Tryptophanyl-Trna Synthetase As a Potential Therapeutic Target

International Journal of Molecular Sciences

Review Tryptophanyl-tRNA Synthetase as a Potential Therapeutic Target

Young Ha Ahn 1,2 , Se-Chan Oh 2,3 , Shengtao Zhou 1,* and Tae-Don Kim 2,3,*

1 Department of Obstetrics and Gynecology, Key Laboratory of Birth Defects and Related Diseases of Women and Children of MOE, State Key Laboratory of Biotherapy, West China Second University Hospital, Sichuan University and Collaborative Innovation Center, Chengdu 610041, China; [email protected] 2 Immunotherapy Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea; [email protected] 3 Department of Functional Genomics, KRIBB School of Bioscience, Korea University of Science and Technology, Daejeon 34113, Korea * Correspondence: [email protected] (S.Z.); [email protected] (T.-D.K.)

Abstract: Tryptophanyl-tRNA synthetase (WRS) is an essential enzyme that catalyzes the ligation of tryptophan (Trp) to its cognate tRNAtrp during translation via aminoacylation. Interestingly, WRS also plays physiopathological roles in diseases including sepsis, cancer, and autoimmune and brain diseases and has potential as a pharmacological target and therapeutic. However, WRS is still generally regarded simply as an enzyme that produces Trp in polypeptides; therefore, studies of the pharmacological effects, therapeutic targets, and mechanisms of action of WRS are still at an emerging stage. This review summarizes the involvement of WRS in human diseases. We hope that this will encourage further investigation into WRS as a potential target for drug development in various pathological states including infection, tumorigenesis, and autoimmune and brain diseases.

Keywords: tryptophanyl-tRNA synthetase; sepsis; cancer; Alzheimer’s disease; IFN-γ; kynurenine pathway; tryptophan metabolism Citation: Ahn, Y.H.; Oh, S.-C.; Zhou, S.; Kim, T.-D. Tryptophanyl-tRNA Synthetase as a Potential Therapeutic Target. Int. J. Mol. Sci. 2021, 22, 4523. 1. Introduction https://doi.org/10.3390/ijms22094523 Aminoacyl-tRNA synthetases (ARSs) are essential enzymes that ligate amino acids to cognate tRNAs in protein synthesis. The catalytic activities of ARSs play essential roles in Academic Editor: Amedeo Amedei maintaining cell viability; however, they are also versatile and multifunctional proteins regulated by diverse control mechanisms [1–4]. Several features distinguish eukaryotic Received: 29 March 2021 ARSs from their prokaryotic homologs. The addition of extra domains and sequence Accepted: 23 April 2021 Published: 26 April 2021 adaptations contribute to cellular functions, including cytokine activity associated with inﬂammation, apoptosis, angiogenesis, and tumorigenesis in mammalian synthetases [5,6].

Publisher’s Note: MDPI stays neutral This review focuses on the roles of eukaryotic tryptophanyl-tRNA synthetase (WRS) in with regard to jurisdictional claims in pathological states and its clinical potential as a pharmacological target. published maps and institutional affil- Elevated levels of WRS are expressed in the bovine pancreas, and its various truncated iations. forms are secreted into bovine pancreatic fluid. Thus, an important role of WRS beyond protein translation has long been suspected [7–9]. Eukaryotic WRS exists as a free cytosolic enzyme that self-aggregates to form large homo-oligomers, rather than associate with a multi-tRNA synthetase complex (MSC). This property of WRS has been identified in higher eukaryotes, but not in prokaryotes [10,11]. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Evolutionarily, ARSs have added new domains that have no apparent connection This article is an open access article with aminoacylation. Human WRS has a unique N-terminal extension domain of about distributed under the terms and 150 amino acids that is not present in its prokaryotic counterpart. The extension domain conditions of the Creative Commons is composed of a vertebrate-specific extension of about 50 amino acids, also known as Attribution (CC BY) license (https:// WHEP domain, named after initials of (underlined) WRS, histidyl-tRNA synthetase (HRS), creativecommons.org/licenses/by/ and glutamyl-prolyl-tRNA synthetase (EPRS). WHEP domain includes a specific helix- 4.0/). turn-helix motif and is involved with diverse interactions with other proteins [5,6,12–14]

Int. J. Mol. Sci. 2021, 22, 4523. https://doi.org/10.3390/ijms22094523 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 4523 2 of 13

