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et al., Global insights into cellular organization and genome function require comprehensive understanding of the interactome networks that mediate genotype–phenotype relationships1,2. Here we present a human ‘all-by-all’ reference interactome map of human binary protein interactions, or ‘HuRI’. With approximately 53,000 protein–protein interactions, HuRI has approximately four times as many such interactions as there are high-quality curated interactions from small-scale studies. The integration of HuRI with genome3, transcriptome4 and proteome5 data enables cellular function to be studied within most physiological or pathological cellular contexts. We demonstrate the utility of HuRI in identifying the specific subcellular roles of protein–protein interactions. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms that might underlie tissue-specific phenotypes of Mendelian diseases. HuRI is a systematic proteome-wide reference that links genomic variation to phenotypic outcomes., This work was primarily supported by the National Institutes of Health (NIH) National Human Genome Research Institute (NHGRI) grant U41HG001715 (M.V., F.P.R., D.E.H., M.A.C., G.D.B. and J.T.) with additional support from NIH grants P50HG004233 (M.V. and F.P.R.), U01HL098166 (M.V.), U01HG007690 (M.V.), R01GM109199 (M.A.C.), Canadian Institute for Health Research (CIHR) Foundation Grants (F.P.R. and J. Rak), the Canada Excellence Research Chairs Program (F.P.R.) and an American Heart Association grant 15CVGPS23430000 (M.V.). D.-K.K. was supported by a Banting Postdoctoral Fellowship through the Natural Sciences and Engineering Research Council (NSERC) of Canada and by the Basic Science Research Program through the National Research Foundation (NRF) of Korea funded by the Ministry of Education (2017R1A6A3A03004385). C. Pons was supported by a Ramon Cajal fellowship (RYC-2017-22959). G.M.S. was supported by NIH Training Grant T32CA009361. M.V. is a Chercheurv Qualifié Honoraire from the Fonds de la Recherche Scientifique (FRS-FNRS, Wallonia-Brussels Federation, Belgium).

Global insights into cellular organization and function require comprehensive understanding of interactome networks. Similar to how a reference genome sequence revolutionized human genetics, a reference map of the human interactome network is critical to fully understand genotype-phenotype relationships. Here we present the first human “all-by-all” binary reference interactome map, or “HuRI”. With ~53,000 high-quality protein-protein interactions (PPIs), HuRI is approximately four times larger than the information curated from small-scale studies available in the literature. Integrating HuRI with genome, transcriptome and proteome data enables the study of cellular function within essentially any physiological or pathological cellular context. We demonstrate the use of HuRI in identifying specific subcellular roles of PPIs and protein function modulation via splicing during brain development. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms underlying tissue-specific phenotypes of Mendelian diseases. HuRI thus represents an unprecedented, systematic reference linking genomic variation to phenotypic outcomes.

PMCID: PMC4266588.-- et al., Just as reference genome sequences revolutionized human genetics, reference maps of interactome networks will be critical to fully understand genotype-phenotype relationships. Here, we describe a systematic map of ∼14,000 high-quality human binary protein-protein interactions. At equal quality, this map is ∼30% larger than what is available from small-scale studies published in the literature in the last few decades. While currently available information is highly biased and only covers a relatively small portion of the proteome, our systematic map appears strikingly more homogeneous, revealing a >broader> human interactome network than currently appreciated. The map also uncovers significant interconnectivity between known and candidate cancer gene products, providing unbiased evidence for an expanded functional cancer landscape, while demonstrating how high-quality interactome models will help >connect the dots> of the genomic revolution., This work was supported primarily by NHGRI grant R01/U01HG001715 awarded to M.V., D.E.H., F.P.R., and J.T. and in part by the following grants and agencies: NHGRI P50HG004233 to M.V., F.P.R., and A.-L.B.; NHLBI U01HL098166 subaward to M.V.; NHLBI U01HL108630 subaward to A.-L.B.; NCI U54CA112962 subaward to M.V.; NCI R33CA132073 to M.V.; NIH RC4HG006066 to M.V., D.E.H., and T.H.; NICHD ARRA R01HD065288, R21MH104766, and R01MH105524 to L.M.I.; NIMH R01MH091350 to L.M.I. and T.H.; NSF CCF-1219007 and NSERC RGPIN-2014-03892 to Y.X.; Canada Excellence Research Chair, Krembil Foundation, Ontario Research Fund–Research Excellence Award, Avon Foundation, grant CSI07A09 from Junta de Castilla y Leon (Valladolid, Spain), grant PI12/00624 from Ministerio de Economia y Competitividad (AES 2012, ISCiii, Madrid, Spain), and grant i-Link0398 from Consejo Superior de Investigaciones Científicas (CSIC, Madrid, Spain) to J.D.L.R.; Spanish Ministerio de Ciencia e Innovación (BIO2010-22073) and the European Commission through the FP7 project SyStemAge grant agreement n: 306240 to P.A.; Group-ID Multidisciplinary Research Partnerships of Ghent University, grant FWO-V G.0864.10 from the Fund for Scientific Research-Flanders and ERC Advanced Grant N° 340941 to J.T.; EMBO long-term fellowship to A.K.; Institute Sponsored Research funds from the Dana-Farber Cancer Institute Strategic Initiative to M.V. I.L. is a postdoctoral fellow with the FWO-V. M.V. is a “Chercheur Qualifié Honoraire” from the Fonds de la Recherche Scientifique (FRS-FNRS, Wallonia-Brussels Federation, Belgium). Since performing the work described, C. Fontanillo has become an employee of Celgene Research SL, part of the Celgene Corporation.

