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Multi-omics Characterization of Interaction-mediated Control of Human Protein Abundance levels*

  • Abel Sousa
    Affiliations
    Instituto de Investigação e Inovação em Saúde da Universidade do Porto (i3s), Rua Alfredo Allen 208, 4200–135, Porto, Portugal

    Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Rua Júlio Amaral de Carvalho 45, 4200–135, Porto, Portugal

    Graduate Program in Areas of Basic and Applied Biology (GABBA), Abel Salazar Biomedical Sciences Institute, University of Porto, Rua de Jorge Viterbo Ferreira 228, 4050–313, Porto, Portugal

    European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, CB10 1SD, Cambridge, UK
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  • Emanuel Gonçalves
    Affiliations
    Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, UK
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  • Bogdan Mirauta
    Affiliations
    European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, CB10 1SD, Cambridge, UK
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  • David Ochoa
    Affiliations
    European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, CB10 1SD, Cambridge, UK
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  • Oliver Stegle
    Affiliations
    European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, CB10 1SD, Cambridge, UK

    ‡European Molecular Biology Laboratory, Genome Biology Unit, 69117 Heidelberg, Germany

    §Division of Computational Genomics and Systems Genetics, German Cancer Research Center (DKFZ), 69120, Heidelberg, Germany
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  • Pedro Beltrao
    Correspondence
    To whom correspondence should be addressed.
    Affiliations
    European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, CB10 1SD, Cambridge, UK
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  • Author Footnotes
    * The authors declare that they have no conflict of interest.
    This article contains supplemental material Tables 1–4 and Figs. S1–S7.
Open AccessPublished:June 25, 2019DOI:https://doi.org/10.1074/mcp.RA118.001280
      Proteogenomic studies of cancer samples have shown that copy-number variation can be attenuated at the protein level for a large fraction of the proteome, likely due to the degradation of unassembled protein complex subunits. Such interaction-mediated control of protein abundance remains poorly characterized. To study this, we compiled genomic, (phospho)proteomic and structural data for hundreds of cancer samples and find that up to 42% of 8,124 analyzed proteins show signs of post-transcriptional control. We find evidence of interaction-dependent control of protein abundance, correlated with interface size, for 516 protein pairs, with some interactions further controlled by phosphorylation. Finally, these findings in cancer were reflected in variation in protein levels in normal tissues. Importantly, expression differences due to natural genetic variation were increasingly buffered from phenotype differences for highly attenuated proteins. Altogether, this study further highlights the importance of posttranscriptional control of protein abundance in cancer and healthy cells.

