A fluorescent probe for cysteine depalmitoylation reveals dynamic APT signaling

Abstract

Hundreds of human proteins are modified by reversible palmitoylation of cysteine residues (S-palmitoylation), but the regulation of depalmitoylation is poorly understood. Here, we develop “depalmitoylation probes” (DPPs), small molecule fluorophores to monitor the endogenous activity levels of “erasers” of S-palmitoylation, acyl-protein thioesterases (APTs). Live-cell analysis with DPPs reveals rapid growth factor-mediated inhibition of the depalmitoylation activity of APTs, exposing a novel regulatory mechanism of dynamic lipid signaling.

The molecular era of protein S-acylation: spotlight on structure, mechanisms, and dynamics

“…Finally, promising fluorescent in vivo depalmitoylation activity probes, named DPPs, were recently developed by the Dickinson group (Kathayat et al 2017;Qiu et al 2018;Kathayat et al 2018). DPPs feature a profluorescent molecule tethered through a carbamate linkage to an S-acylated peptide substrate and have been tuned to localize to specific compartments (DPP-2 and DPP-3 in cytosol, and mito-DPP-2 and mito-DPP-3 in mitochondria), to react better against APT1 than APT2 (DPP-3 versus DPP-2 variants), or to have increased water solubility (DPP-5) (Figure 4).…”

Active and dynamic mitochondrial S-depalmitoylation revealed by targeted fluorescent probes

“…In this work, we sought to experimentally test whether there is active S -depalmitoylation in the mitochondria of mammalian cells, whether APT1 is active in the mitochondria, and whether changes to mitochondrial homeostasis result in alterations of mitochondrial S -depalmitoylation activity. To accomplish this, we expanded our recently reported strategy for the creation of fluorescent “depalmitoylation probes” (DPPs) 34 , which readout endogenous cysteine deacylation activities in live cells, to generate the first mitochondrial-targeted S -deacylase probes (“mitoDPP-2” and “mitoDPP-3”). After synthesis, we validated that mitoDPPs localize to mitochondria and report on endogenous cysteine S -deacylase activities.…”

Engineering an NIR rhodol derivative with spirocyclic ring-opening activation for high-contrast photoacoustic imaging

“…Spirocyclic xanthene dyes, such as uorescein and rhodol, are known to be present in equilibrium between a 'ringopening' form and a 'closed' spirocyclic structure with distinct p-conjugation systems. [19][20][21][22] The 'ring-opening' form exhibits a red-shied and enhanced absorption band as compared to the 'closed' structure, holding great potential for designing highcontrast PA probes. There have been a few studies using spirocyclic xanthene dyes for PA imaging, including a visible light absorbing dye, seminaphthorhodauor-5F, for pH imaging, 23,24 and an NIR absorbing dye, silicon-rhodamine, for hypochlorous acid imaging.…”