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Utilizing split-NanoLuc luciferase fragments as luminescent probes for protein solubility in living cells.

ABSTRACT: Protein misfolding and aggregation is now recognized as a hallmark of numerous human diseases. Standard bioanalytical techniques for monitoring protein aggregation generally rely on small molecules that provide an optical readout of fibril formation. While these methods have been useful for mechanistic studies, additional approaches are required to probe the equilibrium between soluble and insoluble protein within living systems. Such approaches could provide platforms for the identification of inhibitors of protein aggregation as well as a means to investigate the effect of mutations on protein aggregation in model systems. In this chapter, we provide detailed protocols for employing split-NanoLuc luciferase (Nluc) fragments to monitor changes in protein solubility in bacterial and mammalian cells. This sensitive luminesce-based assay can report upon changes in protein solubility induced by inhibitors and disease-relevant mutations.

SUBMITTER: Nelson TJ

PROVIDER: S-EPMC6567984 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Utilizing split-NanoLuc luciferase fragments as luminescent probes for protein solubility in living cells.

Nelson Travis J TJ Zhao Jia J Stains Cliff I CI

Methods in enzymology 20190309

Protein misfolding and aggregation is now recognized as a hallmark of numerous human diseases. Standard bioanalytical techniques for monitoring protein aggregation generally rely on small molecules that provide an optical readout of fibril formation. While these methods have been useful for mechanistic studies, additional approaches are required to probe the equilibrium between soluble and insoluble protein within living systems. Such approaches could provide platforms for the identification of ...[more]

PMID: 31155065

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Project description:The dimerization of RAF kinases represents a key event in their activation cycle and in RAS/ERK pathway activation. Genetic, biochemical and structural approaches provided key insights into this process defining RAF signaling output and the clinical efficacy of RAF inhibitors (RAFi). However, methods reporting the dynamics of RAF dimerization in living cells and in real time are still in their infancy. Recently, split luciferase systems have been developed for the detection of protein-protein-interactions (PPIs), incl. proof-of-concept studies demonstrating the heterodimerization of the BRAF and RAF1 isoforms. Due to their small size, the Nanoluc luciferase moieties LgBiT and SmBiT, which reconstitute a light emitting holoenzyme upon fusion partner promoted interaction, appear as well-suited to study RAF dimerization. Here, we provide an extensive analysis of the suitability of the Nanoluc system to study the homo- and heterodimerization of BRAF, RAF1 and the related KSR1 pseudokinase. We show that KRASG12V promotes the homo- and heterodimerization of BRAF, while considerable KSR1 homo- and KSR1/BRAF heterodimerization already occurs in the absence of this active GTPase and requires a salt bridge between the CC-SAM domain of KSR1 and the BRAF-specific region. We demonstrate that loss-of-function mutations impairing key steps of the RAF activation cycle can be used as calibrators to gauge the dynamics of heterodimerization. This approach identified the RAS-binding domains and the C-terminal 14-3-3 binding motifs as particularly critical for the reconstitution of RAF mediated LgBiT/SmBiT reconstitution, while the dimer interface was less important for dimerization but essential for downstream signaling. We show for the first time that BRAFV600E, the most common BRAF oncoprotein whose dimerization status is controversially portrayed in the literature, forms homodimers in living cells more efficiently than its wildtype counterpart. Of note, Nanoluc activity reconstituted by BRAFV600E homodimers is highly sensitive to the paradox-breaking RAFi PLX8394, indicating a dynamic and specific PPI. We report the effects of eleven ERK pathway inhibitors on RAF dimerization, incl. third-generation compounds that are less-defined in terms of their dimer promoting abilities. We identify Naporafenib as a potent and long-lasting dimerizer and show that the split Nanoluc approach discriminates between type I, I1/2 and II RAFi. Video Abstract.

Dataset Information

Utilizing split-NanoLuc luciferase fragments as luminescent probes for protein solubility in living cells.

Publications

Utilizing split-NanoLuc luciferase fragments as luminescent probes for protein solubility in living cells.

Similar Datasets

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