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DNA nanodevices map enzymatic activity in organelles

Abstract

Cellular reporters of enzyme activity are based on either fluorescent proteins or small molecules. Such reporters provide information corresponding to wherever inside cells the enzyme is maximally active and preclude minor populations present in subcellular compartments. Here we describe a chemical imaging strategy to selectively interrogate minor, subcellular pools of enzymatic activity. This new technology confines the detection chemistry to a designated organelle, enabling imaging of enzymatic cleavage exclusively within the organelle. We have thus quantitatively mapped disulfide reduction exclusively in endosomes in Caenorhabditis elegans and identified that exchange is mediated by minor populations of the enzymes PDI-3 and TRX-1 resident in endosomes. Impeding intra-endosomal disulfide reduction by knocking down TRX-1 protects nematodes from infection by Corynebacterium diphtheriae, revealing the importance of this minor pool of endosomal TRX-1. TRX-1 also mediates endosomal disulfide reduction in human cells. A range of enzymatic cleavage reactions in organelles are amenable to analysis by this new reporter strategy.

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Fig. 1: Design and in vitro characterization of TDX reporter.
Fig. 2: Spatiotemporal detection of the thiol–disulfide exchange reaction in the endo-lysosomal compartment of C. elegans coelomocytes.
Fig. 3: PDI-3 and TRX-1 are responsible for the disulfide exchange reaction inside endocytic vesicles.
Fig. 4: Thioredoxin-1 offers protection against diphtheria toxin infection.
Fig. 5: TRX-1 mediates endosomal disulfide reduction in mammalian cells.

Data availability

The data that support the plots within this paper and other finding of this study are available from the corresponding author upon reasonable request.

References

  1. 1.

    Huang, S.-H., Li, Y., Zhang, J., Rong, J. & Ye, S. Epidermal growth factor receptor-containing exosomes induce tumor-specific regulatory T cells. Cancer Invest. 31, 330–335 (2013).

    CAS  Google Scholar 

  2. 2.

    Efeyan, A., Comb, W. C. & Sabatini, D. M. Nutrient-sensing mechanisms and pathways. Nature 517, 302–310 (2015).

    CAS  Google Scholar 

  3. 3.

    Dean, N. & Pelham, H. R. Recycling of proteins from the Golgi compartment to the ER in yeast. J. Cell Biol. 111, 369–377 (1990).

    CAS  Google Scholar 

  4. 4.

    Bhuniya, S. et al. An activatable theranostic for targeted cancer therapy and imaging. Angew. Chem. Int. Ed. 53, 4469–4474 (2014).

    CAS  Google Scholar 

  5. 5.

    Lee, M. H. et al. Hepatocyte-targeting single galactose-appended naphthalimide: a tool for intracellular thiol imaging in vivo. J. Am. Chem. Soc. 134, 1316–1322 (2012).

    CAS  Google Scholar 

  6. 6.

    Crivat, G. & Taraska, J. W. Imaging proteins inside cells with fluorescent tags. Trends Biotechnol. 30, 8–16 (2012).

    CAS  Google Scholar 

  7. 7.

    Los, G. V. et al. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 3, 373–382 (2008).

    CAS  Google Scholar 

  8. 8.

    Gething, M. J. & Sambrook, J. Protein folding in the cell. Nature 355, 33–45 (1992).

    CAS  Google Scholar 

  9. 9.

    Mesecke, N. et al. A disulfide relay system in the intermembrane space of mitochondria that mediates protein import. Cell 121, 1059–1069 (2005).

    CAS  Google Scholar 

  10. 10.

    Burgoyne, J. R. et al. Cysteine redox sensor in PKGIa enables oxidant-induced activation. Science 317, 1393–1397 (2007).

    CAS  Google Scholar 

  11. 11.

    Mills, J. E. et al. A novel disulfide bond in the SH2 domain of the C-terminal Src kinase controls catalytic activity. J. Mol. Biol. 365, 1460–1468 (2007).

    CAS  Google Scholar 

  12. 12.

    Collins, D. S., Unanue, E. R. & Harding, C. V. Reduction of disulfide bonds within lysosomes is a key step in antigen processing. J. Immunol. 147, 4054–4059 (1991).

    CAS  Google Scholar 

  13. 13.

    Guermonprez, P. et al. ER-phagosome fusion defines an MHC class I cross-presentation compartment in dendritic cells. Nature 425, 397–402 (2003).

    CAS  Google Scholar 

  14. 14.

