Visual detection of binary, ternary, and quaternary protein interactions in fission yeast by Pil1 co-tethering assay

Protein-protein interactions are vital for executing nearly all cellular processes. To facilitate the detection of protein-protein interactions in living cells of the fission yeast Schizosaccharomyces pombe, here we present an efficient and convenient method termed the Pil1 co-tethering assay. In its basic form, we tether a bait protein to mCherry-tagged Pil1, which forms cortical filamentary structures, and examine whether a GFP-tagged prey protein colocalizes with the bait. We demonstrate that this assay is capable of detecting pairwise protein-protein interactions of cytosolic proteins and nuclear proteins. Furthermore, we show that this assay can be used for detecting not only binary protein-protein interactions, but also ternary and quaternary protein-protein interactions. Using this assay, we systematically characterized the protein-protein interactions in the Atg1 complex and in the phosphatidylinositol 3-kinase (PtdIns3K) complexes and found that Atg38 is incorporated into the PtdIns3K complex I via an Atg38-Vps34 interaction. Our data show that this assay is a useful and versatile tool and should be added to the routine toolbox of fission yeast researchers.

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Visual detection of binary, ternary, and quaternary protein-protein interactions in fission yeast by Pil1 co-tethering assay

10.1101/2021.04.13.439745 ◽

2021 ◽

Author(s):

Zhong-Qiu Yu ◽

Xiao-Man Liu ◽

Dan Zhao ◽

Dan-Dan Xu ◽

Li-Lin Du

Keyword(s):

Fission Yeast ◽

Protein Interactions ◽

Visual Detection ◽

Protein Protein Interactions ◽

Phosphatidylinositol 3 Kinase ◽

Basic Form ◽

Bait Protein ◽

Cellular Processes ◽

Prey Protein ◽

Versatile Tool

AbstractProtein-protein interactions are vital for executing nearly all cellular processes. To facilitate the detection of protein-protein interactions in living cells of the fission yeast Schizosaccharomyces pombe, here we present an efficient and convenient method termed the Pil1 co-tethering assay. In its basic form, we tether a bait protein to mCherry-tagged Pil1, which forms cortical filamentary structures, and examine whether a GFP-tagged prey protein colocalizes with the bait. We demonstrate that this assay is capable of detecting pairwise protein-protein interactions of cytosolic proteins, transmembrane proteins, and nuclear proteins. Furthermore, we show that this assay can be used for detecting not only binary protein-protein interactions, but also ternary and quaternary protein-protein interactions. Using this assay, we systematically characterized the protein-protein interactions in the Atg1 complex and in the phosphatidylinositol 3-kinase (PtdIns3K) complexes and found that Atg38 is incorporated into the PtdIns3K complex I via an Atg38-Vps34 interaction. Our data show that this assay is a useful and versatile tool and should be added to the routine toolbox of fission yeast researchers.

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

10.1101/2020.03.09.983270 ◽

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.

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

10.1101/2021.06.01.446599 ◽

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.

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

Genetics ◽

10.1093/genetics/iyab163 ◽

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.

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Structure, Function, Involvement in Diseases and Targeting of 14-3-3 Proteins: An Update

Current Medicinal Chemistry ◽

10.2174/0929867324666170426095015 ◽

2018 ◽

Vol 25 (1) ◽

pp. 5-21 ◽

Cited By ~ 25

Author(s):

Ylenia Cau ◽

Daniela Valensin ◽

Mattia Mori ◽

Sara Draghi ◽

Maurizio Botta

Keyword(s):

