Proteins play important roles in many processes in cells. Some of these proteins can be modified by the addition of a molecule called palmitate. This process, termed “palmitoylation”, helps direct these proteins to the compartments within the cell where they are needed to carry out their roles. One target of palmitoylation is N-Ras, which is a protein that can promote the development of cancer.

We understand quite a lot about how palmitate is added to proteins, but much less about how it is removed. So far, researchers have only identified two enzymes – known as APT1 and APT2 – that can remove palmitate from proteins, but it is possible that there are others. Identifying other “depalmitoylase” enzymes could help us find ways to block the removal of palmitate from N-Ras, which could lead to new treatments for some cancers.

Lin and Conibear used several biochemical techniques to search for depalmitoylase enzymes in human cells. The experiments reveal that although APT1 and APT2 are important for removing palmitate from some proteins, they are not needed to remove palmitate from N-Ras. Instead, Lin and Conibear found that an enzyme called ABHD17 removes palmitate from N-Ras. The next step following on from this work will be to find out what other proteins ABHD17 acts on in cells. A longer-term challenge will be to develop specific chemicals that inhibit ABHD17 activity and test if they are able to reduce the growth of cancer cells.

Protein S-palmitoylation involves the post-translational attachment of the 16-carbon fatty acid palmitate to cysteine residues (Conibear and Davis, 2010; Salaun et al., 2010). While a survey of palmitoylation dynamics indicated the bulk of the palmitoyl-proteome is stably palmitoylated (Martin et al., 2011), rapid and constitutive palmitate turnover has been shown for several proteins, including the Ras GTPases, heterotrimeric G proteins, the neuronal post-synaptic density protein PSD95, and the Lck kinase (Magee et al., 1987; Degtyarev et al., 1993; El-Husseini et al., 2002; Zhang et al., 2010). Dynamic changes in palmitoylation modulate protein localization and trafficking and can be regulated in response to cellular signaling (Conibear and Davis, 2010).

Palmitoylation is mediated by a family of DHHC (Asp-His-His-Cys) proteins (Greaves and Chamberlain, 2011a), whereas the only enzymes identified to date that remove palmitate from cytosolic cysteines, the acyl-protein thioesterases (APTs) APT1 and APT2, are related members of the metabolic serine hydrolase (mSH) superfamily (Duncan and Gilman, 1998; Tomatis et al., 2010; Long and Cravatt, 2011). The β-lactone core-containing compound Palmostatin B (PalmB) potently inhibits these enzymes and blocks depalmitoylation of N-Ras and other proteins (Dekker et al., 2010; Rusch et al., 2011). Hexadecyl fluorophosphonate (HDFP) inhibits a subset of mSHs including APT1 and APT2 and also suppresses palmitate turnover (Martin et al., 2011). However, it is unclear if APT1 and APT2 are the only palmitoylthioesterases responsible for the depalmitoylation of cytosolic proteins (Davda and Martin, 2014).

Here, we show that APT1 and APT2 inhibition or knockdown reduces palmitate turnover on some substrates but has no effect on N-Ras and PSD95. We identified members of the ABHD17 family as novel PalmB targets that depalmitoylate N-Ras and promote its relocalization to internal membranes. This demonstrates the enzymes responsible for protein depalmitoylation are more diverse than previously believed, which has important implications for understanding the selectivity and regulation of dynamic palmitate turnover.

APT1 and APT2 were proposed to act universally and constitutively to remove mislocalized proteins from intracellular membranes and allow their re-palmitoylation at the Golgi (Rocks et al., 2010). Reported rates of palmitate turnover on different substrates vary dramatically (Qanbar and Bouvier, 2004; Martin et al., 2011). We used a dual-click chemistry pulse-chase scheme to simultaneously measure palmitate and protein turnover of proteins expressed in COS-7 cells and labeled with the palmitate analogue 17-octadecynoic acid (17-ODYA) and the methionine surrogate L-azidohomoalanine (L-AHA) (Martin and Cravatt, 2009; Zhang et al., 2010). N-Ras had a rapid palmitate turnover as previously reported (Figure 1A; Magee et al., 1987). SNAP25 turned over slowly, whereas the glutamate decarboxylase subunit GAD65 and PSD95 had intermediate rates of depalmitoylation, demonstrating that these neuronal proteins undergo palmitate turnover at comparable rates in COS-7 cells or neuronal lines (Greaves and Chamberlain, 2011b; El-Husseini et al., 2002). A palmitoylated N-terminal fragment of Huntingtin (N-HTT) implicated in the pathogenesis of Huntington’s disease (Yanai et al., 2006) also showed an intermediate palmitate turnover (Figure 1B). Treatment with the APT1/2 inhibitor PalmB inhibited the depalmitoylation of these substrates without affecting protein turnover (Figure 1A,B). In contrast, we found three proteins identified in a global palmitoyl-proteomics analysis (SPRED2, GOLIM4, and ITM2B) (Martin et al., 2011) did not undergo significant palmitate turnover, suggesting the apparent PalmB-resistant decline in palmitate labeling was due to protein instability (Figure 1B). These results confirm that proteins have inherently distinct rates of depalmitoylation, potentially reflecting differential recognition by APTs (Lin and Conibear, 2015). In all cases examined, PalmB inhibited the palmitate turnover of dynamically palmitoylated proteins.

