of bait plasmid
<ol>
	<li>Inoculate 5 ml of YPD with a colony of Mav203 (Invitrogen), shake overnight at 30&deg;C.</li>
	<li>Dilute the overnight culture in 50 ml of YPD. Grow an additional 2-4 hours.</li>
	<li>Pellet the cells at 3000 rpm for 5 min at RT. Resuspend the pellet in 40 ml of autoclaved, distilled water.</li>
	<li>Re-pellet the cells. Resuspend in 2 ml of solution I, incubate at RT 10 min. 
	Solution I: 0.5 ml 10xLiAc, 0.5 ml 10xTE, 4 ml H2O</li>
	<li>Dispense the DNA to tubes: 2-3 &mu;l of pDBLeu-PGRN (0.25&mu;g/&mu;l) and 10 &mu;l of denatured, sheared salmon sperm DNA (10&mu;g/&mu;l). Add 100&mu;l yeast cells, mix well.</li>
	<li>Add 700&mu;l solution II, mix well. Incubate at 30&deg;C for 30 min. Solution II: 0.2 ml 10xLiAc, 0.2 ml 10xTE, 1.6 ml 50% PEG3350.</li>
	<li>Heat shock at 42&deg;C for 15 min.</li>
	<li>Pellet for 2 min. Discard the supernatant. Resuspend the pellet in 200 &mu;l autoclaved, distilled water.</li>
	<li>Plate the suspension onto SD-Leu plates with serial dilutions. Incubate the plate at 30&deg;C for 2-3 days.</li>
</ol>
5. Bait validation
Before performing yeast two-hybrid screen, test pDBLeu-PGRN for self-activation and determine the basal expression levels of the HIS3 reporter gene. This test determines whether baits activate transcription and whether the self-activation can be neutralized by inhibitors. 3-Amino-1, 2, 4-triazole (3-AT) is a competitive inhibitor of the HIS3-gene product and can be used to titrate the minimum level of HIS3 expression required for growth on histidine-deficient media.
<ol>
	<li>Transform the pDBLeu-PGRN into yeast Mav203 strain, plate the transformation onto SD-Leu plates, and incubate for 48-72 h at 30&deg;C.</li>
	<li>Patch colonies from each transformation using autoclaved toothpicks onto SD-Leu-His plates containing 3-AT at concentrations of 10 mM, 25 mM, 50 mM, 75 mM, and 100 mM. 3-AT is a competitive inhibitor of the HIS3 enzyme and only baits that exhibit self-activation will be able to grow in the presence of the 3-AT.</li>
	<li>Bait strains that grow on plates containing 100 mM 3-AT are not suitable for use in the two-hybrid screen. For baits that can be used, use the lowest 3-AT concentration that inhibits cell growth. In many cases, 25 mM of 3-AT is used for screening.</li>
</ol>
6. cDNA library screening
The pDBLeu-PGRN plasmid is introduced into MaV203 using a small-scale transformation as described above. To introduce pPC86-library (Invitrogen) into MaV203 (pDBLeu-PGRN), the procedure described below typically gives ~4 x 104 colonies with 0.5 &mu;g of plasmid library DNA. Hence, 2.5 x 106 yeast transformants will require ~30.0 &mu;g pPC86-cDNA library plasmid DNA, 25 transformations, and fifty 10-cm plates (SD-Leu-Trp-His-Ura+3AT).
<ol>
	<li>Prepare the appropriate number of 10-cm SD-Leu-Trp-His-Ura+3AT plates (50 for the procedure below). Also prepare at least four 10-cm SD-Leu-Trp plates for estimating the number of transformants.</li>
	<li>Transform the library into the MaV203 containing pDBLeu-PGRN according to the procedure described below.
	<ol>
		<li>Suspend several isolated colonies of MaV203 (pDBLeu-PGRN) in ~100&mu;l autoclaved, distilled water and spread them onto a 10-cm SD-Leu plate. Repeat procedure for a second SD-Leu plate. Incubate both plates for 18-24 h at 30&deg;C.</li>
