Chemoselective ratiometric imaging of protein S-sulfenylation.

Here we report a ratiometric fluorescent probe for chemoselective conjugation to sulfenic acids in living cells. Our approach couples an α-fluoro-substituted dimedone to an aminonaphthalene fluorophore (F-DiNap), which upon sulfenic acid conjugation is locked as the 1,3-diketone, changing the fluorophore excitation. F-DiNap reacts with S-sulfenylated proteins at equivalent rates to current probes, but the α-fluorine substitution blocks side-reactions with biological aldehydes.

APT2 Inhibition Restores Scribble Localization and S-Palmitoylation in Snail-Transformed Cells.

The multidomain scaffolding protein Scribble (Scrib) organizes key signaling complexes to specify basolateral cell polarity and suppress aberrant growth. In many human cancers, genetically normal Scrib mislocalizes from cell-cell junctions to the cytosol, correlating with enhanced growth signaling and malignancy. Here we confirm that expression of the epithelial-to-mesenchymal transcription factor (EMT-TF) Snail in benign epithelial cells leads to Scrib displacement from the plasma membrane, mimicking the mislocalization observed in aggressive cancers. Upon further examination, Snail promotes a transcriptional program that targets genes in the palmitoylation cycle, repressing many protein acyl transferases and elevating expression and activity of protein acyl thioesterase 2 (APT2). APT2 isoform-selective inhibition or knockdown rescued Scrib membrane localization and palmitoylation while attenuating MEK activation. Overall, inhibiting APT2 restores balance to the Scrib palmitoylation cycle, promoting membrane re-localization and growth attenuation. These findings emphasize the importance of S-palmitoylation as a post-translational gatekeeper of cell polarity-mediated tumor suppression.

Correlated S-palmitoylation profiling of Snail-induced epithelial to mesenchymal transition.

Epithelial cells form spatially-organized adhesion complexes that establish polarity gradients, regulate cell proliferation, and direct wound healing. As cells accumulate oncogenic mutations, these key tumor suppression mechanisms are disrupted, eliminating many adhesion complexes and bypassing contact inhibition. The transcription factor Snail is often expressed in malignant cancers, where it promotes transcriptional reprogramming to drive epithelial-mesenchymal transition (EMT) and establishes a more invasive state. S-Palmitoylation describes the fatty-acyl post-translational modification of cysteine residues in proteins, and is required for membrane anchoring, trafficking, localization and function of hundreds of proteins involved in cell growth, polarity, and signaling. Since Snail-expression disrupts apico-basolateral cell polarity, we asked if Snail-dependent transformation induces proteome-wide changes in S-palmitoylation. MCF10A breast cancer cells were retrovirally transduced with Snail and correlated proteome-wide changes in protein abundance and S-palmitoylation were profiled by using stable isotope labeling in cell culture with amino acid (SILAC) mass spectrometry. This analysis identified increased levels of proteins involved in migration, glycolysis, and cell junction remodeling, and decreased levels of proteins involved in cell adhesion. Overall, protein S-palmitoylation is highly correlated with protein abundance, yet for a subset of proteins, this correlation is uncoupled. These findings suggest that Snail-overexpression affects the S-palmitoylation cycle of some proteins, which may participate in cell polarity and tumor suppression.

Profiling targets of the irreversible palmitoylation inhibitor 2-bromopalmitate.

2-Bromohexadecanoic acid, or 2-bromopalmitate, was introduced nearly 50 years ago as a nonselective inhibitor of lipid metabolism. More recently, 2-bromopalmitate re-emerged as a general inhibitor of protein S-palmitoylation. Here, we investigate the cellular targets of 2-bromopalmitate through the synthesis and application of click-enabled analogues. In cells, 2-bromopalmitate is converted to 2-bromopalmitoyl-CoA, although less efficiently than free palmitate. Once conjugated to CoA, probe reactivity is dramatically enhanced. Importantly, both 2-bromopalmitate and 2-bromopalmitoyl-CoA label DHHC palmitoyl acyl transferases (PATs), the enzymes that catalyze protein S-palmitoylation. Mass spectrometry analysis of enriched 2-bromopalmitate targets identified PAT enzymes, transporters, and many palmitoylated proteins, with no observed preference for CoA-dependent enzymes. These data question whether 2-bromopalmitate (or 2-bromopalmitoyl-CoA) blocks S-palmitoylation by inhibiting protein acyl transferases, or by blocking palmitate incorporation by direct covalent competition. Overall, these findings highlight the promiscuous reactivity of 2BP and validate clickable 2BP analogues as activity-based probes of diverse membrane associated enzymes.

Profiling and inhibiting reversible palmitoylation.

Protein palmitoylation describes the posttranslational modification of cysteines by a thioester-linked long-chain fatty acid. This modification is critical for membrane association, spatial organization, and the proper activity of hundreds of membrane-associated proteins. Palmitoylation is continuously remodeled, both by spontaneous hydrolysis and enzyme-mediated de-palmitoylation. Bioorthogonal pulse-chase labeling approaches have highlighted the role of protein thioesterases as key regulators of palmitoylation dynamics. Importantly, thioesterases are critical for regulating the spatial organization of key oncogenic proteins, such as Ras GTPases. New inhibitors, probes, and proteomics methods have put a spotlight on this emerging posttranslational modification. These tools promise to advance our understanding the enzymatic regulation of dynamic palmitoylation, and present new opportunities for drug development.

The intrinsic electrophysiological properties of neurons derived from mouse embryonic stem cells overexpressing neurogenin-1.

A mouse embryonic stem (ES) cell line containing an inducible transgene for the proneural gene Neurog1 has been used to generate glutamatergic neurons at a high efficiency. The present study used in vitro electrophysiology to establish the timeline for acquiring a functional neuronal phenotype in Neurog1-induced cells exhibiting a neuronal morphology. TTX-sensitive action potentials could be evoked from over 80% of the cells after only 4.5 days in vitro (DIV). These cells uniformly showed rapidly adapting responses to current injection, firing one to three action potentials at the onset of the stimulus. In the absence of Neurog1, a limited number of ES cells adopted a neuronal morphology, but these cells displayed slow calcium depolarizations rather than sodium-based spikes. Voltage-gated Na(+), K(+), and Ca(2+) currents were present in nearly all induced cells as early as 4.5 DIV. The voltage-dependent properties of these currents changed little from 4 to 12 DIV with half-activation voltage varying by <10 mV for any current type throughout the culture period. This study demonstrates that forced expression of proneural genes can induce ES cells to quickly acquire a functional neuronal phenotype with mature electrophysiological properties. Transient overexpression of Neurog1 may be used in neural repair strategies that require the rapid induction of functional neurons from pluripotent stem cells.