Molecular basis of fatty acid selectivity in the zDHHC family of S-acyltransferases revealed by click chemistry

Abstract

S-acylation is a major posttranslational modification, catalyzed by the zinc finger DHHC domain containing (zDHHC) enzyme family. S-acylated proteins can be modified by different fatty acids; however, very little is known about how zDHHC enzymes contribute to acyl chain heterogeneity. Here, we used fatty acid-azide/alkyne labeling of mammalian cells, showing their transformation into acyl-CoAs and subsequent click chemistry-based detection, to demonstrate that zDHHC enzymes have marked differences in their fatty acid selectivity. This difference in selectivity was apparent even for highly related enzymes, such as zDHHC3 and zDHHC7, which displayed a marked difference in their ability to use C18:0 acyl-CoA as a substrate. Furthermore, we identified isoleucine-182 in transmembrane domain 3 of zDHHC3 as a key determinant in limiting the use of longer chain acyl-CoAs by this enzyme. This study uncovered differences in the fatty acid selectivity profiles of cellular zDHHC enzymes and mapped molecular determinants governing this selectivity.-acylation is a reversible posttranslational modification (PTM) involving the attachment of fatty acids onto cysteines (1, 2). This PTM occurs on both soluble and transmembrane (TM) proteins and exerts a number of important effects, including mediating membrane binding (of soluble proteins or soluble loops of TM proteins), regulating protein trafficking and targeting to cholesterol-rich membrane microdomains, and modulating protein stability (3, 4). These actions of S-acylation on a diverse array of cellular proteins affect many important physiological pathways, and defects in this process are linked to a number of major diseases and disorders (2, 5).S-acylation is mediated by the opposing actions of acyltransferases and thioesterases. S-acyltransferase enzymes belong to the zinc finger DHHC domain containing (zDHHC) protein family, which are encoded by 24 distinct genes (6-8). zDHHC enzymes are thought to share the same overall membrane topology, with four to six transmembrane domains and the N and C termini present in the cytosol (9). The catalytic DHHC cysteinerich domain (CRD) of the enzymes lies in a cytosolic loop (9), allowing zDHHC enzymes to modify substrate cysteines present at the cytosol-membrane interface. The S-acylation reaction is thought to proceed through an enzyme-acyl intermediate, where the acyl chain is attached to the cysteine of the DHHC motif via a thioester linkage (often referred to as enzyme "autoacylation") (10, 11). The S-acyl chain is then transferred to a cysteine residue of a substrate protein (10,11). This overall process is referred to as a "ping-pong" reaction mechanism. There has been progress in identifying the zDHHC enzymes that are active against many substrate proteins (2), although we lack a detailed understanding of the protein substrate profiles of individual enzymes and how enzyme-substrate interaction specificity is achieved. Coexpression experiments have suggested that individual zDHHC enzymes might exhibit a level of overlap in their ...

Structural Basis for Substrate Recognition by the Ankyrin Repeat Domain of Human DHHC17 Palmitoyltransferase

“…However, in our experiments we notice that DHHC17 does not become autopalmitoylated to the same extent as DHHC15. This behavior is consistent with what was recently observed comparing the autoacylation of several DHHCs and their ability to transfer palmitate to Snap25b (Greaves et al, 2017). We confirmed this by comparing the reactivity of DHHC17 and DHHC15 to NBD-palmitoyl-CoA.…”

“…DHHC15 is a typical DHHC palmitoyl transferase that reacts quickly with palmitoyl-CoA to form a palmitoylated enzyme intermediate. This intermediate is capable of transferring palmitate to a protein substrate such as Snap25b (Greaves et al, 2017). Although DHHC17 does not quickly react with palmitoyl-CoA to form the acylated enzyme intermediate, it binds strongly to its substrate and it is capable of transferring palmitate.…”

Is the Nutritional Value of Fish Fillet Related to Fish Maturation or Fish Age? Integrated Analysis of Transcriptomics and Metabolomics in Blunt Snout Bream (Megalobrama amblycephala)

“…TTN is the major myofibrillar component of vertebrate and invertebrate striated muscle [57]. Moreover, we found ZDHHC, which has been shown to affect fatty acid content [58]. Both TTN and ZDHHC were up-regulated in group II to group III.…”

CYRI (FAM49) proteins are local inhibitors of Scar/WAVE induced lamellipodia that bind directly to active Rac1

“…C14:0-azide was synthesised as previously described 9 . Transfected HEK293T cells were incubated with 100 μM of C14:0-azide (in DMEM with 1 mg/mL defatted BSA) for 4 h at 37 °C.…”

“…Rac1 is one of the activators of Scar/WAVE, but studies in live cells have revealed much faster dynamics of Scar/WAVE recruitment and turnover at the leading edge than Rac [4][5][6] . At least three negative regulators of Arp2/3 complex have been described, Gadkin 7 which sequesters Arp2/3, PICK1 (whose role as an Arp2/3 inhibitor is still under debate) 8,9 and Arpin, which mimics the tail of WASP proteins but inhibits rather than stimulating the Arp2/3 complex 10 . Here, we describe the first negative regulator of Scar/WAVE complex activation by Rac1, CYRI (encoded by the FAM49 gene), an evolutionarily conserved protein that mimics the Rac1 interaction domain of CYFIP/PIR121 and thus acts as a direct local competitor for activated Rac1 at the plasma membrane.…”