(Table1) . Elimination of WHEP domain does not markedly inhibit in vitro aminoacylation activities of several synthetases, indicating that the WHEP domains themselves may provide a non-canonical function unrelated to aminoacylation [15,16]. Among 20 ARSs, EPRS and WRS have similarities, in that they have WHEP domains and are regulated by interferon (IFN)-γ. Human EPRS contains three WHEP domains involved in the formation of the IFN-γ activated inhibitor of translation (GAIT) complex, and controlling the translation of vascular endothelial growth factor A (VEGFA) and ceruloplasmin (Cp) [17,18]. Notably, WRS is the only ARS whose expression is induced by IFN-γ [19–23], the action of which is supposedly mediated by WHEP domain. WRS is also rapidly secreted from immune cells in response to both bacterial and viral infections, suggesting a critical role in inﬂammatory response. In contrast, other ARSs were not secreted from monocytes upon in vitro microbial infections [24,25]. Full-length WRS (FL-WRS) is alternatively spliced or truncated into mini-WRS (residues 48–471). Proteolytic digestion of WRS by extracellular proteases also produces the N- Int. J. Mol. Sci. 2021terminally, 22, x FOR truncated PEER REVIEW variants T1-WRS (residues 71–471) and T2-WRS (residues 94–471) [20, 3 of 14 Int. J. Mol. Sci. 2021 , 22, x FOR PEER22, 26REVIEW]. The expression of these truncated variants is stimulated by IFN-γ, which plays3 aof 14

central regulatory role in anti-angiogenesis [27–29]. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEWUnderstanding of the biological functions of truncated WRS variants in vascular home-3 of 14 Table 1. Schematic structure of ARSs containing WHEP domain. EPRS and MRS are components of the multi-tRNA syn- Int. J. TableMol. Sci. 1. 2021 Schematic, 22thetase, x FOR structure PEERcomplexostasis REVIEW of ARSs(MSC) [26, 30 containing –and32] the as wellcell WHEP regula as the domain.tory structure activities EPRS and andare propertiescontrolledMRS are components by of specif secretedic phosphorylationof WRSthe multi-tRNA has progressed [5,35–39]. syn-3 of 14WRS, HRS,

