Visualization Of Protein-protein Interactions Research Articles

307 Revealing Candidate Inherited Retinal Disease Candidates via Genome-wide Screening of Knockout Mice

OBJECTIVES/GOALS: The goal of this study is to reveal strong candidate genes for inherited retinal diseases (IRDs) in humans to better understand the mechanisms behind IRD development and reveal potential therapeutic targets. We hope these findings will help improve our understanding of IRDs and, subsequently, diagnostic accuracy and prognosis for IRD patients. METHODS/STUDY POPULATION: The goal of the International Mouse Phenotyping Consortium is to identify the function of all protein-coding genes in the mouse genome via generation and phenotypic characterization of single gene knockout (KO) mice. Using this database, we identified all KO strains associated with abnormal retinal phenotypes, removed all RNA coding genes and pseudogenes, converted to human orthologues, and conducted a literature search for existing research regarding candidate IRD genes and retinal function/abnormalities. A similar process was used for RetNet genes. Subsequently, we performed bioinformatics analysis, including functional annotation (e.g. panther-db), pathway analysis (e.g. KEGG), and string-db to visualize known and predicted protein-protein interactions between the two data sets. RESULTS/ANTICIPATED RESULTS: Analysis of the IMPC database revealed, out of 8481 phenotyped genes, 572 unique protein coding genes were associated with 14 categories of retinal abnormalities such as abnormal retinal vasculature and abnormal retinal thickness, 377 of which have never been associated with retinal pathology in humans or mice. Pathway analysis of the IMPC database highlighted a general metabolism pathway as well as PI3K-Akt and MAPK pathways, not found in RetNet pathway results. Unique clusters from functional annotation clustering of the IMPC include DNA methylation and protein ubiquination. Visualization of protein-protein interactions in string-db between the IMPC (mouse) and RetNet (human) revealed 4 clusters of interest with gold standard RetNet IRD proteins interacting with candidate IMPC IRD proteins. DISCUSSION/SIGNIFICANCE: IMPC analysis revealed 572 candidate IRD genes, 377 of which are novel with no existing independent research related to the retina outside of the IMPC. Bioinformatic analysis reveals 4 strong clusters of interest through string-db where a gold standard RetNet gene interacts with candidate IRD genes as well as many functional pathways of interest.

Visualization of the Association of Dimeric Protein Complexes on Specific Enhancers in the Salivary Gland Nuclei of Drosophila Larva.

Transcription factors (TFs) regulate gene expression by recognizing specific target enhancers in the genome. The DNA-binding and regulatory activity of TFs depend on the presence of additional protein partners, leading to the formation of versatile and dynamic multimeric protein complexes. Visualizing these protein-protein interactions (PPIs) in the nucleus is key for decrypting the molecular cues underlying TF specificity in vivo. Over the last few years, Bimolecular Fluorescence Complementation (BiFC) has been developed in several model systems and applied in the analysis of different types of PPIs. In particular, BiFC has been applied when analyzing PPIs with hundreds of TFs in the nucleus of live Drosophila embryos. However, the visualization of PPIs at the level of specific target enhancers or genomic regions of interest awaits the advent of DNA-labelling methods that can be coupled with BiFC. Here, we present a novel experimental strategy that we have called BiFOR and that is based on the coupling of BiFC with the bacterial ANCHOR DNA-labelling system. We demonstrate that BiFOR enables the precise quantification of the enrichment of specific dimeric protein complexes on target enhancers in Drosophila salivary gland nuclei. Given its versatility and sensitivity, BiFOR could be applied more widely to other tissues during Drosophila development. Our work sets up the experimental basis for future applications of this strategy.

In silico Prediction of Molecular Docking between Chicken BRD2 and Newcastle Disease Virus Matrix Protein to Elucidate Host-pathogen Interaction

