The linear ubiquitin chain assembly complex LUBAC generates heterotypic ubiquitin chains

  1. Alan Rodriguez Carvajal
  2. Irina Grishkovskaya
  3. Carlos Gomez Diaz
  4. Antonia Vogel
  5. Adar Sonn-Segev
  6. Manish S Kushwah
  7. Katrin Schodl
  8. Luiza Deszcz
  9. Zsuzsanna Orban-Nemeth
  10. Shinji Sakamoto
  11. Karl Mechtler
  12. Philipp Kukura
  13. Tim Clausen
  14. David Haselbach  Is a corresponding author
  15. Fumiyo Ikeda  Is a corresponding author
  1. Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Austria
  2. Research Institute of Molecular Pathology, Austria
  3. University of Oxford, United Kingdom
  4. JT Inc., Japan
  5. Medical Institute of Bioregulation (MIB), Kyushu University, Japan

Abstract

The linear ubiquitin chain assembly complex (LUBAC) is the only known ubiquitin ligase for linear/Met1-linked ubiquitin chain formation. One of the LUBAC components, HOIL-1L, was recently shown to catalyse oxyester bond formation between ubiquitin and some substrates. However, oxyester bond formation in the context of LUBAC has not been directly observed. Here, we present the first 3D reconstruction of human LUBAC obtained by electron microscopy and report its generation of heterotypic ubiquitin chains containing linear linkages with oxyester-linked branches. We found that this event depends on HOIL-1L catalytic activity. By cross-linking mass spectrometry showing proximity between the catalytic RBR domains, a coordinated ubiquitin relay mechanism between the HOIP and HOIL-1L ligases is suggested. In mouse embryonic fibroblasts, these heterotypic chains were induced by TNF, which is reduced in cells expressing an HOIL-1L catalytic inactive mutant. In conclusion, we demonstrate that LUBAC assembles heterotypic ubiquitin chains by the concerted action of HOIP and HOIL-1L.

Data availability

All data besides the structural data and the mass spec data (see below) generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Supplementary Tables 1-3.wwPDB deposition with dataset ID: D_1292108794ProteomeXchange with identifier PXD019771.

Article and author information

Author details

  1. Alan Rodriguez Carvajal

    Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria
    Competing interests
    No competing interests declared.
  2. Irina Grishkovskaya

    Research Institute of Molecular Pathology, Vienna, Austria
    Competing interests
    No competing interests declared.
  3. Carlos Gomez Diaz

    Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6416-806X
  4. Antonia Vogel

    Research Institute of Molecular Pathology, Vienna, Austria
    Competing interests
    No competing interests declared.
  5. Adar Sonn-Segev

    Department of Chemistry, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
  6. Manish S Kushwah

    Department of Chemistry, University of Oxford, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
  7. Katrin Schodl

    Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria
    Competing interests
    No competing interests declared.
  8. Luiza Deszcz

    Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria
    Competing interests
    No competing interests declared.
  9. Zsuzsanna Orban-Nemeth

    Research Institute of Molecular Pathology, Vienna, Austria
    Competing interests
    No competing interests declared.
  10. Shinji Sakamoto

    Pharmaceutical Frontier Research Labs, JT Inc., Yokohama, Japan
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2480-2940
  11. Karl Mechtler

    Research Institute of Molecular Pathology, Vienna, Austria
    Competing interests
    No competing interests declared.
  12. Philipp Kukura

    Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford, United Kingdom
    Competing interests
    Philipp Kukura, academic founder, consultant, and shareholder in Refeyn Ltd..
  13. Tim Clausen

    Research Institute of Molecular Pathology, Vienna, Austria
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1582-6924
  14. David Haselbach

    Research Institute of Molecular Pathology, Vienna, Austria
    For correspondence
    david.haselbach@IMP.AC.AT
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5276-5633
  15. Fumiyo Ikeda

    Department of Molecular and Cellular Biology, Medical Institute of Bioregulation (MIB), Kyushu University, Fukuoka, Japan
    For correspondence
    fumiyo.ikeda@bioreg.kyushu-u.ac.jp
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0407-2768

Funding

Japan Society for the Promotion of Science (JP 18K19959)

  • Fumiyo Ikeda

Japan Society for the Promotion of Science (JP 21H04777)

  • Fumiyo Ikeda

Japan Society for the Promotion of Science (JP 21H00288)

  • Fumiyo Ikeda

Austrian Academy of Sciences

  • Fumiyo Ikeda

Boehringer Ingelheim

  • David Haselbach

FFG (Headquarter Grant 852936)

  • Tim Clausen

Boehringer Ingelheim

  • Tim Clausen

European Research Counsil (PHOTOMASS 819593)

  • Philipp Kukura

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

Ethics

Animal experimentation: All mice were bred and maintained in accordance with ethical animal license protocols complying with the Austrian and European legislation. Animal procedures were covered by the license 568809/2013/18.

