Abstract
Protein-biomolecule interactions play pivotal roles in almost all biological processes. For a biomolecule of interest, the identification of the interacting protein(s) is essential. For this need, although many assays are available, highly robust and reliable methods are always desired. By combining a substrate-based proximity labeling activity from the pupylation pathway of Mycobacterium tuberculosis and the streptavidin (SA)-biotin system, we developed the Specific Pupylation as IDEntity Reporter (SPIDER) method for identifying protein-biomolecule interactions. Using SPIDER, we validated the interactions between the known binding proteins of protein, DNA, RNA, and small molecule. We successfully applied SPIDER to construct the global protein interactome for m6A and mRNA, identified a variety of uncharacterized m6A binding proteins, and validated SRSF7 as a potential m6A reader. We globally identified the binding proteins for lenalidomide and CobB. Moreover, we identified SARS-CoV-2-specific receptors on the cell membrane. Overall, SPIDER is powerful and highly accessible for the study of protein-biomolecule interactions.
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Data and materials availability
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX (Ma et al., 2019) partner repositor with dataset identifiers as follows: PXD026509: Pupylation sites on CheA identified by SPIDER assay;
PXD026527: Intramolecular pupylation of GFP-PupE identified by mass spectrometry;
PXD026478: Pupylation sites on protein GFP-PupE;
PXD026511: Pupylation sites on protein FKBP12;
PXD037952: Side-by-side comparison of AP-MS
(PXD037953), Tri-functional affinity probe (PXD037952), BioID (PXD037952), PUP-IT (PXD037952) and SPIDER (PXD037952);
PXD026514: CobB interacting proteins identified by SPIDER assay;
PXD026517: Pupylation sites on protein Sox2;
PXD026519: m6A binding proteins identified by the SPIDER assay;
PXD026521: mRNA-protein interactome of THP-1 cells;
PXD037881: m6A binding proteins identified by biotinylated m6A probe assay;
PXD026518: Pupylation sites on SARS-CoV-2 N;
PXD031035: SARS-CoV-2 receptor on the cell surface identified by SPIDER assay;
PXD026523: Lenalidomide binding proteins identified by SPIDER assay.
Additional data related to this paper may be requested from the authors.
References
Backus, K.M., Correia, B.E., Lum, K.M., Forli, S., Horning, B.D., González-Páez, G.E., Chatterjee, S., Lanning, B.R., Teijaro, J.R., Olson, A.J., et al. (2016). Proteome-wide covalent ligand discovery in native biological systems. Nature 534, 570–574.
Baltz, A.G., Munschauer, M., Schwanhäusser, B., Vasile, A., Murakawa, Y., Schueler, M., Youngs, N., Penfold-Brown, D., Drew, K., Milek, M., et al. (2012). The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts. Mol Cell 46, 674–690.
Bürckstümmer, T., Bennett, K.L., Preradovic, A., Schütze, G., Hantschel, O., Superti-Furga, G., and Bauch, A. (2006). An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells. Nat Methods 3, 1013–1019.
Butter, F., Scheibe, M., Mörl, M., and Mann, M. (2009). Unbiased RNA-protein interaction screen by quantitative proteomics. Proc Natl Acad Sci USA 106, 10626–10631.
Cantwell, B.J., and Manson, M.D. (2009). Protein domains and residues involved in the CheZ/CheAS interaction. J Bacteriol 191, 5838–5841.
Castaño-Cerezo, S., Bernal, V., Post, H., Fuhrer, T., Cappadona, S., Sánchez-Díaz, N.C., Sauer, U., Heck, A.J., Altelaar, A.M., and Cánovas, M. (2014). Protein acetylation affects acetate metabolism, motility and acid stress response in Escherichia coli. Mol Syst Biol 10, 762.
Castello, A., Fischer, B., Eichelbaum, K., Horos, R., Beckmann, B.M., Strein, C., Davey, N.E., Humphreys, D.T., Preiss, T., Steinmetz, L.M., et al. (2012). Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. Cell 149, 1393–1406.
