scholarly journals A Method of Lysate Preparation to Improve the Isolation Efficiency of Protein Partners for Target Proteins Encoded by the Genes of Human Chromosome 18

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
P.V. Ershov ◽  
Y.V. Mezentsev ◽  
E.O. Yablokov ◽  
L.A. Kaluzhskiy ◽  
I.V. Vakhrushev ◽  
...  
Keyword(s):  
Human Chromosome ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Binding Capacity ◽  
Standard Protocol ◽  
Chromosome 18 ◽  
Bait Protein ◽  
Cell Lysate ◽  
Affinity Isolation ◽  
Protein Partners

The aim of this work was to test modifications of the standard protocol for the sample preparation of cell/tissue lysate before performing the affinity isolation of lysate protein partners for the target protein (bait protein) which is covalently immobilized on an inert sorbent (e.g. BrCN-, SH-Sepharose 4B) or a carrier (e.g. paramagnetic nanoparticles). The series of our previous works on applying the approach to direct molecular fishing procedure with combination of affinity chromatography and LC-MS/MS analysis using a number of proteins, encoded by the genes of human chromosome 18, have shown that there are at least two problems affecting the specificity and the effectiveness of this procedure. These include: (i) redundancy of the background proteins in the eluates from an affinity sorbent (carrier) due to isolation of multiprotein complexes “labeled” with a direct protein partner which binds with a bait protein immobilized on the sorbent; (ii) low enrichment of the eluates with appropriate protein partners due to the fact that some direct protein partners in the lysate exist in stable “wild type” complexes with the bait protein itself. This means that latter group of protein partners will not be sufficiently isolated from lysate. Therefore, in order to increase the specificity and efficiency of affinity isolation of protein partners for the bait protein, we modified the standard protocol of lysate preparation and the preliminary step on dissociation of lysate protein complexes was added. Several model experiments for the choice of regeneration solution, assessment of their efficiency in the dissociation of lysate protein complexes as well as the stability and binding capacity of proteins were performed under the control of surface plasmon resonance (SPR) biosensor Biacore 3000 using HepG2 cell lysate. It was shown that acid treatment and incubation of the cell lysate for one min on ice (final lysate dilution 20 times) and subsequent neutralization (pH shift from 2.0 to 7.4) resulted in maximal dissociation of the lysate protein complexes without significant negative effects on the protein-protein interactions tested.

Direct Molecular Fishing of Protein Partners for Proteins Encoded by Genes of Human Chromosome 18 in HepG2 Cell Lysate

2018 ◽  
Vol 44 (6) ◽  
pp. 759-768 ◽  
Author(s):  
P. V. Ershov ◽  
Yu. V. Mezentsev ◽  
E. O. Yablokov ◽  
L. A. Kaluzhskiy ◽  
A. V. Florinskaya ◽  
...  
Keyword(s):  
Human Chromosome ◽  
Hepg2 Cell ◽  
Chromosome 18 ◽  
Cell Lysate ◽  
Protein Partners ◽  
Molecular Fishing

scholarly journals Stitching the synapse: Cross-linking mass spectrometry into resolving synaptic protein interactions

2020 ◽  
Vol 6 (8) ◽  
pp. eaax5783 ◽  
Author(s):  
M. A. Gonzalez-Lozano ◽  
F. Koopmans ◽  
P. F. Sullivan ◽  
J. Protze ◽  
G. Krause ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Conformational Dynamics ◽  
Synaptic Proteins ◽  
Cross Linking ◽  
Synaptic Protein ◽  
Protein Partners ◽  
Associated Proteins ◽  
Model Protein

Synaptic transmission is the predominant form of communication in the brain. It requires functionally specialized molecular machineries constituted by thousands of interacting synaptic proteins. Here, we made use of recent advances in cross-linking mass spectrometry (XL-MS) in combination with biochemical and computational approaches to reveal the architecture and assembly of synaptic protein complexes from mouse brain hippocampus and cerebellum. We obtained 11,999 unique lysine-lysine cross-links, comprising connections within and between 2362 proteins. This extensive collection was the basis to identify novel protein partners, to model protein conformational dynamics, and to delineate within and between protein interactions of main synaptic constituents, such as Camk2, the AMPA-type glutamate receptor, and associated proteins. Using XL-MS, we generated a protein interaction resource that we made easily accessible via a web-based platform (http://xlink.cncr.nl) to provide new entries into exploration of all protein interactions identified.

