scholarly journals An atlas of protein-protein interactions across mammalian tissues

2018 ◽  
Author(s):  
Michael A. Skinnider ◽  
Nichollas E. Scott ◽  
Anna Prudova ◽  
Nikolay Stoynov ◽  
R. Greg Stacey ◽  
...  
Keyword(s):  
High Throughput ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Cellular Mechanisms ◽  
Stable Isotope Labelling ◽  
Proteomic Approach ◽  
Mouse Tissues ◽  
Mammalian Tissues ◽  
Genetically Manipulated

SummaryCellular processes arise from the dynamic organization of proteins in networks of physical interactions. Mapping the complete network of biologically relevant protein-protein interactions, the interactome, has therefore been a central objective of high-throughput biology. Yet, because widely used methods for high-throughput interaction discovery rely on heterologous expression or genetically manipulated cell lines, the dynamics of protein interactions across physiological contexts are poorly understood. Here, we use a quantitative proteomic approach combining protein correlation profiling with stable isotope labelling of mammals (PCP SILAM) to map the interactomes of seven mouse tissues. The resulting maps provide the first proteome-scale survey of interactome dynamics across mammalian tissues, revealing over 27,000 unique interactions with an accuracy comparable to the highest-quality human screens. We identify systematic suppression of cross-talk between the evolutionarily ancient housekeeping interactome and younger, tissue-specific modules. Rewiring of protein interactions across tissues is widespread, and is poorly predicted by gene expression or coexpression. Rewired proteins are tightly regulated by multiple cellular mechanisms and implicated in disease. Our study opens up new avenues to uncover regulatory mechanisms that shape in vivo interactome responses to physiological and pathophysiological stimuli in mammalian systems.

The study of protein–protein interactions in bacteria

2012 ◽  
Vol 58 (11) ◽  
pp. 1241-1257 ◽  
Author(s):  
Roberto Velasco-García ◽  
Rocío Vargas-Martínez
Keyword(s):  
Hybrid System ◽  
High Throughput ◽  
Protein Interactions ◽  
Specific Protein ◽  
False Positives ◽  
Interaction Networks ◽  
Protein Protein Interactions ◽  
Extensive Data ◽  
False Positives And Negatives

Many of the functions fulfilled by proteins in the cell require specific protein–protein interactions (PPI). During the last decade, the use of high-throughput experimental technologies, primarily based on the yeast 2-hybrid system, generated extensive data currently located in public databases. This information has been used to build interaction networks for different species. Unfortunately, due to the nature of the yeast 2-hybrid system, these databases contain many false positives and negatives, thus they require purging. A method for confirming these PPI is to test them using a technique that operates in vivo and detects binary PPI. This article comprises an overview of the study of PPI and describes the main techniques that have been used to identify bacterial PPI, prioritizing those that can be used for their verification, and it also mentions a number of PPI that have been identified or confirmed using these methods.

scholarly journals Rapamycin-dependent delocalization as a novel tool to reveal protein-protein interactions in plants

2020 ◽  
Author(s):  
Joanna Winkler ◽  
Evelien Mylle ◽  
Andreas De Meyer ◽  
Benjamin Pavie ◽  
Julie Merchie ◽  
...  
Keyword(s):  
Plant Cell ◽  
High Throughput ◽  
Protein Interactions ◽  
Biological Process ◽  
Higher Order ◽  
Protein Protein Interactions ◽  
Bait Protein ◽  
Versatile Tool ◽  
Protein Tagging

AbstractIdentifying protein-protein interactions (PPI) is crucial to understand any type of biological process. Many PPI tools are available, yet only some function within the context of a plant cell. Narrowing down even further, only few PPI tools allow visualizing higher order interactions. Here, we present a novel and conditional in vivo PPI tool for plant research. Knocksideways in plants (KSP) uses the ability of rapamycin to alter the localization of a bait protein and its interactors via the heterodimerization of FKBP and FRB domains. KSP is inherently free from many limitations, which other PPI systems hold. It is an in vivo tool, it is flexible concerning the orientation of protein tagging as long as this does not interfere with the interaction and it is compatible with a broad range of fluorophores. KSP is also a conditional tool and therefore does not require additional controls. The interactions can be quantified and in high throughput by the scripts that we provide. Finally, we demonstrate that KSP can visualize higher-order interactions. It is therefore a versatile tool, complementing the PPI methods field with unique characteristics and applications.

scholarly journals PDZ Motifs in PTP-BL and RIL Bind to Internal Protein Segments in the LIM Domain Protein RIL

1998 ◽  
Vol 9 (3) ◽  
pp. 671-683 ◽  
Author(s):  
Edwin Cuppen ◽  
Herlinde Gerrits ◽  
Barry Pepers ◽  
Bé Wieringa ◽  
Wiljan Hendriks
Keyword(s):  
Protein Interactions ◽  
Tyrosine Phosphatase ◽  
Specific Interaction ◽  
Protein Protein Interactions ◽  
Lim Domain ◽  
Binding Interface ◽  
Mouse Tissues