Background: Newcastle disease is one of the most contagious viral diseases caused by the Newcastle disease virus (NDV) which causes a huge loss in the terms of economy as well as mortality level in the poultry sector. The viral matrix protein is one of the proteins which helps in the attachment of virus, budding and assembly of progeny virions in the host cell. Host-pathogen interaction plays a very important role in the changeover of genetic diversity in organisms. Methods: The amino acid sequences of chicken BRD2 gene and matrix protein of NDV were used for structure prediction and visualization of protein-protein interaction between BRD2 and matrix protein by using Discovery Studio software. The nucleotide sequences of both genes were used for evolutionary analysis using MEGA7 software followed by selection pressure analysis using online Datamonkey server. Result: The results indicated that the ligand was attached with the matrix protein, which acts as a receptor on the virus surface. The purifying selection has been evident for the viral matrix protein gene. The docking of viral protein with host BRD2 protein has been detected in the study which further warrants experimental validation. In conclusion, the viral matrix protein interacts at amino acid position Tyrosine-148, Valine-339, Alanine-341 with ligand present on the chicken BRD2 protein. The evolutionary analysis revealed that the matrix protein of NDV R2B strain was the closest to that of Peacock, pigeon, duck and quail however, it exhibited maximum distance with the chicken NDV from Ireland, Vietnam and Haryana state of India.

Simultaneous Determination and Subcellular Localization of Protein-Protein Interactions in Plant Cells Using Bimolecular Fluorescence Complementation Assay.

The bimolecular fluorescence complementation (BiFC) assay allows the visualization of protein-protein interactions in their native state within living systems. The BiFC assay is based on the in vivo complementation of nonfluorescent component parts of a fluorescent protein through the interaction or proximity target proteins, each fused to a different component of the fluorescent protein. Expansion of the BiFC toolkit with an increasing spectrum of fluorescence markers and catalog of Gateway-compatible vectors for high-throughput screening, has made BiFC an exceedingly powerful tool in discovering new protein interactions or providing backup evidence for known ones. Apart from the validation of protein-protein interactions, BiFC offers the additional benefit of providing information on the subcellular localization of protein interaction complexes. Subcellular localization to a specific subcellular compartment or organelle may be further validated by the coexpression of a fluorescence-labeled protein marker. Here we describe an efficient yet simple protocol for simultaneous determination and subcellular localization of protein-protein interactions in plant cells.

The Visualization of Protein-Protein Interactions in Breast Cancer: Deployment Study in Pathological Examination.

The therapeutic strategy is determined by protein expression using immunohistochemistry of estrogen receptor (ER), progesterone receptor, and human epidermal growth factor receptor 2 (HER2) in formalin-fixed paraffin-embedded (FFPE) breast cancer tissues. However, few proteins function independently, and many of them functions due to protein-protein interactions (PPIs) with other proteins. Therefore, it is important to focus on PPIs. This review summarizes the PPIs of ER and HER2 in breast cancer, especially those using a proximity ligation assay that can visualize PPIs in FFPE tissues. In particular, assessing the interaction of CEACAM6 with HER2 may serve as a surrogate marker for the efficacy of trastuzumab in patients with breast cancer. Therefore, in this review, the technique used to detect the interaction of CEACAM6 and HER2 in routinely processed pathological specimens will be applied to the clinical practice of drug selection. We showed the possibility as a novel pathological examination method using PPIs.

TagBiFC technique allows long-term single-molecule tracking of protein-protein interactions in living cells

Protein-protein interactions (PPIs) are critical for cellular activity regulation. Visualization of PPIs using bimolecular fluorescence complementation (BiFC) techniques helps to understand how PPIs implement their functions. However, current BiFC is based on fluorescent proteins and the brightness and photostability are suboptimal for single molecule tracking experiments, resulting in either low spatiotemporal resolution or incapability of tracking for extended time course. Here, we developed the TagBiFC technique based on split HaloTag, a self-labeling tag that could conjugate an organic dye molecule and thus offered better brightness and photostability than fluorescent proteins for PPI visualization inside living cells. Through screening and optimization, we demonstrated that the reconstituted HaloTag exhibited higher localization precision and longer tracking length than previous methods. Using TagBiFC, we reveal that the dynamic interactions of transcription factor dimers with chromatin DNA are distinct and closely related to their dimeric states, indicating a general regulatory mechanism for these kinds of transcription factors. In addition, we also demonstrated the advantageous applications of TagBiFC in single nucleosome imaging, light-burden imaging of single mRNA, low background imaging of cellular structures. We believe these superior properties of our TagBiFC system will have broad applications in the studies of single molecule imaging inside living cells.