scholarly journals Trapping mammalian protein complexes in viral particles

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Sven Eyckerman ◽  
Kevin Titeca ◽  
Emmy Van Quickelberghe ◽  
Eva Cloots ◽  
Annick Verhee ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Protein Complexes ◽  
Gag Protein ◽  
Bait Protein ◽  
Protein Partners ◽  
Complementary Approach ◽  
Viral Particles ◽  
Interaction Partners ◽  
Enrichment Protocol

Abstract Cell lysis is an inevitable step in classical mass spectrometry–based strategies to analyse protein complexes. Complementary lysis conditions, in situ cross-linking strategies and proximal labelling techniques are currently used to reduce lysis effects on the protein complex. We have developed Virotrap, a viral particle sorting approach that obviates the need for cell homogenization and preserves the protein complexes during purification. By fusing a bait protein to the HIV-1 GAG protein, we show that interaction partners become trapped within virus-like particles (VLPs) that bud from mammalian cells. Using an efficient VLP enrichment protocol, Virotrap allows the detection of known binary interactions and MS-based identification of novel protein partners as well. In addition, we show the identification of stimulus-dependent interactions and demonstrate trapping of protein partners for small molecules. Virotrap constitutes an elegant complementary approach to the arsenal of methods to study protein complexes.

scholarly journals From Affinity to Proximity Techniques to Investigate Protein Complexes in Plants

2021 ◽  
Vol 22 (13) ◽  
pp. 7101
Author(s):  
Sandra M. Kerbler ◽  
Roberto Natale ◽  
Alisdair R. Fernie ◽  
Youjun Zhang
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Hydrophobic Interactions ◽  
Protein Complexes ◽  
Specific Binding ◽  
Affinity Purification ◽  
Interaction Network ◽  
Unicellular Eukaryote ◽  
Bait Protein ◽  
Advantages And Disadvantages

The study of protein–protein interactions (PPIs) is fundamental in understanding the unique role of proteins within cells and their contribution to complex biological systems. While the toolkit to study PPIs has grown immensely in mammalian and unicellular eukaryote systems over recent years, application of these techniques in plants remains under-utilized. Affinity purification coupled to mass spectrometry (AP-MS) and proximity labeling coupled to mass spectrometry (PL-MS) are two powerful techniques that have significantly enhanced our understanding of PPIs. Relying on the specific binding properties of a protein to an immobilized ligand, AP is a fast, sensitive and targeted approach used to detect interactions between bait (protein of interest) and prey (interacting partners) under near-physiological conditions. Similarly, PL, which utilizes the close proximity of proteins to identify potential interacting partners, has the ability to detect transient or hydrophobic interactions under native conditions. Combined, these techniques have the potential to reveal an unprecedented spatial and temporal protein interaction network that better understands biological processes relevant to many fields of interest. In this review, we summarize the advantages and disadvantages of two increasingly common PPI determination techniques: AP-MS and PL-MS and discuss their important application to plant systems.

scholarly journals Prediction of Protein–Protein Binding Interactions in Dimeric Coiled Coils by Information Contained in Folding Energy Landscapes

2021 ◽  
Vol 22 (3) ◽  
pp. 1368
Author(s):  
Panagiota S. Georgoulia ◽  
Sinisa Bjelic
Keyword(s):  
Protein Interactions ◽  
Protein Complexes ◽  
Coiled Coil ◽  
Extensive Study ◽  
Coiled Coils ◽  
Folding Energy ◽  
Energy Landscapes ◽  
Binding Interactions ◽  
Interacting Protein ◽  
Protein Partners

Coiled coils represent the simplest form of a complex formed between two interacting protein partners. Their extensive study has led to the development of various methods aimed towards the investigation and design of complex forming interactions. Despite the progress that has been made to predict the binding affinities for protein complexes, and specifically those tailored towards coiled coils, many challenges still remain. In this work, we explore whether the information contained in dimeric coiled coil folding energy landscapes can be used to predict binding interactions. Using the published SYNZIP dataset, we start from the amino acid sequence, to simultaneously fold and dock approximately 1000 coiled coil dimers. Assessment of the folding energy landscapes showed that a model based on the calculated number of clusters for the lowest energy structures displayed a signal that correlates with the experimentally determined protein interactions. Although the revealed correlation is weak, we show that such correlation exists; however, more work remains to establish whether further improvements can be made to the presented model.