Reviewing Editor

  1. Andreas Martin, University of California, Berkeley, United States

Version history

  1. Received: July 2, 2020
  2. Accepted: June 17, 2021
  3. Accepted Manuscript published: June 18, 2021 (version 1)
  4. Version of Record published: June 30, 2021 (version 2)

Copyright

© 2021, Rodriguez Carvajal 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

  • 4,620
    Page views
  • 520
    Downloads
  • 34
    Citations

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

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. Alan Rodriguez Carvajal
  2. Irina Grishkovskaya
  3. Carlos Gomez Diaz
  4. Antonia Vogel
  5. Adar Sonn-Segev
  6. Manish S Kushwah
  7. Katrin Schodl
  8. Luiza Deszcz
  9. Zsuzsanna Orban-Nemeth
  10. Shinji Sakamoto
  11. Karl Mechtler
  12. Philipp Kukura
  13. Tim Clausen
  14. David Haselbach
  15. Fumiyo Ikeda
(2021)
The linear ubiquitin chain assembly complex LUBAC generates heterotypic ubiquitin chains
eLife 10:e60660.
https://doi.org/10.7554/eLife.60660

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Sevim Kahraman, Kimitaka Shibue ... Rohit N Kulkarni
    Tools and Resources

    Pancreatic a-cells secrete glucagon, an insulin counter-regulatory peptide hormone critical for the maintenance of glucose homeostasis. Investigation of the function of human a-cells remains a challenge due to the lack of cost-effective purification methods to isolate high-quality a-cells from islets. Here, we use the reaction-based probe diacetylated Zinpyr1 (DA-ZP1) to introduce a novel and simple method for enriching live a-cells from dissociated human islet cells with ~ 95% purity. The a-cells, confirmed by sorting and immunostaining for glucagon, were cultured up to 10 days to form a-pseudoislets. The a-pseudoislets could be maintained in culture without significant loss of viability, and responded to glucose challenge by secreting appropriate levels of glucagon. RNA-sequencing analyses (RNA-seq) revealed that expression levels of key a-cell identity genes were sustained in culture while some of the genes such as DLK1, GSN, SMIM24 were altered in a-pseudoislets in a time-dependent manner. In conclusion, we report a method to sort human primary a-cells with high purity that can be used for downstream analyses such as functional and transcriptional studies.

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Valentin Chabert, Geun-Don Kim ... Andreas Mayer
    Research Article

    Eukaryotic cells control inorganic phosphate to balance its role as essential macronutrient with its negative bioenergetic impact on reactions liberating phosphate. Phosphate homeostasis depends on the conserved INPHORS signaling pathway that utilizes inositol pyrophosphates and SPX receptor domains. Since cells synthesize various inositol pyrophosphates and SPX domains bind them promiscuously, it is unclear whether a specific inositol pyrophosphate regulates SPX domains in vivo, or whether multiple inositol pyrophosphates act as a pool. In contrast to previous models, which postulated that phosphate starvation is signaled by increased production of the inositol pyrophosphate 1-IP7, we now show that the levels of all detectable inositol pyrophosphates of yeast, 1-IP7, 5-IP7, and 1,5-IP8, strongly decline upon phosphate starvation. Among these, specifically the decline of 1,5-IP8 triggers the transcriptional phosphate starvation response, the PHO pathway. 1,5-IP8 inactivates the cyclin-dependent kinase inhibitor Pho81 through its SPX domain. This stimulates the cyclin-dependent kinase Pho85-Pho80 to phosphorylate the transcription factor Pho4 and repress the PHO pathway. Combining our results with observations from other systems, we propose a unified model where 1,5-IP8 signals cytosolic phosphate abundance to SPX proteins in fungi, plants, and mammals. Its absence triggers starvation responses.