Protein acetylation (and deacetylation) areacetylation reactions that occur within living cells asdrug metabolism, by enzymes in the liver and other organs (e. g., the brain). Pharmaceuticals frequently employ acetylation to enable such esters to cross theblood–brain barrier (andplacenta), where they are deacetylated by enzymes (carboxylesterases) in a manner similar toacetylcholine. Examples of acetylated pharmaceuticals arediacetylmorphine (heroin),acetylsalicylic acid (aspirin),THC-O-acetate, anddiacerein. Conversely, drugs such asisoniazid are acetylated within the liver during drug metabolism. A drug that depends on such metabolic transformations in order to act is termed aprodrug.

Acetylation is an important modification of proteins incell biology; and proteomics studies have identified thousands of acetylated mammalian proteins.^[1][2][3] Acetylation occurs as a co-translational andpost-translational modification ofproteins, for example,histones,p53, andtubulins. Among these proteins,chromatin proteins and metabolic enzymes are highly represented, indicating that acetylation has a considerable impact ongene expression andmetabolism. Inbacteria, 90% of proteins involved in central metabolism ofSalmonella enterica are acetylated.^[4][5]

N-terminal acetylation is one of the most common co-translational covalent modifications of proteins ineukaryotes, and it is crucial for the regulation and function of different proteins. N-terminal acetylation plays an important role in the synthesis, stability and localization of proteins. About 85% of all human proteins and 68% inyeast are acetylated at their Nα-terminus.^[6] Several proteins fromprokaryotes andarchaea are also modified by N-terminal acetylation.

N-terminal Acetylation is catalyzed by a set of enzyme complexes, theN-terminal acetyltransferases (NATs). NATs transfer an acetyl group fromacetyl-coenzyme A (Ac-CoA) to the α-amino group of the firstamino acid residue of the protein. Different NATs are responsible for the acetylation of nascent protein N-terminal, and the acetylation was found to be irreversible so far.^[7]

To date, seven different NATs have been found in humans - NatA, NatB, NatC, NatD, NatE, NatF and NatH. Each of these different enzyme complexes is specific for different amino acids or amino acid sequences which is shown in the following table.

Table 1. The Composition and Substrate specificity of NATs.

NatA is composed of two subunits, the catalyticsubunit Naa10 and the auxiliary subunit Naa15. NatA subunits are more complex in highereukaryotes than in lower eukaryotes. In addition to the genesNAA10 andNAA15, the mammal-specific genesNAA11 andNAA16, make functional gene products, which form different active NatA complexes. Four possible hNatA catalytic-auxiliary dimers are formed by these four proteins. However, Naa10/Naa15 is the most abundant NatA.^[9]

NatA acetylatesSer,Ala-,Gly-, Thr-,Val- andCysN-termini after the initiatormethionine is removed by methionine amino-peptidases. These amino acids are more frequently expressed in the N-terminal of proteins in eukaryotes, so NatA is the major NAT corresponding to the whole number of its potential substrates.^[10]

Several different interaction partners are involved in the N-terminal acetylation by NatA. Huntingtin interacting protein K (HYPK) interacts with hNatA on theribosome to affect the N-terminal acetylation of a subset of NatA substrates. Subunits hNaa10 and hNaa15 will increase the tendency for aggregation of Huntingtin if HYPK is depleted.Hypoxia-inducible factor (HIF)-1α has also been found to interact with hNaa10 to inhibit hNaa10-mediated activation of β-catenin transcriptional activity.^[11]

NatB complexes are composed of the catalytic subunit Naa20p and the auxiliary subunit Naa25p, which are both found in yeast and humans. Inyeast, all the NatB subunits are ribosome-associated; but in humans, NatB subunits are both found to be ribosome-associated and non-ribosomal form. NatB acetylates the N-terminal methionine of substrates starting withMet-Glu-,Met-Asp-,Met-Asn- orMet-Gln- N termini.

NatC complex consists of one catalytic subunit Naa30p and two auxiliary subunits Naa35p and Naa38p. All three subunits are found on the ribosome in yeast, but they are also found in non-ribosomal NAT forms like Nat2. NatC complex acetylates the N-terminal methionine of substratesMet-Leu-,Met-Ile-,Met-Trp- orMet-Phe N-termini.

