Skip to main content
Advertisement

Main menu

  • Home
  • Articles
    • Newest Articles
    • Current Issue
    • Methods & Resources
    • Archive
    • Subjects
  • Collections
  • Submit
    • Submit a Manuscript
    • Author Guidelines
    • License, Copyright, Fee
    • FAQ
    • Why Submit
  • About
    • About Us
    • Editors & Staff
    • Board Members
    • Licensing and Reuse
    • Reviewer Guidelines
    • Privacy Policy
    • Advertise
    • Contact Us
    • LSA LLC
  • Alerts
  • Other Publications
    • EMBO Press
    • The EMBO Journal
    • EMBO reports
    • EMBO Molecular Medicine
    • Molecular Systems Biology
    • Rockefeller University Press
    • Journal of Cell Biology
    • Journal of Experimental Medicine
    • Journal of General Physiology
    • Cold Spring Harbor Laboratory Press
    • Genes & Development
    • Genome Research

User menu

  • My alerts

Search

  • Advanced search
Life Science Alliance
  • Other Publications
    • EMBO Press
    • The EMBO Journal
    • EMBO reports
    • EMBO Molecular Medicine
    • Molecular Systems Biology
    • Rockefeller University Press
    • Journal of Cell Biology
    • Journal of Experimental Medicine
    • Journal of General Physiology
    • Cold Spring Harbor Laboratory Press
    • Genes & Development
    • Genome Research
  • My alerts
Life Science Alliance

Advanced Search

  • Home
  • Articles
    • Newest Articles
    • Current Issue
    • Methods & Resources
    • Archive
    • Subjects
  • Collections
  • Submit
    • Submit a Manuscript
    • Author Guidelines
    • License, Copyright, Fee
    • FAQ
    • Why Submit
  • About
    • About Us
    • Editors & Staff
    • Board Members
    • Licensing and Reuse
    • Reviewer Guidelines
    • Privacy Policy
    • Advertise
    • Contact Us
    • LSA LLC
  • Alerts
  • Follow lsa Template on Twitter
Research Article
Transparent Process
Open Access

Dimerization and auto-processing induce caspase-11 protease activation within the non-canonical inflammasome

Connie Ross, Amy H Chan, Jessica Von Pein, Dave Boucher, View ORCID ProfileKate Schroder  Correspondence email
Connie Ross
Institute for Molecular Bioscience (IMB), IMB Centre for Inflammation and Disease Research, The University of Queensland, St Lucia, Australia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Amy H Chan
Institute for Molecular Bioscience (IMB), IMB Centre for Inflammation and Disease Research, The University of Queensland, St Lucia, Australia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jessica Von Pein
Institute for Molecular Bioscience (IMB), IMB Centre for Inflammation and Disease Research, The University of Queensland, St Lucia, Australia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Dave Boucher
Institute for Molecular Bioscience (IMB), IMB Centre for Inflammation and Disease Research, The University of Queensland, St Lucia, Australia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Kate Schroder
Institute for Molecular Bioscience (IMB), IMB Centre for Inflammation and Disease Research, The University of Queensland, St Lucia, Australia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Kate Schroder
  • For correspondence: K.Schroder@imb.uq.edu.au
Published 6 December 2018. DOI: 10.26508/lsa.201800237
  • Article
  • Figures & Data
  • Info
  • Metrics
  • Reviewer Comments
  • PDF
Loading

Article Figures & Data

Figures

  • Figure 1.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 1. LPS-induced caspase-11 processing is independent of the NLRP3 and caspase-1 inflammasome.

    (A) Domain structure of caspase-11 showing potential caspase cleavage sites, the CDL, IDL and the catalytic cysteine (C254), and the relative predicted molecular weights of caspase-11 fragments. (B–E) BMMs were primed for 12 h with Pam3CSK4 (1 μg/ml) and then transfected with ultrapure K12 E. coli LPS (10 μg/ml) using FuGene HD. MCC950 (10 μM) was added to cells 30 min before transfection. Supernatants and cell extracts were collected at 8 h post-transfection, or over a time course as indicated. (B) Cell death was assessed by quantifying lactate dehydrogenase (LDH) release into the culture medium, compared with a full lysis (Triton X100) control. (C) Secretion of mature IL-1β into the culture medium was assessed by ELISA. Data in (B–C) are the mean + SEM of three biological replicates, and significance was assessed by two-way ANOVA using the Pam3CSK4+LPS transfection sample as a reference. (D) WT BMM or (E) WT versus caspase-1 enzyme-dead (C284A) BMM were analysed by immunoblot of the cell culture medium (SUP) and cell extracts (XT). Western blots are representative of three biological replicate experiments.

