Lu TH y: a double‐readout bioluminescence‐based two‐hybrid technology for quantitative mapping of protein–protein interactions in mammalian cells

Abstract

Information on protein–protein interactions (PPIs) is of critical importance for studying complex biological systems and developing therapeutic strategies. Here, we present a double‐readout bioluminescence‐based two‐hybrid technology, termed LuTHy, which provides two quantitative scores in one experimental procedure when testing binary interactions. PPIs are first monitored in cells by quantification of bioluminescence resonance energy transfer (BRET) and, following cell lysis, are again quantitatively assessed by luminescence‐based co‐precipitation (LuC). The double‐readout procedure detects interactions with higher sensitivity than traditional single‐readout methods and is broadly applicable, for example, for detecting the effects of small molecules or disease‐causing mutations on PPIs. Applying LuTHy in a focused screen, we identified 42 interactions for the presynaptic chaperone CSPα, causative to adult‐onset neuronal ceroid lipofuscinosis (ANCL), a progressive neurodegenerative disease. Nearly 50% of PPIs were found to be affected when studying the effect of the disease‐causing missense mutations L115R and ∆L116 in CSPα with LuTHy. Our study presents a robust, sensitive research tool with high utility for investigating the molecular mechanisms by which disease‐associated mutations impair protein activity in biological systems.

Two protein/protein interaction assays in one go

“…On the other, co‐purification assays require an interaction that stays intact during incubation and wash steps, whereas the tag location is unlikely to affect the signal. These differences sound intuitively reasonable, and the control experiments performed by Trepte et al () nicely confirm this notion.…”

“…This is where the work of Trepte et al () comes in. They have developed LuTHy, a novel method which combines co‐purification and proximity methods in one system to assay binary PPIs (Fig ).…”

“…Therefore, it seems rather intuitive that combining multiple assays is beneficial for robust and reliable discovery of interactions. Along those lines, in their recent study, Wanker and colleagues (Trepte et al , ) combined two complementary and quantitative interaction assays in one pot. One assay is luminescence‐based and depends on protein proximity in living cells, while the other relies on formation of more stable complexes detected by co‐precipitation with a luminescence‐based readout, which facilitates confident identification and quantitation of interactions in high throughput.…”

“… Combining complementary assays is beneficial for identifying protein‐protein interactions. In their recent work, Wanker and colleagues (Trepte et al , ) combine a luminescence‐based assay depending on protein proximity in living cells with a co‐precipitation assay that detects more stable complexes.

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Structured Methods for reproducible science

“…The precise description of reagents and procedures is especially important for allowing the widespread adoption of new methodologies, and therefore, we have decided to pilot Structured Methods in our Methods section. The first Method published using this format is the study by Wanker and colleagues (Trepte et al , ) describing a luminescence‐based two‐hybrid approach for quantitatively mapping protein–protein interactions. Until we formally implement Structured Methods across all article types, we also strongly encourage the authors of research Articles and Reports to use this format.…”