Yeast 2-hybrid Assay Research Articles

Informatic Analysis of Sequence Data from Batch Yeast 2-Hybrid Screens.

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing high-throughput short-read DNA sequencing. The resulting sequence datasets can not only track what genes in a population that are enriched during selection for positive yeast 2-hybrid interactions, but also give detailed information about the relevant subdomains of proteins sufficient for interaction. Here, we describe a full suite of stand-alone software programs that allow non-experts to perform all the bioinformatics and statistical steps to process and analyze DNA sequence fastq files from a batch yeast 2-hybrid assay. The processing steps covered by these software include: 1) mapping and counting sequence reads corresponding to each candidate protein encoded within a yeast 2-hybrid prey library; 2) a statistical analysis program that evaluates the enrichment profiles; and 3) tools to examine the translational frame and position within the coding region of each enriched plasmid that encodes the interacting proteins of interest.

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions.

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the discovery of high-affinity interactors that are highly enriched in the library of interacting candidates. In a traditional format, the yeast 2-hybrid assay can yield too many colonies to analyze when conducted at low stringency where low affinity interactors might be found. Moreover, without a comprehensive and complete interrogation of the same library against different bait plasmids, a comparative analysis cannot be achieved. Although some of these problems can be addressed using arrayed prey libraries, the cost and infrastructure required to operate such screens can be prohibitive. As an alternative, we have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing a strategy termed DEEPN (Dynamic Enrichment for Evaluation of Protein Networks), which incorporates high-throughput DNA sequencing and computation to follow the evolution of a population of plasmids that encode interacting partners. Here, we describe customized reagents and protocols that allow a DEEPN screen to be executed easily and cost-effectively.

Bovine adenovirus‐3 protein VIII associates with eukaryotic initiation factor‐6 during infection

Adenovirus protein VIII appears to connect core with the inner surface of the adenovirus capsid. Because protein-protein interactions are central to virus replication, identification of proteins interacting with protein VIII may help in understanding their role in adenovirus infection. Our yeast 2-hybrid assay indicated that protein VIII interacts with eukaryotic initiation factor 6 (eIF6). These findings were confirmed by Glutathione S-transferase-pull down assay, bimolecular fluorescent complementation assay, and coimmunoprecipitation assay in plasmid DNA transfected and bovine adenovirus-3 (BAdV-3) infected cells. The C-terminus amino acids 147 to 174 of protein VIII and N-terminus amino acids 44 to 97 of eIF6 are involved in these interactions. Polysome analysis demonstrated increased level of 60S ribosomal subunit and decreased level of 80S complex in protein VIII expressing cells or BAdV-3 infected cells. Our results suggest that formation of functional 80S ribosome appears impaired in the presence of protein VIII at late times post infection. We speculate that this impaired ribosome assembly may be responsible for the inhibition of cellular mRNA translation observed late in adenovirus infected cells. Moreover, analysis of recombinant BAdV-3 expressing mutant protein VIII (deletion of eIF6 interacting domain) suggests that interaction of protein VIII and eIF6 may help in preferential translation of adenovirus genes during late phase of adenovirus infection.

A yeast two-hybrid system for the screening and characterization of small-molecule inhibitors of protein\u2013protein interactions identifies a novel putative Mdm2-binding site in p53

