NGPINT: A Next-generation protein-protein interaction software

Mapping protein-protein interactions at a proteome scale is critical to understanding how cellular signaling networks respond to stimuli. Since eukaryotic genomes encode thousands of proteins, testing their interactions one-by-one is a challenging prospect. High-throughput yeast-two hybrid (Y2H) assays that employ next-generation sequencing to interrogate cDNA libraries represent an alternative approach that optimizes scale, cost, and effort. We present NGPINT, a robust and scalable software to identify all putative interactors of a protein using Y2H in batch culture. NGPINT combines diverse tools to align sequence reads to target genomes, reconstruct prey fragments and compute gene enrichment under reporter selection. Central to this pipeline is the identification of fusion reads containing sequences derived from both the Y2H expression plasmid and the cDNA of interest. To reduce false positives, these fusion reads are evaluated as to whether the cDNA fragment forms an in-frame translational fusion with the Y2H transcription factor. NGPINT successfully recognized 95% of interactions in simulated test runs. As proof of concept, NGPINT was tested using published data sets and recognized all validated interactions. NGPINT can be used in any organism with an available reference, thus facilitating the discovery of protein-protein interactions in non-model organisms.

[1]  Bernhard Suter,et al.  Next-Generation Sequencing for Binary Protein–Protein Interactions , 2015, Front. Genet..

[2]  Pascal Braun,et al.  History of protein–protein interactions: From egg‐white to complex networks , 2012, Proteomics.

[3]  Jae-Seong Yang,et al.  rec-YnH enables simultaneous many-by-many detection of direct protein–protein and protein–RNA interactions , 2018, Nature Communications.

[4]  A. Barabasi,et al.  A disease module in the interactome explains disease heterogeneity, drug response and captures novel pathways and genes in asthma. , 2015, Human molecular genetics.

[5]  J. Turkson,et al.  Discovery of JSI-124 (cucurbitacin I), a selective Janus kinase/signal transducer and activator of transcription 3 signaling pathway inhibitor with potent antitumor activity against human and murine cancer cells in mice. , 2003, Cancer research.

[6]  F. Kaye,et al.  Identification of cellular proteins that can interact specifically with the T/ElA-binding region of the retinoblastoma gene product , 1991, Cell.

[7]  Darrell Desveaux,et al.  Quantitative Interactor Screening with next-generation Sequencing (QIS-Seq) identifies Arabidopsis thaliana MLO2 as a target of the Pseudomonas syringae type III effector HopZ2 , 2012, BMC Genomics.

[8]  Julie M. Sahalie,et al.  Supplementary Figure and Table Legends , 2022 .

[9]  Damian Szklarczyk,et al.  STRING v11: protein–protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets , 2018, Nucleic Acids Res..

[10]  S. Fields,et al.  Protein-protein interactions: methods for detection and analysis , 1995, Microbiological reviews.

[11]  D. Inzé,et al.  NINJA connects the co-repressor TOPLESS to jasmonate signalling , 2010, Nature.

[12]  M. Kabbage,et al.  An inhibitor of apoptosis (SfIAP) interacts with SQUAMOSA promoter‐binding protein (SBP) transcription factors that exhibit pro‐cell death characteristics , 2018, bioRxiv.

[13]  V. S. Rao,et al.  Protein-Protein Interaction Detection: Methods and Analysis , 2014, International journal of proteomics.

[14]  Klaus Schulten,et al.  Regulation of the protein-conducting channel by a bound ribosome. , 2009, Structure.

[15]  A. Galarneau,et al.  β-Lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein–protein interactions , 2002, Nature Biotechnology.

[16]  S. Fields,et al.  The yeast two-hybrid assay: still finding connections after 25 years , 2014, Nature Methods.

[17]  Roger P. Wise,et al.  Y2H-SCORES: A statistical framework to infer protein-protein interactions from next-generation yeast-two-hybrid sequence data , 2020 .

[18]  A. Barabasi,et al.  An empirical framework for binary interactome mapping , 2008, Nature Methods.

[19]  Nicolas Servant,et al.  A comprehensive evaluation of normalization methods for Illumina high-throughput RNA sequencing data analysis , 2013, Briefings Bioinform..

[20]  Patrick Breheny,et al.  DEEPN as an Approach for Batch Processing of Yeast 2-Hybrid Interactions. , 2016, Cell reports.