      Graphical Abstract

      Cancer cells can harbor a large number of somatic DNA alterations ranging from point mutations to gene copy changes that can occur from deletion or amplification of small regions or whole chromosomes. While these events are the source of the genetic variation that can confer a selective advantage and lead to cancer, large changes in gene numbers can be detrimental and cause imbalances in the corresponding protein levels. Several studies have shown that the majority of changes in gene copy number will propagate to changes in the corresponding protein levels (
      • Dephoure N.
      • Hwang S.
      • O'Sullivan C.
      • Dodgson S.E.
      • Gygi S.P.
      • Amon A.
      • Torres E.M.
      Quantitative proteomic analysis reveals posttranslational responses to aneuploidy in yeast.
      ,
      • Stingele S.
      • Stoehr G.
      • Peplowska K.
      • Cox J.
      • Mann M.
      • Storchova Z.
      Global analysis of genome, transcriptome and proteome reveals the response to aneuploidy in human cells.
      ,
      • Pavelka N.
      • Rancati G.
      • Zhu J.
      • Bradford W.D.
      • Saraf A.
      • Florens L.
      • Sanderson B.W.
      • Hattem G.L.
      • Li R.
      Aneuploidy confers quantitative proteome changes and phenotypic variation in budding yeast.
      ). However, models of aneuploidy of different species and analysis of gene copy-number variation (CNV) in cancer have shown that CNVs of protein-coding genes belonging to protein complexes tend to be attenuated at the protein level (
      • Dephoure N.
      • Hwang S.
      • O'Sullivan C.
      • Dodgson S.E.
      • Gygi S.P.
      • Amon A.
      • Torres E.M.
      Quantitative proteomic analysis reveals posttranslational responses to aneuploidy in yeast.
      ,
      • Gonçalves E.
      • Fragoulis A.
      • Garcia-Alonso L.
      • Cramer T.
      • Saez-Rodriguez J.
      • Beltrao P.
      Widespread post-transcriptional attenuation of genomic copy-number variation in cancer.
      ,
      • Ishikawa K.
      • Makanae K.
      • Iwasaki S.
      • Ingolia N.T.
      • Moriya H.
      Post-translational dosage compensation buffers genetic perturbations to stoichiometry of protein complexes.
      ). In addition, we have shown that some complex members can act as rate-limiting subunits and indirectly control the degradation level of attenuated complex members (
      • Gonçalves E.
      • Fragoulis A.
      • Garcia-Alonso L.
      • Cramer T.
      • Saez-Rodriguez J.
      • Beltrao P.
      Widespread post-transcriptional attenuation of genomic copy-number variation in cancer.
      ). These results are in-line with pulse-chase degradation measurements showing that several complex subunits have a two-state degradation profile that is compatible with a model in which they are expressed above the required levels and have a higher degradation rate when unbound from the complex (
      • McShane E.
      • Sin C.
      • Zauber H.
      • Wells J.N.
      • Donnelly N.
      • Wang X.
      • Hou J.
      • Chen W.
      • Storchova Z.
      • Marsh J.A.
      • Valleriani A.
      • Selbach M.
      Kinetic analysis of protein stability reveals age-dependent degradation.
      ). The attenuation of changes at the protein level also justifies why protein complex subunits show higher correlation of protein abundances than the corresponding mRNA levels (
      • Ryan C.J.
      • Kennedy S.
      • Bajrami I.
      • Matallanas D.
      • Lord C.J.
      A compendium of co-regulated protein complexes in breast cancer reveals collateral loss events.
      ,
      • Wang J.
      • Ma Z.
      • Carr S.A.
      • Mertins P.
      • Zhang H.
      • Zhang Z.
      • Chan D.W.
      • Ellis M.J.
      • Townsend R.R.
      • Smith R.D.
      • McDermott J.E.
      • Chen X.
      • Paulovich A.G.
      • Boja E.S.
      • Mesri M.
      • Kinsinger C.R.
      • Rodriguez H.
      • Rodland K.D.
      • Liebler D.C.
      • Zhang B.
      Proteome profiling outperforms transcriptome profiling for coexpression based gene function prediction.
      ) and why correlation analysis can be used to identify cancer-specific interaction networks (
      • Lapek Jr., J.D.
      • Greninger P.
      • Morris R.
      • Amzallag A.
      • Pruteanu-Malinici I.
      • Benes C.H.
      • Haas W.
      Detection of dysregulated protein-association networks by high-throughput proteomics predicts cancer vulnerabilities.
      ,
      • Roumeliotis T.I.
      • Williams S.P.
      • Gonçalves E.
      • Alsinet C.
      • Del Castillo Velasco-Herrera M.
      • Aben N.
      • Ghavidel F.Z.
      • Michaut M.
      • Schubert M.
      • Price S.
      • Wright J.C.
      • Yu L.
      • Yang M.
      • Dienstmann R.
      • Guinney J.
      • Beltrao P.
      • Brazma A.
      • Pardo M.
      • Stegle O.
      • Adams D.J.
      • Wessels L.
      • Saez-Rodriguez J.
      • McDermott U.
      • Choudhary J.S.
      Genomic determinants of protein abundance variation in colorectal cancer cells.
      ).
      These results support a long-standing view that protein complex formation can set the total amount of protein levels (
      • Abovich N.
      • Gritz L.
      • Tung L.
      • Rosbash M.
      Effect of RP51 gene dosage alterations on ribosome synthesis in Saccharomyces cerevisiae.
      ). The degradation of unbound subunits may be due to a requirement of avoiding free hydrophobic interface surfaces that can be prone to aggregate (
      • Young L.
      • Jernigan R.L.
      • Covell D.G.
      A role for surface hydrophobicity in protein–protein recognition.
      ). In eukaryotic species, this appears to be achieved by degrading excess production, while in bacterial and archaeal species genes coding for protein complexes subunits tend to occur within operon structures such that they will be expressed at similar levels (
      • Mushegian A.R.
      • Koonin E.V.
      Gene order is not conserved in bacterial evolution.
      ). This link between appropriate expression and complex formation is further emphasized by the preferential ordering of subunits in operons starting from the subunits that tend to assemble first (
      • Wells J.N.
      • Bergendahl L.T.
      • Marsh J.A.
      Operon gene order is optimized for ordered protein complex assembly.
      ).
      While this phenomenon of gene dosage attenuation in protein complexes has been well documented, we still do not understand (i) what protein properties are associated with the propensity for a protein to be attenuated or (ii) if the characteristics of the attenuation process are seen in noncancerous cells. Here we have extended on a previous analysis (
      • Gonçalves E.
      • Fragoulis A.
      • Garcia-Alonso L.
      • Cramer T.
      • Saez-Rodriguez J.
      • Beltrao P.
      Widespread post-transcriptional attenuation of genomic copy-number variation in cancer.
      ), performing a multi-omics study of protein-level attenuation of gene dosage that combines genomics, (phospho)proteomics, and structural data. Analyzing 8,124 genes/proteins we observed that up to 42% of proteins show evidence of posttranscriptional regulation. Over 500 protein–protein interactions show indirect control of degradation of one subunit via physical associations, 32 of which may be further controlled by phosphorylation. Using structural models for 3,082 interfaces, we find that a higher fraction of interface residues is associated with a higher degree of attenuation. Finally, we studied the impact of these findings on noncancerous systems. We find that protein interaction-mediated control of protein abundances have an impact of the variation of protein levels across tissues and that the degree of attenuation correlates with the probability that natural variation with an impact on gene expression may result in a phenotypic consequence.