Figure 1

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APT1 and APT2 are reported to have differential substrate specificity (Tomatis et al., 2010; Tian et al., 2012). We found that the selective inhibitors C83 and C115, which target APT1 and APT2 respectively (Adibekian et al., 2012), had little effect on N-HTT depalmitoylation when used individually but achieved significant inhibition when applied together (Figure 2A,B). A similar effect was observed on GAD65 (Figure 2—figure supplement 1A). Surprisingly, these inhibitors had no effect on PSD95 or N-Ras depalmitoylation when used alone (Figure 2—figure supplement 1B,C) or together (Figure 2C,D). Double RNAi knockdown of APT1 and APT2 significantly inhibited N-HTT depalmitoylation (Figure 2B) and also reduced palmitate turnover on GAD65 (Figure 2—figure supplement 1D) but not PSD95 or N-Ras (Figure 2C,D). These findings, which are consistent with a recent report showing APT1/2-independent depalmitoylation of R7BP (Jia et al., 2014), strongly suggest that although APT1 and APT2 are responsible for depalmitoylating some proteins (N-HTT, GAD65), depalmitoylation of other cellular substrates, including PSD95 and N-Ras, involves other enzymes.

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Previous studies suggested that APT1, APT2, and PPT1 were the sole mSHs targeted by PalmB (Rusch et al., 2011), whereas HDFP inhibited additional mSHs (Martin et al., 2011). In pulse-chase experiments, we found HDFP robustly inhibited the depalmitoylation of N-Ras, PSD95, and N-HTT (Figure 3A-C). Because palmitate removal from N-Ras and PSD95 does not require APT1 or APT2, their depalmitoylation may be mediated by a distinct mSH that is a common target of both PalmB and HDFP. To identify overlapping targets, we defined a set of 19 candidate mSHs that showed >25% inhibition by HDFP (Supplementary file 1; Martin et al., 2011) but excluded known proteases and mSHs with established luminal activity. We added to this list APT1L, which was previously implicated in BK channel depalmitoylation (Tian et al., 2012) but whose HDFP sensitivity was unknown. The PalmB sensitivity of each enzyme was evaluated by a competitive activity-based protein profiling (cABPP) assay, in which binding of an inhibitor occludes the enzyme active site and prevents labeling with the activity probe fluorophosphonate-rhodamine (FP-rho) (Figure 3D; Kidd et al., 2001). As expected, PalmB significantly reduced FP-rho labeling of both APT1 and APT2 (Figure 3E,H). In contrast, it had little effect on the labeling of seven candidates (Figure 3F,H), highlighting the distinct substrate specificities of PalmB and HDFP. Four mSHs did not label with FP-Rho due to low activity or expression and could not be assessed (Supplementary file 1). Notably, PalmB potently inhibited seven candidates: FASN, PNPLA6, ABHD6, ABHD16A, and ABHD17A/B/C (Figure 3G,H). Thus, PalmB has additional serine hydrolase targets beyond APT1 and APT2 that may function as protein depalmitoylases.

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The set of candidates inhibited by both PalmB and HDFP (Figure 3G,H) includes ABHD6, which associates with PSD95-containing complexes at synapses (Schwenk et al., 2014), and FASN, which functions in palmitoyl-CoA synthesis (Wakil, 1989). However, treatment with the ABHD6 inhibitor WWL70 (Li et al., 2007) or the FASN inhibitor C75 (Kuhajda et al., 2000) did not alter PSD95 depalmitoylation (Figure 3—figure supplement 1A,C). Palmitate turnover on PSD95 was also unaffected by RHC-80267, which moderately inhibited ABHD6 and PNPLA6 (Figure 3—figure supplement 1B,D; Hoover et al., 2008). Thus, ABHD6, PNPLA6, and FASN are unlikely to play a primary role in PSD95 depalmitoylation.

Selective inhibitors that target the remaining four candidates have not been identified. Therefore, we used pulse-chase click chemistry to test if increased expression of these enzymes enhances palmitate turnover. High levels of ABHD16A, ABHD6, or APT1/2 had little effect on N-Ras (Figure 4A) or PSD95 (Figure 4—figure supplement 1A) depalmitoylation. Strikingly, however, expression of ABHD17A, ABHD17B, or ABHD17C accelerated palmitate cycling on these proteins (Figure 4A, Figure 4—figure supplement 1A), strongly suggesting the uncharacterized ABHD17 family of mSHs are novel protein depalmitoylases.

