A single domain antibody fragment that recognizes the adaptor ASC defines the role of ASC domains in inflammasome assembly

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Abstract

Ploegh et al. raised an alpaca single-domain antibody (VHH) against the inflammasome adaptor ASC. VHH _ASC blocks inflammasome activation in vitro and in living cells, and demonstrates a role of the ASC CARD domain in cross-linking ASC Pyrin domain filaments.

Abstract

Myeloid cells assemble inflammasomes in response to infection or cell damage; cytosolic sensors activate pro–caspase-1, indirectly for the most part, via the adaptors ASC and NLRC4. This leads to secretion of proinflammatory cytokines and pyroptosis. To explore complex formation under physiological conditions, we generated an alpaca single domain antibody, VHH _ASC, which specifically recognizes the CARD of human ASC via its type II interface. VHH _ASC not only impairs ASC ^CARD interactions in vitro , but also inhibits inflammasome activation in response to NLRP3, AIM2, and NAIP triggers when expressed in living cells, highlighting a role of ASC in all three types of inflammasomes. VHH _ASC leaves the Pyrin domain of ASC functional and stabilizes a filamentous intermediate of inflammasome activation. Incorporation of VHH _ASC-EGFP into these structures allowed the visualization of endogenous ASC ^PYD filaments for the first time. These data revealed that cross-linking of ASC ^PYD filaments via ASC ^CARD mediates the assembly of ASC foci.

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Quantitative analysis of HSP90-client interactions reveals principles of substrate recognition.

Mikko Taipale, Irina Krykbaeva, Martina Koeva … (2012)

HSP90 is a molecular chaperone that associates with numerous substrate proteins called clients. It plays many important roles in human biology and medicine, but determinants of client recognition by HSP90 have remained frustratingly elusive. We systematically and quantitatively surveyed most human kinases, transcription factors, and E3 ligases for interaction with HSP90 and its cochaperone CDC37. Unexpectedly, many more kinases than transcription factors bound HSP90. CDC37 interacted with kinases, but not with transcription factors or E3 ligases. HSP90::kinase interactions varied continuously over a 100-fold range and provided a platform to study client protein recognition. In wild-type clients, HSP90 did not bind particular sequence motifs, but rather associated with intrinsically unstable kinases. Stabilization of the kinase in either its active or inactive conformation with diverse small molecules decreased HSP90 association. Our results establish HSP90 client recognition as a combinatorial process: CDC37 provides recognition of the kinase family, whereas thermodynamic parameters determine client binding within the family. Copyright © 2012 Elsevier Inc. All rights reserved.

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Modulation of protein properties in living cells using nanobodies.

Axel Kirchhofer, Jonas Helma, Katrin Schmidthals … (2010)

Protein conformation is critically linked to function and often controlled by interactions with regulatory factors. Here we report the selection of camelid-derived single-domain antibodies (nanobodies) that modulate the conformation and spectral properties of the green fluorescent protein (GFP). One nanobody could reversibly reduce GFP fluorescence by a factor of 5, whereas its displacement by a second nanobody caused an increase by a factor of 10. Structural analysis of GFP-nanobody complexes revealed that the two nanobodies induce subtle opposing changes in the chromophore environment, leading to altered absorption properties. Unlike conventional antibodies, the small, stable nanobodies are functional in living cells. Nanobody-induced changes were detected by ratio imaging and used to monitor protein expression and subcellular localization as well as translocation events such as the tamoxifen-induced nuclear localization of estrogen receptor. This work demonstrates that protein conformations can be manipulated and studied with nanobodies in living cells.

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Redundant roles for inflammasome receptors NLRP3 and NLRC4 in host defense against Salmonella

Petr Broz, Kim Newton, Mohamed Lamkanfi … (2010)

Intracellular pathogens and endogenous danger signals in the cytosol engage NOD-like receptors (NLRs), which assemble inflammasome complexes to activate caspase-1 and promote the release of proinflammatory cytokines IL-1β and IL-18. However, the NLRs that respond to microbial pathogens in vivo are poorly defined. We show that the NLRs NLRP3 and NLRC4 both activate caspase-1 in response to Salmonella typhimurium. Responding to distinct bacterial triggers, NLRP3 and NLRC4 recruited ASC and caspase-1 into a single cytoplasmic focus, which served as the site of pro–IL-1β processing. Consistent with an important role for both NLRP3 and NLRC4 in innate immune defense against S. typhimurium, mice lacking both NLRs were markedly more susceptible to infection. These results reveal unexpected redundancy among NLRs in host defense against intracellular pathogens in vivo.

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Author and article information

Journal

Journal ID (nlm-ta): J Exp Med

Journal ID (iso-abbrev): J. Exp. Med

Journal ID (publisher-id): jem

Journal ID (hwp): jem

Title: The Journal of Experimental Medicine

Publisher: The Rockefeller University Press

ISSN (Print): 0022-1007

ISSN (Electronic): 1540-9538

Publication date (Print): 2 May 2016

Volume: 213

Issue: 5

Pages: 771-790

Affiliations

[1 ]Whitehead Institute for Biomedical Research, Cambridge, MA 02142

[2 ]Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115

[3 ]Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115

[4 ]Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139

Author notes

Correspondence to Hao Wu: wu@ 123456crystal.harvard.edu ; or Hidde L. Ploegh: ploegh@ 123456wi.mit.edu

[*]

F.I. Schmidt and A. Lu contributed equally to this paper.

Article

Publisher ID: 20151790

DOI: 10.1084/jem.20151790

PMC ID: 4854733

PubMed ID: 27069117

SO-VID: ef2d0725-2a02-4460-aa64-c054386c5215

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This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

History

Date received : 13 November 2015

Date accepted : 01 March 2016

Funding

Funded by: National Institutes of Health http://dx.doi.org/10.13039/100000002

Funded by: Swiss National Science Foundation http://dx.doi.org/10.13039/501100001711

A single domain antibody fragment that recognizes the adaptor ASC defines the role of ASC domains in inflammasome assembly

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Most cited references 31

Quantitative analysis of HSP90-client interactions reveals principles of substrate recognition.

Modulation of protein properties in living cells using nanobodies.

Redundant roles for inflammasome receptors NLRP3 and NLRC4 in host defense against Salmonella

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