scholarly journals Evolutionary diversification of protein–protein interactions by interface add-ons

2017 ◽  
Vol 114 (40) ◽  
pp. E8333-E8342 ◽  
Author(s):  
Maximilian G. Plach ◽  
Florian Semmelmann ◽  
Florian Busch ◽  
Markus Busch ◽  
Leonhard Heizinger ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Evolutionary Strategy ◽  
Structural Level ◽  
Protein Protein Interactions ◽  
Protein Protein Interaction ◽  
Evolutionary Diversification

Cells contain a multitude of protein complexes whose subunits interact with high specificity. However, the number of different protein folds and interface geometries found in nature is limited. This raises the question of how protein–protein interaction specificity is achieved on the structural level and how the formation of nonphysiological complexes is avoided. Here, we describe structural elements called interface add-ons that fulfill this function and elucidate their role for the diversification of protein–protein interactions during evolution. We identified interface add-ons in 10% of a representative set of bacterial, heteromeric protein complexes. The importance of interface add-ons for protein–protein interaction specificity is demonstrated by an exemplary experimental characterization of over 30 cognate and hybrid glutamine amidotransferase complexes in combination with comprehensive genetic profiling and protein design. Moreover, growth experiments showed that the lack of interface add-ons can lead to physiologically harmful cross-talk between essential biosynthetic pathways. In sum, our complementary in silico, in vitro, and in vivo analysis argues that interface add-ons are a practical and widespread evolutionary strategy to prevent the formation of nonphysiological complexes by specializing protein–protein interactions.

scholarly journals Interfacial Peptides as Affinity Modulating Agents of Protein-Protein Interactions

Biomolecules ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 106
Author(s):  
Pavel V. Ershov ◽  
Yuri V. Mezentsev ◽  
Alexis S. Ivanov
Keyword(s):  
Protein Interactions ◽  
Targeted Delivery ◽  
Protein Complexes ◽  
Protein Protein Interactions ◽  
Good Efficiency ◽  
Cellular Targets ◽  
Proteolytic Stability ◽  
Clinically Significant

The identification of disease-related protein-protein interactions (PPIs) creates objective conditions for their pharmacological modulation. The contact area (interfaces) of the vast majority of PPIs has some features, such as geometrical and biochemical complementarities, “hot spots”, as well as an extremely low mutation rate that give us key knowledge to influence these PPIs. Exogenous regulation of PPIs is aimed at both inhibiting the assembly and/or destabilization of protein complexes. Often, the design of such modulators is associated with some specific problems in targeted delivery, cell penetration and proteolytic stability, as well as selective binding to cellular targets. Recent progress in interfacial peptide design has been achieved in solving all these difficulties and has provided a good efficiency in preclinical models (in vitro and in vivo). The most promising peptide-containing therapeutic formulations are under investigation in clinical trials. In this review, we update the current state-of-the-art in the field of interfacial peptides as potent modulators of a number of disease-related PPIs. Over the past years, the scientific interest has been focused on following clinically significant heterodimeric PPIs MDM2/p53, PD-1/PD-L1, HIF/HIF, NRF2/KEAP1, RbAp48/MTA1, HSP90/CDC37, BIRC5/CRM1, BIRC5/XIAP, YAP/TAZ–TEAD, TWEAK/FN14, Bcl-2/Bax, YY1/AKT, CD40/CD40L and MINT2/APP.

scholarly journals Mapping of the Hepatitis B Virus Reverse Transcriptase TP and RT Domains by Transcomplementation for Nucleotide Priming and by Protein-Protein Interaction

1999 ◽  
Vol 73 (3) ◽  
pp. 1885-1893 ◽  
Author(s):  
Robert E. Lanford ◽  
Young-Ho Kim ◽  
Helen Lee ◽  
Lena Notvall ◽  
Burton Beames
Keyword(s):  
Amino Acids ◽  
Reverse Transcriptase ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Rnase H ◽  
Genome Replication ◽  
Protein Protein Interactions ◽  
Stem Loop ◽  
Protein Protein Interaction

ABSTRACT Hepadnavirus polymerases initiate reverse transcription in a protein-primed reaction. We previously described a complementation assay for analysis of the roles of the TP and RT domains of HBV reverse transcriptase (pol) in the priming reaction. Independently expressed TP and RT domains form a complex functional for in vitro priming reactions. To map the minimal functional TP and RT domains, we prepared baculoviruses expressing amino- and carboxyl-terminal deletions of both the TP and RT domains and analyzed the proteins for the ability to participate in transcomplementation for the priming reaction. The minimal TP domain spanned amino acids 20 to 175; however, very little activity was observed without a TP domain spanning amino acids 1 to 199. The minimal RT domain spanned amino acids 300 to 775; however, little activity was observed unless the carboxyl end of the RT domain extended to amino acid 800. Thus, most of the RNase H domain was required. In previous studies, we observed a TP inhibitory domain between amino acids 199 and 344. The current analysis narrowed this domain to residues 300 to 334, which is a portion of the minimal RT domain. In addition, the ability of TP and RT deletion mutants to form stable TP-RT complexes was examined in coimmunoprecipitation assays. The minimal TP and RT domains capable of protein-protein interaction were considerably smaller than the domains required for functional interaction in the transcomplementation assays, and unlike priming activity, TP-RT interaction did not require the epsilon RNA stem-loop. These studies help to further define the complex protein-protein interactions required in HBV genome replication.