  • Figure 2.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 2. Caspase-11 requires IDL but not CDL processing for inducing cell death and IL-1β release.

    Caspase-11 WT, catalytic mutant (C254A), CDL mutant (CDLuncl), or IDL mutant (IDLuncl) were retrovirally expressed in Casp11−/− BMM. Cells were primed for 12 h with Pam3CSK4 or 4 h with LPS, and transfected with ultrapure K12 E. coli LPS 10 μg/ml for 6 h or exposed to 5 μM nigericin for 2 h. (A) Western blot assessed expression of caspase-11 mutants in cell extracts of Pam3CSK4-primed, untransfected BMM. (B) Immunoblot detected mature IL-1β and the caspase-11 or caspase-1 large subunits in the culture medium (SUP) and cell extracts (XT) of Pam3CSK4-primed BMMs transfected with LPS for 6 h. (C) Cell death and (D) IL-1β secretion was assessed 6 h after LPS transfection or 2 h after nigericin exposure. Western blots are representative of three biological replicate experiments. Graphs are mean + SEM of four biological replicate experiments, with significance assessed using a Mann–Whitney test.

  • Figure 3.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 3. Caspase-11 dimerization is necessary and sufficient for auto-cleavage to the p32/p10 species, and protease activity.

    HEK293T cells were transiently transfected with pEF6-DmrB-Caspase-11 constructs depicted in (A) to mimic the p43 dimers and p32/p10 species of caspase-11 (actual predicted molecular weights of DmrB fusions are 46 kD and 35/10 kD, respectively). Transfected HEK293T cells were pre-incubated with the dimerizer drug, AP20187, for 30 min before substrate addition. (B) Cleavage of AcLEHD-afc by DmrB-Caspase-11 in digitonin-lysed HEK293T cells was monitored over 30 min. Data are mean of technical quadruplicates, and are representative of at least three biological replicate experiments. (C) HEK293T cells expressing DmrB-Caspase-11 were incubated with AP20187 for 30 min to induce dimerization. Cells were lysed with digitonin, and incubated with lysates from HEK293T expressing V5-GSDMD for 1 h at 37°C. Samples were precipitated and analysed using an immunoblot. Data are representative of three biological replicate experiments.

  • Figure 4.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 4. The MEA/D (D285) cleavage site within the IDL is critical for caspase-11 auto-processing and full protease activity.

    HEK293T cells were transfected with either DmrB alone (empty vector, EV) or DmrB-caspase-11 mutants: WT, C254A (catalytic mutant), IDLuncl (IDL triple cleavage mutant; E266A/D277A/D285A), and single IDL mutants: E266A, D277A, and D285A. Cells were exposed to 500 nM AP20187 for 30 min and lysed with digitonin for quantification of proteolytic activity, either by (A) kinetics of AcLEHD-afc cleavage over 20 min, or (B) reaction rate (upper), and incubation with full-length GSDMD-V5 to assess the extent of cleavage to p30 after 1 h (lower). Data in (B upper) are mean + SEM of four biological replicates. Data were analysed for normality using the Shapiro–Wilk normality test, and tested for significance using parametric paired t tests (two-sided). All other data are representative of at least three biological replicates.

  • Figure 5.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 5. Cleavage of the IDL in trans promotes caspase-11 cleavage of GSDMD and AcLEHD-afc.