BackgroundProtein–protein interactions (PPIs) are fundamental to the growth and survival of cells and serve as excellent targets to develop inhibitors of biological processes such as host-pathogen interactions and cancer cell proliferation. However, isolation of PPI inhibitors is extremely challenging. While several in vitro assays to screen for PPI inhibitors are available, they are often expensive, cumbersome, and require large amounts of purified protein. In contrast, limited in vivo assays are available to screen for small-molecule inhibitors of PPI.MethodsWe have engineered a yeast strain that is suitable for screening of small-molecule inhibitors of protein-protein interaction using the Yeast 2-hybrid Assay. We have optimised and validated the assay using inhibitors of the p53-Mdm2 interaction and identified a hitherto unreported putative Mdm2-binding domain in p53.ResultsWe report a significantly improved and thoroughly validated yeast two-hybrid (Y2H) assay that can be used in a high throughput manner to screen for small-molecule PPI inhibitors. Using the p53-Mdm2 interaction to optimize the assay, we show that the p53-Mdm2 inhibitor nutlin-3 is a substrate for the yeast ATP-binding cassette (ABC) transporter Pdr5. By deleting nine ABC transporter-related genes, we generated a ABC9Δ yeast strain that is highly permeable to small molecules. In the ABC9Δ strain, p53-Mdm2 interaction inhibitors, like AMG232 and MI-773, completely inhibited the p53-Mdm2 interaction at nanomolar concentrations in the Y2H assay. In addition, we identified a conserved segment in the core DNA-binding domain of p53 that facilitates stable interaction with Mdm2 in yeast cells and in vitro.ConclusionThe Y2H assay can be utilized for high-throughput screening of small-molecule inhibitors of PPIs and to identify domains that stabilize PPIs.

Disrupted IRF6-NME1/2 Complexes as a Cause of Cleft Lip/Palate

Mutations and common polymorphisms in interferon regulatory factor 6 (IRF6) are associated with both syndromic and nonsyndromic forms of cleft lip/palate (CLP). To date, much of the focus on this transcription factor has been on identifying its direct targets and the gene regulatory network in which it operates. Notably, however, IRF6 is found predominantly in the cytoplasm, with its import into the nucleus tightly regulated like other members of the IRF family. To provide further insight into the role of IRF6 in the pathogenesis of CLP, we sought to identify direct IRF6 protein interactors using a combination of yeast 2-hybrid screens and co-immunoprecipitation assays. Using this approach, we identified NME1 and NME2, well-known regulators of Rho-type GTPases, E-cadherin endocytosis, and epithelial junctional remodeling, as bona fide IRF6 partner proteins. The NME proteins co-localize with IRF6 in the cytoplasm of primary palatal epithelial cells in vivo, and their interaction with IRF6 is significantly enhanced by phosphorylation of key serine residues in the IRF6 C-terminus. Furthermore, CLP associated IRF6 missense mutations disrupt the ability of IRF6 to bind the NME proteins and result in elevated activation of Rac1 and RhoA, compared to wild-type IRF6, when ectopically expressed in 293T epithelial cells. Significantly, we also report the identification of 2 unique missense mutations in the NME proteins in patients with CLP (NME1 R18Q in an IRF6 and GRHL3 mutation-negative patient with van der Woude syndrome and NME2 G71V in a patient with nonsyndromic CLP). Both variants disrupted the ability of the respective proteins to interact with IRF6. The data presented suggest an important role for cytoplasmic IRF6 in regulating the availability or localization of the NME1/2 complex and thus the dynamic behavior of epithelia during lip/palate development.