scholarly journals Prediction of Protein-Protein Interactions Related to Protein Complexes Based on Protein Interaction Networks

2015 ◽  
Vol 2015 ◽  
pp. 1-9
Author(s):  
Peng Liu ◽  
Lei Yang ◽  
Daming Shi ◽  
Xianglong Tang
Keyword(s):  
Protein Interaction ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Statistical Tests ◽  
Interaction Network ◽  
Biological Data ◽  
Protein Protein Interactions ◽  
Protein Protein Interaction ◽  
Small Parts

A method for predicting protein-protein interactions based on detected protein complexes is proposed to repair deficient interactions derived from high-throughput biological experiments. Protein complexes are pruned and decomposed into small parts based on the adaptivek-cores method to predict protein-protein interactions associated with the complexes. The proposed method is adaptive to protein complexes with different structure, number, and size of nodes in a protein-protein interaction network. Based on different complex sets detected by various algorithms, we can obtain different prediction sets of protein-protein interactions. The reliability of the predicted interaction sets is proved by using estimations with statistical tests and direct confirmation of the biological data. In comparison with the approaches which predict the interactions based on the cliques, the overlap of the predictions is small. Similarly, the overlaps among the predicted sets of interactions derived from various complex sets are also small. Thus, every predicted set of interactions may complement and improve the quality of the original network data. Meanwhile, the predictions from the proposed method replenish protein-protein interactions associated with protein complexes using only the network topology.

scholarly journals In silico analysis of protein-protein interfaces: Tools and approaches

2020 ◽  
Vol 11 (4) ◽  
pp. 7539-7548
Author(s):  
Christina Nilofer ◽  
Arumugam Mohanapriya
Keyword(s):  
Protein Interaction ◽  
Protein Interactions ◽  
In Silico Analysis ◽  
Physicochemical Characteristics ◽  
Structural Features ◽  
Interface Residue ◽  
Protein Protein Interactions ◽  
Protein Protein Interaction ◽  
Molecular Functions

Two or more proteins interact in vivo to perform complex molecular functions including catalysis, regulation, assembly, immunity and inhibition through the formation of stable interfaces. This interaction is governed by several factors that are selective, sensitive and specific in nature. Several interface features has been documented since 1975. The study of these interface features of proteins and their dynamicity during interaction with different proteins help understanding the mechanisms underlying diverse molecular functions and its biological processes. Computational tools greatly assist in studying such interface features that determine the interaction between two or more proteins, and in this context, this review enumerates the different interface features reported thus far along with the tools that aid in deciphering protein features (physicochemical characteristics, binding site and interface residue prediction and hotspot residues) along with their approaches that are employed in the prediction these features. Also, the review discusses the advantages and limitations of experimental techniques and computational biological tools deployed for deciphering the protein-protein interactions. Altogether, the review will provide insights into the optimal tools and different strategies involved in protein interaction studies that would facilitate the researchers to understand the protein structural features and molecular principles of protein-protein interaction with known functions.

scholarly journals Photoproximity Profiling of Protein-Protein Interactions in Cells

2019 ◽  
Author(s):  
David C. McCutcheon ◽  
Gihoon Lee ◽  
Anthony Carlos ◽  
Jeffrey E. Montgomery ◽  
Raymond E. Moellering
Keyword(s):  
Protein Interaction ◽  
Protein Interactions ◽  
Live Cells ◽  
High Temporal Resolution ◽  
Protein Protein Interactions ◽  
Redox Stress ◽  
Covalent Interaction ◽  
Protein Protein Interaction ◽  
In Cells

ABSTRACTWe report a novel photoproximity protein interaction (PhotoPPI) profiling method to map protein-protein interactions in vitro and in live cells. This approach utilizes a bioorthogonal, multifunctional chemical probe that can be targeted to a genetically encoded protein of interest (POI) through a modular SNAP-Tag/benzylguanine covalent interaction. A first generation photoproximity probe, PP1, responds to 365 nm light to simultaneously cleave a central nitroveratryl linker and a peripheral diazirine group, resulting in diffusion of a highly reactive carbene nucleophile away from the POI. We demonstrate facile probe loading, and subsequent interaction- and light-dependent proximal labeling of a model protein-protein interaction (PPI) in vitro. Integration of the PhotoPPI workflow with quantitative LC-MS/MS enabled un-biased interaction mapping for the redox regulated sensor protein, KEAP1, for the first time in live cells. We validated known and novel interactions between KEAP1 and the proteins PGAM5 and HK2, among others, under basal cellular conditions. By contrast, comparison of PhotoPPI profiles in cells experiencing metabolic or redox stress confirmed that KEAP1 sheds many basal interactions and becomes associated with known lysosomal trafficking and proteolytic proteins like SQSTM1, CTSD and LGMN. Together, these data establish PhotoPPI as a method capable of tracking the dynamic sub-cellular and protein interaction “social network” of a redox-sensitive protein in cells with high temporal resolution.SYNOPSIS TOC

scholarly journals CeLINC, a fluorescence-based protein-protein interaction assay in C. elegans