[21]  Jonathan D. G. Jones,et al.  Evidence for Network Evolution in an Arabidopsis Interactome Map , 2011, Science.

[22]  T. Zal,et al.  Using live FRET imaging to reveal early protein-protein interactions during T cell activation. , 2004, Current opinion in immunology.

[23]  CHEN CHEN,et al.  Construction and analysis of protein-protein interaction networks based on proteomics data of prostate cancer , 2016, International journal of molecular medicine.

[24]  Rob Patro,et al.  Salmon provides fast and bias-aware quantification of transcript expression , 2017, Nature Methods.

[25]  G. Patterson,et al.  Photoswitching FRET to monitor protein–protein interactions , 2018, Proceedings of the National Academy of Sciences.

[26]  Kara Dolinski,et al.  The BioGRID interaction database: 2019 update , 2018, Nucleic Acids Res..

[27]  Kai Li,et al.  A DnaJ protein that interacts with soybean mosaic virus coat protein serves as a key susceptibility factor for viral infection. , 2020, Virus research.

[28]  Sara Linse,et al.  Methods for the detection and analysis of protein–protein interactions , 2007, Proteomics.

[29]  W. Huber,et al.  which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. MAnorm: a robust model for quantitative comparison of ChIP-Seq data sets , 2011 .

[30]  Takayuki Tohge,et al.  A protein–protein interaction network linking the energy-sensor kinase SnRK1 to multiple signaling pathways in Arabidopsis thaliana , 2016 .

[31]  Thomas R. Gingeras,et al.  STAR: ultrafast universal RNA-seq aligner , 2013, Bioinform..

[32]  Ulrich Stelzl,et al.  A Y2H-seq approach defines the human protein methyltransferase interactome , 2013, Nature Methods.

[33]  Günter Mayer,et al.  Systematic evaluation of error rates and causes in short samples in next-generation sequencing , 2018, Scientific Reports.

[34]  Gonçalo R. Abecasis,et al.  The Sequence Alignment/Map format and SAMtools , 2009, Bioinform..

[35]  Joseph R. Ecker,et al.  CrY2H-seq: a massively-multiplexed assay for deep coverage interactome mapping , 2017, Nature Methods.

[36]  D. Hasselquist,et al.  No evidence that carotenoid pigments boost either immune or antioxidant defenses in a songbird , 2018, Nature Communications.

[37]  M. Vidal,et al.  High-throughput yeast two-hybrid assays for large-scale protein interaction mapping. , 2001, Methods.

[38]  Alyssa C. Frazee,et al.  Polyester: Simulating RNA-Seq Datasets With Differential Transcript Expression , 2014, bioRxiv.

[39]  W. Huber,et al.  Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 , 2014, Genome Biology.

[40]  Martin Vingron,et al.  IntAct: an open source molecular interaction database , 2004, Nucleic Acids Res..

[41]  Mi Zhou,et al.  Current Experimental Methods for Characterizing Protein–Protein Interactions , 2016, ChemMedChem.

[42]  Alain Goossens,et al.  A user-friendly platform for yeast two-hybrid library screening using next generation sequencing , 2018, PloS one.

[43]  A. Vinayagam,et al.  A Directed Protein Interaction Network for Investigating Intracellular Signal Transduction , 2011, Science Signaling.

[44]  Bridget E. Begg,et al.  A Proteome-Scale Map of the Human Interactome Network , 2014, Cell.

[45]  David R. Kelley,et al.  Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks , 2012, Nature Protocols.

[46]  C. Thermes,et al.  Ten years of next-generation sequencing technology. , 2014, Trends in genetics : TIG.

[47]  S. Michnick,et al.  A highly sensitive protein-protein interaction assay based on Gaussia luciferase , 2006, Nature Methods.

[48]  Salvador Ventura,et al.  Protein complementation assays: Approaches for the in vivo analysis of protein interactions , 2009, FEBS letters.

[49]  Michael I. Love,et al.  Differential analysis of count data – the DESeq2 package , 2013 .

[50]  David E Hill,et al.  next-generation sequencing to generate interactome datasets , 2011 .

[51]  Haiyuan Yu,et al.  Three-dimensional reconstruction of protein networks provides insight into human genetic disease , 2012, Nature Biotechnology.