Corrigendum

New PhytologistVolume 215, Issue 4 p. 1623-1624 CorrigendumFree Access Corrigendum This article corrects the following: The Arabidopsis thaliana mitogen-activated protein kinases MPK3 and MPK6 target a subclass of ‘VQ-motif’-containing proteins to regulate immune responses Pascal Pecher, Lennart Eschen-Lippold, Siska Herklotz, Katja Kuhle, Kai Naumann, Gerit Bethke, Joachim Uhrig, Martin Weyhe, Dierk Scheel, Justin Lee, Volume 203Issue 2New Phytologist pages: 592-606 First Published online: April 22, 2014 First published: 24 July 2017 https://doi.org/10.1111/nph.14708 Author for correspondence: Justin Lee Tel: +49 345 5582 1410 Email: [email protected] AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL New Phytologist203 (2014), 592–606. Since its publication, it has been drawn to our attention that there are errors in Fig. 3 of Pecher et al. (2014). A number of yeast colonies were mistakenly duplicated and used to depict different interaction pairs. Several of the errors have similar-looking numbers (e.g. MVQ5 with WRKY33 or WRKY13; MVQ7 with WRKY26 or WRKY28; MVQ9 with WRKY58 or WRKY28) and since the yeast colonies were not always perfectly aligned on the original plates, the error presumably arose during the assembly of the figure through the thousands (24 × 59 times) of ‘cut & paste’ steps. To validate the reported data, the authors have repeated the complete set of the yeast 2-hybrid (Y2H) assay two further times. In order to minimize the potential for error during figure assembly, the colonies were plated – within the space limitation of a standard 12-cm square plate – in the depicted sequential format (in blocks of 10–12 × 12 colonies using a 12-channel pipette). Transformation efficiency was monitored on selection drop-out (SD) media lacking leucine and tryptophan (SD-LW) and protein interaction assessed after selection on SD-LWAH (lacking leucine, tryptophan, adenine and histidine) or SD-LWAH supplemented with the indicated concentrations of 3-amino-1,2,4-triazole (+3AT). A complete set of the experiment showing the growth of yeast on the different selection plates can be viewed in in Figshare (doi: https://doi.org/10.6084/m9.figshare.5086942). Figure 3Open in figure viewerPowerPoint Yeast-two-hybrid analysis of WRKY-MVQ interactions. (MVQ, mitogen-activated protein kinase 3/6 (MPK3/6)-targeted VQ-motifcontaining protein). Y2H analysis was carried out as described in Fig 1c and positive interactions are indicated by the yeast growth on -His/-Ade selection media. The top half of the panel depicts interactions with wild-type MVQs while the lower half covers the MVQs with the “VQ” residues mutated to “DL” (MVQDL). Note that substantial auto-activation in the Y2H assay precluded the assessment of whether MVQ9 (= VQ14 or IKU1) interacted with WRKYs. pDEST22/32 are the corresponding empty vector controls for the WRKYs and VQ-motif-containing proteins (VQPs), respectively. The majority of the previously reported interaction (mainly between MVQs with Group I and Group IIc WRKYs) could be confirmed. Where there are discrepancies to the previously data, selected pairs were re-tested in a third transformation study and furthermore, identities of the plasmid clones used for transformation were re-verified by DNA restriction or sequencing analysis. Thus, the current figure represents a more confident representation of the MVQ–WRKY interactions. Nevertheless, it should be emphasized that it remains only a guideline for further confirmation by independent protein–protein interaction analytical methods. Some of the differences to the previous interaction dataset include the following: No interaction detected in the current experiment compared to the previous strong interaction: WRKY11 with MVQ4–6. No interaction detected now compared to interactions previously considered as weak or ambiguous: MVQ4/WRKY1; MVQ3/WRKY75; MVQ5/WRKY60; MVQ4/WRKY29; MVQ1/WRKY35; MVQ6/WRKY30; MVQ5/WRKY63. New interactions detected now, which were not in the previous report: MVQ4/WRKY4; MVQ6/WRKY26; MVQ6/WRKY33; MVQ6/WRKY8; MVQ7/WRKY1; MVQ7/WRKY58; MVQ5/WRKY28; MVQ6/WRKY28. The revised Fig. 3 is shown below. We apologize to our readers for this mistake. Reference Pecher P, Eschen-Lippold L, Herklotz S, Kuhle K, Naumann K, Bethke G, Uhrig J, Weyhe M, Scheel D, Lee J. 2014. The Arabidopsis thaliana mitogen-activated protein kinases MPK3 and MPK6 target a subclass of ‘VQ-motif’-containing proteins to regulate immune responses. New Phytologist 203: 592– 606. Volume215, Issue4September 2017Pages 1623-1624 FiguresReferencesRelatedInformation

2BC Non-Structural Protein of Enterovirus A71 Interacts with SNARE Proteins to Trigger Autolysosome Formation.

