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Figure 1. Two-step aminoacylation and mis-aminoacylation reactions by ARSs. (i): aminoacylation. The first reversible step involves activation of an amino acid (aa) by ATP with the release of inorganic pyrophosphate (PPi). In the second step the amino acid is transferred to the tRNA with release of AMP. (ii): mis-aminoacylation. Formation of mis-activated and mis-charged tRNA in the presence of non-cognate aminoacid.

Aminoacyl-tRNA synthetases (ARSs) are highly conserved group of ancient enzymes that are critical components of the cellular protein synthesis machinery [2, 3]. They catalyze the two-step aminoacylation reaction during protein synthesis. First step involves activation of amino acid by an ATP molecule resulting in the formation of aminoacyl adenylate and release of inorganic pyrophosphate (PPi) (Figure 1(i)). During the second step the activated amino acid is transferred to the 2'- or 3'-OH group of the terminal adenosine of its cognate tRNA while releasing AMP [4]. Each ARS catalyzes the covalent attachment of a single amino acid to one or more tRNA isoacceptors to form charged tRNAs. Molecular elements within the tRNAs serve as determinants or anti-determinants that aid in selection by cognate ARSs [5].

Typically, cells contain twenty ARSs for activating twenty different amino acids. They have been classified into two groups (Table 1) based on chemical properties of their primary sequence homologies [6, 7] and architectures of their secondary structures (catalytic domains) [8]. The ARSs belonging to Class I are mostly monomers while Class II ARSs are typically dimers or tetramers. The catalytic site of Class I ARSs comprises of a characteristic Rossmann dinucleotide binding fold marked by HIGH and KMSKS signature peptides [9, 10] unlike Class II ARSs that possess a more unique catalytic core. The aminoacylation reaction catalyzed by Class I enzymes is mechanistically different from that of class II [2].

Class I ARSs utilize a large portion of the enzyme to bind to tRNA [11]. The N-terminal catalytic Rossmann fold [12] binds the acceptor stem of the tRNA from the minor groove side, and therefore orients the 2'-OH group of the tRNA A76 ribose for attachment to the amino acid [13, 14]. ATP (in an extended conformation) binding is facilitated by interactions with the conserved KMSKS and HIGH consensus sequences within the Rossmann fold while amino acid binding is facilitated by a conserved aspartate that makes a salt bridge with the α-NH3+ group of the bound amino acid via hydrogen bonds. Amino acid is activated by nucleophilic attack of the α-carboxylate oxygen of the amino acid onto the α-phosphorus atom of ATP. This is followed by the cleavage of the phosphoanhydride linkage between the α- and β-phosphates of ATP releasing PPi. Second step involves the nucleophilic attack of the 2'-OH of the tRNA’s A76 ribose onto the carboxyl carbon of the aminoacyl adenylate intermediate to cleave the mixed anhydride. This process releases AMP along with the formation of charged tRNA [15-17].

The catalytic core of class II ARS is comprised of seven anti-parallel β sheets flanked by α-helices and harbors three conserved motifs [7, 8, 18]. Motif 1 forms a long α-helix followed by a short strand at the dimer interface [19]. Motifs 2 and 3 are found in the active site and form a pair of antiparallel β-strands connected by a loop [7, 8]. Motif 3 binds ATP in bent conformation in which the α-phosphate is positioned over the adenine base, which stacks on a conserved phenylalanine of motif 2. Hence the bound ATP molecule interacts with both motifs 2 and 3. Similar to Class I ARSs, the amino acid activation proceeds by nucleophilic displacement mechanism. But the second step in Class II involves a nucleophilic attack by the 3'-OH of the A76 ribose on the carboxyl carbon of the aminoacyl adenylate intermediate, forming aminoacyl-tRNA and AMP. This occurs because Class II ARSs bind to tRNAs via their major groove and therefore charge the 3'-OH of the terminal adenosine.