A tandem near-infrared fluorescence complementation system with enhanced fluorescence for imaging protein–protein interactions in vivo

Bimolecular fluorescence complementation (BiFC) is an effective tool for visualizing protein–protein interactions (PPIs). However, a BiFC system with long wavelength and high fluorescence intensity is yet to be developed for in vivo imaging. In this study, we constructed a tandem near-infrared BiFC (tBiFC) system by splitting a near-infrared phytochrome, IFP2.0. This system allows the identification and visualization of PPIs in live cells and living mice. The photophysical properties of the complementary fluorescence of the tBiFC system were similar to those of its parent protein IFP2.0, but the intensity was twice that of a single-copy IFP2.0-based BiFC system. Compared with previously reported near infrared BiFC systems—iRFP-BiFC and IFP1.4-BiFC—the signal intensity of the tBiFC system increased by ~1.48- and ~400-fold for weak PPIs in living cells, and ~1.51- and ~8-fold for strong PPIs, respectively. When applied to imaging PPIs in live mice, the complementary fluorescence intensity of the tBiFC system was also significantly higher than that of the other near-infrared BiFC systems. The use of this bright phytochrome in a tandem arrangement constitutes a powerful tool for imaging PPIs in the near infrared region.

Simultaneous measurement of p53:Mdm2 and p53:Mdm4 protein-protein interactions in whole cells using fluorescence labelled foci

In this report we describe the development of a Fluorescent Protein-Protein Interaction-visualization (FLUOPPI) to enable the simultaneous measurement of both Mdm2:p53 and Mdm4:p53 interactions in order to assess the relative efficiencies of mimetic molecules of the p53 peptide helix against both PPIs. Mdm2 and Mdm4 overexpression frequently leads to the inactivation of non-mutated p53 in human cancers, via inhibition of its transcriptional activity, enhancing its degradation by the proteasome or by preventing its nuclear import. Development of inhibitors to disrupt the binding of one or both of these protein interactions have been the subject of intensive pharmaceutical development for anti-cancer therapies. Using the bimodal FLUOPPI system we have characterised compounds that were either monospecific for Mdm2 or bispecific for both Mdm2 and Mdm4. We have also demonstrated that the FLUOPPI assay can reliably differentiate between specific and non-specific disruption of these protein complexes via accurate assessment and normalization to the cell population under measurement. We envision that this methodology will increase the efficiency of identifying compounds that are either specific against a single PPI from a closely related family of interactions or compounds that interact across multiple related PPI pairs, depending on which is more desirable.

MicroRNA expression in cervical cancer: Novel diagnostic and prognostic biomarkers.

Growing evidence has shown that a large number of miRNAs are abnormally expressed in cervical cancer (CC) tissues and play irreplaceable roles in tumorigenesis, progression, and metastasis. This study aimed to identify new biomarkers and pivotal genes associated with CC prognosis through comprehensive bioinformatics analysis. At first, the data of gene expression microarray (GSE30656) was downloaded from GEO database and differential miRNAs were obtained. Additionally, 4 miRNAs associated with the survival time of patients with CC were screened through TCGA differential data analysis, Kaplan-Meier, and Landmark analysis. Among them, the low expression of miR-188 and high expression of miR-223 correlated with the short survival of CC patients, while the down-regulation of miR-99a and miR-125b was closely related to the 5-year survival rate of patients. Then, based on the correspondence between the differentially expressed genes (DEGs) in CC from the TCGA data and the 4 miRNAs target genes, 58 target genes were screened to perform the analysis of function enrichment and the visualization of protein-protein interaction (PPI) networks. The seven pivotal genes of the PPI network as the target genes of four miRNAs related to prognosis, they were directly or indirectly involved in the development of CC. In this study, based on high-throughput data mining, differentially expressed miRNAs and related target genes were analyzed to provide an effective bioinformatics basis for further understanding of the pathogenesis and prognosis of CC. And the results may be a promising biomarker for the early screening of high-risk populations and early diagnosis of cervical cancer.

Development of bimolecular fluorescence complementation using rsEGFP2 for detection and super-resolution imaging of protein-protein interactions in live cells.