Protein Interactions ◽

Molecular Mechanisms ◽

Physiological Role ◽

Specific Protein ◽

Protein Protein Interactions ◽

Cellular Processes ◽

Protein Partners ◽

High Sequence Homology ◽

Small Molecule Modulators

14-3-3 is a class of proteins able to interact with a multitude of targets by establishing protein-protein interactions (PPIs). They are usually found in all eukaryotes with a conserved secondary structure and high sequence homology among species. 14-3-3 proteins are involved in many physiological and pathological cellular processes either by triggering or interfering with the activity of specific protein partners. In the last years, the scientific community has collected many evidences on the role played by seven human 14-3-3 isoforms in cancer or neurodegenerative diseases. Indeed, these proteins regulate the molecular mechanisms associated to these diseases by interacting with (i) oncogenic and (ii) pro-apoptotic proteins and (iii) with proteins involved in Parkinson and Alzheimer diseases. The discovery of small molecule modulators of 14-3-3 PPIs could facilitate complete understanding of the physiological role of these proteins, and might offer valuable therapeutic approaches for these critical pathological states.

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Protein–protein interactions in bacteria: a promising and challenging avenue towards the discovery of new antibiotics

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.14.267 ◽

2018 ◽

Vol 14 ◽

pp. 2881-2896 ◽

Cited By ~ 7

Author(s):

Laura Carro

Keyword(s):

Protein Interactions ◽

Bacterial Infections ◽

Multidrug Resistant ◽

Resistant Bacteria ◽

Protein Protein Interactions ◽

Lead Discovery ◽

Antibiotic Development ◽

Cellular Processes ◽

Ppi Networks ◽

New Antibiotics

Antibiotics are potent pharmacological weapons against bacterial infections; however, the growing antibiotic resistance of microorganisms is compromising the efficacy of the currently available pharmacotherapies. Even though antimicrobial resistance is not a new problem, antibiotic development has failed to match the growth of resistant pathogens and hence, it is highly critical to discover new anti-infective drugs with novel mechanisms of action which will help reducing the burden of multidrug-resistant microorganisms. Protein–protein interactions (PPIs) are involved in a myriad of vital cellular processes and have become an attractive target to treat diseases. Therefore, targeting PPI networks in bacteria may offer a new and unconventional point of intervention to develop novel anti-infective drugs which can combat the ever-increasing rate of multidrug-resistant bacteria. This review describes the progress achieved towards the discovery of molecules that disrupt PPI systems in bacteria for which inhibitors have been identified and whose targets could represent an alternative lead discovery strategy to obtain new anti-infective molecules.

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Assay Systems for Profiling Deubiquitinating Activity

International Journal of Molecular Sciences ◽

10.3390/ijms21165638 ◽

2020 ◽

Vol 21 (16) ◽

pp. 5638

Author(s):

Jinhong Cho ◽

Jinyoung Park ◽

Eunice EunKyeong Kim ◽

Eun Joo Song

Keyword(s):

Protein Degradation ◽

Protein Interactions ◽

Live Cells ◽

Deubiquitinating Enzyme ◽

Protein Protein Interactions ◽

Deubiquitinating Enzymes ◽

Cell Lysates ◽

Cellular Processes

Deubiquitinating enzymes regulate various cellular processes, particularly protein degradation, localization, and protein–protein interactions. The dysregulation of deubiquitinating enzyme (DUB) activity has been linked to several diseases; however, the function of many DUBs has not been identified. Therefore, the development of methods to assess DUB activity is important to identify novel DUBs, characterize DUB selectivity, and profile dynamic DUB substrates. Here, we review various methods of evaluating DUB activity using cell lysates or purified DUBs, as well as the types of probes used in these methods. In addition, we introduce some techniques that can deliver DUB probes into the cells and cell-permeable activity-based probes to directly visualize and quantify DUB activity in live cells. This review could contribute to the development of DUB inhibitors by providing important information on the characteristics and applications of various probes used to evaluate and detect DUB activity in vitro and in vivo.

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Deep learning based prediction of reversible HAT/HDAC-specific lysine acetylation

Briefings in Bioinformatics ◽

10.1093/bib/bbz107 ◽

2019 ◽

Vol 21 (5) ◽

pp. 1798-1805 ◽

Cited By ~ 1

Author(s):

Kai Yu ◽

Qingfeng Zhang ◽

Zekun Liu ◽

Yimeng Du ◽

Xinjiao Gao ◽

...