Viruses have evolved unique strategies to evade or subvert autophagy machinery. Enterovirus A71 (EV-A71) induces autophagy during infection in vitro and in vivo. In this study, we report that EV-A71 triggers autolysosome formation during infection in human rhabdomyosarcoma (RD) cells to facilitate its replication. Blocking autophagosome-lysosome fusion with chloroquine inhibited virus RNA replication, resulting in lower viral titres, viral RNA copies and viral proteins. Overexpression of the non-structural protein 2BC of EV-A71 induced autolysosome formation. Yeast 2-hybrid and co-affinity purification assays showed that 2BC physically and specifically interacted with a N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein, syntaxin-17 (STX17). Co-immunoprecipitation assay further showed that 2BC binds to SNARE proteins, STX17 and synaptosome associated protein 29 (SNAP29). Transient knockdown of STX17, SNAP29, and microtubule-associated protein 1 light chain 3B (LC3B), crucial proteins in the fusion between autophagosomes and lysosomes) as well as the lysosomal-associated membrane protein 1 (LAMP1) impaired production of infectious EV-A71 in RD cells. Collectively, these results demonstrate that the generation of autolysosomes triggered by the 2BC non-structural protein is important for EV-A71 replication, revealing a potential molecular pathway targeted by the virus to exploit autophagy. This study opens the possibility for the development of novel antivirals that specifically target 2BC to inhibit formation of autolysosomes during EV-A71 infection.

Hypoxia-Induced Mitogenic Factor Acts as a Nonclassical Ligand of Calcium-Sensing Receptor, Therapeutically Exploitable for Intermittent Hypoxia-Induced Pulmonary Hypertension.

Hypoxia-induced mitogenic factor (HIMF) is an inflammatory cytokine playing important role(s) in the development of hypoxic pulmonary hypertension. The molecular target mediating HIMF-stimulated downstream events remains unclear. The coimmunoprecipitation screen identified extracellular calcium-sensing receptor (CaSR) as the binding partner for HIMF in pulmonary artery smooth muscle cells. The yeast 2-hybrid assay then revealed the binding of HIMF to the intracellular, not the extracellular, domain of extracellular CaSR. The binding of HIMF enhanced the activity of extracellular CaSR and mediated hypoxia-evoked proliferation of pulmonary artery smooth cells and the development of pulmonary vascular remodeling and pulmonary hypertension, all of which was specifically attenuated by a synthesized membrane-permeable peptide flanking the core amino acids of the intracellular binding domain of extracellular CaSR. Thus, HIMF induces pulmonary hypertension as a nonclassical ligand of extracellular CaSR, and the binding motif of extracellular CaSR can be therapeutically exploitable.

Armc5 deletion causes developmental defects and compromises T-cell immune responses

Armadillo repeat containing 5 (ARMC5) is a cytosolic protein with no enzymatic activities. Little is known about its function and mechanisms of action, except that gene mutations are associated with risks of primary macronodular adrenal gland hyperplasia. Here we map Armc5 expression by in situ hybridization, and generate Armc5 knockout mice, which are small in body size. Armc5 knockout mice have compromised T-cell proliferation and differentiation into Th1 and Th17 cells, increased T-cell apoptosis, reduced severity of experimental autoimmune encephalitis, and defective immune responses to lymphocytic choriomeningitis virus infection. These mice also develop adrenal gland hyperplasia in old age. Yeast 2-hybrid assays identify 16 ARMC5-binding partners. Together these data indicate that ARMC5 is crucial in fetal development, T-cell function and adrenal gland growth homeostasis, and that the functions of ARMC5 probably depend on interaction with multiple signalling pathways.