Chen, N., Zhou, S., and Palmisano, M. (2017). Clinical pharmacokinetics and pharmacodynamics of lenalidomide. Clin Pharmacokinet 56, 139–152.
Cheng, Z.F., and Deutscher, M.P. (2002). Purification and characterization of the Escherichia coli exoribonuclease RNase R—comparison with RNase II. J Biol Chem 277, 21624–21629.
Chodosh, L.A. (2001). UV crosslinking of proteins to nucleic acids. Curr Protoc Mol Biol 36.
Cox, J., and Mann, M. (2008). MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26, 1367–1372.
Cun, Y., An, S., Zheng, H., Lan, J., Chen, W., Luo, W., Yao, C., Li, X., Huang, X., Sun, X., et al. (2021). Specific regulation of m6A by SRSF7 promotes the progression of glioblastoma. Genomics Proteomics Bioinformatics doi: https://doi.org/10.1016/j.gpb.2021.11.001.
Dinesh, D.C., Chalupska, D., Silhan, J., Koutna, E., Nencka, R., Veverka, V., and Boura, E. (2020). Structural basis of RNA recognition by the SARS-CoV-2 nucleocapsid phosphoprotein. PloS Pathog 16, e100910.
Edupuganti, R.R., Geiger, S., Lindeboom, R.G.H., Shi, H., Hsu, P.J., Lu, Z., Wang, S.Y., Baltissen, M.P.A., Jansen, P.W.T.C., Rossa, M., et al. (2017). N6-methyladenosine (m6A) recruits and repels proteins to regulate mRNA homeostasis. Nat Struct Mol Biol 24, 870–878.
Fischer, E.S., Bohm, K., and Thoma, N.H. (2014). Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide: insights into CRL4 inhibition by small molecules. FEBS J 281, 248–249.
Gomes, A.F., and Gozzo, F.C. (2010). Chemical cross-linking with a diazirine photoactivatable cross-linker investigated by MALDI- and ESI-MS/MS. J Mass Spectrom 45, 892–899.
Gururaj, A.E., Singh, R.R., Rayala, S.K., Holm, C., den Hollander, P., Zhang, H., Balasenthil, S., Talukder, A.H., Landberg, G., and Kumar, R. (2006). MTA1, a transcriptional activator of breast cancer amplified sequence 3. Proc Natl Acad Sci USA 103, 6670–6675.
Han, X., Zhou, Z., Fei, L., Sun, H., Wang, R., Chen, Y., Chen, H., Wang, J., Tang, H., Ge, W., et al. (2020). Construction of a human cell landscape at single-cell level. Nature 581, 303–309.
Hatakeyama, S., Yada, M., Matsumoto, M., Ishida, N., and Nakayama, K.I. (2001). U box proteins as a new family of ubiquitin-protein ligases. J Biol Chem 276, 33111–33120.
Ho, Y., Gruhler, A., Heilbut, A., Bader, G.D., Moore, L., Adams, S.L., Millar, A., Taylor, P., Bennett, K., Boutilier, K., et al. (2002). Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415, 180–183.
Hou, L., Wei, Y., Lin, Y., Wang, X., Lai, Y., Yin, M., Chen, Y., Guo, X., Wu, S., Zhu, Y., et al. (2020). Concurrent binding to DNA and RNA facilitates the pluripotency reprogramming activity of Sox2. Nucleic Acids Res 48, 3869–3887.
Huang, H., Weng, H., Sun, W., Qin, X., Shi, H., Wu, H., Zhao, B.S., Mesquita, A., Liu, C., Yuan, C.L., et al. (2018). Recognition of RNA N6-methyladenosine by IGF2BP proteins enhances mRNA stability and translation. Nat Cell Biol 20, 285–295.