Molecular elements within tRNAs that contribute to their recognition by ARSs can be determinants (augment the selection) or anti-determinants. These recognition elements could be in the form of individual nucleotides, base pairs/modifications, or structural motifs. For example, discriminator base N73 within the acceptor stem is a crucial recognition factor for most ARSs. Also, the first few base pairs of acceptor stem often serve as determinants for many tRNAs. Modified nucleotides can act as determinants, as in the case of E. coli tRNAIle, tRNAGlu, tRNALys, and yeast tRNAIle. The long variable loop of tRNASer serves as an identity element interacting specifically with SerRS. Some examples of anti-determinants that prevent mis-aminoacylation of the tRNA by a noncognate ARS include U34 in yeast tRNAIle (blocks MetRS), A73 in human tRNALeu (hinders aminoacylation by SerRS) and a G3:U70 base pair in yeast tRNAAla (blocks ThrRS) [5].

Aminoacylation reaction is an error prone mechanism. There is a possibility of mis-activation and transfer of structurally similar amino acids by a single ARS [20] (Figure 1(ii)). If this phenomenon is prevalent in cells, it would lead to mis-incorporation of amino acids generating defective proteins that might affect cellular viability [21]. In order to ensure fidelity, some of the synthetases have evolved to perform editing functions to reduce the occurrence of errors during protein synthesis. Therefore, ARSs have evolved to perform editing functions to generate higher proportion of accurately synthesized proteins in cells.

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Figure 2. Editing mechanisms of ARSs.

According to Pauling, theoretically, valine can be mis-incorporated once for every 5 correct isoleucine amino acids based on the selection predictions for IleRS [22]. However, the prevention of such high rates of mis-incorporations is achieved by an efficient mechanism of proofreading for at least ten of the ARSs. This process can occur at two stages: pre-transfer (correction of mis-activated amino acid) or post-transfer (correction of mis-charged tRNA) of amino acid to the tRNA (Figure 2). In general, editing ARSs seem to employ both pathways, although one may overshadow the other.

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Figure 3. Amino acid editing can occur via pre-transfer or post-transfer editing pathway. Amino acid analogues of leucine. Amino acids that differ from leucine by distinct arrangements of their methyl groups in their side chains. The red dashed circle indicates the γ-branched methyl on the leucine molecule. This figure has been adapted from [1].

Editing ARSs mostly rely on a hydrolytic active site that has been integrated into the protein. Class I editing synthetases are monomeric with two distinct domains. The aminoacylation occurs in the catalytic site that is comprised of the Rossmann dinucleotide fold in the main body of the enzyme. Appended to the main body, by two β-strand linkers, is a distinct domain called the connective polypeptide 1 (CP1) [23-28], which splits the Rossmann fold [29]. It is located approximately 35 Å from the aminoacylation active site [29] and harbors a distinct catalytic site for post-transfer editing and hydrolysis of mis-charged tRNA [27]. Among Class I ARSs for example, LeuRS, ValRS and IleRS that activate structurally similar aliphatic amino acids are grouped under subclass A. These ARSs can mis-activate non-cognate aliphatic amino acids that differ by as little as a single methyl group. For example, isoleucine and valine which differ by one methyl group can be activated by LeuRS [30] (Figure 3). So a critical task for these enzymes is to distinguish between cognate and non-cognate substrates thereby making the process of protein synthesis less error prone [1]. On the contrary, Class II ARS editing domains are highly diverse.

Every organism does not contain all the 20 ARSs required to activate the 20 amino acids. Some archaeal, bacterial and organellar genomes lack complete set of ARS and therefore carryout indirect aminoacylation pathways that involve non-discriminating (ND) synthetases. These enzymes display broad specificity to form a mis-charged canonical amino acid-tRNA pair, which is then further modified by RNA dependent enzymes, changing the tRNA-bound amino acid. For example, GlnRS and/or AsnRS are missing from many bacteria, archaea, and some eukaryotic organelles [31]. In such cases ND-GluRS or ND-AspRS first generate mis-charged cognate tRNAs (Glu-tRNAGln or Asp-tRNAAsn respectively) following which charged tRNA species are amidated by the appropriate amidotransferases [32-34].