Identification of candidates for interacting partners of the tail domain of DcNMCP1, a major component of the Daucus carota nuclear lamina-like structure

ABSTRACTNMCP/CRWN (NUCLEAR MATRIX CONSTITUENT PROTEIN/CROWDED NUCLEI) is a major component of a protein fibrous meshwork (lamina-like structure) on the plant inner nuclear membrane. NMCP/CRWN contributes to regulating nuclear shape and nuclear functions. An NMCP/CRWN protein in Daucus carota (DcNMCP1) is localized to the nuclear periphery in interphase cells, and surrounds chromosomes in cells in metaphase and anaphase. The N-terminal region and the C-terminal region of DcNMCP1 are both necessary for localizing DcNMCP1 to the nuclear periphery. Here candidate interacting partners of the amino acid position 975–1053 of DcNMCP1 (T975–1053), which is present in the C-terminal region and contains a conserved sequence that plays a role in localizing DcNMCP1 to the nuclear periphery, are screened for. Arabidopsis thaliana nuclear proteins were subjected to far-Western blotting with GST-fused T975–1053 as a probe, and signals were detected at the positions corresponding to ∼70, ∼40, and ∼18 kDa. These ∼70, ∼40, and ∼18 kDa nuclear proteins were identified by mass spectrometry, and subjected to a yeast 2-hybrid (Y2H) analysis with T975–1053 as bait. In this analysis, the ∼40 kDa protein ARP7, which is a nuclear actin-related protein possibly involved in regulating chromatin structures, was confirmed to interact with T975–1053. Independently of the far-Western blotting, a Y2H screen was performed using T975–1053 as bait. Targeted Y2H assays confirmed that 3 proteins identified in the screen, MYB3, SINAT1, and BIM1, interact with T975–1053. These proteins might have roles in NMCP/CRWN protein-mediated biologic processes.

Mapping Protein-Protein Interaction Using High-Throughput Yeast 2-Hybrid.

A tremendous asset to the analysis of protein-protein interactions is the yeast-2-hybrid (Y2H) method. The Y2H assay is a heterologous system that is expanding network biology knowledge via in vivo investigations of binary protein-protein interactions. Traditionally, the Y2H protocol entails the mating or co-transformation of yeast in solid agar media followed by visual analysis for diploid selection. Having played a key role in identifying protein-protein interactions for nearly three decades in a wide range of biological systems, the Y2H system assays the interaction between two proteins of interest which results in a reconstituted and/or activation of transcription factor allowing a reporter gene to be transcribed. Overall, the Y2H method takes advantage of two factors: (1) the auxotrophic yeast requires expression of the reporter gene to grow in media purposefully designed to lack one or more essential amino acids, and (2) the DNA-binding (DB) domain of transcription factor GAL4 is unable to initiate transcription unless it is physically associated with an activating domain (AD), which, together, DBs and ADs are fused to proteins of interest that must interact with each other to reconstitute the transcription factor and activate the reporter gene. The applications of Y2H are broad, entailing fields such as drug discovery, clinical trials for human disease including cancer and neurodegenerative disease, and extend even into synthetic biology applications and cellular engineering. This chapter begins with an introduction to the fundamental mechanics of Y2H utilizing a genetically engineered strain of yeast and proceeds with an in-depth look at the different types of Y2H and turn our focus particularly to the GAL4-based Y2H system to map protein-protein interactions. We will then provide a step-by-step protocol for the Y2H experimentation preceded by a materials listing while simultaneously including key notes throughout the entire experimental process of biological-mechanistic and historical understandings of the steps.

Genetic Analysis of Mismatch Repair Genes Alterations in Extramammary Paget Disease.

