Structure and Function of a Key Flexible Loop in Controlling the Biological Function of Acyl Protein Thioesterase 1

Acyl Protein Thioesterase 1 (APT1) is a depalmitoylase involved in removing and reusing palmitoylated proteins from the plasma membrane. This biological function requires that APT1 be localized to and have controlled catalytic activity at the plasma membrane. The main focus of this study was to investigate the role of a surface exposed loop in APT1, which may provide dual control over its membrane binding and catalytic activity. To explore this surface exposed loop, I used a classic protein structure-function approach of introducing alanine scanning substitutions across nine residues in this flexible loop. Specifically, nine variants of APT1 were assembled by site-directed mutagenesis and purified to uniformity through nickel affinity protein purification. The catalytic and membrane binding activity of the loop modifications was then compared to wild-type APT1’s activity to identify the hotspots controlling each of these biological functions. Based off of this analysis, differential locations within this flexible loop were assigned that control the catalytic and membrane binding activity of APT1. Given the role of APT1 in controlling Ras dependent cancers, these results provide a novel mechanism for regulating the structure, function, and properties of APT1