Protocol to measure and analyze protein interactions in mammalian cells using bioluminescence resonance energy transfer.

Bioluminescence resonance energy transfer (BRET) allows to quantitate protein interactions in intact cells. Here, we present a protocol for measuring BRET due to transient interactions of oncogenic K-RasG12V in plasma membrane nanoclusters of HEK293-EBNA cells. We describe steps for seeding, transfecting, and replating cells.

Identification of an H-Ras nanocluster disrupting peptide.

Hyperactive Ras signalling is found in most cancers. Ras proteins are only active in membrane nanoclusters, which are therefore potential drug targets. We previously showed that the nanocluster scaffold galectin-1 (Gal1) enhances H-Ras nanoclustering via direct interaction with the Ras binding domain (RBD) of Raf.

An acyl-CoA thioesterase is essential for the biosynthesis of a key dauer pheromone in C. elegans.

Methyl ketone (MK)-ascarosides represent essential components of several pheromones in Caenorhabditis elegans, including the dauer pheromone, which triggers the stress-resistant dauer larval stage, and the male-attracting sex pheromone. Here, we identify an acyl-CoA thioesterase, ACOT-15, that is required for the biosynthesis of MK-ascarosides. We propose a model in which ACOT-15 hydrolyzes the β-keto acyl-CoA side chain of an ascaroside intermediate during β-oxidation, leading to decarboxylation and formation of the MK.

Eliminating oncogenic RAS: back to the future at the drawing board.

RAS drug development has made enormous strides in the past ten years, with the first direct KRAS inhibitor being approved in 2021. However, despite the clinical success of covalent KRAS-G12C inhibitors, we are immediately confronted with resistances as commonly found with targeted drugs. Previously believed to be undruggable due to its lack of obvious druggable pockets, a couple of new approaches to hit this much feared oncogene have now been carved out.