Non-standard and unusual amino acids like selenocysteine (Sec) and pyrrolysine (Pyl) are also incorporated by indirect aminoacylation mechanisms [35]. A promiscuous orthogonal aminoacyl-tRNA synthetase, ORS p-CNF-RS, has been explored to incorporate noncanonical amino acids, like the novel substrate p-cyanopyridylalanine (p-CNpyrA), to enable a pyridine-thiazoline (pyr-thn) macrocyclization in mRNA display in order to quickly identify potent peptide binders of therapeutic protein targets [36]. Romei MG et al incorporated site-specific substitutions of 10 non-canonical amino acids into photoswitchable green fluorescent protein Dronpa2 using five different aminoacyl-tRNA synthetases (aaRSs) and their corresponding tRNA_CUA to study photoisomerization in proteins [37]. D Cervettini et al discovered and evolved aminoacyl-tRNA synthetase–tRNA pairs for incorporating noncanonical amino acids into proteins [38]. S Mondal et al engineered E. coli-derived leucyl tRNA synthetase-tRNA pair to incorporate a photocaged-citrulline into proteins in response to a nonsense codon [39].

In bacteria, mis-charged Ser-tRNASec generated by SerRS is converted to Sec-tRNASec by Sec synthase (or in eukaryotes and archaea O-phosphoseryl-tRNA kinase and Sep-tRNA:Sec-tRNA synthase) following which Sec is inserted co-translationally at an in-frame UGA codon upstream of an RNA stem-loop on a stalled ribosome. The insertion is aided by a second protein called SelB that recognizes specific mRNA sequences located 3' to a UGA codon [35]. Another example is of MetRS that mis-charges tRNAfMet with methionine (Met-tRNAfMet). A formyl group from N10-formyl tetrahydrofolate is subsequently transferred to the charged methionine by the methionyl-tRNA formyltransferase enzyme to produce fMet-tRNAfMet [40]. Pyrrolysine is another non-standard amino acid incorporated into a Methanosarcina barkeri protein possibly by two different mechanisms – a direct aminoacylation of tRNAPyl with pyrrolysine by PylRS or an indirect pathway in which LysRS-I and LysRS-II form a complex with tRNAPyl and attach lysine to it [41-43]. The conversion of mis-charged Lys-tRNAPyl to Pyl-tRNAPyl is not understood.

In eukaryotic cells the ARSs assemble to form multi-synthetase complexes (MSC) unlike the free prokaryotic synthetases. For example, in lower eukaryotes small MSCs have been identified in archaea Methanothermobacter hermautotrophicus (comprising LeuRS, LysRS, and ProRS) [44, 45]. A primitive simple ternary complex consisting of GluRS and MetRS and an auxiliary factor (Arc1p) has been detected in S. cerevisiae which is a unicellular eukaryote [46, 47]. The complexity of MSCs increases with the complexity of the organism. In mammals, the MSCs comprise of nine synthetases namely, ArgRS, AspRS, GlnRS, GluRS, IleRS, LeuRS, LysRS, MetRS and ProRS. Also part of the MSC are three scaffold proteins, aminoacyl-tRNA synthetase interacting multi-functional proteins 1, 2, and 3 (AIMP1, 2, and 3) that promote the assembly of the 1.5 million Da MSC. Specifically, AIMP2 interacts with most of the MSC components, including LysRS, GlnRS, AspRS, GluProRS, and IleRS as well as AIMP1 and AIMP3 [48-50]. The advantages of complex assembly are unclear but it has been proposed to facilitate channeling of charged tRNA to the ribosome during protein synthesis [51]. These assemblies could also act as cellular reservoirs of various non-canonical functions of their components which are released in response to certain stimulation or environmental cues [52]. For example, MSC associated LysRS is released upon IgE induced mast cell activation [53].

Sequence comparisons revealed presence of extensions or insertions in eukaryotic cytoplasmic ARSs that were absent in their prokaryotic counterparts. These modules form well folded structures such as leucine-zippers, glutathione S-transferase (GST) domains (widely found in other human proteins), WHEP and EMAPII domains (found in more than one ARSs or ARS-associated protein factors) and several unique domains (found in a single ARS). Remarkably, the addition of these domains in evolution has been correlated with increasing the complexity of organisms [54].

Leucine zippers exist in ArgRS of higher eukaryotes (insects to humans), and also in scaffold proteins AIMP1 and AIMP2, suggesting that they play critical roles in the assembly of the MSC. GST domains have been identified in MetRS (Saccharomyces cerevisiae, insects to humans), ValRS (vertebrate) [54-56] and three ARS-associated proteins (the yeast Arc1p, AIMP2 and AIMP3) [57]. GST domains have been implicated in the aminoacylation activities of human and yeast MetRS [58].