Direct visualization of protein-protein interactions (PPIs) at high spatial and temporal resolution in live cells is crucial for understanding the intricate and dynamic behaviors of signaling protein complexes. Recently, bimolecular fluorescence complementation (BiFC) assays have been combined with super-resolution imaging techniques including PALM and SOFI to visualize PPIs at the nanometer spatial resolution. RESOLFT nanoscopy has been proven as a powerful live-cell super-resolution imaging technique. With regard to the detection and visualization of PPIs in live cells with high temporal and spatial resolution, here we developed a BiFC assay using split rsEGFP2, a highly photostable and reversibly photoswitchable fluorescent protein previously developed for RESOLFT nanoscopy. Combined with parallelized RESOLFT microscopy, we demonstrated the high spatiotemporal resolving capability of a rsEGFP2-based BiFC assay by detecting and visualizing specifically the heterodimerization interactions between Bcl-xL and Bak as well as the dynamics of the complex on mitochondria membrane in live cells.

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay.

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins. Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

CapsidMaps: protein-protein interaction pattern discovery platform for the structural analysis of virus capsids using Google Maps.

Structural analysis and visualization of protein-protein interactions is a challenging task since it is difficult to appreciate easily the extent of all contacts made by the residues forming the interfaces. In the case of viruses, structural analysis becomes even more demanding because several interfaces coexist and, in most cases, these are formed by hundreds of contacting residues that belong to multiple interacting coat proteins. CapsidMaps is an interactive analysis and visualization tool that is designed to benefit the structural virology community. Developed as an improved extension of the φ-ψ Explorer, here we describe the details of its design and implementation. We present results of analysis of a spherical virus to showcase the features and utility of the new tool. CapsidMaps also facilitates the comparison of quaternary interactions between two spherical virus particles by computing a similarity (S)-score. The tool can also be used to identify residues that are solvent exposed and in the process of locating antigenic epitope regions as well as residues forming the inside surface of the capsid that interact with the nucleic acid genome. CapsidMaps is part of the VIPERdb Science Gateway, and is freely available as a web-based and cross-browser compliant application at http://viperdb.scripps.edu.

Bimolecular fluorescence complementation (BiFC) technique in yeast Saccharomyces cerevisiae and mammalian cells.

Visualization of protein-protein interactions in vivo offers a powerful tool to resolve spatial and temporal aspects of cellular functions. The bimolecular fluorescence complementation (BiFC) makes use of nonfluorescent fragments of green fluorescent protein or its variants that are added as "tags" to target proteins under study. Only upon target protein interaction is a fluorescent protein complex assembled, and the site of interaction can be monitored by microscopy. In this chapter, we describe the method and tools for the use of BiFC in the yeast Saccharomyces cerevisiae and in mammalian cells.

Detection of Protein-protein Interactions by FRET and BRET Methods

Nowadays, in vivo protein-protein interaction studies have become preferable detecting meth-ods that enable to show or specify (already known) protein interactions and discover their inhibitors. They also facilitate detection of protein conformational changes and discovery or specification of signaling pathways in living cells. One group of in vivo methods enabling these findings is based on fluorescent resonance energy transfer (FRET) and its bio-luminescent modification (BRET). They are based on visualization of protein-protein interactions via light or enzymatic excitation of fluorescent or bio-luminescent proteins. These methods allow not only protein localization within the cell or its organelles (or small animals) but they also allow us to quantify fluorescent signals and to discover weak or strong interaction partners. In this review, we explain the principles of FRET and BRET, their applications in the characterization of protein-protein interactions and we describe several findings using these two methods that clarify molecular and cellular mechanisms and signals related to cancer biology.

Comparison of Computational Tools for Protein-Protein Interaction (PPI) Mapping and Analysis

Several visualization tools for the mapping of protein-protein interactions have been developed in recent years. However, a systematic comparison of the virtues and limitations of different PPI visualization tools has not been carried out so far. In this study, we compare seven commonly used visualization tools, based on input and output file format, layout algorithm, database integration, Gene Ontology annotation and accessibility of each tool. The assessment was carried out based on brain disease datasets. Our suggested tools, NAViGaTOR, Cytoscape and Gephi perform competitively as PPI network visualization tools, can be a reference for future researches on PPI mapping and analysis. 

