scholarly journals Application of Skyline for Analysis of Protein–Protein Interactions In Vivo

Molecules ◽  
2021 ◽  
Vol 26 (23) ◽  
pp. 7170
Author(s):  
Arman Kulyyassov
Keyword(s):  
Protein Interactions ◽  
Mammalian Cells ◽  
Protein Composition ◽  
Shotgun Proteomics ◽  
Cell Protein ◽  
Protein Protein Interactions ◽  
Proteomics Data ◽  
Liquid Chromatography Tandem Mass ◽  
High Level

Quantitative and qualitative analyses of cell protein composition using liquid chromatography/tandem mass spectrometry are now standard techniques in biological and clinical research. However, the quantitative analysis of protein–protein interactions (PPIs) in cells is also important since these interactions are the bases of many processes, such as the cell cycle and signaling pathways. This paper describes the application of Skyline software for the identification and quantification of the biotinylated form of the biotin acceptor peptide (BAP) tag, which is a marker of in vivo PPIs. The tag was used in the Proximity Utilizing Biotinylation (PUB) method, which is based on the co-expression of BAP-X and BirA-Y in mammalian cells, where X or Y are interacting proteins of interest. A high level of biotinylation was detected in the model experiments where X and Y were pluripotency transcription factors Sox2 and Oct4, or heterochromatin protein HP1γ. MRM data processed by Skyline were normalized and recalculated. Ratios of biotinylation levels in experiment versus controls were 86 ± 6 (3 h biotinylation time) and 71 ± 5 (9 h biotinylation time) for BAP-Sox2 + BirA-Oct4 and 32 ± 3 (4 h biotinylation time) for BAP-HP1γ + BirA-HP1γ experiments. Skyline can also be applied for the analysis and identification of PPIs from shotgun proteomics data downloaded from publicly available datasets and repositories.

scholarly journals Monitoring protein–protein interactions in mammalian cells by trans-SUMOylation

2011 ◽  
Vol 438 (3) ◽  
pp. 495-503 ◽  
Author(s):  
Ratnesh K. Srivastav ◽  
Susan Schwede ◽  
Malte Klaus ◽  
Jessica Schwermann ◽  
Matthias Gaestel ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
Glutathione Transferase ◽  
Mitogen Activated Protein Kinase ◽  
Protein Protein Interactions ◽  
Sumoylation Site ◽  
Endogenous Expression ◽  
Mitogen Activated Protein

Protein–protein interactions are essential for almost all cellular processes, hence understanding these processes mainly depends on the identification and characterization of the relevant protein–protein interactions. In the present paper, we introduce the concept of TRS (trans-SUMOylation), a new method developed to identify and verify protein–protein interactions in mammalian cells in vivo. TRS utilizes Ubc9-fusion proteins that trans-SUMOylate co-expressed interacting proteins. Using TRS, we analysed interactions of 65 protein pairs co-expressed in HEK (human embryonic kidney)-293 cells. We identified seven new and confirmed 16 known protein interactions, which were determined via endogenous SUMOylation sites of the binding partners or by using SUMOylation-site tags respectively. Four of the new protein interactions were confirmed by GST (glutathione transferase) pull-down and the p38α–Edr2 interaction was verified by co-localization analysis. Functionally, this p38α–Edr2 interaction could possibly be involved in the recruitment of p38α to the polycomb chromatin-remodelling complex to phosphorylate Bmi1. We also used TRS to characterize protein-interaction domains of the protein kinase pairs p38α–MK2 [MK is MAPK (mitogen-activated protein kinase)-activated protein kinase] and ERK3 (extracellular-signal-regulated kinase 3)–MK5 and of the p38α–p53 complex. The ability of TRS to monitor protein interactions in mammalian cells in vivo at levels similar to endogenous expression makes it an excellent new tool that can help in defining the protein interactome of mammalian cells.

scholarly journals Protein-Protein Interactions in the Cytoplasmic Membrane of Escherichia coli: Influence of the Overexpression of Diverse Transporter-Encoding Genes on the Activities of PTS Sugar Uptake Systems

2020 ◽  
Vol 30 (1-6) ◽  
pp. 36-49
Author(s):  
Mohammad Aboulwafa ◽  
Zhongge Zhang ◽  
Milton H. Saier Jr.
Keyword(s):  
Protein Interactions ◽  
Transport Systems ◽  
Sugar Uptake ◽  
Protein Protein Interactions ◽  
Phosphotransferase System ◽  
Uptake Rates ◽  
Enhanced Expression ◽  
High Level