Itoh, S., and Navia, M.A. (1995). Structure comparison of native and mutant human recombinant FKBP12 complexes with the immunosuppressant drug FK506 (tacrolimus). Protein Sci 4, 2261–2268.
Kulkarni, P.M., Kulkarni, A.R., Korde, A., Tichkule, R.B., Laprairie, R.B., Denovan-Wright, E.M., Zhou, H., Janero, D.R., Zvonok, N., Makriyannis, A., et al. (2016). Novel electrophilic and photoaffinity covalent probes for mapping the cannabinoid 1 receptor allosteric site (s). J Med Chem 59, 44–60.
Liang, W., Malhotra, A., and Deutscher, M.P. (2011). Acetylation regulates the stability of a bacterial protein: growth stage-dependent modification of RNase R. Mol Cell 44, 160–166.
Liao, S., Shang, Q., Zhang, X., Zhang, J., Xu, C., and Tu, X. (2009). Pup, a prokaryotic ubiquitin-like protein, is an intrinsically disordered protein. Biochem J 422, 207–215.
Linares, A.J., Lin, C.H., Damianov, A., Adams, K.L., Novitch, B.G., and Black, D.L. (2015). The splicing regulator PTBP1 controls the activity of the transcription factor Pbx1 during neuronal differentiation. eLife 4, e09268.
Liu, H., Lorenzini, P.A., Zhang, F., Xu, S., Wong, M.S.M., Zheng, J., and Roca, X. (2018a). Alternative splicing analysis in human monocytes and macrophages reveals MBNL1 as major regulator. Nucleic Acids Res 46, 6069–6086.
Liu, Q., Zheng, J., Sun, W., Huo, Y., Zhang, L., Hao, P., Wang, H., and Zhuang, M. (2018b). A proximity-tagging system to identify membrane protein-protein interactions. Nat Methods 15, 715–722.
Ma, J., Chen, T., Wu, S., Yang, C., Bai, M., Shu, K., Li, K., Zhang, G., Jin, Z., He, F., et al. (2019). iProX: an integrated proteome resource. Nucleic Acids Res 47, D1211–D1217.
MacKinnon, A.L., and Taunton, J. (2009). Target identification by diazirine photo-cross-linking and click chemistry. Curr Protoc Chem Biol 1, 55–73.
Mathew, B., Bathla, S., Williams, K.R., and Nairn, A.C. (2022). Deciphering spatial protein-protein interactions in brain using proximity labeling. Mol Cell Proteomics 21, 100422.
McHugh, C.A., Chen, C.K., Chow, A., Surka, C.F., Tran, C., McDonel, P., Pandya-Jones, A., Blanco, M., Burghard, C., Moradian, A., et al. (2015). The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3. Nature 521, 232–236.
Paul, B.J., Barker, M.M., Ross, W., Schneider, D.A., Webb, C., Foster, J. W., and Gourse, R.L. (2004). DksA: a critical component of the transcription initiation machinery that potentiates the regulation of rRNA promoters by ppGpp and the initiating NTP. Cell 118, 311–322.
Pearce, M.J., Mintseris, J., Ferreyra, J., Gygi, S.P., and Darwin, K.H. (2008). Ubiquitin-like protein involved in the proteasome pathway of Mycobacterium tuberculosis. Science 322, 1104–1107.
Rhee, H.W., Zou, P., Udeshi, N.D., Martell, J.D., Mootha, V.K., Carr, S.A., and Ting, A.Y. (2013). Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. Science 339, 1328–1331.
Roux, K.J., Kim, D.I., Raida, M., and Burke, B. (2012). A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J Cell Biol 196, 801–810.
Schwende, H., Fitzke, E., Ambs, P., and Dieter, P. (1996). Differences in the state of differentiation of THP-1 cells induced by phorbol ester and 1,25-dihydroxyvitamin D3. J Leukoc Biol 59, 555–561.
Shannon, P., Markiel, A., Ozier, O., Baliga, N.S., Wang, J.T., Ramage, D., Amin, N., Schwikowski, B., and Ideker, T. (2003). Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13, 2498–2504.