		<li>Scrape and completely suspend the cells in 10 ml autoclaved, distilled water. Add a sufficient volume of cell suspension to 500 ml of liquid YPD medium in a flask to give an OD600 of ~0.1.</li>
		<li>Verify that the OD is ~0.1 after inoculation.</li>
		<li>Shake at 30&deg;C until the OD600 reaches ~0.4.</li>
		<li>Prepare fresh:
		<ol type="a">
			<li>110 ml 1X TE/LiAc by combining 11 ml 10X TE, 11 ml 10X LiAc, and 88 ml autoclaved water.</li>
			<li>16 ml PEG/LiAc by combining 1.6 ml 10X TE, 1.6 ml 10X LiAc, and 12.8 ml 50% PEG-3350.</li>
		</ol>
		</li>
		<li>Pellet the cells at 3000g for 5 min at room temperature.</li>
		<li>Discard the supernatants and gently resuspend the pellet by pipetting up and down in 100 ml autoclaved, distilled water at room temperature.</li>
		<li>Centrifuge at 3000 g for 5 min at room temperature.</li>
		<li>Discard the supernatant of the centrifuged cells and resuspend the cell pellet in 50 ml 1X TE/LiAc solution.</li>
		<li>Centrifuge at 3000 g for 5 min at room temperature.</li>
		<li>Remove the supernatants and resuspend the pellet in a final volume of 2.5 ml 1X TE/LiAc solution.</li>
		<li>Perform 25 transformations. Combine 2.5 ml cells, 125 &mu;l (10&mu;g/&mu;l) denatured, sheared salmon sperm DNA, and 30 &mu;g cDNA library. Mix gently by pipetting up and down. Add 15 ml PEG/LiAc solution and mix gently. Aliquot into 25 autoclaved 1.5-ml microcentrifuge tubes of 700 &mu;l each.</li>
		<li>Incubate for 30 min in a 30&deg;C water bath.</li>
		<li>Heat shock for 15 min in a 42&deg;C water bath.</li>
		<li>Centrifuge at 6,000 g for 1 min at room temperature. Carefully remove the supernatant. Gently resuspend each pellet in 400 &mu;l autoclaved, distilled water by pipetting up and down.</li>
	</ol>
	</li>
	<li>Plate the 200 &mu;l transformation mixture from each transformation onto single 10-cm SD-Leu-Trp-His-Ura+3AT (3AT concentration: 25 mM) plates using a spread bar, so 25 autoclaved 1.5-ml microcentrifuge tubes can make 50 plates (SD-Leu-Trp-His-Ura +3AT).</li>
	<li>Incubate plates for 5-10 days at 30&deg;C.</li>
	<li>To estimate the transformation efficiency of the reaction, serial dilutions of one reaction (1:40; 1:400 and 1:4000) are plated on SD-Leu-Trp plate. After incubating for 3 days at 30&deg;C, the number of colonies is counted and the total number of transformants calculated.</li>
</ol>
7. X-gal assay
<ol>
	<li>Transfer colonies to YPD plate; incubate at 30&deg;C overnight.</li>
	<li>Place rounded Nitrocellulose membrane on the YPD plate; make sure there are no bubbles between the membrane and the YPD plate. After 1-2 min, make sure all colonies are transferred to membranes (nitrocellulose paper).</li>
	<li>Place the membrane colony side up in boat made with aluminum foil paper.</li>
	<li>Put the membrane into liquid nitrogen, wait twenty seconds, and submerge into liquid nitrogen for a couple of minutes.</li>
	<li>Take out membrane to thaw.</li>
	<li>Meanwhile mix the following reagent: 
	1.5ml Z buffer 
	20&mu;l X-gal (20 mg/ml)</li>
	<li>Drop Z buffer/X-gal in a petri dish, place whatman paper on top of that.</li>
	<li>Carefully place nitrocellulose paper on top of whatman paper.</li>
	<li>Incubate at 37&deg; until blue colonies appear.</li>
</ol>
8. Retransformation assay