    Stolf, B. S. et al. Protein disulfide isomerase and host–pathogen interaction. ScientificWorldJournal 11, 1749–1761 (2011).

    CAS  Google Scholar 

  15. 15.

    Maiti, S. et al. Gemcitabine–coumarin–biotin conjugates: a target specific theranostic anticancer prodrug. J. Am. Chem. Soc. 135, 4567–4572 (2013).

    CAS  Google Scholar 

  16. 16.

    Feener, S. E., Shene, W.-C. & Ryser, H. Cleavage of disulfide bonds in endocytosed macromolecules. J. Biol. Chem. 265, 18780–18785 (1990)..

  17. 17.

    Shen, W. C., Ryser, H. J. & LaManna, L. Disulfide spacer between methotrexate and poly(d-lysine). A probe for exploring the reductive process in endocytosis. J. Biol. Chem. 260, 10905–10908 (1985).

    CAS  Google Scholar 

  18. 18.

    Meyer, A. J. & Dick, T. P. Fluorescent protein-based redox probes. Antioxid. Redox. Signal. 13, 621–650 (2010).

    CAS  Google Scholar 

  19. 19.

    Chakraborty, K., Veetil, A. T., Jaffrey, S. R. & Krishnan, Y. Nucleic acid-based nanodevices in biological imaging. Annu. Rev. Biochem. 85, 349–373 (2016).

    CAS  Google Scholar 

  20. 20.

    Liu, J., Cao, Z. & Lu, Y. Functional nucleic acid sensors. Chem. Rev. 109, 1948–1998 (2009).

    CAS  Google Scholar 

  21. 21.

    Modi, S. et al. A DNA nanomachine that maps spatial and temporal pH changes inside living cells. Nat. Nanotechnol. 4, 325–330 (2009).

    CAS  Google Scholar 

  22. 22.

    Modi, S., Nizak, C., Surana, S., Halder, S. & Krishnan, Y. Two DNA nanomachines map pH changes along intersecting endocytic pathways inside the same cell. Nat. Nanotechnol. 8, 459–467 (2013).

    CAS  Google Scholar 

  23. 23.

    Saha, S., Prakash, V., Halder, S., Chakraborty, K. & Krishnan, Y. A pH-independent DNA nanodevice for quantifying chloride transport in organelles of living cells. Nat. Nanotechnol. 10, 645–651 (2015).

    CAS  Google Scholar 

  24. 24.

    Surana, S., Bhat, J. M., Koushika, S. P. & Krishnan, Y. An autonomous DNA nanomachine maps spatiotemporal pH changes in a multicellular living organism. Nat. Commun. 2, 340 (2011).

    Google Scholar 

  25. 25.

    Narayanaswamy, N. et al. A pH-correctable, DNA-based fluorescent reporter for organellar calcium. Nat. Methods 16, 95–102 (2019).

    CAS  Google Scholar 

  26. 26.

    Gutscher, M. et al. Real-time imaging of the intracellular glutathione redox potential. Nat. Methods 5, 553–559 (2008).

    CAS  Google Scholar 

  27. 27.

    Romero-Aristizabal, C., Marks, D. S., Fontana, W. & Apfeld, J. Regulated spatial organization and sensitivity of cytosolic protein oxidation in Caenorhabditis elegans. Nat. Commun. 5, 5020 (2014).

    CAS  Google Scholar 

  28. 28.

    Chakraborty, K., Leung, K. & Krishnan, Y. High lumenal chloride in the lysosome is critical for lysosome function. eLife 6, e28862 (2017).

    Google Scholar 

  29. 29.

    Yang, J., Chen, H., Vlahov, I. R., Cheng, J.-X. & Low, P. S. Evaluation of disulfide reduction during receptor-mediated endocytosis by using FRET imaging. Proc. Natl Acad. Sci. USA 103, 13872–13877 (2006).

    CAS  Google Scholar 

  30. 30.

    Bhatia, D. et al. Icosahedral DNA nanocapsules by modular assembly. Angew. Chem. Int. Ed. 48, 4134–4137 (2009).

    CAS  Google Scholar 

  31. 31.

    Veetil, A. T., Jani, M. S. & Krishnan, Y. Chemical control over membrane-initiated steroid signaling with a DNA nanocapsule. Proc. Natl Acad. Sci. USA 115, 9432–9437 (2018).

    CAS  Google Scholar 

  32. 32.

    Sevier, C. S. & Kaiser, C. A. Formation and transfer of disulphide bonds in living cells. Nat. Rev. Mol. Cell Biol. 3, 836–847 (2002).