WHEP domain, {initially discovered in TrpRS(W), HisRS(H), GluProRS(EP)}, is ~50 aa long and shares sequence homology among different ARSs [59]. The three consecutive WHEP domains of human GluProRS aid in translational gene silencing of target mRNAs. They help in the assembly of the gamma-IFN-activated inhibitor of translation (GAIT) complex [60-62] which interacts with eIF4G and blocks 43S recruitment to target mRNAs [62, 63]. Other ARSs that contain WHEP domain include GlyRS, MetRS and human TrpRS [57]. Natural proteolysis or alternative splicing removes the N-terminal WHEP domain of human TrpRS generating T2-TrpRS and mini-TrpRS fragments that can interact with the extracellular domain of vascular endothelial (VE) cadherin to exhibit non-canonical angiostatic activity [64-66].

Metazoan TyrRSs, AIMP1 and yeast Arc1p contain tRNA binding EMAPII (endothelial monocyte activating polypeptide II) domains [57]. Natural proteolysis eliminates EMAPII domain from human TyrRS generating mini-TyrRS with cytokine-like function [67, 68] by exposing a masked tri-peptide cytokine motif [69]. It is interesting to note that EMAPII and WHEP domains only exists within ARS genes which is suggestive of a special selective pressure to develop new functions for ARS genes during the evolution of higher eukaryotes.

Unique domains, specific to particular ARS, have been identified in eukaryotic IleRS (UNE-I1 and UNE-I2), LeuRS (UNE-L), PheRS (UNE-F), GlnRS (UNE-Q), CysRS (UNE-C1 and UNE-C2), vertebrate SerRS (UNE-S) and metazoan and fungal AsnRS (UNE-N) [54, 57]. They share no detectable sequence similarity to other common structural modules. The functional significance of these unique domains is yet to be investigated. UNE-I2 has been shown to interact with the WHEP domains of GluProRS in the MSC [70, 71]. UNE-S containing a nuclear localization signal (NLS) enables SerRS nuclear localization and regulation of VEGFA expression essential for vascular development [72].

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Figure 4. Non-canonical activities of ARSs.

Several scientific investigations reported the broad usage of ARSs as essential proteins or co-factors in multiple cellular metabolic activities [54, 73]. Throughout their long evolutionary history, ARSs have adapted to carry out dual roles in the cell that includes non-protein synthesis activities (Figure 4). The secondary activities of ARSs capitalize on their existing binding sites for RNA, amino acid, and ATP. They also take advantage of inserted domains or peptides for their alternate functions [57]. The range of diverse non-canonical functions encompass amino acid and metabolite biosynthesis, tRNA modification, RNA splicing, translational silencing, transcriptional and translational control, viral assembly, cytokine-like activity, cell signaling, anti-angiogenesis etc. Small ncRNAs derived from tRNAs, such as those derived from mitochondrial tRNAs (mitosRNAs), may also play cell signaling roles [74].

Evolutionary selective pressures might have exploited ARSs for novel functions, perhaps by leveraging their RNA binding properties. Examples from lower organisms include, two different mitochondrial ARSs that have adapted to support group I intron splicing, by promoting folding of introns into an active conformation [76-78]. The most extensively studied and well characterized ARS essential for RNA splicing is the mitochondrial TyrRS from Neurospora crassa [79]. Also S. cerevisiae mitochondrial LeuRS is required for excision of several group I introns [80]. In E. coli, AlaRS and ThrRS aid in the regulation of gene expression at transcription and translational levels. Biochemical studies showed that AlaRS binds to a palindromic sequence that flanks its promoter site to repress transcription of its own gene [81]. Also, ThrRS regulates its production at the translational level by preventing ribosome binding for translation [82].

It has been hypothesized that multi-component complexes act as storehouses for regulatory proteins that are released in response to a trigger and consequently exhibit new auxiliary functions [52]. The MSC is a multi-protein complex that contains nine aminoacyl-tRNA synthetases (ArgRS, AspRS, GlnRS, GluRS, IleRS, LeuRS, LysRS, MetRS and ProRS) and three non-synthetase proteins [50]. Several of these synthetases have non-canonical functions unrelated to aminoacylation.