The specificity of protein–protein interactions in cellular signaling cascades is dependent on the sequence and intramolecular location of distinct amino acid motifs. We used the two-hybrid interaction trap to identify proteins that can associate with the PDZ motif-rich segment in the protein tyrosine phosphatase PTP-BL. A specific interaction was found with the Lin-11, Isl-1, Mec-3 (LIM) domain containing protein RIL. More detailed analysis demonstrated that the binding specificity resides in the second and fourth PDZ motif of PTP-BL and the LIM domain in RIL. Immunohistochemistry on various mouse tissues revealed a submembranous colocalization of PTP-BL and RIL in epithelial cells. Remarkably, there is also an N-terminal PDZ motif in RIL itself that can bind to the RIL-LIM domain. We demonstrate here that the RIL-LIM domain can be phosphorylated on tyrosine in vitro and in vivo and can be dephosphorylated in vitro by the PTPase domain of PTP-BL. Our data point to the presence of a double PDZ-binding interface on the RIL-LIM domain and suggest tyrosine phosphorylation as a regulatory mechanism for LIM-PDZ associations in the assembly of multiprotein complexes. These findings are in line with an important role of PDZ-mediated interactions in the shaping and organization of submembranous microenvironments of polarized cells.

Probing the 14-3-3 Isoform-Specificity Profile of Interactions Stabilized by Fusicoccin A

2020 ◽  
Author(s):  
James Frederich ◽  
Ananya Sengupta ◽  
Josue Liriano ◽  
Ewa A. Bienkiewicz ◽  
Brian G. Miller
Keyword(s):  
Protein Interactions ◽  
Neurological Diseases ◽  
Adapter Proteins ◽  
Protein Protein Interactions ◽  
Specific Expression ◽  
Individual Client ◽  
Isoform Specificity ◽  
Fusicoccin A

Fusicoccin A (FC) is a fungal phytotoxin that stabilizes protein–protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. In recent years, FC has emerged as an important chemical probe of human 14-3-3 PPIs implicated in cancer and neurological diseases. These previous studies have established the structural requirements for FC-induced stabilization of 14-3-3·client phosphoprotein complexes; however, the effect of different 14-3-3 isoforms on FC activity has not been systematically explored. This is a relevant question for the continued development of FC variants because there are seven distinct isoforms of 14-3-3 in humans. Despite their remarkable sequence and structural similarities, a growing body of experimental evidence supports both tissue-specific expression of 14-3-3 isoforms and isoform-specific functions <i>in vivo</i>. Herein, we report the isoform-specificity profile of FC <i>in vitro</i>using recombinant human 14-3-3 isoforms and a focused library of fluorescein-labeled hexaphosphopeptides mimicking the C-terminal 14-3-3 recognition domains of client phosphoproteins targeted by FC in cell culture. Our results reveal modest isoform preferences for individual client phospholigands and demonstrate that FC differentially stabilizes PPIs involving 14-3-3s. Together, these data provide strong motivation for the development of non-natural FC variants with enhanced selectivity for individual 14-3-3 isoforms.

scholarly journals Interactome Analysis of KIN (Kin17) Shows New Functions of This Protein

2021 ◽  
Vol 43 (2) ◽  
pp. 767-781
Author(s):  
Vanessa Pinatto Gaspar ◽  
Anelise Cardoso Ramos ◽  
Philippe Cloutier ◽  
José Renato Pattaro Junior ◽  
Francisco Ferreira Duarte Junior ◽  
...  
Keyword(s):  
Dna Replication ◽  
Protein Interactions ◽  
Ribosome Biogenesis ◽  
Cell Metabolism ◽  
Cell Types ◽  
Protein Protein Interactions ◽  
Cellular Mechanisms ◽  
Cancerous Cell ◽  
First Time

KIN (Kin17) protein is overexpressed in a number of cancerous cell lines, and is therefore considered a possible cancer biomarker. It is a well-conserved protein across eukaryotes and is ubiquitously expressed in all cell types studied, suggesting an important role in the maintenance of basic cellular function which is yet to be well determined. Early studies on KIN suggested that this nuclear protein plays a role in cellular mechanisms such as DNA replication and/or repair; however, its association with chromatin depends on its methylation state. In order to provide a better understanding of the cellular role of this protein, we investigated its interactome by proximity-dependent biotin identification coupled to mass spectrometry (BioID-MS), used for identification of protein–protein interactions. Our analyses detected interaction with a novel set of proteins and reinforced previous observations linking KIN to factors involved in RNA processing, notably pre-mRNA splicing and ribosome biogenesis. However, little evidence supports that this protein is directly coupled to DNA replication and/or repair processes, as previously suggested. Furthermore, a novel interaction was observed with PRMT7 (protein arginine methyltransferase 7) and we demonstrated that KIN is modified by this enzyme. This interactome analysis indicates that KIN is associated with several cell metabolism functions, and shows for the first time an association with ribosome biogenesis, suggesting that KIN is likely a moonlight protein.

