Nanobody-thioesterase chimeras to specifically target protein palmitoylation

Abstract The complexity of the cellular proteome is massively expanded by a repertoire of chemically distinct reversible post-translational modifications (PTMs) that control protein localisation, interactions, and function. The temporal and spatial control of these PTMs is central to organism physiology, and mis-regulation of PTMs is a hallmark of many diseases. Here we present an approach to manipulate PTMs on target proteins using nanobodies fused to enzymes that control these PTMs. Anti-GFP nanobodies fused to thioesterases (which depalmitoylate protein cysteines) depalmitoylate GFP tagged substrates. A chemogenetic approach to enhance nanobody affinity for its target enables temporal control of target depalmitoylation. Using a thioesterase fused to a nanobody directed against the Ca(v)1.2 beta subunit we reduce palmitoylation of the Ca(v)1.2 alpha subunit, modifying the channel’s voltage dependence and arrhythmia susceptibility in stem cell derived cardiac myocytes. We conclude that nanobody enzyme chimeras represent an approach to specifically manipulate PTMs, with applications in both the laboratory and the clinic.