LysRS, an integral MSC component is secreted in response to tumor necrosis factor-α. It triggers a pro-inflammatory response in target macrophages by enhancing their migration and TNF-α production in a positive feedback loop [83]. Also human LysRS generates diadenosine tetraphosphate (Ap4A), a critical signaling molecule, in order to activate mast cells. In quiescent cells, transcription factors microphthalmia-associated transcription factor (MITF) and upstream transcription factor 2 (USF2) are inhibited by the histidine triad nucleotide-binding protein 1 (Hint-1). Upon extracellular stimulation, phosphorylated form of LysRS dissociates from the MSC localizes to the nucleus where it forms a multiprotein complex with MITF/USF2 and Hint [83, 84]. Ap4A binds to Hint-1 causing its dissociation from MITF/USF2, allowing transcription of their target genes [84, 85].

MetRS, another MSC component harboring the conserved WHEP domain as well as two nuclear localization signals at the C-terminus, translocates to the nucleolus in response to growth factor stimulation and enhances rRNA synthesis [86, 87]. GlnRS also dissociates from the MSC acts as anti-apoptotic factor by inhibiting apoptosis signal-regulating kinase 1 activity (ASK1, a kinase involved in the apoptotic signaling cascade) [88].

The heterodimeric GluProRS is also released from MSC upon phosphorylation in response to IFN-γ and is involved in the translational suppression of multiple IFN-γ inducible mRNAs by assembling into GAIT complex [61]. Another component of the MSC, LeuRS functions as a leucine sensor for the mTOR pathway and mediates the regulation of leucine metabolism by glucose [75]. Importantly, human LeuRS activates the RagD GTPase of mTORC1 in a leucine-dependent manner and the C-terminal portion of LeuRS comprising of ~220 residues (including UNE-L and a LeuRS specific domain) is shown to be essential for interaction with RagD [89-91].

Many ARSs that are not associated with MSC also function in cellular signaling. For example, HisRS and AsnRS interact with cell surface chemokine receptors resulting in pro-inflammatory responses [92]. Vertebrate SerRS acts as a regulator of vascular development and its C-terminal appendage is implicated in this non-canonical role [72, 93, 94]. Natural proteolysis of inactive human TyrRS generates two fragments with distinct cytokine activities and C terminally appended EMAP II-like domain is shown to regulate its cell signaling function [67, 68]. In case of human TrpRS also, alternative splicing or IFN-γ induction [95-97] generates two isoforms (Full-length, 471aa and mini-TrpRS, 424aa). Mini-TrpRS with the N-terminal WHEP domain truncation exhibits angiostatic activity by inhibiting VEGF induced angiogenesis and endogenous vascularization [65, 66].

The three scaffold proteins, AIMP1, AIMP2, and AIMP3, associated with the MSC also possess signaling activities when freed from the complex like ARSs. For example, AIMP1 has dual roles in angiogenesis where it promotes migration of endothelial cells at low levels, but induces apoptosis at high doses [98]. Also AIMP1 is secreted from endothelial and immune cells where it activates monocytes/macrophages to induce synthesis of pro-inflammatory cytokines and plays a role in inflammation [99]. In response to DNA damage, AIMP2 acts as a pro-apoptotic factor via p53 and therefore is involved in regulation of cell death and proliferation [100]. AIMP3 translocates to the nucleus and upregulates p53 through activation of ATM and ATR kinases and functions as a tumor suppressor [101]. Thus the scaffold proteins have developed expanded functions similar to the ARSs and dissociate from the MSC to exhibit these properties.

The expanded functions of ARSs and their expression may be pathologically associated with various human diseases. Some disease states including neurodegenerative and autoimmune disorders have implicated or identified ARS alternate functions when they have been compromised or failed [102].

Charcot Marie Tooth (CMT) disease is a most common heritable disorder of the peripheral nervous system (affecting ~ 1 in 2,500). CMT is characterized by progressive degeneration of distal motor and sensory neuronal function resulting in muscular weakness and atrophy in the distal extremities, stoppage gait, high arched foot, diminished deep-tendon reflexes and impaired sensation. Type I CMTs involve axonal demyelination while type II CMTs show no demyelination with decreased amplitudes of evoked motor and sensory nerve responses respectively [103]. This heterogeneous group of autosomal-dominant peripheral neuropathies is caused by mutations in cytoplasmic ARSs and specifically mutations in GlyRS [104], TyrRS [105] LysRS and AlaRS genes have been discovered and investigated.