    HEK293T cells were transiently transfected with constructs containing DmrB-Caspase-11, WT, C254A, and IDLthr, in which a thrombin cleavage site replaces the caspase-11 IDL auto-processing site, as depicted in (A), to allow generation of unprocessed dimers (analogous to caspase-11 p43; actual predicted weight for the DmrB-caspase-11 fusion, ∼46 kD) and IDL-cleaved dimers (analogous to caspase-11 p32/p10, actual predicted weight 35/10 kD). (B) Cells were incubated with AP20187 (500 nM) for 30 min, and then AcLEHD-afc cleavage was measured with and without the addition of thrombin (20 U/ml) to the reaction. Data are mean + SEM of four biological replicates. Data were analysed for normality using the Shapiro–Wilk normality test, and tested for significance using parametric paired t tests (two-sided). (C) AcLEHD-afc kinetic trace of AP20187-treated cells expressing DmrB-Caspase-11 WT versus IDLthr, in the presence and absence of thrombin (20 U/ml) in the reaction. (D) HEK293T expressing the DmrB-Caspase-11 constructs were exposed to AP20187 for 30 min, and then lysed and incubated for 15 min with thrombin (20 U/ml) before the addition of V5-GSDMD for 1 h. (E) Model for LPS-induced caspase-11 dimerization, auto-processing, and activation.

  • Figure S1.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure S1. The D59 and D80 residues of caspase-11 are buried within α helices of the CARD domain.

    A homology model of the caspase-11 CARD domain and its linker sequence was generated using RaptorX. Two putative cleavage sites, D59 (red) and D80 (orange), are highlighted.

PreviousNext
Back to top
Download PDF
Article Alerts
Sign In to Email Alerts with your Email Address
Email Article

Thank you for your interest in spreading the word on Life Science Alliance.

NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

Enter multiple addresses on separate lines or separate them with commas.
Dimerization and auto-processing induce caspase-11 protease activation within the non-canonical inflammasome
(Your Name) has sent you a message from Life Science Alliance
(Your Name) thought you would like to see the Life Science Alliance web site.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Citation Tools
Caspase-11 is activated by dimerization and auto-cleavage
Connie Ross, Amy H Chan, Jessica Von Pein, Dave Boucher, Kate Schroder
Life Science Alliance Dec 2018, 1 (6) e201800237; DOI: 10.26508/lsa.201800237

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Share
Caspase-11 is activated by dimerization and auto-cleavage
Connie Ross, Amy H Chan, Jessica Von Pein, Dave Boucher, Kate Schroder
Life Science Alliance Dec 2018, 1 (6) e201800237; DOI: 10.26508/lsa.201800237
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Tweet Widget
  • Facebook Like
Issue Cover

In this Issue

Volume 1, No. 6
December 2018
  • Table of Contents
  • Cover (PDF)
  • About the Cover
  • Masthead (PDF)
Advertisement

Jump to section

  • Article
    • Abstract
    • Introduction
    • Results
    • Discussion
    • Materials and Methods
    • Acknowledgments
    • References
  • Figures & Data
  • Info
  • Metrics
  • Reviewer Comments
  • PDF

Subjects

  • Molecular Biology
  • Microbiology, Virology & Host Pathogen Interaction
  • Immunology

Related Articles

  • No related articles found.

Cited By...

  • Function and Regulation of Noncanonical Caspase-4/5/11 Inflammasome
  • Recent Insights on Inflammasomes, Gasdermin Pores, and Pyroptosis
  • Caspase-1 interdomain linker cleavage is required for pyroptosis
  • Cytokines Focus: Inflammasome signaling and regulation of interleukin-1 family cytokines
  • Human caspase-1 autoproteolysis is required for ASC-dependent and -independent inflammasome activation
  • Google Scholar

More in this TOC Section

  • Structure of mEAK-7 bound to V-ATPase
  • MD-related proteins at the centrosome
  • Prion-like α-synuclein toxicity via proteoglycans in vivo
Show more Research Article

Similar Articles

EMBO Press LogoRockefeller University Press LogoCold Spring Harbor Logo

Content

  • Home
  • Newest Articles
  • Current Issue
  • Archive
  • Subject Collections

For Authors

  • Submit a Manuscript
  • Author Guidelines
  • License, copyright, Fee

Other Services

  • Alerts
  • Twitter
  • RSS Feeds

More Information

  • Editors & Staff
  • Reviewer Guidelines
  • Feedback
  • Licensing and Reuse
  • Privacy Policy

ISSN: 2575-1077
© 2022 Life Science Alliance LLC

Life Science Alliance is registered as a trademark in the U.S. Patent and Trade Mark Office and in the European Union Intellectual Property Office.