    CAS  Google Scholar 

  33. 33.

    Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).

    CAS  Google Scholar 

  34. 34.

    Hawkins, H. C., Blackburn, E. C. & Freedman, R. B. Comparison of the activities of protein disulphide-isomerase and thioredoxin in catalysing disulphide isomerization in a protein substrate. Biochem. J. 275, 349–353 (1991).

    CAS  Google Scholar 

  35. 35.

    Eschenlauer, S. C. P. & Page, A. P. The Caenorhabditis elegans ERp60 homolog protein disulfide isomerase-3 has disulfide isomerase and transglutaminase-like cross-linking activity and is involved in the maintenance of body morphology. J. Biol. Chem. 278, 4227–4237 (2003).

    CAS  Google Scholar 

  36. 36.

    Smith, C. V., Jones, D. P., Guenthner, T. M., Lash, L. H. & Lauterburg, B. H. Compartmentation of glutathione: implications for the study of toxicity and disease. Toxicol. Appl. Pharmacol. 140, 1–12 (1996).

    CAS  Google Scholar 

  37. 37.

    Rubartelli, A., Bajetto, A., Allavena, G., Wollman, E. & Sitia, R. Secretion of thioredoxin by normal and neoplastic cells through a leaderless secretory pathway. J. Biol. Chem. 267, 24161–24164 (1992).

    CAS  Google Scholar 

  38. 38.

    Dihazi, H. et al. Secretion of ERP57 is important for extracellular matrix accumulation and progression of renal fibrosis, and is an early sign of disease onset. J. Cell Sci. 126, 3649–3663 (2013).

    CAS  Google Scholar 

  39. 39.

    Pacello, F., D’Orazio, M. & Battistoni, A. An ERp57-mediated disulphide exchange promotes the interaction between Burkholderia cenocepacia and epithelial respiratory cells. Sci. Rep. 6, 21140 (2016).

    CAS  Google Scholar 

  40. 40.

    Lasecka, L. & Baron, M. D. The nairovirus nairobi sheep disease virus/ganjam virus induces the translocation of protein disulphide isomerase-like oxidoreductases from the endoplasmic reticulum to the cell surface and the extracellular space. PLoS One 9, e94656 (2014).

    Google Scholar 

  41. 41.

    Ott, L. et al. Evaluation of invertebrate infection models for pathogenic corynebacteria. FEMS Immunol. Med. Microbiol. 65, 413–421 (2012).

    CAS  Google Scholar 

  42. 42.

    Dunbar, T. L., Yan, Z., Balla, K. M., Smelkinson, M. G. & Troemel, E. R. C. elegans detects pathogen-induced translational inhibition to activate immune signaling. Cell Host Microbe 11, 375–386 (2012).

    CAS  Google Scholar 

  43. 43.

    Moskaug, J. O., Sandvig, K. & Olsnes, S. Cell-mediated reduction of the interfragment disulfide in nicked diphtheria toxin. A new system to study toxin entry at low pH. J. Biol. Chem. 262, 10339–10345 (1987).

    CAS  Google Scholar 

  44. 44.

    Patel, P. C. et al. Scavenger receptors mediate cellular uptake of polyvalent oligonucleotide-functionalized gold nanoparticles. Bioconjug. Chem. 21, 2250–2256 (2010).

    CAS  Google Scholar 

  45. 45.

    Karala, A.-R., Lappi, A.-K. & Ruddock, L. W. Modulation of an active-site cysteine pKa allows PDI to act as a catalyst of both disulfide bond formation and isomerization. J. Mol. Biol. 396, 883–892 (2010).

    CAS  Google Scholar 

  46. 46.

    Wu, C. et al. Thioredoxin 1-mediated post-translational modifications: reduction, transnitrosylation, denitrosylation, and related proteomics methodologies. Antioxid. Redox Signal. 15, 2565–2604 (2011).

    CAS  Google Scholar 

  47. 47.

    Thekkan, S. et al. A DNA-based fluorescent reporter maps HOCl production in the maturing phagosome. Nat. Chem. Biol. https://doi.org/10.1038/s41589-018-0176-3 (2018).

  48. 48.

    Leung, K., Chakraborty, K., Saminathan, A. & Krishnan, Y. A DNA nanomachine chemically resolves lysosomes in live cells. Nat. Nanotechnol. https://doi.org/10.1038/s41565-018-0318-5 (2018).

  49. 49.

    Futerman, A. H. & van Meer, G. The cell biology of lysosomal storage disorders. Nat. Rev. Mol. Cell Biol. 5, 554–565 (2004).