NatD is only composed with the catalytic unit Naa40p and Naa40p and it is conceptually different form the other NATs. At first, only two substrates, H2A and H4 have been identified in yeast and humans. Secondly, the substrate specificity of Naa40p lies within the first 30-50 residues which are quite larger than the substrate specificity of other NATs. The acetylation of histones by NatD is partially associate with ribosomes and the amino acids substrates are the very N-terminal residues, which makes it different fromlysine N-acetyltransferases (KATs).^[12]

NatE complex consists with subunit Naa50p and two NatA subunits, Naa10p and Naa15p. The N terminus of Naa50p substrates is different from those acetylated by the NatA activity of Naa10p.^[13] NAA50 in plants is essential to control plant growth, development, and stress responses and NAA50 function is highly conserved between humans and plants.^{[14][15][16][17]}

NatF is a NAT that is composed of the Naa60 enzyme. Initially, it was thought that NatF was only found in higher eukaryotes, since it was absent from yeast.^[18] However, it was later found that Naa60 is found throughout the eukaryotic domain, but was secondarily lost in the fungi lineage.^[19] Compared to yeast, NatF contributes to the higher abundance of N-terminal acetylation in humans. NatF complex acetylates the N-terminal methionine of substratesMet-Lys-,Met-Leu-,Met-Ile-,Met-Trp- andMet-Phe N termini which are partly overlapping with NatC and NatE.^[6] NatF has been shown to have an organellar localization and acetylates cytosolic N-termini of transmembrane proteins.^[20] The organellar localization of Naa60 is mediated by its unique C-terminus, which consists of two alpha helices that peripherally associate with the membrane and mediate interactions withPI(4)P.^[21]

NAA80/NatH is an N-terminal acetyltransferase that specifically acetylates the N-terminus ofactin.^[22]

N-terminal acetylation of proteins can affect protein stability, but the results and mechanism were not very clear until now.^[23] It was believed that N-terminal acetylation protects proteins from being degraded as Nα-acetylation N-termini were supposed to block N-terminal ubiquitination and subsequentprotein degradation.^[24] However, several studies have shown that the N-terminal acetylated protein have a similar degradation rate as proteins with a non-blocked N-terminus.^[25]

N-terminal acetylation has been shown that it can steer the localization of proteins. Arl3p is one of the 'Arf-like' (Arl)GTPases, which is crucial for the organization of membrane traffic.^[26] It requires its Nα-acetyl group for its targeting to the Golgi membrane by the interaction with Golgi membrane-residing protein Sys1p. If thePhe or Tyr is replaced by anAla at the N-terminal of Arl3p, it can no longer localized to the Golgi membrane, indicating that Arl3p needs its natural N-terminal residues which could be acetylated for proper localization.^[27]

Protein N-terminal acetylation has also been proved to relate with cell cycle regulation and apoptosis with protein knockdown experiments. Knockdown of the NatA or the NatC complex leads to the induction ofp53-dependentapoptosis, which may indicate that the anti-apoptotic proteins were less or no longer functional because of reduced protein N-terminal acetylation.^[28] But in contrast, thecaspase-2, which is acetylated by NatA, can interact with the adaptor protein RIP associated Ich-1/Ced-3 homologous protein with a death domain (RAIDD). This could activate caspase-2 and inducecell apoptosis.^[29]

Ribosome proteins play an important role in the protein synthesis, which could also be N-terminal acetylated. The N-terminal acetylation of the ribosome proteins may have an effect on protein synthesis. A decrease of 27% and 23% in the protein synthesis rate was observed with NatA and NatB deletion strains. A reduction of translation fidelity was observed in the NatA deletion strain and a defect in ribosome was noticed in the NatB deletion strain.^[30]

NATs have been suggested to act as both onco-proteins and tumor suppressors in human cancers, and NAT expression may be increased and decreased in cancer cells. Ectopic expression of hNaa10p increasedcell proliferation and up regulation of gene involved in cell survival proliferation andmetabolism. Overexpression of hNaa10p was in the urinarybladder cancer,breast cancer andcervical carcinoma.^[31] But a high level expression of hNaa10p could also suppress tumor growth and a reduced level of expressed hNaa10p is associated with a poor prognosis, large tumors and more lymph node metastases.

Table 2. Overview of the expression of NatA subunits in various cancer tissues^[32]

Proteins are typically acetylated onlysine residues and this reaction relies onacetyl-coenzyme A as the acetyl group donor.Inhistone acetylation and deacetylation, histone proteins are acetylated and deacetylated on lysine residues in the N-terminal tail as part ofgene regulation. Typically, these reactions are catalyzed byenzymes withhistone acetyltransferase (HAT) orhistone deacetylase (HDAC) activity, although HATs and HDACs can modify the acetylation status of non-histone proteins as well.^[33]

The regulation of transcription factors, effector proteins,molecular chaperones, and cytoskeletal proteins by acetylation and deacetylation is a significant post-translational regulatory mechanism^[34] These regulatory mechanisms are analogous to phosphorylation and dephosphorylation by the action ofkinases andphosphatases. Not only can the acetylation state of a protein modify its activity but there has been recent suggestion that this post-translational modification may also crosstalk withphosphorylation,methylation,ubiquitination, sumoylation, and others for dynamic control of cellular signaling.^[35] The regulation oftubulin protein is an example of this in mouse neurons and astroglia.^[36][37] Atubulin acetyltransferase is located in theaxoneme, and acetylates the α-tubulin subunit in an assembled microtubule. Once disassembled, this acetylation is removed by another specific deacetylase in the cell cytosol. Thus axonemal microtubules, which have a long half-life, carry a "signature acetylation," which is absent from cytosolic microtubules that have a shorter half-life.

