Identification of pharmacological agents that induce HMGB1 release

Peng Liu,Heng Zhou,Liwei Zhao,Gaelle Boncompain,Oliver Kepp,Franck Perez,Guido Kroemer,Lucillia Bezu,Guo Chen,Kristina Iribarren,Laurence Zitvogel,Lígia C Gomes-Da-Silva,Sylvie Lachkar,Friedemann Loos

doi:10.1038/s41598-017-14848-1

Abstract

The translocation of the protein high mobility group box 1 (HMGB1) from the nucleus to the cytoplasm and its secretion or passive release through the permeabilized plasma membrane, constitutes a major cellular danger signal. Extracellular HMGB1 can interact with pattern recognition receptors to stimulate pro-inflammatory and immunostimulatory pathways. Here, we developed a screening assay to identify pharmacological agents endowed with HMGB1 releasing properties. For this, we took advantage of the “retention using selective hooks” (RUSH) system in which a streptavidin-NLS3 fusion protein was used as a nuclear hook to sequestrate streptavidin-binding peptide (SBP) fused with HMGB1 and green fluorescent protein (GFP). When combined with biotin, which competitively disrupts the interaction between streptavidin-NLS3 and HMGB1-SBP-GFP, immunogenic cell death (ICD) inducers such as anthracyclines were able to cause the nucleo-cytoplasmic translocation of HMGB1-SBP-GFP. This system, was used in a high-content screening (HCS) campaign for the identification of HMGB1 releasing agents. Hits fell into three functional categories: known ICD inducers, microtubule inhibitors and epigenetic modifiers. These agents induced ICD through a panoply of distinct mechanisms. Their effective action was confirmed by multiple methods monitoring nuclear, cytoplasmic and extracellular HMGB1 pools, both in cultured human or murine cells, as well as in mouse plasma.

Highlights

High mobility group box 1 (HMGB1) is a protein that is normally localized in the nucleus, where it is the most abundant non-histone chromatin-binding protein
The protein of interest is fused to a streptavidin-binding peptide (SBP) as well as a green fluorescent protein (GFP) to facilitate monitoring of its subcellular localization by fluorescence videomicroscopy. Such a construct is stably expressed in cells together with streptavidin that is targeted towards a specific subcellular compartment, here to the nucleus by means of three nuclear localization sequences (Str-NLS3) motif (Fig. S1)
Biotin has a subnanomolar affinity to streptavidin and can outcompete its binding to SBP, causing rapid release of the high mobility group box 1 (HMGB1)-SBP-GFP fusion protein from its interaction with streptavidin-NLS3

Summary

Introduction

High mobility group box 1 (HMGB1) is a protein that is normally localized in the nucleus, where it is the most abundant non-histone chromatin-binding protein. Under homeostatic conditions HMGB1 bidirectionally shuttles between the cytoplasm and the nucleus, yet predominantly resides in the nucleus due to two nuclear localization and two non-classical nuclear export signals[1,2]. JAK-STAT-dependent lysine hyperacetylation within the NLS sites blocks nuclear re-import and leads to a cytoplasmic aggregation of HMGB14, as it occurs in monocytes responding to inflammatory signals including lipopolysaccharide (LPS) and tumor necrosis factor (TNF)[5]. HMGB1 binds to chromatin and changes the architecture of the DNA, thereby enhancing transcription and replication from chromatin templates. HMGB1 contributes to danger signaling in a variety of contexts, thereby exerting pro-inflammatory and immunostimulatory effects

Methods

Results

Conclusion