High-resolution protein–protein interaction mapping using all-versus-all sequencing (AVA-Seq)

Andrews, Simeon; Schaefer-Ramadan, Stephanie; Al-Thani, Nayra M; Ahmed, Ikhlak; Mohamoud, Yasmin Ali; Malek, Joel A.

doi:10.1074/jbc.ra119.008792

Cited by 12 publications

(40 citation statements)

References 18 publications

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“…Coupling Y2H and HTS can be mainly performed using two strategies. An all-versus-all strategy using libraries recombination approaches or the use of barcode indexing which enables simultaneous sequencing of interacting preys from multiple separate assays in a single Illumina paired-end run [46,47,49,55]. In our case, the latter proved more appropriate since Bam35 has a limited number of viral genes, that were already available and tested for autoactivation in Y2H [20].…”

Section: Discussionmentioning

confidence: 99%

“…However, it should be noted that the unconfirmed interactions by this method should not be excluded from biological relevant interactions. Indeed, although some interactions that require full-length proteins could be missed [55], the use of protein fragments can increase the sensitivity of the screening as shown in Yang et al [47], where some known interactions were only detected when domains or domains vs. full-length proteins were assayed. Likewise, the previously described interaction between LexA and P7 [29] was detected in our screen with a fragment covering 42% of the protein.…”

Section: Discussionmentioning

confidence: 99%

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Unraveling protein interactions between the temperate virus Bam35 and its Bacillus host using an integrative Yeast two hybrid-High throughput sequencing approach

Lechuga

Lood

Berjón-Otero

et al. 2021

Preprint

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Bacillus virus Bam35 is the model Betatectivirus and member of the Tectiviridae family, which is composed of tailless, icosahedral, and membrane-containing bacteriophages. The interest in these viruses has greatly increased in recent years as they are thought to be an evolutionary link between diverse groups of prokaryotic and eukaryotic viruses. Additionally, betatectiviruses infect bacteria of the Bacillus cereus group, known for their applications in industry and notorious since it contains many pathogens. Here, we present the first protein-protein interactions network for a tectivirus-host system by studying the Bam35- Bacillus thuringiensis model using a novel approach that integrates the traditional yeast two-hybrid system and Illumina high-throughput sequencing. We generated and thoroughly analyzed a genomic library of Bam35’s host B. thuringiensis HER1410 and screened interactions with all the viral proteins using different combinations of bait-prey couples. In total, this screen resulted in the detection of over 4,000 potential interactions, of which 183 high-confidence interactions were defined as part of the core virus-host interactome. Overall, host metabolism proteins and peptidases are particularly enriched within the detected interactions, distinguishing this host-phage system from the other reported host-phage protein-protein interaction networks (PPIs). Our approach also suggests biological roles for several Bam35 proteins of unknown function, resulting in a better understanding of the Bam35- B. thuringiensis interaction at the molecular level.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Unraveling protein interactions between the temperate virus Bam35 and its Bacillus host using an integrative Yeast two hybrid-High throughput sequencing approach

Lechuga

Lood

Berjón-Otero

et al. 2021

Preprint

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“…By uniting gene synthesis with a mapping step and a barcode readout, our system allows high throughput characterization of any binary PPI. Previous high-throughput studies used highly constrained libraries--either the ORFome [21][22][23][24] of one of a handful of reference genomes, targeted single residue mutations which only explore a sliver of sequence space around a primary sequence 25,26 or several randomly sheared coding sequences 27 . Using the capabilities of DNA synthesis broadens the testable sequence space which facilitates investigations of a variety of areas such as protein domains, extant genetic variation, evolutionary trajectories or epistatic effects.…”

Section: Discussionmentioning

confidence: 99%

“…Deconvoluting library diversity has also been a challenge for other multiplexed assays. Other multiplexed methods involved picking colonies and sanger sequencing them 21 , mapping the beginning of reading frames to reference genomes [22][23][24] or manually BLASTing obtained reads 27 . Our explicit mapping step allows for the high-throughput creation of a library to map arbitrary proteins to DNA barcodes, and because it is a separate step it could use long read sequencing to overcome the length limitations of Illumina sequencing.…”