The most extensively studied GlyRS harbors CMT causing mutations at the dimer interface, and presumably affect interactions between the two subunits [106]. These mutations cause CMT type IID, a subtype of the disease characterized by a slowly progressing neuropathy affecting mostly the distal extremities. Secondly, mutations in TyrRS result in dominant-intermediate CMT disease (DI-CMT) characterized by slow progressive neuropathy, intermediate nerve conduction velocities, axonal degeneration, and demyelination of peripheral motor and sensory neurons. In neurons expressing mutant TyrRS proteins, their normal function and axonal distribution is disrupted leading to axonal loss, degeneration, and peripheral neuropathy [105].

In case of LysRS, out of three variants identified two severely inhibit enzymatic activity. An editing-defective, recessive missense mutation in AlaRS in a mouse model of ataxia was also reported [21]. The mutant enzyme mischarges tRNAAla with Gly or Ser, leading to amino acid misincorporation and protein misfolding, causing cerebellar Purkinje cell loss and ataxia, but not peripheral axon degeneration.

CMT mutations within in the catalytic domains result in reduced aminoacylation activity either due to mutated active sites or altered dimerization and thereby causing defective global protein synthesis. But this rationale is not applicable in all cases because several TyrRS, LysRS and AlaRS mutants associated with CMT retain full catalytic activity. Hence the exact molecular mechanisms causing CMT pathology are not clearly understood.

Amyotrophic lateral sclerosis is a progressive neurodegenerative disorder of motor neurons. A mutation in Cu/Zn superoxide dismutase 1 (SOD1) gene is sometimes associated with amyotrophic lateral sclerosis [107, 108]. Interestingly, LysRS associates with mutant SOD1 although no such association has been detected with wild-type protein [109]. The SOD1 mutation enhances oligomerization of SOD1 to form aggregates with other proteins including LysRS. But whether this aggregation with SOD1 inhibits its LysRS’s protein synthesis activity contributing to motor neuronal apoptosis and further neurodegeneration is not understood.

‘Anti-synthetase syndrome’ is a condition that involves idiopathic inflammatory myopathies (IIM), interstitial lung diseases (ILD), rheumatoid and erosive arthritis, and Reynaud’s phenomenon resulting from production of antibodies that bind and inhibit ARSs. IIM characterized by chronic muscle inflammation includes three distinct clinic pathologic subgroups – polymyositis, dermatomyositis and inclusion body myositis. In about 30% of all autoimmune patients, autoantibodies against different ARSs (HisRS, ThrRS, AlaRS, IleRS, PheRS, GlyRS and AsnRS) have been found [110, 111]. For example, autoantibodies against HisRS are associated with chronic muscle inflammatory disease or polymyositis, a form of idiopathic inflammatory myopathy [112]. Antibodies to AsnRS are linked to autoimmune interstitial lung disease [113, 114]. IleRS and GlyRS were also shown to be antigenic in screens for autoantibodies generated in myositis patients [115-117].

A lot of emerging scientific evidence is indicating that ARSs play a key role in regulating cancer. The disease manifestation may be due to mis-regulation or altered expression of ARSs. In some forms of cancer, ARS itself is mutated, while in others regulation of ARS expression levels or its function seems to play a role. Some examples are discussed below.

Although LeuRS’s direct association with cancer has not been detected, its upregulation has been reported in lung cancer cells and in case of acute myeloid leukemia [118]. This connection could be possibly through the mTORC1 signaling pathway which is implicated in the process of cancer development where in LeuRS acts as a regulator of mTORC1 signaling [90, 91]. Low expression levels of TrpRS have been associated with recurrence of colorectal cancer [119]. Several other ARSs including AlaRS, α subunit of PheRS, GlyRS, AsnRS, ThrRS, HisRS, and TrpRS have been reported to be over expressed in prostate cancer tissues [120]. Overexpression of LysRS was reported in breast cancer, metastatic cell lines, and glioma-derived stem cells [83, 121, 122]. Also PheRS α-subunit is overexpressed in lung solid tumors and acute phase chronic myeloid leukemia [123, 124]. In human colon cancer, an elevated aminoacylation activity of MetRS has been reported [125], and its overexpression was also studied in several types of cancers, such as malignant fibrous histiocytomas, lipoma, osteosarcomas, malignant gliomas, and glioblastomas [126-129].