The prokaryotic phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) concomitantly transports and phosphorylates its substrate sugars. In a recent publication, we provided evidence that protein-protein interactions of the fructose-specific integral membrane transporter (FruAB) with other PTS sugar group translocators regulate the activities of the latter systems in vivo and sometimes in vitro. In this communication, we examine the consequences of the overexpression of several different transport systems on the activities of selected PTS and non-PTS permeases. We report that high levels of these transport systems enhance the in vivo activities of several other systems in a fairly specific fashion. Thus, (1) overexpression of <i>ptsG</i> (glucose porter) selectively enhanced mannitol, N-acetylglucosamine, and 2-deoxyglucose (2DG) uptake rates; (2) overexpression of <i>mtlA</i> (mannitol porter) promoted methyl α-glucoside (αMG) and 2DG uptake; (3) <i>manYZ</i> (but not <i>manY</i> alone) (mannose porter) overexpression enhanced αMG uptake; (4) <i>galP</i> (galactose porter) overexpression enhanced mannitol and αMG uptake; and (5) <i>ansP</i> (asparagine porter) overexpression preferentially enhanced αMG and 2DG uptake, all presumably as a result of direct protein-protein interactions. Thus, it appears that high level production of several integral membrane permeases enhances sugar uptake rates, with the PtsG and ManXYZ systems being most consistently stimulated, but the MtlA and NagE systems being more selectively stimulated and to a lesser extent. Neither enhanced expression nor in vitro PEP-dependent phosphorylation activities of the target PTS systems were appreciably affected. The results are consistent with the suggestion that integral membrane transport proteins form an interacting network in vivo with physiological consequences, dependent on specific transporters and their concentrations in the membrane.

scholarly journals Tumor Suppressor p53 Can Participate in Transcriptional Induction of the GADD45 Promoter in the Absence of Direct DNA Binding

1998 ◽  
Vol 18 (5) ◽  
pp. 2768-2778 ◽  
Author(s):  
Qimin Zhan ◽  
I-Tsuen Chen ◽  
Michael J. Antinore ◽  
Albert J. Fornace
Keyword(s):  
Protein Interactions ◽  
Mammalian Cells ◽  
Dominant Negative ◽  
Proximal Promoter ◽  
Expression Vectors ◽  
Breast Carcinoma Cell Line ◽  
Protein Protein Interactions ◽  
Dna Damaging Agents ◽  
Transcriptional Induction

ABSTRACT The GADD45 gene is a growth arrest-associated gene that is induced by certain DNA-damaging agents and other stresses, such as starvation, in all mammalian cells. In addition to a strong p53-binding element in an intronic sequence, we have recently found that p53, while not required or sufficient alone, may contribute to the stress responsiveness of the promoter. Much of the responsiveness was localized to a GC-rich motif in the proximal promoter which contains multiple Egr1 sites and a larger WT1 site; this 20-bp WT1 motif is identical to the WT1-binding site in the PDGF-A gene. In extracts from a human breast carcinoma cell line expressing p53 and WT1, which is known to associate with p53 in vivo, evidence was obtained that these proteins are in a complex that binds this 20-bp element. A combination of p53 and WT1 expression vectors strongly induced a GADD45-reporter construct, while mutation of the WT1-Egr1 site in the promoter prevented this induction. Abrogation of p53 function by a dominant-negative vector or abrogation of WT1 function by an antisense vector markedly reduced the induction of this promoter. Since p53 does not bind directly to the promoter, these results indicate that p53 can contribute to the positive regulation of a promoter by protein-protein interactions.

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 RNA-Centric Approaches to Profile the RNA–Protein Interaction Landscape on Selected RNAs

2021 ◽  
Vol 7 (1) ◽  
pp. 11 ◽  
Author(s):  
André P. Gerber
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Regulatory Networks ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Protein Complexes ◽  
Cell Protein ◽  
Transcriptional Regulatory Networks ◽  
Technological Advances

RNA–protein interactions frame post-transcriptional regulatory networks and modulate transcription and epigenetics. While the technological advances in RNA sequencing have significantly expanded the repertoire of RNAs, recently developed biochemical approaches combined with sensitive mass-spectrometry have revealed hundreds of previously unrecognized and potentially novel RNA-binding proteins. Nevertheless, a major challenge remains to understand how the thousands of RNA molecules and their interacting proteins assemble and control the fate of each individual RNA in a cell. Here, I review recent methodological advances to approach this problem through systematic identification of proteins that interact with particular RNAs in living cells. Thereby, a specific focus is given to in vivo approaches that involve crosslinking of RNA–protein interactions through ultraviolet irradiation or treatment of cells with chemicals, followed by capture of the RNA under study with antisense-oligonucleotides and identification of bound proteins with mass-spectrometry. Several recent studies defining interactomes of long non-coding RNAs, viral RNAs, as well as mRNAs are highlighted, and short reference is given to recent in-cell protein labeling techniques. These recent experimental improvements could open the door for broader applications and to study the remodeling of RNA–protein complexes upon different environmental cues and in disease.

IL-14 MAPPIT: a versatile METHOD to study protein-protein interactions in mammalian cells

2003 ◽  
Vol 16 (5) ◽  
pp. 577-577
Author(s):  
J. Tavernier ◽  
S. Eyckerman ◽  
I. Lemmens ◽  
S. Lievens ◽  
J. Vandekerckhove ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Mammalian Cells ◽  
Protein Protein Interactions

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.

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