Prey fusion proteins (AD-Y) isolated from library screening should retain the interaction with bait fusion protein (DB-X) to induce the report genes, and the retransformation of prey clones and bait construct into yeast can further eliminate false positives and facilitate additional analysis.
<ol>
	<li>Isolate plasmid DNA from yeast strains potentially containing interacting proteins.</li>
	<li>Transform DNA into E. coli DH5&alpha; cells, plate the transformations on LB+100 &mu;g/ml ampicillin plates to selectively isolate the pPC86-cDNA library, and incubate overnight at 37&deg;C.</li>
	<li>Pick several colonies from the plate to put into LB+100 &mu;g/ml ampicillin broth.</li>
	<li>Prepare miniprep DNA and examine by double restriction analysis with Sal I and Not I.</li>
	<li>Co-transform the prey plasmid and bait plasmid into MaV203, plate the transformation mixtures on SC-Leu-Trp plates, and incubate for 2-3 days at 30&deg;C.</li>
	<li>Pick three different colonies from the SC-Leu-Trp plate, perform X-gal Assay.</li>
</ol>
9. Sequencing and bioinformatics analysis
Sequence the plasmid DNA that was isolated from likely true positive clones, compare these sequences to those in the GenBank using the BLAST program, and identify those two-hybrid clones that correspond to known genes. Sequencing data showed that two of the 12 positives obtained above were cell surface TNFR2 (TNFRSF1B/CD120b; Accession #NM_130426). In addition, the interaction between PGRN and TNFR was verified using various protein-protein interaction assays, including in vitro Solid-phase binding assay, Co-immunoprecipitation, Surface plasmon resonance analysis, and Flow cytometry assay 14.
10. Representative Results
The flow chart of the screening is outlined in Fig. 3. Typically 50-100 positive clone candidates will be obtained at this step. We initially isolated 54 positive clone candidates among 2.5 million transformants screened with the PGRN bait. Positive clone candidates were then verified by performing X-gal assay. Typically, approximately 50% false positive clones are removed via X-gal assay. We obtained 23 positive clone candidates at this step for the PGRN bait (Fig. 4). Retransformation of the prey clones and bait construct into yeast further eliminate false positives and clones that still activate the reporter genes likely represent the true positives. Typically, approximately 50% positive clone candidates will be removed. We finally isolated 12 positive clones that interact with PGRN in yeast.
<img alt="Figure 1" src="/files/ftp_upload/3562/3562fig1.jpg" /> 
Figure 1. Principle of yeast two-hybrid system
<img alt="Figure 2" src="/files/ftp_upload/3562/3562fig2.jpg" /> 
Figure 2. Pipeline of identifying protein binding partners using yeast two-hybrid system
<img alt="Figure 3" src="/files/ftp_upload/3562/3562fig3.jpg" /> 
Figure 3. Flow chart of screening yeast two-hybrid library. <a href="https://www.jove.com/files/ftp_upload/3562/3562fig3large.jpg" target="_blank">Click here to view a full-sized version of this image.</a>
<img alt="Figure 4" src="/files/ftp_upload/3562/3562fig4.jpg" /> 
Figure 4. Beta-Galactosidase assay of positive clone candidates. A, Positive clones obtained from library screen were transferred to YPD plate and incubated at 30&deg;C overnight; B, All colonies on YPD plate were transferred to nitrocellulose membranes and beta-galactosidase assay performed.