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We focused on ABHD17A, which showed the strongest effect in promoting palmitate turnover on N-Ras and PSD95. The ABHD17 proteins are targeted to membranes by a palmitoylated N-terminal cysteine cluster (Kang et al., 2008; Martin and Cravatt, 2009). We found ABHD17A localized to the plasma membrane and to Rab5- and Rab11-positive endosomes (Figure 4—figure supplement 2A). Mutation of the predicted active site serine (S211A) (Figure 4B) abolished ABHD17A activity (Figure 4C) but did not alter its localization (Figure 4—figure supplement 2C), whereas removing the N-terminal amino acid residues 1-19 (∆N; Figure 4B) shifted it to the cytosol (Figure 4—figure supplement 2B,C) and reduced its catalytic activity (Figure 4C). Importantly, neither mutant stimulated N-Ras or PSD95 depalmitoylation (Figure 4D, Figure 4—figure supplement 1B), suggesting both the catalytic activity and membrane localization of ABHD17A are functionally important.

We next examined the cellular consequences of ABHD17A expression. Disrupting N-Ras palmitoylation by mutating the palmitoylated residue (C181S) or treating cells with the inhibitor 2-bromopalmitate (2-BP) relocalized N-Ras from the plasma membrane to internal organelles, as previously described (Choy et al., 1999; Goodwin et al., 2005) (Figure 4E,F). Overexpression of APT1 or APT2 had little effect on N-Ras localization (Figure 4E,F), consistent with a recent report (Agudo-Ibáñez et al., 2015). In contrast, overexpression of ABHD17A, but not catalytically dead or cytosolic mutant forms, redistributed N-Ras from the plasma membrane to intracellular compartments consistent with its altered palmitoylation status (Figure 4E,F). Taken together, these findings demonstrate the membrane-localized pool of ABHD17A depalmitoylates N-Ras and alters its subcellular targeting.

To determine if the endogenous ABHD17 proteins regulate palmitate cycling in vivo, we investigated the effect of ABHD17 knockdown on N-Ras depalmitoylation in HEK293T cells. RT-qPCR (Reverse transcription quantitative polymerase chain reaction) showed efficient silencing of ABHD17A alone, or ABHD17A, ABHD17B, and ABHD17C in concert, after 72 hr with siRNA treatment (Figure 5A). ABHD17A knockdown had a slight effect on N-Ras depalmitoylation (p=0.084). In contrast, N-Ras palmitate turnover was significantly inhibited when all three ABHD17 proteins were simultaneously downregulated (p=0.0083), and this was not further enhanced by the APT1 and APT2 inhibitors C83 and C115 (Figure 5B). Knockdown was less effective than PalmB treatment, which could be due to activity of the residual ABHD17 enzymes. PalmB may also inhibit additional factors that either directly or indirectly affect N-Ras palmitate cycling. Taken together, these results demonstrate that ABHD17 proteins redundantly mediate palmitate turnover on N-Ras.

Figure 5

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Our discovery that ABHD17 proteins are novel protein depalmitoylases expands the current repertoire of cellular APTs, and suggests depalmitoylation occurs in a substrate-selective and compartment-specific manner. Whereas APT1 and APT2 were proposed to act ubiquitously (Rocks et al., 2010; Vartak et al., 2014), ABHD17-mediated depalmitoylation of N-Ras at the plasma membrane may specifically attenuate oncogenic signaling pathways (Song et al., 2013). ABHD17 proteins are also active in the brain (Bachovchin et al., 2010), where palmitoylated PSD95 regulates AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor nanodomain assemblies linked to synaptic plasticity (Fukata et al., 2013). miRNA-138 targets APT1 to alter dendritic spine size (Siegel et al., 2009), whereas the Caenorhabditis elegans ABHD17 homologue AHO-3 regulates starvation-induced thermotactic plasticity (Nishio et al., 2012). Thus, functionally specialized APTs may prove to be critical modulators of palmitoyl-proteins in distinct cellular processes.

The total number of cellular depalmitoylases is not known. We identified new PalmB targets, consistent with a recent report showing PalmB inhibits ABHD12 and monoacylglyerol lipase (Savinainen et al., 2014). As the mSH superfamily consists of >110 members, only half of which are functionally annotated (Simon and Cravatt, 2010), a comprehensively survey the mSH proteome may uncover yet more depalmitoylases. APTs are a critical element of the dynamic palmitoylation cycle, thus it will be imperative to identify the complete set of cellular APTs and determine how they contribute to the regulation of dynamic palmitoylation.

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Author details

1. David Tse Shen Lin

   1. Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver, Canada
   2. Department of Medical Genetics, University of British Columbia, Vancouver, Canada

   Contribution

   DTSL, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0001-5695-9446

2. Elizabeth Conibear

   1. Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver, Canada
   2. Department of Medical Genetics, University of British Columbia, Vancouver, Canada

   Contribution

   EC, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   conibear@cmmt.ubc.ca

   Competing interests

   The authors declare that no competing interests exist.

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