    CAS  Google Scholar 

  50. 50.

    Olson, O. C. & Joyce, J. A. Cysteine cathepsin proteases: regulators of cancer progression and therapeutic response. Nat. Rev. Cancer 15, 712–729 (2015).

    CAS  Google Scholar 

  51. 51.

    Vassar, R., Kovacs, D. M., Yan, R. & Wong, P. C. The beta-secretase enzyme BACE in health and Alzheimer’s disease: regulation, cell biology, function, and therapeutic potential. J. Neurosci. 29, 12787–12794 (2009).

    CAS  Google Scholar 

  52. 52.

    Seidah, N. G. & Prat, A. The biology and therapeutic targeting of the proprotein convertases. Nat. Rev. Drug Discov. 11, 367–383 (2012).

    CAS  Google Scholar 

  53. 53.

    Nomura, D. K., Dix, M. M. & Cravatt, B. F. Activity-based protein profiling for biochemical pathway discovery in cancer. Nat. Rev. Cancer 10, 630–638 (2010).

    CAS  Google Scholar 

  54. 54.

    Prifti, E. et al. A fluorogenic probe for SNAP-tagged plasma membrane proteins based on the solvatochromic molecule Nile Red. ACS Chem. Biol. 9, 606–612 (2014).

    CAS  Google Scholar 

  55. 55.

    Presolski, S. I., Hong, V. P. & Finn, M. G. Copper-catalyzed azide-alkyne click chemistry for bioconjugation. Curr. Protoc. Chem. Biol. 3, 153–162 (2011).

    Google Scholar 

  56. 56.

    Bhatia, D., Surana, S., Chakraborty, S., Koushika, S. P. & Krishnan, Y. A synthetic icosahedral DNA-based host–cargo complex for functional in vivo imaging. Nat. Commun. 2, 339 (2011).

    Google Scholar 

  57. 57.

    Veetil, A. T. et al. Cell-targetable DNA nanocapsules for spatiotemporal release of caged bioactive small molecules. Nat. Nanotechnol. 12, 1183–1189 (2017).

    CAS  Google Scholar 

  58. 58.

    Kamath, R. S. & Ahringer, J. Genome-wide RNAi screening in Caenorhabditis elegans. Methods 30, 313–321 (2003).

    CAS  Google Scholar 

  59. 59.

    Rual, J.-F. et al. Toward improving Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi library. Genome Res. 14, 2162–2168 (2004).

    CAS  Google Scholar 

  60. 60.

    Xu, D., Perez, R. E., Rezaiekhaligh, M. H., Bourdi, M. & Truog, W. E. Knockdown of ERp57 increases BiP/GRP78 induction and protects against hyperoxia and tunicamycin-induced apoptosis. Am. J. Physiol. Lung Cell. Mol. Physiol. 297, L44–L51 (2009).

    CAS  Google Scholar 

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Acknowledgements

The authors thank J. Clardy, J. Kuriyan, S. Modi and A. Lin Chun for valuable comments on this work. The authors thank the Integrated Light Microscopy facility at the University of Chicago, the Caenorhabditis Genetic Center (CGC), J. Fares and M. Edgley for C. elegans strains, M. Glotzer and F. M. Ausubel for RNAi clones and valuable discussions, S. Crosson at the BSL facility for C. diphtheriae work and C. Cui for BMDMs and flow cytometry. This work was supported by the University of Chicago Women’s Board, Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust, C-084 as well as a CBC post-doctoral research grant (PDR-073), Pilot and Feasibility award from an NIDDK Center grant P30DK42086 to the University of Chicago Digestive Diseases Research Core Center and University of Chicago start-up funds to Y.K. Y.K. is a Brain Research Foundation Fellow.

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K.D. and Y.K. designed the project. K.D. developed tripartite TDX reporters and performed all experiments related to TDX reporter in C. elegans and mammalian cells. A.T.V. prepared the dextran encapsulated icosahedron and in vitro experiments related to icosahedron. K.C. contributed to cathepsin-related experiments. K.D., A.T.V., K.C. and Y.K. analysed the data. K.D., A.T.V. and Y.K. wrote the paper. All authors discussed the results and provided input on the manuscript.

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Correspondence to Yamuna Krishnan.

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Dan, K., Veetil, A.T., Chakraborty, K. et al. DNA nanodevices map enzymatic activity in organelles. Nat. Nanotechnol. 14, 252–259 (2019). https://doi.org/10.1038/s41565-019-0365-6

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