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Wnt signalling is essential for regulation of embryonic development and adult tissue homeostasis 1 – 3 , and aberrant Wnt signalling is frequently associated with cancers 4 . Wnt signalling requires palmitoleoylation on a hairpin 2 motif by the endoplasmic reticulum-resident membrane-bound O -acyltransferase Porcupine 5 – 7 (PORCN). This modification is indispensable for Wnt binding to its receptor Frizzled, which triggers signalling 8 , 9 . Here we report four cryo-electron microscopy structures of human PORCN: the complex with the palmitoleoyl-coenzyme A (palmitoleoyl-CoA) substrate; the complex with the PORCN inhibitor LGK974, an anti-cancer drug currently in clinical trials 10 ; the complex with LGK974 and WNT3A hairpin 2 (WNT3Ap); and the complex with a synthetic palmitoleoylated WNT3Ap analogue. The structures reveal that hairpin 2 of WNT3A, which is well conserved in all Wnt ligands, inserts into PORCN from the lumenal side, and the palmitoleoyl-CoA accesses the enzyme from the cytosolic side. The catalytic histidine triggers the transfer of the unsaturated palmitoleoyl group to the target serine on the Wnt hairpin 2, facilitated by the proximity of the two substrates. The inhibitor-bound structure shows that LGK974 occupies the palmitoleoyl-CoA binding site to prevent the reaction. Thus, this work provides a mechanism for Wnt acylation and advances the development of PORCN inhibitors for cancer treatment. Cryo-electron microscopy structures of human Porcupine in complex with palmitoleoyl-coenzyme A, the inhibitor LGK974 and its peptide substrate suggest a mechanism for Wnt acylation.

Most eukaryotic proteins are N-terminally acetylated, but the functional impact on a global scale has remained obscure. Using genome-wide CRISPR knockout screens in human cells, we reveal a strong genetic dependency between a major N-terminal acetyltransferase and specific ubiquitin ligases. Biochemical analyses uncover that both the ubiquitin ligase complex UBR4-KCMF1 and the acetyltransferase NatC recognize proteins bearing an unacetylated N-terminal methionine followed by a hydrophobic residue. NatC KO-induced protein degradation and phenotypes are reversed by UBR knockdown, demonstrating the central cellular role of this interplay. We reveal that loss of Drosophila NatC is associated with male sterility, reduced longevity, and age-dependent loss of motility due to developmental muscle defects. Remarkably, muscle-specific overexpression of UbcE2M, one of the proteins targeted for NatC KO-mediated degradation, suppresses defects of NatC deletion. In conclusion, NatC-mediated N-terminal acetylation acts as a protective mechanism against protein degradation, which is relevant for increased longevity and motility. The most common protein modification in eukaryotes is N-terminal acetylation, but its functional impact has remained enigmatic. Here, the authors find that a key role for N-terminal acetylation is shielding proteins from ubiquitin ligase-mediated degradation, mediating motility and longevity.

Glycine decarboxylase (GLDC) is a key enzyme of glycine cleavage system that converts glycine into one-carbon units. GLDC is commonly up-regulated and plays important roles in many human cancers. Whether and how GLDC is regulated by post-translational modifications is unknown. Here we report that mechanistic target of rapamycin complex 1 (mTORC1) signal inhibits GLDC acetylation at lysine (K) 514 by inducing transcription of the deacetylase sirtuin 3 (SIRT3). Upon inhibition of mTORC1, the acetyltransferase acetyl-CoA acetyltransferase 1 (ACAT1) catalyzes GLDC K514 acetylation. This acetylation of GLDC impairs its enzymatic activity. In addition, this acetylation of GLDC primes for its K33-linked polyubiquitination at K544 by the ubiquitin ligase NF-X1, leading to its degradation by the proteasomal pathway. Finally, we find that GLDC K514 acetylation inhibits glycine catabolism, pyrimidines synthesis and glioma tumorigenesis. Our finding reveals critical roles of post-translational modifications of GLDC in regulation of its enzymatic activity, glycine metabolism and tumorigenesis, and provides potential targets for therapeutics of cancers such as glioma. An increase in glycine decarboxylase (GLDC) activity, a key enzyme for glycine catabolism, has been associated to tumourigenesis. Here, the authors show that mTORC1 activation induces GLDC deacetylation which impairs its ubiquitin-associated degradation leading to increased GLDC activity and tumourigenesis.