Rat muscle was extracted at pH 4 and submitted to gel-filtration on Sephadex G-75 and to chromatography on DEAE-Sephadex. Gel-filtration gave a large peak of activity towards Bz-Arg-NNap with an estimated molecular weight of 25 500. Activity towards Bz-Arg-NH2 was present in this peak and in another peak of molecular weight 45 000. The second peak also hydrolysed benzoyl-glycyl- L -arginine. DEAE-Sephadex gave five peaks of Bz-Arg-NNap hydrolysing activity; all showed thiol dependence. Peaks III, IV and V hydrolysed Z-Ala-Arg-Arg-NNap-OMe rapidly; they also inactivated aldolase and were strongly inhibited by leupeptin. They are probably isoenzymes of cathepsin B1. Peak I showed these properties to a relatively small extent. 7-(N- Benzoyl - DL - argininamide )-4- methylcoumarin appears to be an alternative substrate for cathepsin B1; it was hydrolysed also by peak I, but relatively less rapidly. Peaks I and II were inhibited more than peaks III, IV and V by a muscle extract. Total activity of the Bz-Arg-NH2-hydrolysing enzyme in extensor digitorum longus muscle increased after denervation.

1. Adult rabbit skeletal-muscle myosin has been shown to contain 1.0 residue of mono-N-methyl-lysine and 3.3 residues of tri-N-methyl-lysine per molecule of molecular weight 500000. 2. The methyl-lysines appear to be located in the subfragment 1 portion of the myosin molecule. 3. Methyl-lysines are not present in actin, tropomyosin, inhibitory factor and calcium-sensitizing factor. 4. Enzymic methylation of histidine and lysine residues of myosin has been demonstrated in vitro. 5. The methylation of histidine and lysine of the total myofibrillar protein occurs after peptide-bond synthesis. 6. Although methylated lysines and 3-methyl-histidine could not be detected by analysis of hydrolysates, radiochemical evidence is provided for the presence of these residues in the soluble-protein fraction of rabbit skeletal muscle.

3-METHYL HISTIDINE is a normal amino-acid component of myosin from adult muscle1 and of actin1,2. But whereas 3-methyl histidine occurs in actin isolated from adult and foetal muscle it is absent from myosin isolated from skeletal muscle of the foetal rabbit. After birth the amount of this amino-acid in myosin from rabbit skeletal muscle increases until it reaches the adult value3. This implies that development in muscle is accompanied by the methylation of histidine residues of myosin. Nothing is known of the mechanism of this process and, although it has not been directly demonstrated that the methyl group of methionine is a precursor of the methyl group of 3-methyl histidine found in actin, the results of Asatoor and Armstrong2, obtained with whole animals, are compatible with such a mechanism. We have studied in vitro the methylation of muscle proteins with S-adenosyl methionine labelled with 14C in the methyl group in an attempt to elucidate the mechanism of formation of the 3-methyl histidine present in myosin and actin.