Mechanisms and functions of protein S-acylation

Abstract

Over the past two decades, protein S-acylation (often referred to as S-palmitoylation) has emerged as an important regulator of vital signalling pathways. S-Acylation is a reversible post-translational modification that involves the attachment of a fatty acid to a protein. Maintenance of the equilibrium between protein S-acylation and deacylation has demonstrated profound effects on various cellular processes, including innate immunity, inflammation, glucose metabolism and fat metabolism, as well as on brain and heart function. This Review provides an overview of current understanding of S-acylation and deacylation enzymes, their spatiotemporal regulation by sophisticated multilayered mechanisms, and their influence on protein function, cellular processes and physiological pathways. Furthermore, we examine how disruptions in protein S-acylation are associated with a broad spectrum of diseases from cancer to autoinflammatory disorders and neurological conditions.

Fig. 1 ∣. S-Acylation enzymes and cycles.

a, Overview of protein S-acylation regulation. A target protein of interest is acylated at the thiol of a target cysteine residue by zinc-finger and aspartate–histidine–histidine–cysteine (DHHC) motif-containing (ZDHHC) enzymes, which use acyl-coenzyme A (acyl-CoA) molecules (such as palmitoyl-CoA, shown) as an acyl donor. The thioester-tethered lipid modification can be removed through hydrolysis mediated by deacylation enzymes such as acyl-protein thioesterases (APTs). ZDHHC and APT inhibitors, as well as imaging probes, enable the perturbation and study of this dynamic regulatory process. b, Phylogenetic tree of ZDHHC S-acyltransferases. The 23 human ZDHHCs are categorized into subfamilies on the basis of their full-length amino acid sequences. c, Core membrane topology and conserved sequence motifs. ZDHHC proteins contain at least four transmembrane domains. The conserved DHHC cysteine-rich domain (green) includes the canonical DHHC motif (magenta) situated on the cytoplasmic face of the lipid bilayer. Additional conserved features include an aspartic acid–proline–glycine (DPG) motif of unknown function that lies adjacent to transmembrane domain 2 on the cytoplasmic face of the membrane (yellow); a threonine–threonine–x–glutamic acid (TTxE) motif that interacts with the catalytic DHHC and is required for robust enzymatic activity (red); and a palmitoyltransferase conserved carboxy-terminal (PaCCT) motif, part of an amphipathic helix that interacts with transmembrane domains 3 and 4 and contributes to stabilization of the C-terminal domain (blue). In a subset of ZDHHCs, cysteine residues within the PaCCT motif are S-acylated^,,. d, Structures of ZDHHC20 (Protein Database (PDB) entry [PDB:6BML]) and the ZDHHC9–golgin subfamily A (GOLGA) member 7 (GCP16) heterodimer [PDB:8HF3]. The four transmembrane domain helices in ZDHHC20 and ZDHHC9 (blue) form a tepee-like cavity into which the acyl chain of the acyl-CoA co-substrate is inserted. Cytoplasmic regions flanking the green DHHC cysteine-rich domain are shown in white. Two zinc ions (brown spheres) are positioned between β-hairpins, each coordinated by three cysteines and a histidine. The zinc ions do not coordinate the active-site cysteine in the DHHC motif and are not involved in catalysis but, rather, serve a structural role. In the ZDHHC9–GCP16 complex, GCP16 (red) lacks a transmembrane domain but has two α-helices that insert into the membrane and interact with transmembrane domains 2 and 3. The position of these helices is analogous to that of the hydrophobic loop in ZDHHC20 (red-loop), which stabilizes the structure to fully support enzyme activity. The hydrophobic loop of GCP16 probably fulfils a similar function and might partly account for the requirement of ZDHHC9 for an accessory protein. Three additional interfaces drive the main interactions of GCP16 and ZDHHC9: two involve the zinc-finger motifs and the third contacts a polyproline helix near the end of the cytoplasmic domain. Together, these interactions stabilize the structure of ZDHHC9 and promote its catalytic activity. CMA, cyano-myracrylamide.

Fig. 2 ∣. Consequences of protein S-acylation.