Several antibiotics have been developed against ARSs from pathogenic microorganisms [130, 131]. For example, mupirocin is once such inhibitor of eubacterial and archaeal IleRS used for the treatment of Gram-positive pathogens [28]. Tavaborole is used an antifungal agent that targets the LeuRS.

Recent studies have suggested that targeting human ARSs may provide new strategies for developing therapies for human diseases. Halofuginone is a derivative of febrifugine, acts as a prolyladenylate-mimetic, inhibits aminoacylation activity of ProRS and might be applicable for the treatment of cancer [132, 133]. Borrelidin, isolated from Streptomyces [134] inhibits bacterial ThrRS was initially used in the treatment of infectious diseases such as malaria. It also inhibits eukaryotic ThrRS, suggesting that ThrRS could be a useful target for therapeutics.

The tRNAs used in assays are either in vitro transcribed or purified from cells harboring a tRNA expression plasmid. In vitro transcription generates large yields of tRNA and involves transcription from a tRNA gene inserted downstream of a T7 promoter. The plasmid is linearized using Fok I or Bst N1 restriction digestion at the end of the tRNA gene to generate terminal CCA end in the transcript [135-137]. Transcription is initiated from a linearized plasmid under optimized reaction conditions followed by fractionation and purification on 8M urea-10% polyacrylamide gels. The RNA gel bands are further excised, crushed, precipitated using ammonium acetate and butanol extracted to concentrate the tRNA [137]. Alternatively, tRNA can also be prepared from overexpressing strains containing plasmids with tRNA genes under a highly transcribed promoter. The tRNA is purified from cells by phenol extraction [38] and fractionation by native polyacrylamide gel electrophoresis [138]. The tRNA thus generated contains the base modifications characteristic of natural tRNAs but the yields are low compared to in vitro transcription.

The aminoacylation reaction is traditionally conducted to monitor the formation of radiolabeled ³H/¹⁴C-charged tRNA over time. Typically the assay contains 60 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, 1–50 nM ARS enzyme, and a range of amino acid, ATP, and tRNA concentrations. The reactions are initiated by the addition of 2-4 mM ATP and incubated at 37 °C. Reactions are then quenched at specific time points by transferring to filter pads presoaked in 5% trichloroacetic acid. The pads are further washed with cold 5% trichloroacetic acid and 70% ethanol for 10 min. The pads are dried under a heat lamp prior to processing in a liquid scintillation counter.

This assay is conducted to investigate the ARS dependent amino acid activation independent of tRNA. Reaction is conducted in 50 mM Hepes (pH 8.0), 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 1 mM ³²P-PPi, and aminoacid in varying concentrations. It is initiated by the addition 1-50 nM ARS enzyme. Subsequently, 2 µl aliquots are quenched by spotting on polyethyleneimine (PEI) cellulose thin layer chromatography pre-run in deionized H2O. The phosphate and adenosine products are separated in 750 mM KH2PO4 (pH 3.5) and 4 M urea and visualized by phosphoroimaging. These assays may be carried out to determine which amino acids are mis-activated by the ARS [139].

Amino acid editing in ARSs can occur via pre- or post-transfer mechanism. A typical post-transfer editing assay involves presenting mis-charged tRNA substrate to the synthetase to detect its hydrolysis in the presence of specific ARS. Firstly, mis-charged tRNA is prepared in reaction similar to aminoacylation but containing non-cognate amino acid. In a second step the hydrolysis of mis-charged tRNA/hydrolytic editing is carried out in 60 mM Tris (pH 7.5), 10 mM MgCl2, and the mis-charged tRNA. The reactions are initiated with 50-100 nM enzyme and quenched as described above [1].

The ancient housekeeping ARSs primarily involved in cellular protein synthesis have served as remarkable proteins to understand several molecular and cellular mechanisms ranging from protein-protein interactions, RNA–protein interactions, enzyme catalysis, cell signaling events as well as evolutionary milestones. This functional adaptability can be attributed to their structural and functional resilience achieved by incorporating new domains and motifs. Therefore many of the novel functions associated with these enzymes can be exploited for development of therapeutics to combat human diseases.