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High-Throughput Quantification of Surface Protein Internalization and Degradation


Stüber, Jakob C; Kast, Florian; Plückthun, Andreas (2019). High-Throughput Quantification of Surface Protein Internalization and Degradation. ACS Chemical Biology, 14(6):1154-1163.

Abstract

Cell surface proteins are key regulators of fundamental cellular processes and, therefore, often at the root of human diseases. Thus, a large number of targeted drugs which are approved or under development act upon cell surface proteins. Although down-regulation of surface proteins by many natural ligands is well-established, the ability of drug candidates to cause internalization or degradation of the target is only recently moving into focus. This property is important both for the pharmacokinetics and pharmacodynamics of the drug but may also constitute a potential resistance mechanism. The enormous numbers of drug candidates targeting cell surface molecules, comprising small molecules, antibodies, or alternative protein scaffolds, necessitate methods for the investigation of internalization and degradation in high throughput. Here, we present a generic high-throughput assay protocol, which allows the simultaneous and independent quantification of internalization and degradation of surface proteins on a single-cell level. Because we fuse a HaloTag to the cell surface protein of interest and exploit the differential cell permeability of two fluorescent HaloTag ligands, no labeling of the molecules to be screened is required. In contrast to previously described approaches, our homogeneous assay is performed with adherent live cells in a 96-well format. Through channel rescaling, we are furthermore able to obtain true relative abundances of surface and internal protein. We demonstrate the applicability of our procedure to three major drug targets, EGFR, HER2, and EpCAM, examining a selection of well-investigated but also novel small molecule ligands and protein affinity reagents.

Abstract

Cell surface proteins are key regulators of fundamental cellular processes and, therefore, often at the root of human diseases. Thus, a large number of targeted drugs which are approved or under development act upon cell surface proteins. Although down-regulation of surface proteins by many natural ligands is well-established, the ability of drug candidates to cause internalization or degradation of the target is only recently moving into focus. This property is important both for the pharmacokinetics and pharmacodynamics of the drug but may also constitute a potential resistance mechanism. The enormous numbers of drug candidates targeting cell surface molecules, comprising small molecules, antibodies, or alternative protein scaffolds, necessitate methods for the investigation of internalization and degradation in high throughput. Here, we present a generic high-throughput assay protocol, which allows the simultaneous and independent quantification of internalization and degradation of surface proteins on a single-cell level. Because we fuse a HaloTag to the cell surface protein of interest and exploit the differential cell permeability of two fluorescent HaloTag ligands, no labeling of the molecules to be screened is required. In contrast to previously described approaches, our homogeneous assay is performed with adherent live cells in a 96-well format. Through channel rescaling, we are furthermore able to obtain true relative abundances of surface and internal protein. We demonstrate the applicability of our procedure to three major drug targets, EGFR, HER2, and EpCAM, examining a selection of well-investigated but also novel small molecule ligands and protein affinity reagents.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:21 June 2019
Deposited On:11 Sep 2019 14:05
Last Modified:25 Sep 2019 00:44
Publisher:American Chemical Society (ACS)
ISSN:1554-8929
OA Status:Closed
Publisher DOI:https://doi.org/10.1021/acschembio.9b00016
PubMed ID:31050891

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