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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The data that support the plots within this paper and other finding of this study are available from the corresponding author upon reasonable request.
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).
Efeyan, A., Comb, W. C. & Sabatini, D. M. Nutrient-sensing mechanisms and pathways. Nature 517, 302–310 (2015).
Dean, N. & Pelham, H. R. Recycling of proteins from the Golgi compartment to the ER in yeast. J. Cell Biol. 111, 369–377 (1990).
Bhuniya, S. et al. An activatable theranostic for targeted cancer therapy and imaging. Angew. Chem. Int. Ed. 53, 4469–4474 (2014).
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).
Crivat, G. & Taraska, J. W. Imaging proteins inside cells with fluorescent tags. Trends Biotechnol. 30, 8–16 (2012).
Los, G. V. et al. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 3, 373–382 (2008).
Gething, M. J. & Sambrook, J. Protein folding in the cell. Nature 355, 33–45 (1992).
Mesecke, N. et al. A disulfide relay system in the intermembrane space of mitochondria that mediates protein import. Cell 121, 1059–1069 (2005).
Burgoyne, J. R. et al. Cysteine redox sensor in PKGIa enables oxidant-induced activation. Science 317, 1393–1397 (2007).
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).
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).
Guermonprez, P. et al. ER-phagosome fusion defines an MHC class I cross-presentation compartment in dendritic cells. Nature 425, 397–402 (2003).
Stolf, B. S. et al. Protein disulfide isomerase and host–pathogen interaction. ScientificWorldJournal 11, 1749–1761 (2011).
Maiti, S. et al. Gemcitabine–coumarin–biotin conjugates: a target specific theranostic anticancer prodrug. J. Am. Chem. Soc. 135, 4567–4572 (2013).
Feener, S. E., Shene, W.-C. & Ryser, H. Cleavage of disulfide bonds in endocytosed macromolecules. J. Biol. Chem. 265, 18780–18785 (1990)..
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).
Meyer, A. J. & Dick, T. P. Fluorescent protein-based redox probes. Antioxid. Redox. Signal. 13, 621–650 (2010).
Chakraborty, K., Veetil, A. T., Jaffrey, S. R. & Krishnan, Y. Nucleic acid-based nanodevices in biological imaging. Annu. Rev. Biochem. 85, 349–373 (2016).
Liu, J., Cao, Z. & Lu, Y. Functional nucleic acid sensors. Chem. Rev. 109, 1948–1998 (2009).
Modi, S. et al. A DNA nanomachine that maps spatial and temporal pH changes inside living cells. Nat. Nanotechnol. 4, 325–330 (2009).
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).
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).
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).
Narayanaswamy, N. et al. A pH-correctable, DNA-based fluorescent reporter for organellar calcium. Nat. Methods 16, 95–102 (2019).
Gutscher, M. et al. Real-time imaging of the intracellular glutathione redox potential. Nat. Methods 5, 553–559 (2008).
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).
Chakraborty, K., Leung, K. & Krishnan, Y. High lumenal chloride in the lysosome is critical for lysosome function. eLife 6, e28862 (2017).
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).
Bhatia, D. et al. Icosahedral DNA nanocapsules by modular assembly. Angew. Chem. Int. Ed. 48, 4134–4137 (2009).
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).
Sevier, C. S. & Kaiser, C. A. Formation and transfer of disulphide bonds in living cells. Nat. Rev. Mol. Cell Biol. 3, 836–847 (2002).
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).
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).
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).
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).
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).
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).
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).
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).
Ott, L. et al. Evaluation of invertebrate infection models for pathogenic corynebacteria. FEMS Immunol. Med. Microbiol. 65, 413–421 (2012).
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).
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).
Patel, P. C. et al. Scavenger receptors mediate cellular uptake of polyvalent oligonucleotide-functionalized gold nanoparticles. Bioconjug. Chem. 21, 2250–2256 (2010).
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).
Wu, C. et al. Thioredoxin 1-mediated post-translational modifications: reduction, transnitrosylation, denitrosylation, and related proteomics methodologies. Antioxid. Redox Signal. 15, 2565–2604 (2011).
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).
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).
Futerman, A. H. & van Meer, G. The cell biology of lysosomal storage disorders. Nat. Rev. Mol. Cell Biol. 5, 554–565 (2004).
Olson, O. C. & Joyce, J. A. Cysteine cathepsin proteases: regulators of cancer progression and therapeutic response. Nat. Rev. Cancer 15, 712–729 (2015).
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).
Seidah, N. G. & Prat, A. The biology and therapeutic targeting of the proprotein convertases. Nat. Rev. Drug Discov. 11, 367–383 (2012).
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).
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).
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).
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).
Veetil, A. T. et al. Cell-targetable DNA nanocapsules for spatiotemporal release of caged bioactive small molecules. Nat. Nanotechnol. 12, 1183–1189 (2017).
Kamath, R. S. & Ahringer, J. Genome-wide RNAi screening in Caenorhabditis elegans. Methods 30, 313–321 (2003).
Rual, J.-F. et al. Toward improving Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi library. Genome Res. 14, 2162–2168 (2004).
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).
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.
The authors declare no competing interests.
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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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