Dataset Information

CFTR interactome mapping using the mammalian membrane two-hybrid high-throughput screening system.

ABSTRACT: Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride and bicarbonate channel in secretory epithelia with a critical role in maintaining fluid homeostasis. Mutations in CFTR are associated with Cystic Fibrosis (CF), the most common lethal autosomal recessive disorder in Caucasians. While remarkable treatment advances have been made recently in the form of modulator drugs directly rescuing CFTR dysfunction, there is still considerable scope for improvement of therapeutic effectiveness. Here, we report the application of a high-throughput screening variant of the Mammalian Membrane Two-Hybrid (MaMTH-HTS) to map the protein-protein interactions of wild-type (wt) and mutant CFTR (F508del), in an effort to better understand CF cellular effects and identify new drug targets for patient-specific treatments. Combined with functional validation in multiple disease models, we have uncovered candidate proteins with potential roles in CFTR function/CF pathophysiology, including Fibrinogen Like 2 (FGL2), which we demonstrate in patient-derived intestinal organoids has a significant effect on CFTR functional expression.

SUBMITTER: Lim SH

PROVIDER: S-EPMC8842165 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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CFTR interactome mapping using the mammalian membrane two-hybrid high-throughput screening system.

Lim Sang Hyun SH Snider Jamie J Birimberg-Schwartz Liron L Ip Wan W Serralha Joana C JC Botelho Hugo M HM Lopes-Pacheco Miquéias M Pinto Madalena C MC Moutaoufik Mohamed Taha MT Zilocchi Mara M Laselva Onofrio O Esmaeili Mohsen M Kotlyar Max M Lyakisheva Anna A Tang Priscilla P López Vázquez Lucía L Akula Indira I Aboualizadeh Farzaneh F Wong Victoria V Grozavu Ingrid I Opacak-Bernardi Teuta T Yao Zhong Z Mendoza Meg M Babu Mohan M Jurisica Igor I Gonska Tanja T Bear Christine E CE Amaral Margarida D MD Stagljar Igor I

Molecular systems biology 20220201 2

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride and bicarbonate channel in secretory epithelia with a critical role in maintaining fluid homeostasis. Mutations in CFTR are associated with Cystic Fibrosis (CF), the most common lethal autosomal recessive disorder in Caucasians. While remarkable treatment advances have been made recently in the form of modulator drugs directly rescuing CFTR dysfunction, there is still considerable scope for improvement of therapeutic effect ...[more]

PMID: 35156780

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Project description:BackgroundMost of the biological processes rely on the formation of protein complexes. Investigation of protein-protein interactions (PPI) is therefore essential for understanding of cellular functions. It is advantageous to perform mammalian PPI analysis in mammalian cells because the expressed proteins can then be subjected to essential post-translational modifications. Until now mammalian two-hybrid assays have been performed on individual gene scale. We here describe a new and cost-effective method for the high-throughput detection of protein-protein interactions in mammalian cells that combines the advantages of mammalian two-hybrid systems with those of DNA microarrays.ResultsIn this cell array protein-protein interaction assay (CAPPIA), mixtures of bait and prey expression plasmids together with an auto-fluorescent reporter are immobilized on glass slides in defined array formats. Adherent cells that grow on top of the micro-array will become fluorescent only if the expressed proteins interact and subsequently trans-activate the reporter. Using known interaction partners and by screening 160 different combinations of prey and bait proteins associated with the human androgen receptor we demonstrate that this assay allows the quantitative detection of specific protein interactions in different types of mammalian cells and under the influence of different compounds. Moreover, different strategies in respect to bait-prey combinations are presented.ConclusionWe demonstrate that the CAPPIA assay allows the quantitative detection of specific protein interactions in different types of mammalian cells and under the influence of different compounds. The high number of preys that can be tested per slide together with the flexibility to interrogate any bait of interest and the small amounts of reagents that are required makes this assay currently one of the most economical high-throughput detection assays for protein-protein interactions in mammalian cells.

Project description:BACKGROUND:Yeast two-hybrid (Y2H) screens have been among the most powerful methods to detect and analyze protein-protein interactions. However, they suffer from a significant degree of false negatives, i.e. true interactions that are not detected, and to a certain degree from false positives, i.e. interactions that appear to take place only in the context of the Y2H assay. While the fraction of false positives remains difficult to estimate, the fraction of false negatives in typical Y2H screens is on the order of 70-90%. Here we present novel Y2H vectors that significantly decrease the number of false negatives and help to mitigate the false positive problem. RESULTS:We have constructed two new vectors (pGBKCg and pGADCg) that allow us to make both C-terminal fusion proteins of DNA-binding and activation domains. Both vectors can be combined with existing vectors for N-terminal fusions and thus allow four different bait-prey combinations: NN, CC, NC, and CN. We have tested all approximately 4,900 pairwise combinations of the 70 Varicella-Zoster-Virus (VZV) proteins for interactions, using all possible combinations. About approximately 20,000 individual Y2H tests resulted in 182 NN, 89 NC, 149 CN, and 144 CC interactions. Overlap between screens ranged from 17% (NC-CN) to 43% (CN-CC). Performing four screens (i.e. permutations) instead of one resulted in about twice as many interactions and thus much fewer false negatives. In addition, interactions that are found in multiple combinations confirm each other and thus provide a quality score. This study is the first systematic analysis of such N- and C-terminal Y2H vectors. CONCLUSIONS:Permutations of C- and N-terminal Y2H vectors dramatically increase the coverage of interactome studies and thus significantly reduce the number of false negatives. We suggest that future interaction screens should use such vector combinations on a routine basis, not the least because they provide a built-in quality score for Y2H interactions that can provide a measure of reproducibility without additional assays.

Dataset Information

CFTR interactome mapping using the mammalian membrane two-hybrid high-throughput screening system.

Publications

CFTR interactome mapping using the mammalian membrane two-hybrid high-throughput screening system.

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