In the field ofepigenetics,histone acetylation (anddeacetylation) have been shown to be important mechanisms in the regulation of gene transcription. Histones, however, are not the only proteins regulated byposttranslational acetylation. The following are examples of various other proteins with roles in regulating signal transduction, whose activities are also affected by acetylation and deacetylation.

Thep53 protein is atumor suppressor that plays an important role in the signal transactions in cells, especially in maintaining the stability of thegenome by preventing mutation. Therefore, it is also known as "the guardian of the genome". It also regulates thecell cycle and arrests cell growth by activating a regulator of the cell cycle,p21. Under severeDNA damage, it also initiatesprogrammed cell death.The function ofp53 is negatively regulated byoncoproteinMdm2. Studies suggested thatMdm2 will form a complex withp53 and prevent it from binding to specific p53-responsive genes.^[38][39]

The acetylation of p53 is indispensable for its activation. It has been reported that the acetylation level of p53 will increase significantly when the cell undergoes stress. Acetylation sites have been observed on the DNA binding domain (K164 and K120) and the C terminus.^[40] Acetylation sites demonstrate significant redundancy: if only one acetylation site is inactivated by mutation to arginine, the expression ofp21 is still observed. However, if multiple acetylation sites are blocked, the expression ofp21 and the suppression of cell growth caused byp53 is completely lost. In addition, the acetylation ofp53 prevents its binding to the repressorMdm2 on DNA.^[41] In addition, it is suggested that the p53 acetylation is crucial for its transcription-independentproapoptotic functions.^[42] An acetylation site of the C-terminus was investigated bymolecular dynamics simulations andcircular dichroism spectroscopy, and it was suggested that the acetylation changes the structural ensemble of the C-terminus.^[43]

Since the major function ofp53 istumor suppressor, the idea that activation of p53 is an appealing strategy for cancer treatment.Nutlin-3^[44] is a small molecule designed to targetp53 andMdm2 interaction that keptp53 from deactivation.^[45] Reports also shown that thecancer cell under the Nutilin-3a treatment, acetylation of lys 382 was observed in the c-terminal of p53.^[46][47]

The structure ofmicrotubules is long, hollow cylinder dynamically assembled from α/β-tubulin dimers. They play an essential role in maintaining the structure of the cell as well as cell processes, for example, movement oforganelles.^[48] In addition,microtubule is responsible of formingmitotic spindle ineukaryotic cells to transportchromosomes incell division.^[49][50]

The acetylated residue of α-tubulin is K40, which is catalyzed by α-tubulin acetyl-transferase (α-TAT) in human. The acetylation of K40 on α-tubulin is a hallmark of stablemicrotubules. The active site residues D157 and C120 of α-TAT1 are responsible for the catalysis because of the shape complementary to α-Tubulin. In addition, some unique structural features such as β4-β5hairpin, C-terminal loop, and α1-α2 loop regions are important for specific α-Tubulinmolecular recognition.^[51] The reverse reaction of the acetylation is catalyzed byhistone deacetylase 6.^[52]

Sincemicrotubules play an important role incell division, especially in theG2/M phase of thecell cycle, attempts have been made to impedemicrotubule function using small molecule inhibitors, which have been successfully used in clinics as cancer therapies.^[53] For example, thevinca alkaloids andtaxanes selectively bind and inhibitmicrotubules, leading to cell cycle arrest.^[54] The identification of the crystal structure of acetylation of α-tubulin acetyl-transferase (α-TAT) also sheds a light on the discovery of small molecule that could modulate the stability or de-polymerization oftubulin. In other words, by targeting α-TAT, it is possible to prevent the tubulin from acetylation and result in the destabilization of tubulin, which is a similar mechanism for tubulin destabilizing agents.^[51]

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is phosphorylated by receptor associatedkinases, for example,Janus-family tyrosine kinases, and translocate tonucleus. STAT3 regulates several genes in response togrowth factors andcytokines and play an important role in cell growth. Therefore,STAT3 facilitatesoncogenesis in a variety of cell growth related pathways. On the other hand, it also play a role in thetumor suppressor.^[55]

The acetylation of Lys685 ofSTAT3 is important forSTAT3 homo-dimerization, which is essential for the DNA-binding and the transcriptional activation ofoncogenes. The acetylation ofSTAT3 is catalyzed byhistone acetyltransferasep300, and reversed by type 1histone deacetylase. The lysine acetylation of STAT3 is also elevated in cancer cells.^[56]

Since the acetylation ofSTAT3 is important for itsoncogenic activity and the fact that the level of acetylated STAT3 is high in cancer cells, it is implied that targeting acetylated STAT3 forchemoprevention andchemotherapy is a promising strategy. This strategy is supported by treatingresveratrol, an inhibitor of acetylation of STAT3, in cancer cell line reverses aberrant CpG island methylation.^[57]

• Compendium of protein lysine acetylation
• Glycosylation
• Lipidation
• Proteolysis

• Post-translational modification