Extramammary Paget disease (EMPD) is a rare cutaneous malignant neoplasm. The familial occurrence of EMPD and the high risk of concomitant secondary tumors in EMPD patients have gained much attention. These findings highlight the importance of genetic alterations in the tumorigenesis of this skin cancer. Genetic tests and functional analysis of mismatch repair (MMR) genes were performed in EMPD. The results showed that 8 of 20 cases with germline MMR genes mutations and 5 of them exhibited microsatellite instability (MSI). Immunohistochemical staining showed that the tumor tissues from 20 patients had the normal expression of MLH1 but 5 cases had the reduced expression of MSH2. There is a nearly significant correlation between MSI and germline mutations. In 172 cases, rates of germline and somatic mutations were 34.3% and 13.4%, respectively. The mutations of MLH1 V384D (15.7%), R217C (4.1%), and I219V (5.2%) were common in this cancer. In addition, the yeast 2-hybrid and immunoprecipitation assays exhibited reduced interaction between MLH1 and PMS2 in MLH1 V384D and R217C but not I219V. Moreover, MLH1 V384D and R217C had impaired MMR activity compared with the wild-type and I219V mutation by an in vitro MMR assay. The germline mutations in MMR genes are involved in the pathogenesis of EMPD and partially explain the genetic abnormalities for this disease.

DEEPN as an Approach for Batch Processing of Yeast 2-Hybrid Interactions

We adapted the yeast 2-hybrid assay to simultaneously uncover multiple transient protein interactions within a single screen by using a strategy termed DEEPN (dynamic enrichment for evaluation of protein networks). This approach incorporates high-throughput DNA sequencing and computation to follow competition among a plasmid population encoding interacting partners. To demonstrate the capacity of DEEPN, we identify a wide range of ubiquitin-binding proteins, including interactors that we verify biochemically. To demonstrate the specificity of DEEPN, we show that DEEPN allows simultaneous comparison of candidate interactors across multiple bait proteins, allowing differential interactions to be identified. This feature was used to identify interactors that distinguish between GTP- and GDP-bound conformations of Rab5.

Regulation of transcription by the Arabidopsis UVR8 photoreceptor involves a specific histone modification.

The photoreceptor UV RESISTANCE LOCUS 8 (UVR8) specifically mediates photomorphogenic responses to UV-B wavelengths. UVR8 acts by regulating transcription of a set of genes, but the underlying mechanisms are unknown. Previous research indicated that UVR8 can associate with chromatin, but the specificity and functional significance of this interaction are not clear. Here we show, by chromatin immunoprecipitation, that UV-B exposure of Arabidopsis increases acetylation of lysines K9 and/or K14 of histone H3 at UVR8-regulated gene loci in a UVR8-dependent manner. The transcription factors HY5 and/or HYH, which mediate UVR8-regulated transcription, are also required for this chromatin modification, at least for the ELIP1 gene. Furthermore, sequencing of the immunoprecipitated DNA revealed that all UV-B-induced enrichments in H3K9,14diacetylation across the genome are UVR8-dependent, and approximately 40 % of the enriched loci contain known UVR8-regulated genes. In addition, inhibition of histone acetylation by anacardic acid reduces the UV-B induced, UVR8 mediated expression of ELIP1 and CHS. No evidence was obtained in yeast 2-hybrid assays for a direct interaction between either UVR8 or HY5 and several proteins involved in light-regulated histone modification, nor for the involvement of these proteins in UVR8-mediated responses in plants, although functional redundancy between proteins could influence the results. In summary, this study shows that UVR8 regulates a specific chromatin modification associated with transcriptional regulation of a set of UVR8-target genes.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-016-0522-3) contains supplementary material, which is available to authorized users.

Rab11a Mediates Vascular Endothelial-Cadherin Recycling and Controls Endothelial Barrier Function.