Acetylation contributes to the bioactivity of numerous medicinally important natural products. However, little is known about the acetylation on sugar moieties. Here we report a saponin acetyltransferase from Astragalus membranaceus . AmAT7-3 is discovered through a stepwise gene mining approach and characterized as the xylose C3′/C4′- O -acetyltransferse of astragaloside IV ( 1 ). To elucidate its catalytic mechanism, complex crystal structures of AmAT7-3/ 1 and AmAT7-3 A310G / 1 are obtained, which reveal a large active pocket decided by a specific sequence AADAG. Combining with QM/MM computation, the regiospecificity of AmAT7-3 is determined by sugar positioning modulated by surrounding amino acids including #A310 and #L290. Furthermore, a small mutant library is built using semi-rational design, where variants A310G and A310W are found to catalyze specific C3′- O and C4′- O acetylation, respectively. AmAT7-3 and its variants are also employed to acetylate other bioactive saponins. This work expands the understanding of saponin acetyltransferases, and provide efficient catalytic tools for saponin acetylation. Currently little is known about the acetylation on sugar moieties. Here the authors report a saponin acetyltransferase from Astragalus membranaceus , AmAT7-3, and utilise crystal structures and QM/MM computation to elucidate the catalytic mechanism: they generate mutants for specific site acetylation.

Approximately 40% of the mammalian proteome undergoes N-terminal methionine excision and acetylation, mediated sequentially by methionine aminopeptidase (MetAP) and N-acetyltransferase A (NatA), respectively 1 . Both modifications are strictly cotranslational and essential in higher eukaryotic organisms 1 . The interaction, activity and regulation of these enzymes on translating ribosomes are poorly understood. Here we perform biochemical, structural and in vivo studies to demonstrate that the nascent polypeptide-associated complex 2 , 3 (NAC) orchestrates the action of these enzymes. NAC assembles a multienzyme complex with MetAP1 and NatA early during translation and pre-positions the active sites of both enzymes for timely sequential processing of the nascent protein. NAC further releases the inhibitory interactions from the NatA regulatory protein huntingtin yeast two-hybrid protein K 4 , 5 (HYPK) to activate NatA on the ribosome, enforcing cotranslational N-terminal acetylation. Our results provide a mechanistic model for the cotranslational processing of proteins in eukaryotic cells. Cotranslational N-terminal methionine excision and acetylation of eukaryotic proteins on ribosomes is coordinated by the nascent polypeptide-associated complex.

The properties of single microtubules within the microtubule network can be modulated through post-translational modifications (PTMs), including acetylation within the lumen of microtubules. To access the lumen, the enzymes could enter through the microtubule ends and at damage sites along the microtubule shaft. Here we show that the acetylation profile depends on damage sites, which can be caused by the motor protein kinesin-1. Indeed, the entry of the deacetylase HDAC6 into the microtubule lumen can be modulated by kinesin-1-induced damage sites. In contrast, activity of the microtubule acetylase αTAT1 is independent of kinesin-1-caused shaft damage. On a cellular level, our results show that microtubule acetylation distributes in an exponential gradient. This gradient results from tight regulation of microtubule (de)acetylation and scales with the size of the cells. The control of shaft damage represents a mechanism to regulate PTMs inside the microtubule by giving access to the lumen. Microtubules are acetylated on the inside of their hollow lumen, a modification linked to their lifespan. Here, the authors show that damage holes act as entry points for a deacetylase to access the lumen, thereby locally counteracting acetylation.

