Post-translational S-palmitoylation directs the trafficking and membrane localization of hundreds of cellular proteins, often involving a coordinated palmitoylation cycle that requires both protein acyl transferases (PATs) and acyl protein thioesterases (APTs) to actively re-distribute S-palmitoylated proteins towards different cellular membrane compartments. This process is necessary for the trafficking and oncogenic signaling of S-palmitoylated Ras isoforms, and potentially many peripheral membrane proteins. The de-palmitoylating enzymes APT1 and APT2 are separately conserved in all vertebrates, suggesting unique functional roles for each enzyme. The recent discovery of the APT isoform-selective inhibitors ML348 and ML349 has opened new possibilities to probe the function of each enzyme, yet it remains unclear how each inhibitor achieves orthogonal inhibition. Herein we report the high-resolution structure of human APT2 in complex with ML349 (1.64 Å), as well as the complementary structure of human APT1 bound to ML348 (1.55 Å). Although the overall peptide backbone structures are nearly identical, each inhibitor adopts a distinct conformation within each active site. In APT1, ML348 is positioned above the catalytic triad, but in APT2, the sulfonyl group of ML349 forms hydrogen bonds with active site resident waters to indirectly engage the catalytic triad and oxyanion hole. Reciprocal mutagenesis and activity profiling revealed several differing residues surrounding the active site that serve as critical gatekeepers for isoform accessibility and dynamics. Structural and biochemical analysis suggests the inhibitors occupy a putative acyl-binding region, establishing the mechanism for isoform-specific inhibition, hydrolysis of acyl substrates, and structural orthogonality important for future probe development.

Protein depalmitoylation describes the removal of thioester-linked long chain fatty acids from cysteine residues in proteins. For many S-palmitoylated proteins, this process is promoted by acyl protein thioesterase enzymes, which catalyze thioester hydrolysis to solubilize and displace substrate proteins from membranes. The closely related enzymes acyl protein thioesterase 1 (APT1; LYPLA1) and acyl protein thioesterase 2 (APT2; LYPLA2) were initially identified from biochemical assays as G protein depalmitoylases, yet later were shown to accept a number of S-palmitoylated protein and phospholipid substrates. Leveraging the development of isoform-selective APT inhibitors, several studies report distinct roles for APT enzymes in growth factor and hormonal signaling. Recent crystal structures of APT1 and APT2 reveal convergent acyl binding channels, suggesting additional factors beyond acyl chain recognition mediate substrate selection. In addition to APT enzymes, the ABHD17 family of hydrolases contributes to the depalmitoylation of Ras-family GTPases and synaptic proteins. Overall, enzymatic depalmitoylation ensures efficient membrane targeting by balancing the palmitoylation cycle, and may play additional roles in signaling, growth, and cell organization. In this review, we provide a perspective on the biochemical, structural, and cellular analysis of protein depalmitoylases, and outline opportunities for future studies of systems-wide analysis of protein depalmitoylation.

Summary The multi-domain 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.

S-palmitoylation is required for membrane anchoring, proper trafficking, and the normal function of hundreds of integral and peripheral membrane proteins. Previous bioorthogonal pulse-chase proteomics analyses identified Ras family GTPases, polarity proteins, and G proteins as rapidly cycling S-palmitoylated proteins sensitive to depalmitoylase inhibition, yet the breadth of enzyme regulated dynamic S-palmitoylation largely remains a mystery. Here, we present a pulsed bioorthogonal S-palmitoylation assay for temporal analysis of S-palmitoylation dynamics. Low concentration hexadecylfluorophosphonate (HDFP) inactivates the APT and ABHD17 families of depalmitoylases, which dramatically increases alkynyl-fatty acid labeling and stratifies S-palmitoylated proteins into kinetically distinct subgroups. Most surprisingly, HDFP treatment does not affect steady-state S-palmitoylation levels, despite inhibiting all validated depalmitoylating enzymes. S-palmitoylation profiling of APT1/APT2 mouse brains similarly show no change in S-palmitoylation levels. In comparison with hydroxylamine-switch methods, bioorthogonal alkynyl fatty acids are only incorporated into a small fraction of dynamic S-palmitoylated proteins, raising the possibility that S-palmitoylation is more stable than generally characterized. Overall, disrupting depalmitoylase activity enhances alkynyl fatty acid incorporation, but does not greatly affect steady state S-palmitoylation across the proteome.

