Identification of ubiquitin Ser57 kinases regulating the oxidative stress response in yeast

Abstract

Ubiquitination regulates many different cellular processes, including protein quality control, membrane trafficking, and stress responses. The diversity of ubiquitin functions in the cell is partly due to its ability to form chains with distinct linkages that can alter the fate of substrate proteins in unique ways. The complexity of the ubiquitin code is further enhanced by post-translational modifications on ubiquitin itself, the biological functions of which are not well understood. Here, we present genetic and biochemical evidence that serine 57 (Ser57) phosphorylation of ubiquitin functions in stress responses in Saccharomyces cerevisiae, including the oxidative stress response. We also identify and characterize the first known Ser57 ubiquitin kinases in yeast and human cells, and we report that two Ser57 ubiquitin kinases regulate the oxidative stress response in yeast. These studies implicate ubiquitin phosphorylation at the Ser57 position as an important modifier of ubiquitin function, particularly in response to proteotoxic stress.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. Nathaniel L Hepowit

    Department of Cell and Developmental Biology, Vanderbilt University, Nashville, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Kevin N Pereira

    Department of Cell and Developmental Biology, Vanderbilt University, Nashville, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Jessica M Tumolo

    Department of Cell and Developmental Biology, Vanderbilt University, Nashville, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Walter J Chazin

    Biochemistry, Chemistry, Vanderbilt University, Nashville, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2180-0790
  5. Jason A MacGurn

    Department of Cell and Developmental Biology, Vanderbilt University, Nashville, United States
    For correspondence
    jason.a.macgurn@vanderbilt.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5063-259X

Funding

National Institutes of Health (R21 AG053562)

  • Jason A MacGurn

National Institutes of Health (R01 GM118491)

  • Jason A MacGurn

National Institutes of Health (R35 GM118089)

  • Walter J Chazin

National Institutes of Health (T32 CA119925)

  • Jessica M Tumolo

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Benoît Kornmann, University of Oxford, United Kingdom

Publication history

  1. Received: April 22, 2020
  2. Accepted: October 18, 2020
  3. Accepted Manuscript published: October 19, 2020 (version 1)
  4. Version of Record published: November 6, 2020 (version 2)

Copyright

© 2020, Hepowit et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Nathaniel L Hepowit
  2. Kevin N Pereira
  3. Jessica M Tumolo
  4. Walter J Chazin
  5. Jason A MacGurn
(2020)
Identification of ubiquitin Ser57 kinases regulating the oxidative stress response in yeast
eLife 9:e58155.
https://doi.org/10.7554/eLife.58155

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    Patients with cardiomyopathy of Duchenne Muscular Dystrophy (DMD) are at risk of developing life-threatening arrhythmias, but the mechanisms are unknown. We aimed to determine the role of ion channels controlling cardiac excitability in the mechanisms of arrhythmias in DMD patients.

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    To test whether dystrophin mutations lead to defective cardiac NaV1.5–Kir2.1 channelosomes and arrhythmias, we generated iPSC-CMs from two hemizygous DMD males, a heterozygous female, and two unrelated control males. We conducted studies including confocal microscopy, protein expression analysis, patch-clamping, non-viral piggy-bac gene expression, optical mapping and contractility assays.

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    We provide the first demonstration that iPSC-CMs reprogrammed from skin fibroblasts of DMD patients with cardiomyopathy have a dysfunction of the NaV1.5–Kir2.1 channelosome, with consequent reduction of cardiac excitability and conduction. Altogether, iPSC-CMs from patients with DMD cardiomyopathy have a NaV1.5–Kir2.1 channelosome dysfunction, which can be rescued by the scaffolding protein α1-syntrophin to restore excitability and prevent arrhythmias.

    Funding:

    Supported by National Institutes of Health R01 HL122352 grant; ‘la Caixa’ Banking Foundation (HR18-00304); Fundación La Marató TV3: Ayudas a la investigación en enfermedades raras 2020 (LA MARATO-2020); Instituto de Salud Carlos III/FEDER/FSE; Horizon 2020 - Research and Innovation Framework Programme GA-965286 to JJ; the CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation), and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). American Heart Association postdoctoral fellowship 19POST34380706s to JVEN. Israel Science Foundation to OB and MA [824/19]. Rappaport grant [01012020RI]; and Niedersachsen Foundation [ZN3452] to OB; US-Israel Binational Science Foundation (BSF) to OB and TH [2019039]; Dr. Bernard Lublin Donation to OB; and The Duchenne Parent Project Netherlands (DPPNL 2029771) to OB. National Institutes of Health R01 AR068428 to DM and US-Israel Binational Science Foundation Grant [2013032] to DM and OB.