Cycles of autoubiquitination and deubiquitination regulate the ERAD ubiquitin ligase Hrd1

  1. Brian G Peterson
  2. Morgan L Glaser
  3. Tom A Rapoport
  4. Ryan D Baldridge  Is a corresponding author
  1. University of Michigan Medical School, United States
  2. Harvard Medical School, United States

Abstract

Misfolded proteins in the lumen of the endoplasmic reticulum (ER) are retrotranslocated into the cytosol and polyubiquitinated before being degraded by the proteasome. The multi-spanning ubiquitin ligase Hrd1 forms the retrotranslocation channel and associates with three other membrane proteins (Hrd3, Usa1, Der1) of poorly defined function. The Hrd1 channel is gated by autoubiquitination, but how Hrd1 escapes degradation by the proteasome and returns to its inactive ground state is unknown. Here, we show that autoubiquitination of Hrd1 is counteracted by Ubp1, a deubiquitinating enzyme that requires its N-terminal transmembrane segment for activity towards Hrd1. The Hrd1 partner Hrd3 serves as a brake for autoubiquitination, while Usa1 attenuates Ubp1's deubiquitination activity through an inhibitory effect of its UBL domain. These results lead to a model in which the Hrd1 channel is regulated by cycles of autoubiquitination and deubiquitination, reactions that are modulated by the other components of the Hrd1 complex.

Data availability

Data generated or analysed during this study are included in the manuscript.

Article and author information

Author details

  1. Brian G Peterson

    Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6871-2336
  2. Morgan L Glaser

    Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Tom A Rapoport

    Department of Cell Biology, Harvard Medical School, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9911-4216
  4. Ryan D Baldridge

    Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, United States
    For correspondence
    ryanbald@med.umich.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7158-7812

Funding

National Institute of General Medical Sciences (R35GM128592)

  • Ryan D Baldridge

National Institute of General Medical Sciences (R01GM052586)

  • Tom A Rapoport

Damon Runyon Cancer Research Foundation (DRG-2184-14)

  • Ryan D Baldridge

Damon Runyon Cancer Research Foundation (DFS-26-18)

  • Ryan D Baldridge

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

Reviewing Editor

  1. Ramanujan S Hegde, MRC Laboratory of Molecular Biology, United Kingdom

Publication history

  1. Received: August 6, 2019
  2. Accepted: November 11, 2019
  3. Accepted Manuscript published: November 12, 2019 (version 1)
  4. Version of Record published: December 16, 2019 (version 2)

Copyright

© 2019, Peterson 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.

Metrics

  • 2,827
    Page views
  • 497
    Downloads
  • 21
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Brian G Peterson
  2. Morgan L Glaser
  3. Tom A Rapoport
  4. Ryan D Baldridge
(2019)
Cycles of autoubiquitination and deubiquitination regulate the ERAD ubiquitin ligase Hrd1
eLife 8:e50903.
https://doi.org/10.7554/eLife.50903

Further reading

    1. Biochemistry and Chemical Biology
    Holly Y Chen, Manju Swaroop ... Anand Swaroop
    Research Article

    Ciliopathies manifest from sensory abnormalities to syndromic disorders with multi-organ pathologies, with retinal degeneration a highly penetrant phenotype. Photoreceptor cell death is a major cause of incurable blindness in retinal ciliopathies. To identify drug candidates to maintain photoreceptor survival, we performed an unbiased, high-throughput screening of over 6,000 bioactive small molecules using retinal organoids differentiated from induced pluripotent stem cells (iPSC) of rd16 mouse, which is a model of Leber congenital amaurosis (LCA) type 10 caused by mutations in the cilia-centrosomal gene CEP290. We identified five non-toxic positive hits, including the lead molecule reserpine, which maintained photoreceptor development and survival in rd16 organoids. Reserpine also improved photoreceptors in retinal organoids derived from induced pluripotent stem cells of LCA10 patients and in rd16 mouse retina in vivo. Reserpine-treated patient organoids revealed modulation of signaling pathways related to cell survival/death, metabolism, and proteostasis. Further investigation uncovered dysregulation of autophagy associated with compromised primary cilium biogenesis in patient organoids and rd16 mouse retina. Reserpine partially restored the balance between autophagy and the ubiquitin-proteasome system at least in part by increasing the cargo adaptor p62, resulting in improved primary cilium assembly. Our study identifies effective drug candidates in preclinical studies of CEP290 retinal ciliopathies through cross-species drug discovery using iPSC-derived organoids, highlights the impact of proteostasis in the pathogenesis of ciliopathies, and provides new insights for treatments of retinal neurodegeneration.

    1. Biochemistry and Chemical Biology
    Lisa Goebel, Tonia Kirschner ... Daniel Rauh
    Short Report

    Mutations within Ras proteins represent major drivers in human cancer. In this study, we report the structure-based design, synthesis, as well as biochemical and cellular evaluation of nucleotide-based covalent inhibitors for KRasG13C, an important oncogenic mutant of Ras that has not been successfully addressed in the past. Mass spectrometry experiments and kinetic studies reveal promising molecular properties of these covalent inhibitors, and X-ray crystallographic analysis has yielded the first reported crystal structures of KRasG13C covalently locked with these GDP analogues. Importantly, KRasG13C covalently modified with these inhibitors can no longer undergo SOS-catalysed nucleotide exchange. As a final proof-of-concept, we show that in contrast to KRasG13C, the covalently locked protein is unable to induce oncogenic signalling in cells, further highlighting the possibility of using nucleotide-based inhibitors with covalent warheads in KRasG13C-driven cancer.