The bacterial recombinase, RecA protein, plays a central role in maintenance of genome stability. It generally functions as a RecA nucleoprotein filament formed on DNA (1). RecA has three identified functions in the cell. First, it participates directly in all recombination processes. Filaments of Escherichia coli RecA protein form most readily on ssDNA; these filaments can then catalyze DNA pairing and strand exchange with a homologous duplex DNA (2). A RecA filament complexed with an oligonucleotide can invade duplex DNA and pair the oligonucleotide with a complementary sequence within a much longer duplex, forming a displacement loop (D-loop) (2). Second, RecA filaments play a central role in the induction of the SOS response (3). In brief, RecA filaments assembled on ssDNA generated by stalled replication forks bind and stimulate the autocatalytic cleavage of the LexA repressor. This co-protease function for RecA inactivates LexA, which leads to the induction of SOS. Third, RecA filaments play two roles in activating the translesion DNA synthesis function of DNA polymerase V. If cellular genomic replication is not restored after 30 -60 min of SOS response, polymerase V opens a final, mutagenic phase of SOS. The UmuD subunit of polymerase V is autocatalytically cleaved, again facilitated by interaction with RecA filaments, to create UmuDЈ. The weakly active UmuDЈ 2 ⅐UmuC complex undergoes a final activation step with the transfer of a RecA subunit from the 3Ј-proximal end of a RecA filament to form the activated polymerase V enzyme consisting of UmuDЈ 2 ⅐UmuC/RecA (4, 5). The presence of RecA as a subunit of active polymerase V is the only known activity where RecA exhibits a function when it is not part of a filament.Many other proteins interact with RecA protein filaments on DNA, and many of these serve to regulate almost every aspect of RecA function (6). The regulators include DinI (7), RecX (8 -10), RdgC (11), PsiB (12), RecFOR (13-15), UvrD (16), and RecBCD (17-19), a list that will doubtlessly grow. In all, more than a dozen known proteins interact with RecA and help coordinate its functions with many aspects of DNA metabolism. A number of RecA partner proteins bind within the helical groove of active RecA nucleoprotein filaments. These include LexA (20,21), UmuD (22), RecX (20), and DinI.2 The LexA and UmuD proteins (along with the bacteriophage repressor (23)) undergo autocatalytic cleavage in this environment. The regulation of RecA is not limited to bacteria-encoded functions. The PsiB protein is a product of conjugative F-plasmids, expressed early in conjugation to suppress the SOS response in the recipient cell (12,24,25). Additional proteins that interact with RecA are encoded by bacteriophages, including the bacteriophage P1.Almost from the moment it was described in 1951 (26), bacteriophage P1 has been a workhorse of molecular biology. It is now used largely for generalized transduction applied to strain construction (27,28). Its genome (93.6 kbp; ϳ117 genes (27)) is packaged in phage particl...

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), which promotes 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 prevents 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 using by stable isotope labeling in cell culture with amino acids (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, and may affect cell polarity and tumor suppression.

Activity-based protein profiling (ABPP) has revolutionized the discovery and optimization of active-site ligands across distinct enzyme families, providing a robust platform for in-class selectivity profiling. Nonetheless, this approach is less straightforward for profiling reversible inhibitors and does not access proteins outside the ABPP probe's target profile. While the active-site competitive acyl protein thioesterase 2 inhibitor ML349 (K i = 120 nM) is highly selective within the serine hydrolase enzyme family, it could still interact with other cellular targets. Here we present a chemoproteomic workflow to enrich and profile candidate ML349-binding proteins. In human cell lysates, biotinylated-ML349 enriches a recurring set of proteins, including metabolite kinases and flavin-dependent oxidoreductases that are potentially enhanced by avidity-driven multimeric interactions. Confirmatory assays by native mass spectrometry and fluorescence polarization quickly rank-ordered these weak off-targets, providing justification to explore ligand interactions and stoichiometry beyond ABPP.

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