Section: Discussionmentioning

confidence: 99%

A Multiplexed Bacterial Two-Hybrid for Rapid Characterization of Protein-Protein Interactions and Iterative Protein Design

Wc¹,

Ljubetič

Kim³

et al. 2020

Preprint

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Myriad biological functions require protein-protein interactions (PPIs), and engineered PPIs are crucial for applications ranging from drug design to synthetic cell circuits. Understanding and engineering specificity in PPIs is particularly challenging as subtle sequence changes can drastically alter specificity. Coiled-coils are small protein domains that have long served as a simple model for studying the sequence-determinants of specificity and have been used as modular building blocks to build large protein nanostructures and synthetic circuits. Despite their simple rules and long-time use, building large sets of well-behaved orthogonal pairs that can be used together is still challenging because predictions are often inaccurate, and, as the library size increases, it becomes difficult to test predictions at scale. To address these problems, we first developed a method called the Next-Generation Bacterial Two-Hybrid (NGB2H), which combines gene synthesis, a bacterial two-hybrid assay, and a high-throughput next-generation sequencing readout, allowing rapid exploration of interactions of programmed protein libraries in a quantitative and scalable way. After validating the NGB2H system on previously characterized libraries, we designed, built, and tested large sets of orthogonal synthetic coiled-coils. In an iterative set of experiments, we assayed more than 8,000 PPIs, used the dataset to train a novel linear model-based coiled-coil scoring algorithm, and then characterized nearly 18,000 interactions to identify the largest set of orthogonal PPIs to date with twenty-two on-target interactions.

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“…The all-vs-all sequencing (AVA-Seq) method is based on a bacterial two-hybrid system and with the innovation of a plasmid which allows convergent fusion proteins was recently developed 10 . This system allows the use of next-generation sequencing (NGS) to determine protein interactions on a large scale as well as providing a level of domain-domain interaction information due to the testing of multiple overlapping protein fragments present in the library.…”

Section: Introductionmentioning

confidence: 99%

High-resolution protein fragment interactions using AVA-Seq on a human reference set

Schaefer-Ramadan

Aleksic

Al-Thani

et al. 2021

Preprint

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Protein-protein interactions (PPIs) are important in understanding numerous aspects of protein function. Here, the recently developed all-vs-all sequencing (AVA-Seq) approach to determine protein-protein interactions was tested on a gold-standard human protein interaction set (hsPRS-v2). Initially, these data were interpreted strictly from a binary PPI perspective to compare AVA-Seq to other binary PPI methods tested on the same hsPRS-v2. AVA-Seq recovered 20 of 47 (43%) binary PPIs from this reference set comparing favorably with other methods. The same experimental data allowed for the determination of >500 known and novel PPIs including interactions between wildtype fragments of tumor protein p53 and minichromosomal maintenance complex proteins 2, and 5 (MCM2 and MCM5) that could be of interest in human disease. Additional results gave a better understanding of why interactions might be missed using AVA-Seq and aide future PPI experimental design for maximum recovery of information.

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High-resolution protein–protein interaction mapping using all-versus-all sequencing (AVA-Seq)

Abstract: The authors declare that they have no conflicts of interest with the contents of this article. This article contains Figs. S1 and S2 and Tables S1-S3. The data have been deposited with links to BioProject accession number PRJNA542588 in the DDBJ BioProject database.

Cited by 12 publications

References 18 publications

Unraveling protein interactions between the temperate virus Bam35 and its Bacillus host using an integrative Yeast two hybrid-High throughput sequencing approach

Unraveling protein interactions between the temperate virus Bam35 and its Bacillus host using an integrative Yeast two hybrid-High throughput sequencing approach

A Multiplexed Bacterial Two-Hybrid for Rapid Characterization of Protein-Protein Interactions and Iterative Protein Design

High-resolution protein fragment interactions using AVA-Seq on a human reference set

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