NMT1 and NMT2 are lysine myristoyltransferases regulating the ARF6 GTPase cycle

Abstract

Lysine fatty acylation in mammalian cells was discovered nearly three decades ago, yet the enzymes catalyzing it remain unknown. Unexpectedly, we find that human N-terminal glycine myristoyltransferases (NMT) 1 and 2 can efficiently myristoylate specific lysine residues. They modify ADP-ribosylation factor 6 (ARF6) on lysine 3 allowing it to remain on membranes during the GTPase cycle. We demonstrate that the NAD+-dependent deacylase SIRT2 removes the myristoyl group, and our evidence suggests that NMT prefers the GTP-bound while SIRT2 prefers the GDP-bound ARF6. This allows the lysine myrisotylation-demyristoylation cycle to couple to and promote the GTPase cycle of ARF6. Our study provides an explanation for the puzzling dissimilarity of ARF6 to other ARFs and suggests the existence of other substrates regulated by this previously unknown function of NMT. Furthermore, we identified a NMT/SIRT2-ARF6 regulatory axis, which may offer new ways to treat human diseases.

Targeting N-myristoylation for therapy of B-cell lymphomas

“…Increased ER stress is a pro-apoptotic phenomenon previously shown in cells treated with another NMT inhibitor 59 . We postulate the inhibition of myristoylation of the Arf1 GTPase, whether at its N-terminal glycine residue or nearby lysine residue 36 , 37 , interferes with its membrane targeting and impairs vesicle trafficking thereby detrimentally affecting chronic/tonic or antigen dependent BCR signaling. Loss of proper Arf1 functionality at the ER may also explain in part the increase in ER stress marker Bip 60 upon PCLX-001 treatment (Fig.…”

“…SFKs, Abl, G α subunits, Arf GTPases, caspase truncated (ct-) Bid, and ct-PAK2 are examples of myristoylated proteins that critically regulate cell growth and apoptosis 23 , 29 – 35 . Recently, NMTs were also shown to be responsible for myristoylation of N-terminally located lysine residues of Arf6 GTPase, thereby adding to their roles in cell signaling 36 , 37 . Because NMTs are essential for the viability of parasites, small molecule inhibitors such as DDD85646 were developed as a T. brucei NMT inhibitor to treat African sleeping sickness 38 .…”

Myristoylation, an Ancient Protein Modification Mirroring Eukaryogenesis and Evolution

“…MYR allows ARF6 to remain on membranes during GTPase cycling [85]. In contrast to the irreversible Gly-MYR, several acyl hydrolases targeting Lys-MYR have recently been described including Sirtuin6, HDAC8, and HDAC11 [82], making Lys-MYR a reversible modification dependent on these specific hydrolases.…”

“…This implies that Lys-MYR, as with other reversible modifications (i.e., phosphorylation, N-terminal acetylation), might be intimately related to signal transduction and tightly regulated by as yet unknown biotic or abiotic signals. In the case of ARF6, NMT prefers the GTP-bound form of ARF6 while SIRT2 prefers the GDP-bound form, allowing an NMT/SIRT2-ARF6 regulatory axis of the GTPase cycle [85]. The N-terminal Lys-MYR might be part of an N-degron, so an important role of Lys-MYR deacylases could be to ensure protein quality control by preventing intracellular Lys-MYR accumulation [84].…”

Mitochondria-Targeted Inhibitors of the Human SIRT3 Lysine Deacetylase

“…Based on this initial compound series, the thiomyristoylated analogs were abandoned due to their apparent selectivity towards SIRT2, which was not surprising based on previous literature. 43,44 A small series of substitutions of the N-terminal functional group were selected based on previous SAR studies and X-ray co-crystal structures, which indicated a high degree of freedom for the selection of functionalities at this position ( Figure S2). 32,35 Based on this series, we proceeded with the 3-phenylpropionyl group (c; Figure 2) and the alkyne-containing group (a, Figure 2, Scheme S6), which is amenable for incorporation of fluorophores or other tags using Cu(I)-catalyzed azide-alkyne Huisgen 3+2 cycloaddition "click" chemistry.…”