2021 ◽  
Author(s):  
Jason R Kroll ◽  
Sanne Remmelzwaal ◽  
Mike Boxem
Keyword(s):  
Protein Interaction ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Protein Protein Interactions ◽  
Bait Protein ◽  
C Elegans ◽  
Protein Protein Interaction ◽  
Prey Protein ◽  
Reference Protein

Interactions among proteins are fundamental for life and determining whether two particular proteins physically interact can be essential for fully understanding a protein's function. We present C. elegans light-induced co-clustering (CeLINC), an optical binary protein-protein interaction assay to determine whether two proteins interact in vivo. Based on CRY2/CIB1 light-dependent oligomerization, CeLINC can rapidly and unambiguously identify protein-protein interactions between pairs of fluorescently tagged proteins. A fluorescently tagged bait protein is captured using a nanobody directed against the fluorescent protein (GFP or mCherry) and brought into artificial clusters within the cell. Co-localization of a fluorescently tagged prey protein in the cluster indicates a protein interaction. We tested the system with an array of positive and negative reference protein pairs. Assay performance was extremely robust with no false positives detected in the negative reference pairs. We then used the system to test for interactions among apical and basolateral polarity regulators. We confirmed interactions seen between PAR-6, PKC-3, and PAR-3, but observed no physical interactions among the basolateral Scribble module proteins LET-413, DLG-1, and LGL-1. We have generated a plasmid toolkit that allows use of custom promoters or CRY2 variants to promote flexibility of the system. The CeLINC assay is a powerful and rapid technique that can be widely applied in C. elegans due to the universal plasmids that can be used with existing fluorescently tagged strains without need for additional cloning or genetic modification of the genome.

scholarly journals CeLINC, a fluorescence-based protein-protein interaction assay in C. elegans

Genetics ◽  
2021 ◽  
Author(s):  
Jason R Kroll ◽  
Sanne Remmelzwaal ◽  
Mike Boxem
Keyword(s):  
Protein Interaction ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Protein Protein Interactions ◽  
Bait Protein ◽  
C Elegans ◽  
Protein Protein Interaction ◽  
Prey Protein ◽  
Reference Protein

Abstract Interactions among proteins are fundamental for life and determining whether two particular proteins physically interact can be essential for fully understanding a protein’s function. We present C. elegans light-induced co-clustering (CeLINC), an optical binary protein-protein interaction assay to determine whether two proteins interact in vivo. Based on CRY2/CIB1 light-dependent oligomerization, CeLINC can rapidly and unambiguously identify protein-protein interactions between pairs of fluorescently tagged proteins. A fluorescently tagged bait protein is captured using a nanobody directed against the fluorescent protein (GFP or mCherry) and brought into artificial clusters within the cell. Co-localization of a fluorescently tagged prey protein in the cluster indicates a protein interaction. We tested the system with an array of positive and negative reference protein pairs. Assay performance was extremely robust with no false positives detected in the negative reference pairs. We then used the system to test for interactions among apical and basolateral polarity regulators. We confirmed interactions seen between PAR-6, PKC-3, and PAR-3, but observed no physical interactions among the basolateral Scribble module proteins LET-413, DLG-1, and LGL-1. We have generated a plasmid toolkit that allows use of custom promoters or CRY2 variants to promote flexibility of the system. The CeLINC assay is a powerful and rapid technique that can be widely applied in C. elegans due to the universal plasmids that can be used with existing fluorescently tagged strains without need for additional cloning or genetic modification of the genome.

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.

An efficient transient assays system using Agrobacterium-mediated transformation of onion (Allium cepa) epidermal cells

2020 ◽  
Vol 80 (03) ◽  
Author(s):  
Yu-Miao Zhang ◽  
Jun Wang ◽  
Tao Wu
Keyword(s):  
Subcellular Localization ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Epidermal Cells ◽  
Cyclin Dependent Kinase ◽  
Protein Subcellular Localization ◽  
Protein Protein Interactions ◽  
Efficient System ◽  
Protein Protein Interaction ◽  
Onion Epidermal Cells

In this study, the Agrobacterium infection medium, infection duration, detergent, and cell density were optimized. The sorghum-based infection medium (SbIM), 10-20 min infection time, addition of 0.01% Silwet L-77, and Agrobacterium optical density at 600 nm (OD600), improved the competence of onion epidermal cells to support Agrobacterium infection at >90% efficiency. Cyclin-dependent kinase D-2 (CDKD-2) and cytochrome c-type biogenesis protein (CYCH), protein-protein interactions were localized. The optimized procedure is a quick and efficient system for examining protein subcellular localization and protein-protein interaction.

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.

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