Protein depalmitoylases

Abstract

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.

Keywords: Palmitoylation; inhibitor; post-translational modification; serine hydrolase; thioesterase.

Figure 1. Cellular pathways of protein depalmitoylation

Protein depalmitoylation can proceed either by non-enzymatic hydrolysis, inducible depalmitoylation, or constitutive cycles of palmitoylation and depalmitoylation during trafficking. zDHHC enzymes are shown as either 4 or 6 transmembrane domains. A color version of the figure is available online.

Figure 2. APT inhibitor selectivity index

All listed inhibitors are commercially available, except the promiscuous lipase inhibitor HDFP. HDFP (fluorophosphonate), Palmostatin B (b-lactone), ML378 (N-hydroxyhydantoin carbamate), and ML211 (triazole urea) are covalent inhibitors, although the covalent Palmostatin B adduct is slowly reversible. ML211 inhibits ABHD11 (Adibekian, Martin, Speers, et al. 2010) and PPT1 (unpublished results). ML378 inhibits ABHD6, FAAH, and PPT1 at higher doses (Cognetta et al. 2015). A color version of the figure is available online.

Figure 3. APT2 inhibition restores Scribble plasma membrane localization in Snail-expressing cells

(A) Scribble (Scrib) moves from the plasma membrane to the cytosol in more malignant cells. (B) Scrib is localized at the plasma membrane in MDCK polarized epithelial cells. (C) APT2 inhibition significantly rescues Scrib plasma membrane localization in Snail expressing MDCK cells. Adapted with permission from (Hernandez et al. 2017). A color version of the figure is available online.

Figure 4. Divergence in APT1•ML348 and APT2•ML349 structures

Blue regions signify divergence between APT1 and APT2. The inhibitor selectivity filter APT1-L176M and APT2-M178L is shown in grey sticks in the β8/α4 region. Adapted with permission from (Won et al. 2016). A color version of the figure is available online.

Figure 5. Isoform-selective inhibitor binding in APT1•ML348 and APT2•ML349

Alignment demonstrates inhibitor engagement spanning along a hydrophobic channel towards the catalytic triad. Select residues are highlighted with sequence divergence between APT1 and APT2. Adapted from (Won et al. 2016). A color version of the figure is available online.

Figure 6. Surface polarity of APT1•ML348 and APT2•ML349

Both APT1 and APT2 have similar surface polarity, with similar distributions of polar and hydrophobic surfaces. A color version of the figure is available online.

Figure 7. ABHD17 enzymes are S-palmitoylated and localize to the plasma membrane

(A) ABHD17A-C are S-palmitoylated in a conserved N-terminal cysteine-rich motif separate from the α/β-hydrolase domain. (B) ABHD17A-GFP localization in HeLa cells demonstrates predominant plasma membrane localization. A color version of the figure is available online.

Figure 8. Homology, depalmitoylase activity, and Palmostatin B inhibition of HDFP-sensitive serine hydrolases

HDFP-sensitive seine hydrolases reported from mouse B-cell hybidoma cells (Martin et al. 2011) are shown based on active-site anchored homology. Palmostatin B targets are highlighted in red (Savinainen et al. 2014; Lin and Conibear 2015). Enzymes that reduced PSD-95 S-palmitoylation after over-expression are labeled with a red star (Yokoi et al. 2016). A color version of the figure is available online.