Starai, V.J., Celic, I., Cole, R.N., Boeke, J.D., and Escalante-Semerena, J.C. (2002). Sir2-dependent activation of acetyl-CoA synthetase by deacetylation of active lysine. Science 298, 2390–2392.
Suprewicz, Ł., Swoger, M., Gupta, S., Piktel, E., Byfield, F.J., Iwamoto, D. V., Germann, D., Reszeć, J., Marcińczyk, N., Carroll, R.J., et al. (2022). Extracellular vimentin as a target against SARS-CoV-2 host cell invasion. Small 18, 2105640.
Szklarczyk, D., Gable, A.L., Lyon, D., Junge, A., Wyder, S., Huerta-Cepas, J., Simonovic, M., Doncheva, N.T., Morris, J.H., Bork, P., et al. (2019). STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res 47, D607–D613.
Uhlén, M., Fagerberg, L., Hallström, B.M., Lindskog, C., Oksvold, P., Mardinoglu, A., Sivertsson, Å., Kampf, C., Sjöstedt, E., Asplund, A., et al. (2015). Tissue-based map of the human proteome. Science 347, 1260419.
Vikis, H.G., and Guan, K.L. (2004). Glutathione-S-transferase-fusion based assays for studying protein-protein interactions. In: Fu, H., ed. Protein-Protein Interactions. Methods in Molecular Biology. New York: Humana Press. 175–186.
Wang, H., and Matsumura, P. (1996). Characterization of the CheAS/CheZ complex: a specific interaction resulting in enhanced dephosphorylating activity on CheY-phosphate. Mol Microbiol 19, 695–703.
Wang, X., Lu, Z., Gomez, A., Hon, G.C., Yue, Y., Han, D., Fu, Y., Parisien, M., Dai, Q., Jia, G., et al. (2014). N6-methyladenosine-dependent regulation of messenger RNA stability. Nature 505, 117–120.
Weinert, B.T., Iesmantavicius, V., Wagner, S.A., Schölz, C., Gummesson, B., Beli, P., Nyström, T., and Choudhary, C. (2013). Acetyl-phosphate is a critical determinant of lysine acetylation in E. coli. Mol Cell 51, 265–272.
Xiao, W., Adhikari, S., Dahal, U., Chen, Y.S., Hao, Y.J., Sun, B.F., Sun, H. Y., Li, A., Ping, X.L., Lai, W.Y., et al. (2016). Nuclear m6A reader YTHDC1 regulates mRNA splicing. Mol Cell 61, 507–519.
Zhu, H., Bilgin, M., Bangham, R., Hall, D., Casamayor, A., Bertone, P., Lan, N., Jansen, R., Bidlingmaier, S., Houfek, T., et al. (2001). Global analysis of protein activities using proteome chips. Science 293, 2101–2105.
Acknowledgements
This work was supported by the National Key Research and Development Program of China (2020YFE0202200), the National Natural Science Foundation of China (31900112, 21907065, 31970130 and 31670831). We thank Dr. Heng Zhu for his long-term guidance. We thank Prof. Pilong Li of School of Life Sciences of Tsinghua University for kindly providing the YTHDF family plasmids. We thank Prof. Xichen Bao of Guangzhou Institutes of Biomedicine and Health of Chinese Academy of Sciences for kindly providing the Sox2 plasmid. We thank Prof. Xiaodong Zhao of Shanghai Jiao Tong University and Prof. Jian Yang of Tongji University for critical comments.
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Jiang, HW., Chen, H., Zheng, YX. et al. Specific pupylation as IDEntity reporter (SPIDER) for the identification of protein-biomolecule interactions. Sci. China Life Sci. 66, 1869–1887 (2023). https://doi.org/10.1007/s11427-023-2316-2
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DOI: https://doi.org/10.1007/s11427-023-2316-2