Vascular endothelial (VE)-cadherin is the predominant component of endothelial adherens junctions essential for cell-cell adhesion and formation of the vascular barrier. Endocytic recycling is an important mechanism for maintaining the expression of cell surface membrane proteins. However, little is known about the molecular mechanism of VE-cadherin recycling and its role in maintenance of vascular integrity. Using calcium-switch assay, confocal imaging, cell surface biotinylation, and flow cytometry, we showed that VE-cadherin recycling required Ras-related proteins in brain (Rab)11a and Rab11 family-interacting protein 2. Yeast 2-hybrid assay and coimmunoprecipitation demonstrated that direct interaction of VE-cadherin with family-interacting protein 2 (at aa 453-484) formed a ternary complex with Rab11a in human endothelial cells. Silencing of Rab11a or Rab11 family-interacting protein 2 in endothelial cells prevented VE-cadherin recycling and VE-cadherin expression at endothelial plasma membrane. Furthermore, inactivation of Rab11a signaling blocked junctional reannealing after vascular inflammation. Selective knockdown of Rab11a in pulmonary microvessels markedly increased vascular leakage in mice challenged with lipopolysaccharide or polymicrobial sepsis. Rab11a/Rab11 family-interacting protein 2-mediated VE-cadherin recycling is required for formation of adherens junctions and restoration of VE barrier integrity and hence a potential target for clinical intervention in inflammatory disease.

FTO is necessary for the induction of leptin resistance by high-fat feeding.

ObjectiveLoss of function FTO mutations significantly impact body composition in humans and mice, with Fto-deficient mice reported to resist the development of obesity in response to a high-fat diet (HFD). We aimed to further explore the interactions between FTO and HFD and determine if FTO can influence the adverse metabolic consequence of HFD.MethodsWe studied mice deficient in FTO in two well validated models of leptin resistance (HFD feeding and central palmitate injection) to determine how Fto genotype may influence the action of leptin. Using transcriptomic analysis of hypothalamic tissue to identify relevant pathways affected by the loss of Fto, we combined data from co-immunoprecipitation, yeast 2-hybrid and luciferase reporter assays to identify mechanisms through which FTO can influence the development of leptin resistant states.ResultsMice deficient in Fto significantly increased their fat mass in response to HFD. Fto+/− and Fto−/− mice remained sensitive to the anorexigenic effects of leptin, both after exposure to a HFD or after acute central application of palmitate. Genes encoding components of the NFкB signalling pathway were down-regulated in the hypothalami of Fto-deficient mice following a HFD. When this pathway was reactivated in Fto-deficient mice with a single low central dose of TNFα, the mice became less sensitive to the effect of leptin. We identified a transcriptional coactivator of NFкB, TRIP4, as a binding partner of FTO and a molecule that is required for TRIP4 dependent transactivation of NFкB.ConclusionsOur study demonstrates that, independent of body weight, Fto influences the metabolic outcomes of a HFD through alteration of hypothalamic NFкB signalling. This supports the notion that pharmacological modulation of FTO activity might have the potential for therapeutic benefit in improving leptin sensitivity, in a manner that is influenced by the nutritional environment.

Identification and characterization of a ligand-selective mineralocorticoid receptor coactivator.

The mineralocorticoid receptor (MR) is unique in responding to 2 physiological ligands: aldosterone and cortisol. In epithelial tissues, aldosterone selectivity is determined by the activity of 11β-hydroxysteroid dehydrogenase type 2. In other tissues, cortisol is the primary ligand. To understand the structural determinants of ligand-specific MR activation, we sought to identify coregulatory molecules that interact with the ligand-binding domain (LBD) of the MR. A yeast-2-hybrid (Y2H) kidney cDNA library was screened with the human MR-LBD in the presence of aldosterone and cortisol. One clone, identified as aldosterone-specific in the Y2H assay, exhibited a 7-fold greater response, aldosterone vs. cortisol, in a mammalian-2-hybrid (M2H) assay. This clone encodes the region of the tesmin gene that has 2 leucine-x-x-leucine-leucine (LxxLL) motifs. Full-length tesmin coactivates (>2-fold) MR-mediated transactivation in the presence of aldosterone, but not of cortisol; this specificity is observed with a range of promoters. GST pulldown and coimmunoprecipitation of the MR by tesmin supports a direct interaction, mediated by the 2 LxxLL motifs. Tesmin thus represents a novel MR coregulator that exhibits a differential interaction, providing further evidence of the adoption of ligand-dependent conformations by the MR-LBD.

