Abstract

Post-translational modifications of proteins, such as glycosylation, nitrosylation, or phosphorylation, are widespread phenomena in cellular physiology. Here we report a novel post-translational modification of the transient receptor potential protein, TRPM8, by a polyester comprised of repeated units of R-3-hydroxybutyrate, which forms a polymeric chain, poly-(R)-3-hydroxybutyrate (PHB). We term this modification PHBylation by analogy with the established protein modifications. The homopolymer PHB modifies numerous membrane proteins where the polyester may exert substantial conformational changes on the protein structure and affect its function. Recently we identified that the cold and menthol receptor, mammalian ion channel TRPM8, the member of the transient receptor potential melastatin subfamily, undergoes post-translational modification with PHB. The TRPM8 ion channel is expressed in sensory neurons and is responsible for sensing environmental cues such as cold temperatures and chemical compounds, including menthol and icilin. The channel functional activity is regulated by various physical and chemical factors, and is likely to be pre-conditioned by its molecular composition. Our studies indicate that TRPM8 channel forms a structural-functional complex with PHB. We identified by mass spectrometry a number of PHB-modified peptides in the N-terminus of the TRPM8 protein and in its extracellular S3-S4 linker. Removal of PHB moieties by enzymatic hydrolysis, and site-directed mutagenesis of both the serine residues that serve as covalent anchors for PHB and adjacent hydrophobic residues that interact with the methyl groups of the polymer, resulted in significant inhibition of TRPM8 channel activity. We conclude that the TRPM8 channel undergoes post-translational modification by PHB and that this modification is required for its normal function.