Keyword(s):

Deep Learning ◽

Protein Interactions ◽

Web Server ◽

Data Sets ◽

Lysine Acetylation ◽

Protein Acetylation ◽

Protein Protein Interactions ◽

Cellular Processes ◽

Protein Lysine Acetylation ◽

Specific Regulation

Abstract Protein lysine acetylation regulation is an important molecular mechanism for regulating cellular processes and plays critical physiological and pathological roles in cancers and diseases. Although massive acetylation sites have been identified through experimental identification and high-throughput proteomics techniques, their enzyme-specific regulation remains largely unknown. Here, we developed the deep learning-based protein lysine acetylation modification prediction (Deep-PLA) software for histone acetyltransferase (HAT)/histone deacetylase (HDAC)-specific acetylation prediction based on deep learning. Experimentally identified substrates and sites of several HATs and HDACs were curated from the literature to generate enzyme-specific data sets. We integrated various protein sequence features with deep neural network and optimized the hyperparameters with particle swarm optimization, which achieved satisfactory performance. Through comparisons based on cross-validations and testing data sets, the model outperformed previous studies. Meanwhile, we found that protein–protein interactions could enrich enzyme-specific acetylation regulatory relations and visualized this information in the Deep-PLA web server. Furthermore, a cross-cancer analysis of acetylation-associated mutations revealed that acetylation regulation was intensively disrupted by mutations in cancers and heavily implicated in the regulation of cancer signaling. These prediction and analysis results might provide helpful information to reveal the regulatory mechanism of protein acetylation in various biological processes to promote the research on prognosis and treatment of cancers. Therefore, the Deep-PLA predictor and protein acetylation interaction networks could provide helpful information for studying the regulation of protein acetylation. The web server of Deep-PLA could be accessed at http://deeppla.cancerbio.info.

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Conserved HORMA domain-containing protein Hop1 stabilizes interaction between proteins of meiotic DNA break hotspots and chromosome axis

Nucleic Acids Research ◽

10.1093/nar/gkz754 ◽

2019 ◽

Vol 47 (19) ◽

pp. 10166-10180 ◽

Cited By ~ 2

Author(s):

Ryo Kariyazono ◽

Arisa Oda ◽

Takatomi Yamada ◽

Kunihiro Ohta

Keyword(s):

Fission Yeast ◽

Protein Interactions ◽

Meiotic Recombination ◽

Protein Protein Interactions ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Multiple Protein ◽

Chromatin Immunoprecipitation Sequencing ◽

The One ◽

Chromosome Axis

Abstract HORMA domain-containing proteins such as Hop1 play crucial regulatory roles in various chromosomal functions. Here, we investigated roles of the fission yeast Hop1 in the formation of recombination-initiating meiotic DNA double strand breaks (DSBs). Meiotic DSB formation in fission yeast relies on multiple protein-protein interactions such as the one between the chromosome axial protein Rec10 and the DSB-forming complex subunit Rec15. Chromatin immunoprecipitation sequencing demonstrated that Hop1 is colocalized with both Rec10 and Rec15, and we observed physical interactions of Hop1 to Rec15 and Rec10. These results suggest that Hop1 promotes DSB formation by interacting with both axis components and the DSB-forming complex. We also show that Hop1 binding to DSB hotspots requires Rec15 and Rec10, while Hop1 axis binding requires Rec10 only, suggesting that Hop1 is recruited to the axis via Rec10, and to hotspots by hotspot-bound Rec15. Furthermore, we introduced separation-of-function Rec10 mutations, deficient for interaction with either Rec15 or Hop1. These single mutations and hop1Δ conferred only partial defects in meiotic recombination, while the combining the Rec15-binding-deficient rec10 mutation with hop1Δ synergistically reduced meiotic recombination, at least at a model hotspot. Taken together, Hop1 likely functions as a stabilizer for Rec15–Rec10 interaction to promote DSB formation.

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