scholarly journals A simple and sensitive SYBR Gold-based assay to quantify DNA-protein interactions

2019 ◽  
Author(s):  
Spencer Schreier ◽  
Bhanu Prakash Petla ◽  
Tao Lin ◽  
Suvobrata Chakravarty ◽  
Senthil Subramanian
Keyword(s):  
Protein Interactions ◽  
Protein Complexes ◽  
Binding Capacity ◽  
Relative Strength ◽  
Auxin Response ◽  
Sybr Gold ◽  
Uniform Manner ◽  
Nucleic Acid Stain ◽  
Biology Laboratory ◽  
Dna Protein Interactions

AbstractA simple, accessible, and inexpensive assay to quantify the strength of DNA-protein interactions was developed. The assay relies on capturing DNA-protein complexes using an affinity resin that binds tagged, recombinant proteins. Sequential washes with filtration spin cups and centrifugation remove non-specific interactions in a gentle, uniform manner and a final elution isolates specific DNA-protein complexes. SYBR Gold nucleic acid stain is added to the eluted product and the fluorescence intensity accurately quantifies the amount of captured DNA, ultimately illustrating the relative strength of the DNA-protein interaction. The major utility of the assay resides in the versatility and quantitative nature of the SYBR Gold:nucleic acid interaction, eliminating the need for customized or labeled oligos and permitting relatively inexpensive quantification of binding capacity. The assay also employs DNA-protein complex capture by the very common purification tag, 6xHis, but other tags could likely be utilized. Further, SYBR Gold fluorescence is compatible with a wide variety of instruments, including UV transilluminators, a staple to any molecular biology laboratory. This assay was used to compare the binding capacities of different Auxin Response Factor (ARF) transcription factors to various dsDNA targets, including the classical AuxRE motif and several divergent sequences. Results from dose-response assays suggest that different ARF proteins might show distinct comparative affinities for AuxRE variants, emphasizing that specific ARF-AuxRE binding strengths likely contribute to the complex and fine-tuned cellular auxin response.

scholarly journals Affinity Isolation and Mass Spectrometry Identification of Prostacyclin Synthase (PTGIS) Subinteractome

Biology ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 49 ◽  
Author(s):  
Pavel V. Ershov ◽  
Yuri V. Mezentsev ◽  
Arthur T. Kopylov ◽  
Evgeniy O. Yablokov ◽  
Andrey V. Svirid ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Functional Coupling ◽  
Protein Protein Interactions ◽  
Affinity Isolation ◽  
Protein Partners ◽  
Mass Spectrometry Identification ◽  
Potent Vasodilator ◽  
Rat Tissue ◽  
Prostacyclin Synthase

Prostacyclin synthase (PTGIS; EC 5.3.99.4) catalyzes isomerization of prostaglandin H2 to prostacyclin, a potent vasodilator and inhibitor of platelet aggregation. At present, limited data exist on functional coupling and possible ways of regulating PTGIS due to insufficient information about protein–protein interactions in which this crucial enzyme is involved. The aim of this study is to isolate protein partners for PTGIS from rat tissue lysates. Using CNBr-activated Sepharose 4B with covalently immobilized PTGIS as an affinity sorbent, we confidently identified 58 unique proteins by mass spectrometry (LC-MS/MS). The participation of these proteins in lysate complex formation was characterized by SEC lysate profiling. Several potential members of the PTGIS subinteractome have been validated by surface plasmon resonance (SPR) analysis. SPR revealed that PTGIS interacted with full-length cytochrome P450 2J2 and glutathione S-transferase (GST). In addition, PTGIS was shown to bind synthetic peptides corresponding to sequences of for GSTA1, GSTM1, aldo-keto reductase (AKR1A1), glutaredoxin 3 (GLRX3) and histidine triad nucleotide binding protein 2 (HINT2). Prostacyclin synthase could potentially be involved in functional interactions with identified novel protein partners participating in iron and heme metabolism, oxidative stress, xenobiotic and drugs metabolism, glutathione and prostaglandin metabolism. The possible biological role of the recognized interaction is discussed in the context of PTGIS functioning.