An approach for exploring interaction between two proteins in vivo.

We describe a strategy for exploring the function of protein-protein interactions in striated muscle in vivo. We describe our experience using this strategy to study the interaction of UNC-112 (kindlin) with PAT-4 (integrin linked kinase). Random mutagenesis is used to generate a collection of mutants that are screened for lack of binding or gain of binding using a yeast 2-hybrid assay. The mutant proteins are then expressed in transgenic C. elegans to determine their ability to localize in the sarcomere. We emphasize two advantages of this strategy: (1) for studying the interaction of protein A with protein B, when protein A can interact with multiple proteins, and (2) it explores the function of an interaction rather than the absence of, or reduced level of, a protein as can be obtained with null mutants or knockdown by RNAi. We propose that this method can be generalized for studying the meaning of a protein-protein interaction in muscle for any system in which transgenic animals can be generated and their muscles can be imaged.

ALDOB Acts as a Novel HBsAg-Binding Protein and Its Coexistence Inhibits Cisplatin-Induced HepG2 Cell Apoptosis

Chronic infection with hepatitis B virus is a cause of end-stage liver disease and hepatocellular carcinoma (HCC). We previously screened fructose-bisphosphate aldolase B (ALDOB) as a candidate binding protein of hepatitis B surface antigen (HBsAg) using a yeast 2-hybrid assay. In this study we aimed to confirm ALDOB as a binding protein of the S region of the HbsAg (HBs) and to investigate the function and involved mechanism between its interactions during HCC development. Our results demonstrated that both of exogenous and endogenous ALDOB proteins bind to HBs and colocalize in the cytoplasm in vitro. The coexistence of HBs and ALDOB inhibit apoptosis of cisplatin-induced HepG2 cells. Furthermore, western blot analysis showed the coexistence of HBs and ALDOB enhance the phosphorylations of AKT and its downstream of GSK-3β (phosphorylation); decreased expression of the pro-apoptotic proteins Bax, Bid, Bim, and Puma; and increased expression of the prosurvival proteins Bcl-2, Bcl-xl, and Mcl-1 in HepG2 cells. These findings suggest that interaction between HBs and ALDOB might be applied as a potential therapeutic target during the treatment of HBV-related hepatitis or HCC.

The ABCA1 domain responsible for interaction with HIV-1 Nef is conformational and not linear

HIV-1 Nef is an accessory protein responsible for inactivation of a number of host cell proteins essential for anti-viral immune responses. In most cases, Nef binds to the target protein and directs it to a degradation pathway. Our previous studies demonstrated that Nef impairs activity of the cellular cholesterol transporter, ABCA1, and that Nef interacts with ABCA1. Mutation of the 2226DDDHLK motif in the C-terminal cytoplasmic tail of ABCA1 disrupted interaction with Nef. Here, we tested Nef interaction with the ABCA1 C-terminal cytoplasmic fragment using yeast 2-hybrid system assay and co-immunoprecipitation analysis in human cells. Surprisingly, analysis in a yeast 2-hybrid system did not reveal any interaction between Nef and the C-terminal cytoplasmic fragment of ABCA1. Using co-immunoprecipitation from HEK 293T cells expressing these polypeptides, only a very weak interaction could be detected. The 2226DDDHLK motif in the C-terminal cytoplasmic tail of ABCA1 found previously to be essential for interaction between ABCA1 and Nef is insufficient to bestow strong binding to Nef. Molecular modeling suggested that interaction with Nef may be mediated by a conformational epitope composed of the sequences within the cytoplasmic loop of ABCA1 and the C-terminal cytoplasmic domain. Studies are now underway to characterize this epitope.