      DISCUSSION

      The joint analysis of multi-omics datasets of cancer samples suggests that a very significant fraction of the proteome (up to 42%) is under posttranscriptional control. The set of genes with protein-level buffering of CNVs is enriched in gene products belonging to large protein complexes. In addition, we found that the fraction of interface residues of a protein is a strong determinant of attenuation. Together with experiments on pulse-chase degradation (
      • McShane E.
      • Sin C.
      • Zauber H.
      • Wells J.N.
      • Donnelly N.
      • Wang X.
      • Hou J.
      • Chen W.
      • Storchova Z.
      • Marsh J.A.
      • Valleriani A.
      • Selbach M.
      Kinetic analysis of protein stability reveals age-dependent degradation.
      ), aneuploidy (
      • Dephoure N.
      • Hwang S.
      • O'Sullivan C.
      • Dodgson S.E.
      • Gygi S.P.
      • Amon A.
      • Torres E.M.
      Quantitative proteomic analysis reveals posttranslational responses to aneuploidy in yeast.
      ,
      • Stingele S.
      • Stoehr G.
      • Peplowska K.
      • Cox J.
      • Mann M.
      • Storchova Z.
      Global analysis of genome, transcriptome and proteome reveals the response to aneuploidy in human cells.
      ,
      • Pavelka N.
      • Rancati G.
      • Zhu J.
      • Bradford W.D.
      • Saraf A.
      • Florens L.
      • Sanderson B.W.
      • Hattem G.L.
      • Li R.
      Aneuploidy confers quantitative proteome changes and phenotypic variation in budding yeast.
      ), and the impact of natural genetic variation on protein levels (
      • Chick J.M.
      • Munger S.C.
      • Simecek P.
      • Huttlin E.L.
      • Choi K.
      • Gatti D.M.
      • Raghupathy N.
      • Svenson K.L.
      • Churchill G.A.
      • Gygi S.P.
      Defining the consequences of genetic variation on a proteome-wide scale.
      ,
      • Battle A.
      • Khan Z.
      • Wang S.H.
      • Mitrano A.
      • Ford M.J.
      • Pritchard J.K.
      • Gilad Y.
      Genomic variation. Impact of regulatory variation from RNA to protein.
      ), these results implicate protein complex formation as an important factor in posttranscriptional control, most likely via a high degradation rate of unassembled subunits. We note that this mechanism of CNV buffering at the protein level may be possible with CNV amplifications and deletions. While in the former it would be manifested by an apparent increase in the degradation rate of free complex subunits, in the latter it would result from a decrease in the apparent degradation rate of free subunits. However, it is likely that multiple mechanisms contribute to posttranscriptional control measured in the cancer samples including, for example, the control of protein translation rates by microRNAs or RNA-binding proteins. The extent of posttranscriptional control that is explained by the different processes remains to be studied.
      We observed that the fraction of residues at the interface correlates with the probability that a protein shows gene dosage attenuation. Similarly, the size of the interface correlates with the strength of association between pairs of physical interactions in which one subunit appears to control the abundance level of the interaction partner. The size of the interface typically correlates with increasing binding affinity between proteins as well as larger amounts of hydrophobic residues that are exposed in the absence of interactions. We speculate that either of these consequences could play a role in the attenuation. In particular, larger fraction of hydrophobic regions could increase the propensity to form aggregates, and in some cases hydrophobic regions are known to be recognized for degradation (
      • Xu Y.
      • Anderson D.E.
      • Ye Y.
      The HECT domain ubiquitin ligase HUWE1 targets unassembled soluble proteins for degradation.
      ). This could represent a general mechanism for recognition of unassembled complex subunits. The structural analysis performed here is limited by the current lack of coverage for structures of protein complexes. In the future, additional structures may allow us to study in more detail the interface features that are important for the attenuation mechanism.
      We have used data from cancer samples to identify the attenuated proteins and physical interactions with rate-limiting subunits. We find that most of the controlling–controlled protein–protein associations we predict have a positive relationship. Given the working model that these are explained by protein complex formation, the negative associations could be explained by cases of mutually exclusive complex membership. The fact that few associations predicted are negative are consistent with the idea that most complex members are not mutually exclusive.
      It is still unclear if the same proteins and interactions will have the same posttranscriptional control in other systems and/or species. When studying expression variation in normal tissues and the association of eQTLs with phenotypes, we observed that, in aggregate, the same proteins and interactions show signals consistent with posttranscriptional buffering of mRNA expression variation. Of note, we find that eQTLs are less likely to be linked to phenotypes in highly attenuated proteins. This is in line with studies of mRNA and protein QTLs in human induced pluripotent stem cell lines, showing that genetic variation driving mRNA changes are more likely to be associated to genotype differences when they are observed at the protein level (
      • Mirauta B.
      • Seaton D.D.
      • Bensaddek D.
      • Brenes A.
      • Bonder M.J.
      • Kilpinen H.
      • HipSci Consortium
      • Steigle O.
      • Lamond A.I.
      Population-scale proteome variation in human induced pluripotent stem cells.
      ). These findings highlight the importance of studying the degree of conservation of these posttranscriptional processes in different tissues and systems in the context of human genetics and disease.

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