S-Acylation can be categorized as having primary, secondary and tertiary consequences. a, S-Acylation of a cytosolic protein can promote its association with a membrane, and specifically with a particular type of membrane domain. S-Acylation of a transmembrane protein can move it to a specific membrane nanodomain. b, S-Acylation can trigger changes in a protein’s conformation, locally or globally, including of its transmembrane domains. c,d, These primary consequences can lead proteins to move into regions of the membrane that have a different curvature or thickness (c), or enable a protein to interact with other proteins, thanks to their new proximity or change in conformation (d). e,f, Once the primary or secondary consequences of S-acylation occur, S-acylated proteins can be incorporated into transport vesicles, thereby influencing their subcellular localization (e), or undergo multimerization and/or incorporation into protein complexes (f). g–i, S-Acylation can also modulate signalling pathways (g), affect protein activity and/or function (h) and, perhaps, affect other post-translational modifications of the same protein (i).

Fig. 3 ∣. S-Acylation and innate immunity.

a, S-Acylation can control the surface abundance and specific localization of receptors involved in innate immunity. Some Toll-like receptors (TLRs) localize to the cell surface and to specific domains in an S-acylation-dependent manner, as do tumour necrosis factor 1 receptor (TNFR1) and the death receptor Fas. b, S-Acylation influences the ability of various transmembrane and soluble intracellular innate immunity sensors to signal to their downstream effectors. Zinc-finger and aspartate–histidine–histidine–cysteine (DHHC) motif-containing (ZDHHC) enzymes and acyl-protein thioesterases (APTs) identified in each situation are mentioned in parentheses. Signalling through TLRs requires S-acylation of the downstream effector MYD88 (1). S-Acylation of the transmembrane TNF precursor and TNFR1 ensure that the two transmembrane proteins remain segregated in different regions of the plasma membrane, which prevents premature binding of TNF to its receptor. Binding of soluble TNF to its receptor triggers deacylation and endocytosis, which initiates further intracellular signalling (2). The function and dimerization of cyclic GMP–AMP synthase (cGAS) depends on its S-acylation status (3). The cyclic dinucleotides generated by cGAS bind to STING (stimulator of interferon genes) but its ability to signal to downstream effectors once transported to the Golgi depends on its S-acylation (4). The intracellular danger detectors NOD1 and NOD2 (nucleotide-binding oligomerization domain-containing proteins 1 and 2, respectively) must undergo S-acylation before associating with intracellular bacteria-containing membrane-bound structures and inducing appropriate signalling and inflammatory responses (5). c, S-Acylation accelerates or slows down the lysosomal degradation of innate immunity sensors. Stimulus-provoked assembly of the NLRP3 inflammasome induces caspase-1-mediated proteolysis of the pore-forming protein gasdermin D. However, for gasdermin D to oligomerize and puncture membranes it must undergo S-acylation (1). Given the potency of the NLRP3 inflammasome, it must be switched off by S-acylation of NLRP3, which leads to its targeting to lysosomes via chaperone-mediated autophagy (2). S-Acylation of NOD2 also inhibits its targeting to autophagosomes in a p62-dependent manner (3). LYPLA1, lysophospholipase 1.

Fig. 4 ∣. Effect of S-acylation on physiological pathways.

a, The fatty acid transporter CD36 has two acylation sites at the cytosolic boundary of each of its two transmembrane domains. S-Acylation of these four sites controls trafficking of CD36 through the secretory and endocytic pathways. The zinc-finger and aspartate–histidine–histidine–cysteine (DHHC) motif-containing (ZDHHC) enzymes ZDHHC4, ZDHHC6, and ZDHHC7 are involved in the biogenesis of CD36 and its constitutive delivery to the plasma membrane (5). Constitutive acylation by ZDHHC5 and deacylation by acyl-protein thioesterase 1 (APT1), which are present at the plasma membrane, control the steady-state cell surface abundance of CD36 (1). ZDHHC5-mediated S-acylation of CD36 also enables its regulated, stimulus-induced delivery to the cell surface from an intracellular pool (4). S-Acylation of CD36 is required for its ability to bind fatty acids at the cell surface, which in turn triggers APT1-mediated deacylation (2), endocytosis and intracellular fatty acid delivery (3). The activity of ZDHHC5 can be regulated through insulin signalling (4). b, S-Acylation has several important roles in neurons and oligodendrocytes. Multiple S-acylating enzymes regulate neuronal morphology, the formation of specialized compartments and the degeneration of axons following injury. Numerous S-acylating and deacylation enzymes regulate the formation and plasticity of synaptic connections. Emerging work has demonstrated important roles for S-acylation in oligodendrocyte maturation and myelination. ABHD17, α/β-hydrolase domain-containing protein 17; PPT1, palmitoyl-protein thioesterase 1.