Acetylation of histone H3K23 has emerged as an essential posttranslational modification associated with cancer and learning and memory impairment, yet our understanding of this epigenetic mark remains insufficient. Here, we identify the native MORF complex as a histone H3K23-specific acetyltransferase and elucidate its mechanism of action. The acetyltransferase function of the catalytic MORF subunit is positively regulated by the DPF domain of MORF (MORF DPF ). The crystal structure of MORF DPF in complex with crotonylated H3K14 peptide provides mechanistic insight into selectivity of this epigenetic reader and its ability to recognize both histone and DNA. ChIP data reveal the role of MORF DPF in MORF-dependent H3K23 acetylation of target genes. Mass spectrometry, biochemical and genomic analyses show co-existence of the H3K23ac and H3K14ac modifications in vitro and co-occupancy of the MORF complex, H3K23ac, and H3K14ac at specific loci in vivo. Our findings suggest a model in which interaction of MORF DPF with acylated H3K14 promotes acetylation of H3K23 by the native MORF complex to activate transcription. Acetylation of histone H3K23 has emerged as an essential posttranslational modification, yet this epigenetic mark remains poorly understood. Here, the authors identify the native MORF complex as a histone H3K23-specific acetyltransferase and show that interaction of the MORF subunit with acylated H3K14 promotes acetylation of H3K23 by this complex to activate transcription.

MEC1-7 is long-sought α-tubulin acetyltransferase It has long been known that in a subset of microtubules, α-tubulin is modified post-translationally by acetylation of lysine-40. There is growing evidence that this highly conserved microtubule modification is a key event during cell polarization, especially in the nervous system. The enzyme responsible for this reaction has now been identified as MEC-17, a protein related to the Gcn5 histone receptor acetyltransferase and required for the function of touch receptor neurons in Caenorhabditis elegans . In eukaryotic cells, a subset of microtubules undergo post-translational modifications such as acetylation, which alters microtubule dynamics and trafficking of motors. These authors identify MEC-17 as the enzyme that directly acetylates α-tubulin in vitro and in vivo and in both invertebrates and vertebrates. This is the identification of the long-sought enzyme that acetylates microtubules. In most eukaryotic cells, subsets of microtubules are adapted for specific functions by post-translational modifications (PTMs) of tubulin subunits. Acetylation of the ε-amino group of K40 on α-tubulin is a conserved PTM on the luminal side of microtubules 1 that was discovered in the flagella of Chlamydomonas reinhardtii 2 , 3 . Studies on the significance of microtubule acetylation have been limited by the undefined status of the α-tubulin acetyltransferase. Here we show that MEC-17, a protein related to the Gcn5 histone acetyltransferases 4 and required for the function of touch receptor neurons in Caenorhabditis elegans 5 , 6 , acts as a K40-specific acetyltransferase for α-tubulin. In vitro , MEC-17 exclusively acetylates K40 of α-tubulin. Disruption of the Tetrahymena MEC-17 gene phenocopies the K40R α-tubulin mutation and makes microtubules more labile. Depletion of MEC-17 in zebrafish produces phenotypes consistent with neuromuscular defects. In C. elegans , MEC-17 and its paralogue W06B11.1 are redundantly required for acetylation of MEC-12 α-tubulin, and contribute to the function of touch receptor neurons partly via MEC-12 acetylation and partly via another function, possibly by acetylating another protein. In summary, we identify MEC-17 as an enzyme that acetylates the K40 residue of α-tubulin, the only PTM known to occur on the luminal surface of microtubules.

In eukaryotes, N-terminal acetylation is one of the most common protein modifications involved in a wide range of biological processes. Most N-acetyltransferase complexes (NATs) act co-translationally, with the heterodimeric NatA complex modifying the majority of substrate proteins. Here we show that the Huntingtin yeast two-hybrid protein K (HypK) binds tightly to the NatA complex comprising the auxiliary subunit Naa15 and the catalytic subunit Naa10. The crystal structures of NatA bound to HypK or to a N-terminal deletion variant of HypK were determined without or with a bi-substrate analogue, respectively. The HypK C-terminal region is responsible for high-affinity interaction with the C-terminal part of Naa15. In combination with acetylation assays, the HypK N-terminal region is identified as a negative regulator of the NatA acetylation activity. Our study provides mechanistic insights into the regulation of this pivotal protein modification. N-terminal acetylation is a common eukaryotic protein modification that is primarily catalysed by the N-acetyl transferase complex A (NatA). Here, the authors present the crystal structure of NatA bound to Huntingtin yeast two-hybrid protein K (HypK) and show that HypK is a negative regulator of NatA.