scholarly journals Ways into Understanding HIF Inhibition

Cancers ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 159
Author(s):  
Tina Schönberger ◽  
Joachim Fandrey ◽  
Katrin Prost-Fingerle
Keyword(s):  
Protein Interactions ◽  
Target Genes ◽  
Protein Protein Interactions ◽  
Low Oxygen ◽  
Drug Candidates ◽  
Open Questions ◽  
Wide Range ◽  
Hif Pathway

Hypoxia is a key characteristic of tumor tissue. Cancer cells adapt to low oxygen by activating hypoxia-inducible factors (HIFs), ensuring their survival and continued growth despite this hostile environment. Therefore, the inhibition of HIFs and their target genes is a promising and emerging field of cancer research. Several drug candidates target protein–protein interactions or transcription mechanisms of the HIF pathway in order to interfere with activation of this pathway, which is deregulated in a wide range of solid and liquid cancers. Although some inhibitors are already in clinical trials, open questions remain with respect to their modes of action. New imaging technologies using luminescent and fluorescent methods or nanobodies to complement widely used approaches such as chromatin immunoprecipitation may help to answer some of these questions. In this review, we aim to summarize current inhibitor classes targeting the HIF pathway and to provide an overview of in vitro and in vivo techniques that could improve the understanding of inhibitor mechanisms. Unravelling the distinct principles regarding how inhibitors work is an indispensable step for efficient clinical applications and safety of anticancer compounds.

scholarly journals EEF1A2 interacts with HSP90AB1 to promote lung adenocarcinoma metastasis via enhancing TGF-β/SMAD signalling

2021 ◽  
Author(s):  
Liqing Jia ◽  
Xiaolu Ge ◽  
Chao Du ◽  
Linna Chen ◽  
Yanhong Zhou ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Protein Interactions ◽  
Elongation Factor ◽  
Protein Translation ◽  
Protein Protein Interactions ◽  
Promising Target ◽  
Tgfβ Receptor ◽  
Mesenchymal Transformation

Abstract Background Eukaryotic protein translation elongation factor 1α2 (EEF1A2) is an oncogene that promotes the progression of breast and pancreatic cancer. In this study, we aimed to elucidate the oncogenic function of EEF1A2 in the metastasis of lung adenocarcinoma (LUAD). Methods Immunohistochemistry and western blot were used to study EEF1A2 expression levels in LUAD tissues and cells, respectively. The role of EEF1A2 in LUAD progression were investigated in vitro and in vivo. We identified potential EEF1A2-binding proteins by liquid chromatography-electrospray mass spectrometry (LC-MS)/MS. Protein–protein interactions were determined by immunofluorescence and co-immunoprecipitation (Co-IP). Results In this study, we report that EEF1A2 mediates the epithelial–mesenchymal transformation (EMT), to promote the metastasis of LUAD cells in vitro and in vivo. Moreover, EEF1A2 interacts with HSP90AB1 to increase TGFβ Receptor (TβR)-I, and TβRII expression, followed by enhanced SMAD3 and pSMAD3 expression and nuclear localisation, which promotes the EMT of LUAD cells. Overexpression of EEF1A2 in cancer tissues is associated with poor prognosis and short survival of patients with LUAD. Conclusions These findings underscore the molecular functions of EEF1A2 in LUAD metastasis and indicate that EEF1A2 represents a promising target in the treatment of aggressive LUAD.

scholarly journals Measuring ligand-dependent and ligand-independent interactions between nuclear receptors and associated proteins using Bioluminescence Resonance Energy Transfer (BRET2)

2006 ◽  
Vol 4 (1) ◽  
pp. nrs.04021 ◽  
Author(s):  
Kristen L. Koterba ◽  
Brian G. Rowan
Keyword(s):  
Energy Transfer ◽  
Protein Interactions ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Renilla Luciferase ◽  
Receptor Protein ◽  
Protein Protein Interactions

Bioluminescent resonance energy transfer (BRET2) is a recently developed technology for the measurement of protein-protein interactions in a live, cell-based system. BRET2 is characterized by the efficient transfer of excited energy between a bioluminescent donor molecule (Renilla luciferase) and a fluorescent acceptor molecule (a mutant of Green Fluorescent Protein (GFP2)). The BRET2 assay offers advantages over fluorescence resonance energy transfer (FRET) because it does not require an external light source thereby eliminating problems of photobleaching and autoflourescence. The absence of contamination by light results in low background that permits detection of very small changes in the BRET2 signal. BRET2 is dependent on the orientation and distance between two fusion proteins and therefore requires extensive preliminary standardization experiments to conclude a positive BRET2 signal independent of variations in protein titrations and arrangement in tertiary structures. Estrogen receptor (ER) signaling is modulated by steroid receptor coactivator 1 (SRC-1). To establish BRET2 in a ligand inducible system we used SRC-1 as the donor moiety and ER as the acceptor moiety. Expression and functionality of the fusion proteins were assessed by transient transfection in HEK-293 cells followed by Western blot analysis and measurement of ER-dependent reporter gene activity. These preliminary determinations are required prior to measuring nuclear receptor protein-protein interactions by BRET2. This article describes in detail the BRET2 methodology for measuring interaction between full-length ER and coregulator proteins in real-time, in an in vivo environment.

Sign in / Sign up

Export Citation Format

Share Document