Identification of pharmacological inhibitors of conventional protein secretion

Liwei Zhao,Oliver Kepp,Gaelle Boncompain,Peng Liu,Guido Kroemer,Sylvie Lachkar,Lucillia Bezu,Franck Perez,Guo Chen,Friedemann Loos,Heng Zhou

doi:10.1038/s41598-018-33378-y

Abstract

The retention using selective hooks (RUSH) system allows to withhold a fluorescent biosensor such as green fluorescent protein (GFP) fused to a streptavidin-binding peptide (SBP) by an excess of streptavidin molecules that are addressed to different subcellular localizations. Addition of biotin competitively disrupts this interaction, liberating the biosensor from its hook. We constructed a human cell line co-expressing soluble secretory-SBP-GFP (ss-SBP-GFP) and streptavidin within the endoplasmic reticulum (ER) lumen and then used this system to screen a compound library for inhibitors of the biotin-induced release of ss-SBP-GFP via the conventional Golgi-dependent protein secretion pathway into the culture supernatant. We identified and validated a series of molecularly unrelated drugs including antianginal, antidepressant, anthelmintic, antipsychotic, antiprotozoal and immunosuppressive agents that inhibit protein secretion. These compounds vary in their capacity to suppress protein synthesis and to compromise ER morphology and Golgi integrity, as well as in the degree of reversibility of such effects. In sum, we demonstrate the feasibility and utility of a novel RUSH-based phenotypic screening assay.

Highlights

Fluorescence based biosensors introduced into the cells may be observed by microscopy to detect their abundance as well as their subcellular distribution to obtain morphological information that can be subjected to high-content image analysis[1,2]
The hook consists in a streptavidin molecule that can be addressed to virtually any subcellular compartment by means of suitable targeting moieties, causing the protein for instance to be withhold in the endoplasmic reticulum (ER) lumen or to be located in other organelles including the Golgi apparatus (GA), mitochondria or the nucleus[11,12,13]
Upon addition of excess biotin to the cells, the green fluorescent protein (GFP) fluorescence gradually distributed toward a perinuclear cap, presumably the GA, followed by a strong reduction of the fluorescent signal that was completed around 4 h after the addition of biotin (Fig. 1B)

Summary

Introduction

Fluorescence based biosensors introduced into the cells may be observed by (video-) microscopy to detect their abundance as well as their subcellular distribution to obtain morphological information that can be subjected to high-content image analysis[1,2]. The biosensor is usually composed of a green fluorescent protein (GFP) - streptavidin binding peptide (SBP) module fused to a secretory cargo such as E-Cadherin, TNF or EGFR. The addition of biotin triggers the close-to-immediate, synchronized release of the SBP-GFP tagged biosensor from streptavidin, allowing the fluorescent cargo to move to another subcellular localization[11]. We have used this system in the past to study the unconventional secretion of the chromatin-binding non-histone protein HMGB1 (high mobility group B1), namely by generating an SBP-GFP-HMGB1 fusion protein that was retained by streptavidin in the nucleus (because streptavidin was fused with several nuclear localization sequences and imported in this organelle). We reported in[14] that the RUSH assay can be adapted to high content screening and we show here that it can efficiently be used to identify and characterize novel inhibitors of conventional protein secretion

Methods

Results

Conclusion