Mapping of Protein-Protein Interactions: Web-Based Resources for Revealing Interactomes

2019 ◽  
Vol 26 (21) ◽  
pp. 3890-3910 ◽  
Author(s):  
Branislava Gemovic ◽  
Neven Sumonja ◽  
Radoslav Davidovic ◽  
Vladimir Perovic ◽  
Nevena Veljkovic
Keyword(s):  
Drug Discovery ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Experimental Studies ◽  
Protein Protein Interactions ◽  
Web Based ◽  
Prediction Tools ◽  
Physiological Processes ◽  
Modern Drug ◽  
Ppi Prediction

Background: The significant number of protein-protein interactions (PPIs) discovered by harnessing concomitant advances in the fields of sequencing, crystallography, spectrometry and two-hybrid screening suggests astonishing prospects for remodelling drug discovery. The PPI space which includes up to 650 000 entities is a remarkable reservoir of potential therapeutic targets for every human disease. In order to allow modern drug discovery programs to leverage this, we should be able to discern complete PPI maps associated with a specific disorder and corresponding normal physiology. Objective: Here, we will review community available computational programs for predicting PPIs and web-based resources for storing experimentally annotated interactions. Methods: We compared the capacities of prediction tools: iLoops, Struck2Net, HOMCOS, COTH, PrePPI, InterPreTS and PRISM to predict recently discovered protein interactions. Results: We described sequence-based and structure-based PPI prediction tools and addressed their peculiarities. Additionally, since the usefulness of prediction algorithms critically depends on the quality and quantity of the experimental data they are built on; we extensively discussed community resources for protein interactions. We focused on the active and recently updated primary and secondary PPI databases, repositories specialized to the subject or species, as well as databases that include both experimental and predicted PPIs. Conclusion: PPI complexes are the basis of important physiological processes and therefore, possible targets for cell-penetrating ligands. Reliable computational PPI predictions can speed up new target discoveries through prioritization of therapeutically relevant protein–protein complexes for experimental studies.

Protein Interaction Domains and Post-Translational Modifications: Structural Features and Drug Discovery Applications

2020 ◽  
Vol 27 (37) ◽  
pp. 6306-6355 ◽  
Author(s):  
Marian Vincenzi ◽  
Flavia Anna Mercurio ◽  
Marilisa Leone
Keyword(s):  
Drug Discovery ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Structural Information ◽  
Protein Complexes ◽  
Structural Features ◽  
Protein Protein Interactions ◽  
Modular Architecture ◽  
Post Translational Modifications ◽  
Interaction Domains

Background:: Many pathways regarding healthy cells and/or linked to diseases onset and progression depend on large assemblies including multi-protein complexes. Protein-protein interactions may occur through a vast array of modules known as protein interaction domains (PIDs). Objective:: This review concerns with PIDs recognizing post-translationally modified peptide sequences and intends to provide the scientific community with state of art knowledge on their 3D structures, binding topologies and potential applications in the drug discovery field. Method:: Several databases, such as the Pfam (Protein family), the SMART (Simple Modular Architecture Research Tool) and the PDB (Protein Data Bank), were searched to look for different domain families and gain structural information on protein complexes in which particular PIDs are involved. Recent literature on PIDs and related drug discovery campaigns was retrieved through Pubmed and analyzed. Results and Conclusion:: PIDs are rather versatile as concerning their binding preferences. Many of them recognize specifically only determined amino acid stretches with post-translational modifications, a few others are able to interact with several post-translationally modified sequences or with unmodified ones. Many PIDs can be linked to different diseases including cancer. The tremendous amount of available structural data led to the structure-based design of several molecules targeting protein-protein interactions mediated by PIDs, including peptides, peptidomimetics and small compounds. More studies are needed to fully role out, among different families, PIDs that can be considered reliable therapeutic targets, however, attacking PIDs rather than catalytic domains of a particular protein may represent a route to obtain selective inhibitors.

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.

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