Redistribution of DAT/α-Synuclein Complexes Visualized by “In Situ” Proximity Ligation Assay in Transgenic Mice Modelling Early Parkinson's Disease

Alpha-synuclein, the major component of Lewy bodies, is thought to play a central role in the onset of synaptic dysfunctions in Parkinson's disease (PD). In particular, α-synuclein may affect dopaminergic neuron function as it interacts with a key protein modulating dopamine (DA) content at the synapse: the DA transporter (DAT). Indeed, recent evidence from our “in vitro” studies showed that α-synuclein aggregation decreases the expression and membrane trafficking of the DAT as the DAT is retained into α-synuclein-immunopositive inclusions. This notwithstanding, “in vivo” studies on PD animal models investigating whether DAT distribution is altered by the pathological overexpression and aggregation of α-synuclein are missing. By using the proximity ligation assay, a technique which allows the “in situ” visualization of protein-protein interactions, we studied the occurrence of alterations in the distribution of DAT/α-synuclein complexes in the SYN120 transgenic mouse model, showing insoluble α-synuclein aggregates into dopaminergic neurons of the nigrostriatal system, reduced striatal DA levels and an altered distribution of synaptic proteins in the striatum. We found that DAT/α-synuclein complexes were markedly redistributed in the striatum and substantia nigra of SYN120 mice. These alterations were accompanied by a significant increase of DAT striatal levels in transgenic animals when compared to wild type littermates. Our data indicate that, in the early pathogenesis of PD, α-synuclein acts as a fine modulator of the dopaminergic synapse by regulating the subcellular distribution of key proteins such as the DAT.

Immunoassay for visualization of protein‐protein interactions on Ni‐nitrilotriacetate support: Example of a laboratory exercise with recombinant heterotrimeric Gαi2β1γ2 Tagged by Hexahistidine from sf9 Cells

We have developed an in vitro assay for following the interaction between the α(i2) subunit and β(1γ2) dimer from sf9 cells. This method is suitable for education purposes because it is easy, reliable, nonexpensive, can be applied for a big class of 20 students, and avoid the commonly used kinetic approach, which does not allow visualization of protein-protein interaction.

The PEDANT genome database.

The PEDANT genome database (http://pedant.gsf.de) provides exhaustive automatic analysis of genomic sequences by a large variety of established bioinformatics tools through a comprehensive Web-based user interface. One hundred and seventy seven completely sequenced and unfinished genomes have been processed so far, including large eukaryotic genomes (mouse, human) published recently. In this contribution, we describe the current status of the PEDANT database and novel analytical features added to the PEDANT server in 2002. Those include: (i) integration with the BioRS data retrieval system which allows fast text queries, (ii) pre-computed sequence clusters in each complete genome, (iii) a comprehensive set of tools for genome comparison, including genome comparison tables and protein function prediction based on genomic context, and (iv) computation and visualization of protein-protein interaction (PPI) networks based on experimental data. The availability of functional and structural predictions for 650 000 genomic proteins in well organized form makes PEDANT a useful resource for both functional and structural genomics.

Visualization of Pit-1 transcription factor interactions in the living cell nucleus by fluorescence resonance energy transfer microscopy.

The pituitary-specific transcription factor Pit-1 forms dimers when interacting with specific DNA elements and has been shown to associate with several other nuclear proteins. Recently, techniques have become available that allow visualization of protein-protein interactions as they occur in single living cells. In this study, the technique of fluorescence resonance energy transfer (FRET) microscopy was used to visualize the physical interactions of Pit-1 proteins fused to spectral variants of the jellyfish green fluorescent protein (GFP) that emit green or blue light [blue fluorescent protein (BFP)]. An optimized imaging system was used to discriminate fluorescence signals from single cells coexpressing the BFP- and GFP-fusion proteins, and the contribution of spectral overlap to background fluorescence detected in the FRET images was established. Energy transfer signals from living cells expressing a fusion protein in which GFP was tethered to BFP by short protein linker was used to demonstrate acquisition of FRET signals. Genetic vectors encoding GFP- and BFP-Pit-1 proteins were prepared, and biological function of the fusion proteins was confirmed. FRET microscopy of HeLa cells coexpressing the GFP- and BFP-Pit-1 demonstrated energy transfer, which required the two fluorophores to be separated by less than 100 A. Biochemical studies previously demonstrated that Pit-1 physically interacts with both c-Ets-1 and the estrogen receptor. FRET imaging of cells coexpressing BFP-Pit-1 and GFP-Ets-1 demonstrated energy transfer between these fusion proteins, a result consistent with their association in the nucleus of these living cells. In contrast, there was no evidence for energy transfer between the BFP-Pit-1 and an estrogen receptor-GFP fusion proteins. It is likely that the FRET imaging approach described here can be applied to many different protein-partner pairs in a variety of cellular contexts.