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No conflicts of interest declared.

Yeast two-hybrid screening has proven to be an effective tool in identifying protein interaction 16, 17. Compared with other approaches for identifying protein-binding partners, such as biochemical co-purification and protein chips, yeast two-hybrid system is a sensitive genetic approach that can be used for screening very high numbers of coding sequences in a relatively simple experiment; in addition, it detects the in vivo interaction and does not need complicated protein purification. Of course yeast two-hy...

<table border="1">
<tbody>
 <tr>
	<td>Name of the reagent</td>
	<td>Company</td>
	<td>Catalogue number</td>
 </tr>
 <tr>
	<td>ProQuest two-hybrid system</td>
	<td>Invitrogen</td>
	<td>10835</td>
 </tr>
 <tr>
	<td>YPD Growth Medium</td>
	<td>Clontech</td>
	<td>630409</td>
 </tr>
 <tr>
	<td>YPD Agar Medium</td>
	<td>Clontech</td>
	<td>630410</td>
 </tr>
 <tr>
	<td>Minimal SD agar Base</td>
	<td>Clontech</td>
	<td>630412</td>
 </tr>
 <tr>
	<td>-Leu DO Supplement</td>
	<td>Clontech</td>
	<td>630414</td>
 </tr>
 <tr>
	<td>-Leu/-Trp DO Supplement</td>
	<td>Clontech</td>
	<td>630417</td>
 </tr>
 <tr>
	<td>-His/-Leu/-Trp/-Ura DO Supplement</td>
	<td>Clontech</td>
	<td>630425</td>
 </tr>
 <tr>
	<td>3-Amino-1,2,4-Triazole (3AT)</td>
	<td>Sigma-Aldrich</td>
	<td>A8056</td>
 </tr>
 <tr>
	<td>Luria broth (LB)</td>
	<td>Sigma-Aldrich</td>
	<td>L3022</td>
 </tr>
 <tr>
	<td>X-Gal</td>
	<td>Invitrogen</td>
	<td>15520-034</td>
 </tr>
 <tr>
	<td>Sonicated Salmon Sperm DNA</td>
	<td>Stratagene</td>
	<td>201190</td>
 </tr>
 <tr>
	<td>Ampicillin</td>
	<td>AMRESCO</td>
	<td>0339</td>
 </tr>
 <tr>
	<td>Kanamycin Sulfate</td>
	<td>Invitrogen</td>
	<td>11815-024</td>
 </tr>
 <tr>
	<td>Subcloning Efficiency&trade; DH5&alpha;&trade; Competent Cells</td>
	<td>Invitrogen</td>
	<td>18265-017</td>
 </tr>
 <tr>
	<td>Trizma&reg; base</td>
	<td>Sigma-Aldrich</td>
	<td>T6066</td>
 </tr>
 <tr>
	<td>Lithium acetate</td>
	<td>Sigma-Aldrich</td>
	<td>L4158</td>
 </tr>
 <tr>
	<td>Ethylenediaminetetraacetic acid</td>
	<td>Sigma-Aldrich</td>
	<td>ED</td>
 </tr>
 <tr>
	<td>Nitrocellulose Membrane</td>
	<td>Bio-Rad</td>
	<td>162-0115</td>
 </tr>
 <tr>
	<td>10-cm petri dish</td>
	<td>ITI Scientific</td>
	<td>CT-903</td>
 </tr>
 <tr>
	<td>Incubator (30 &deg;C)</td>
	<td>ATR (Ecotron)</td>
	<td>&nbsp;</td>
 </tr>
</table>

modified yeast-two-hybrid system to identify proteins interacting with the growth factor progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

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Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

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28.6K Views.  NYU Hospital for Joint Diseases. We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

Video: Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Growth Factor-Coated Bead Placement on Dorsal Forebrain Explants

This video demonstrates two methods for preparing and placing beads, which have been coated with growth factor, on explants of the developing cerebral cortex. These beads can be used to induce spatially restricted gene expression on developing neural tissue such as forebrain explants. Methods are given for using both Affi-Gel beads and heparin acryllic beads.

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

Preparing T Cell Growth Factor from Rat Splenocytes

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. Addition of interleukin 2 to the culture media during the expansion phase is necessary to prevent cell death and sufficient to maintain short-term T cell lines but has been shown to increase Th1 polarization 3. Replacement of interleukin 2 by T cell growth factor (TCGF) which contains a mix of cytokines is more effective than interleukin 2 in maintaining long-term T cell lines in vitro 3. Moreover, TCGF can easily be prepared in large amounts in the laboratory and is much cheaper than recombinant interleukin 2. 
Here, we show how to prepare TCGF from rat splenocyte culture supernatants. For this procedure, we harvest spleens from naive Lewis rats euthanized for thymus and blood collection. We prepare single-cell suspensions from the spleens, lyze the red blood cells by osmotic shock, and seed the splenocytes in culture medium. The cells are stimulated with concanavalin A, a mitogen that non-selectively activates all rat T lymphocytes, inducing the production of cytokines. The culture supernantant is collected 48 hours later andexcess concanavalin A is bound to alpha methyl mannoside to prevent it from activating T cell lines to which TCGF will be added. The TCGF is then sterile-filtered, aliquoted, and stored at -20&deg;C.

We describe the preparation of T cell growth factor used for the in vitro expansion of antigen-specific rat T lymphocyte lines.

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. ...

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

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

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

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.

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

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...