Monitoring Protein Secretion in Living Cells With a HiBiT-Integrated RUSH System

Background

The cellular secretory pathway is a fundamental physiological process that refers to the transport of newly synthesized proteins to their target compartments. In eukaryotes, conventional protein secretion is primarily mediated by secretory vesicles, which transport cargo through the endoplasmic reticulum (ER) and the Golgi apparatus to the plasma membrane (PM) or extracellular space. This process is essential because it directs proteins, such as cytokines, chemokines, hormones, and receptors, to their sites of action, where they perform crucial physiological and pathological functions [1].

Several classic methods have been developed to investigate secretory trafficking, with the RUSH (Retention Using Selective Hooks) system being the most advanced technique [2–5]. The RUSH system exploits the high-affinity interaction between streptavidin (Str) and a streptavidin-binding peptide (SBP) to reversibly retain an enhanced GFP (EGFP)-tagged reporter within the ER. Subsequent addition of biotin, which competitively displaces SBP from Str, triggers reporter release. This controlled release enables quantification of secretion kinetics and status by monitoring EGFP localization dynamics via fluorescence microscopy [3].

Researchers have conducted impressive studies using the RUSH system, driving significant progress in the field of cellular trafficking [6–8]. However, the classic RUSH assay requires the acquisition and quantification of numerous fluorescence images, which poses challenges for achieving precision and efficiency, particularly constraining its use in high-throughput screening applications. Boncompain et al. [9] utilized the classic RUSH system to screen and validate three molecules from over 4,000 compounds that inhibit HIV infection by targeting CC chemokine receptor 5 (CCR5) secretion. To our knowledge, this is the only reported study to apply the RUSH technique to high-throughput screening. HiBiT is an 11-amino-acid peptide tag developed by Promega in recent years. When HiBiT-tagged proteins on the cell surface or in the extracellular space bind to the exogenously added LgBiT, a NanoBiT luciferase is generated, enabling the detection and quantification of HiBiT-tagged proteins in living cells [10,11]. Building on this advantage, we propose substituting EGFP in the RUSH reporter with HiBiT, thereby converting the fluorescent signal into a quantifiable luciferase signal, which would facilitate real-time, quantitative monitoring of reporter trafficking.

Here, we combined the HiBiT technology with the classic RUSH system to investigate the secretion process of angiotensin-converting enzyme 2 (ACE2) as a model, which is the receptor for SARS-CoV-2. Using a plate reader, we achieved real-time monitoring of the secretion dynamics, enabling a precise and efficient identification of the trafficking regulators. Notably, this strategy was also applied to monitor the secretion process of programmed death-ligand 1 (PDL1), sodium taurocholate cotransporting polypeptide (NTCP), and CD209 (also known as DC-SIGN), all of which are cell membrane receptors with important physiological functions, highlighting the significant potential of this approach in advancing the field of cellular trafficking research.

Since its introduction in 2012, RUSH has been widely used by researchers and has become a cornerstone method in the field of cellular secretion studies [3]. However, its application still faces several limitations. One of the main challenges is achieving a perfect hook, as fluorescence leakage can compromise result interpretation [12]. In addition, quantitative bioimaging remains a difficult task, especially since a single two-dimensional image cannot accurately reflect the three-dimensional nature of cells [13]. Furthermore, cellular transport is a highly dynamic process, necessitating rapid and precise data acquisition.

Denus et al. also utilized the combination of RUSH and HiBiT to quantitatively measure unconventional protein secretion, demonstrating the feasibility and compatibility of the HiBiT-integrated RUSH system [14]. However, their approach relied on endpoint measurements rather than real-time monitoring, which is essential for capturing the highly dynamic nature of cellular secretion. In this study, we employed the HiBiT-integrated RUSH system, which converts the cellular secretion process into a quantitative curve without the need for stable cell line construction or achieving a perfect hook, thereby providing an objective means for result interpretation. It is particularly effective for studying the transport dynamics of various cell membrane receptors such as ACE2, PDL1, NTCP, and CD209, facilitating the identification of both common and specific regulators of the cellular secretory process. Ongoing studies are focused on screening compound libraries using this strategy to identify potential interventions targeting the secretion of disease-associated proteins.