High-Throughput Quantification of Surface Protein Internalization and Degradation

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.

Engineering an anti-HER2 biparatopic antibody with a multimodal mechanism of action

“…Human Fc regions are colored magenta, the lysosomal marker LAMP1 in green and DAPI as nuclear stain in blue. b Internalization and degradation were quantitatively resolved in a time-dependent manner using the recently developed SPIDA protocol 39 (mean plus error bar SD). 441 led to distinct internalization as early as after 1 h, and thereafter internal HER2 was continuously degraded.…”

HaloFlippers: A General Tool for the Fluorescence Imaging of Precisely Localized Membrane Tension Changes in Living Cells

“…They were also used as covalent long-lived tethers for protein nanomechanics. 67 − 69 Moreover, several biological assays have been established in recent years that employ HaloTagging as the key step in their protocols, 70 − 72 including the chloroalkane penetration assay (CAPA) 73 − 75 for the quantification of cell permeability and cytosolic delivery. In this report, we explore the use of HaloTag technology for force imaging in mechanobiology.…”

Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates

“…Separating pools of the same protein has been achieved before, for instance by using uorogenactivated protein (FAP) 62,63 probes, the uorescence-activating and absorbance-shiing tag (FAST) 52 and Halo-tag. 64 While these previous studies rely on non-covalent labelled protein tags fused to the BK channel or a transmembrane helix, respectively, we report a SNAP-fusion to a widely-drugged GPCR. In addition, our system does not rely on Förster Resonance Energy Transfer (FRET), which adds another layer of complexity and need for additional control experiments, as has been shown for malachite green conjugates.…”