Methods for measuring HMGB1 release during immunogenic cell death

Liwei Zhao, Peng Liu, Oliver Kepp, Guido Kroemer

    Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

    10 Citations (Scopus)

    Abstract

    The exodus of the alarmin high mobility group box 1 (HMGB1) from the nucleus constitutes a crucial cellular danger signal and manifests as a sequential process in which HMGB1 first exits the nucleus into the cytoplasm and then is secreted or passively released through the permeabilized plasma membrane. Extracellular HMGB1 can interact with pattern recognition receptors to stimulate innate immune responses. Here, we describe a discovery pipeline for the identification of pharmacological agents endowed with HMGB1 releasing properties. The “retention using selective hooks” (RUSH) system in which a streptavidin-NLS3 fusion protein serves as a nuclear hook to sequester streptavidin-binding peptide (SBP) fused with HMGB1 and green fluorescent protein (GFP) allowed for synchronizing HMGB1 increase. Thus, exclusively in the presence of biotin, which liberates HMGB1-SBP-GFP from its nuclear hook, immunogenic cell death (ICD) inducers such as anthracyclines are able to cause the nucleo-cytoplasmic translocation of HMGB1-SBP-GFP. This system facilitates the identification of HMGB1 releasing agents in medium- to high-throughput screening assays.

    Original languageEnglish
    Title of host publicationTumor Immunology and Immunotherapy - Molecular Methods
    EditorsLorenzo Galluzzi, Nils-Petter Rudqvist
    PublisherAcademic Press Inc.
    Pages177-193
    Number of pages17
    ISBN (Print)9780128186718
    DOIs
    Publication statusPublished - 1 Jan 2019

    Publication series

    NameMethods in Enzymology
    Volume629
    ISSN (Print)0076-6879
    ISSN (Electronic)1557-7988

    Keywords

    • Anthracyclines
    • Cancer
    • High-throughput screening
    • Immunogenic cell death (ICD)
    • Retention using selective hooks (RUSH) assay

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