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Understanding renal nuclear protein accumulation: an in vitro approach to explain an in vivo phenomenon

  • Molecular Toxicology
  • Published:
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Abstract

Proper subcellular trafficking is essential to prevent protein mislocalization and aggregation. Transport of the peroxisomal enzyme d-amino acid oxidase (DAAO) appears dysregulated by specific pharmaceuticals, e.g., the anti-overactive bladder drug propiverine or a norepinephrine/serotonin reuptake inhibitor (NSRI), resulting in massive cytosolic and nuclear accumulations in rat kidney. To assess the underlying molecular mechanism of the latter, we aimed to characterize the nature of peroxisomal and cyto-nuclear shuttling of human and rat DAAO overexpressed in three cell lines using confocal microscopy. Indeed, interference with peroxisomal transport via deletion of the PTS1 signal or PEX5 knockdown resulted in induced nuclear DAAO localization. Having demonstrated the absence of active nuclear import and employing variably sized mCherry- and/or EYFP-fusion proteins of DAAO and catalase, we showed that peroxisomal proteins ≤134 kDa can passively diffuse into mammalian cell nuclei—thereby contradicting the often-cited 40 kDa diffusion limit. Moreover, their inherent nuclear presence and nuclear accumulation subsequent to proteasome inhibition or abrogated peroxisomal transport suggests that nuclear localization is a characteristic in the lifecycle of peroxisomal proteins. Based on this molecular trafficking analysis, we suggest that pharmaceuticals like propiverine or an NSRI may interfere with peroxisomal protein targeting and import, consequently resulting in massive nuclear protein accumulation in vivo.

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Abbreviations

h/rCAT:

Human/rat catalase

h/rDAAO:

Human/rat d-amino acid oxidase

NES:

Nuclear export signal

NLS:

Nuclear localization signal

NTS:

Nuclear translocation signal

PTS:

Peroxisomal targeting signal

RNI:

Relative nuclear intensity

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Acknowledgements

This work was supported by the University of Konstanz and the Konstanz Research School Chemical Biology (KoRS-CB), the Deutsche Forschungsgemeinschaft (RTG 1331), and the Fondo di Ateneo per la Ricerca. We thank the Bioimaging Center (University of Konstanz) for the use of the imaging equipment. We acknowledge Prof. Dr. Frank Thevenod (University of Witten/Herdecke, Germany) for providing WKPT cells. Special thanks to Johannes Delp and Prof. Dr. Marcel Leist for providing instrumentation and support with Cellomics. We thank Dr. Sascha Beneke and Philipp Secker for helpful discussions and our students Nadja Schlichenmaier, Madeline Walz, and Céline Weller for assistance with cloning.

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Author notes

  1. Lisanne Luks and Marcia Y. Maier contributed equally to this work and are listed in alphabetical order.

Authors and Affiliations

  1. Human and Environmental Toxicology, Department of Biology, University of Konstanz, Universitätsstrasse 10, 78457, Konstanz, Germany

    Lisanne Luks, Marcia Y. Maier & Daniel R. Dietrich

  2. Konstanz Research School Chemical Biology (KoRS-CB), University of Konstanz, Konstanz, Germany

    Marcia Y. Maier & Daniel R. Dietrich

  3. Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy

    Silvia Sacchi & Loredano Pollegioni

  4. Protein Factory Research Center, Politecnico di Milano and University of Insubria, Milan, Italy

    Silvia Sacchi & Loredano Pollegioni

Authors
  1. Lisanne Luks
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  2. Marcia Y. Maier
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  3. Silvia Sacchi
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  4. Loredano Pollegioni
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  5. Daniel R. Dietrich
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Contributions

L. Luks and M. Y. Maier planned the project, designed the experiments, performed all experiments except for the U87 data, analyzed, interpreted and visualized all data, wrote the original draft, and reviewed the manuscript. D. R. Dietrich provided resources, planned the project, and was involved in writing, reviewing, and editing the manuscript. S. Sacchi and L. Pollegioni provided resources and materials, performed the experiments with U87 cells, and were involved in editing the manuscript.

Corresponding author

Correspondence to Daniel R. Dietrich.

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204_2017_1970_MOESM1_ESM.tif

Supplementary material S1 Inhibition of peroxisomal import promotes nuclear localization of DAAO in WKPT cells. Confocal microscopy of transiently transfected WKPT expressing EYFP-tagged a rDAAO, b rDAAO-ΔPTS. Nuclei are labeled with Hoechst33342 and are indicated by dashed lines. Pearson’s correlation coefficient (r) and line plot profile were determined for colocalization analysis. c Representative far-UV (upper figure) and near-UV (lower figure) circular dichroism (CD) spectra of recombinant His-rDAAO and His-rDAAO-ΔPTS. d Enzymatic activity of His-rDAAO and His-rDAAO-ΔPTS1 determined using oxygen consumption. Mean ± SEM, n = 3. Unpaired t test, n.s. = not significant. e Confocal analysis of HEK293 cells expressing EYFP-DAAO 24 h after knockdown of PEX5. rDAAO = EYFP-tagged rat DAAO. PMP70 = peroxisomal membrane protein 70. PTS = peroxisomal targeting signal. PEX5 = peroxisomal biogenesis factor 5. Scale bar 10 μm (TIFF 32713 kb)

204_2017_1970_MOESM2_ESM.tif

Supplementary material S2 Cross-talk between different targeting signals. a Schematic representation of EYFP-DAAO constructs with N-terminal EYFP-tag, C-terminal PTS1 (SHL), and the classical monopartite NLS (PKKKRKVE) at different positions within the DAAO protein sequence. b Confocal microscopy of transiently transfected HEK293 cells expressing different DAAO constructs as listed in a. Nuclei are labeled with Hoechst33342. Same results observed for WKPT cells (data not shown). NLS nuclear localization signal. PTS peroxisomal targeting signal. Scale bar 10 μm (TIFF 18,132 kb)

204_2017_1970_MOESM3_ESM.tif

Supplementary material S3 DAAO size-dependently enters the nucleus via passive diffusion in WKPT cells but is not actively exported. a Native PAGE and Western blot of EYFP constructs. b Quantification of relative nuclear intensity (RNI) of EYFP signal (intensitynucleus/intensitycell) in WKPT cells. Mean ± SEM, n = 5. One-way ANOVA + Tukey’s post-test, different colored bars indicate significant difference at p < 0.05. C) Confocal microscopy of transiently transfected WKPT cells expressing EYFP constructs. Nuclei are indicated by dashed lines. For letter code see b. h/rDAAO = EYFP-tagged human/rat DAAO. h/rCAT = EYFP-tagged human/rat catalase. NLS nuclear localization signal, PTS peroxisomal targeting signal, mCh mCherry-tag. Scale bar 10 μm (TIFF 26,528 kb)

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Luks, L., Maier, M.Y., Sacchi, S. et al. Understanding renal nuclear protein accumulation: an in vitro approach to explain an in vivo phenomenon. Arch Toxicol 91, 3599–3611 (2017). https://doi.org/10.1007/s00204-017-1970-5

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