thetase complexand (MSC) GRS and significantlyexist the in cella free regula [form11,tory20 and,21 activities ,are24, 25induced,33 are]. Numerouscontrolled under various by studies specif pathologicalic havephosphorylation associated conditions [5,35–39]. WRS[12,40–44] with WRS,. The infection, HRS,regulatory activities Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 14 andTable GRS 1. Schematicexist inof aWRS free structure areformcancer, controlled andof ARSs autoimmunity, are inducedbycontaining proteolytic under andWHEP cleavage.various brain domain. diseases.pa Thethological EPRS secreted Consideringand conditions WRSMRS areare cleaved thatcomponents[12,40–44] WRS and. isThe produceof secreted theregulatory multi-tRNA WR duringS activitiesvariants bacte-syn- such mini-, T1- of WRS are controlled byrial proteolytic and viral cleavage. infections, The it secreted has been WRS recently are cleaved identified and produce as a promising WRS variants biomarker such mini-, in patients T1- Int. J. thetaseMol. Sci. complex2021, 22and, x (MSC) FOR T2-WRS. PEER and REVIEW theGST, cell Glutathione regulatory S-transferactivities arease; controlled CD, catalytic by specifdomain;ic phosphorylation ERS, glutamyl-tRNA [5,35–39]. synthetase; WRS, HRS, PRS,3 ofprolyl-tRNA 14 andandTable T2-WRS.GRS 1. Schematic exist GST, synthetase;in a freeGlutathionestructure withform EPRS, andof sepsis ARSsS-transfer glutamyl-prolyl-tRNAare [ induced34containing].ase; This CD,under review WHEP catalytic various summarizesdomain.synthetase; domain; pathological EPRS MRS,ERS, the and conditionsglutam physiopathologicalmethionyl-tRNS MRS areyl-tRNA components[12,40–44] synthetase; synthe. The roles oftase; theregulatory PRS, of HRS,multi-tRNA WRS prolyl-tRNA histidyl-tRNA thatactivities syn- have synthetase; synthetase;ofthetaseTable WRS 1. complexare Schematic EPRS,controlledGRS, (MSC) glutamyl-prolyl-tRNA structure glycyl-tRNA by beenand proteolytic the of reported ARSscell synthetase. regula cleavage.containingto synthetase;tory date activitiesThe WHEP and secreted MRS, discusses domain.are methionyl-tRNS controlledWRS EPRS are WRS cleaved byand as specif MRS asynthe potentialandic are phosphorylationproducetase; components HRS, therapeutic WR histidyl-tRNAS variants of [5,35–39]. the target multi-tRNA such synthetase;WRS, for mini-, human HRS, syn- T1- and GRS exist in a free form and are induced under various pathological conditions [12,40–44]. The regulatory activities GRS,andthetase T2-WRS. glycyl-tRNA complex GST, (MSC) synthetase. Glutathione pathologies,and the cellS-transfer regula especiallyase;tory CD, activities infection, catalytic are domain; cancer,controlled autoimmune,ERS, by specifglutamicyl-tRNA phosphorylation Alzheimer’s synthetase; disease [5,35–39]. PRS, (AD). prolyl-tRNA WRS, HRS, synthetase;ofandTable WRS GRS 1. are Schematicexist EPRS,controlled in a glutamyl-prolyl-tRNA freestructure by form proteolytic andof ARSs are inducedcleavage.containing synthetase; under The WHEP secreted various MRS, domain. methionyl-tRNS WRSpathological EPRS are cleaved and conditions MRS synthe and are producetase; components[12,40–44] HRS, WR histidyl-tRNA.S The variants of theregulatory multi-tRNA such synthetase; mini-, activities syn- T1- and T2-WRS. GST, Glutathione S-transferase; CD, catalytic domain; ERS, glutamyl-tRNAAlternative synthetase; PRS,Proteolytic prolyl-tRNA TableGRS,ofthetase 1.WRS Schematicglycyl-tRNA complexare controlled structure(MSC) synthetase.ARSs by and proteolytic of the ARSs cell containing regula cleavage.tory Schematic WHEPTheactivities secreted domain. areStructure controlledWRS EPRS are cleavedAlternativeand by specif MRS andic are phosphorylationproduce componentsProteolytic WRS variants of [5,35–39]. the multi-tRNA such WRS, mini-, HRS, T1- References andsynthetase; T2-WRS.GRSARSs exist EPRS, GST, in a glutamyl-prolyl-tRNA freeGlutathione form and Schematic S-transfer are induced synthetase;ase;Structure underCD, catalytic variousMRS, methionyl-tRNSdomain; pathological ERS, conditionsglutam syntheyl-tRNAtase; [12,40–44]Variants HRS, synthetase; histidyl-tRNA. The regulatory PRS,Cleavage prolyl-tRNAReferences synthetase; activities synthetase complex (MSC) and the cell regulatory activities are controlled by specificVariants phosphorylation Cleavage [5,35– 39]. WRS, HRS, synthetase;GRS,of WRS glycyl-tRNA are EPRS,controlled glutamyl-prolyl-tRNAsynthetase. by proteolytic cleavage. synthetase; The secreted MRS, methionyl-tRNS WRS are cleaved synthe and producetase; HRS, WR histidyl-tRNAS variants such synthetase; mini-, T1- and GRS exist in a free form and are induced under various pathological conditionsAlternative [12 ,40–44].Proteolytic The regulatory activities GRS,and T2-WRS. glycyl-tRNAARSs GST, synthetase. Glutathione Schematic S-transfer ase;Structure CD, catalytic domain; ERS, glutamyl-tRNA synthetase; PRS, prolyl-tRNAReferences N1 of WRSsynthetase; are controlled EPRS, byglutamyl-prolyl-tRNAEPRS proteolytic 1 cleavage. synthetase; The secreted MRS, WRS methionyl-tRNS are cleaved Alternative Variants1512 and synthe produce EPRStase; WRS HRS,Proteolytic Cleavage variants histidyl-tRNA such mini-, synthetase; T1- [17,18,35,36] EPRS 1 1512 EPRSN1 [17,18,35,36] and GRS, T2-WRS. glycyl-tRNAARSs GST,MCS Glutathione synthetase. S-transferase; Schematic CD,Structure catalytic domain; ERS, glutamyl-tRNA synthetase; PRS, prolyl-tRNAReferences MCS AlternativeVariants ProteolyticCleavage synthetase; ARSs EPRS,form glutamyl-prolyl-tRNA Schematic synthetase; Structure MRS, methionyl-tRNS synthetase; HRS, histidyl-tRNA synthetase;References EPRS 1 1512 EPRSVariantsN1 Cleavage [17,18,35,36] GRS,form glycyl-tRNA synthetase.MRS 1 900 Alternative Proteolytic [37–39] MCS MRSEPRSARSs 1 1 Schematic Structure 900 1512 EPRSN1 [17,18,35,36]References [37–39] AlternativeVariants ProteolyticCleavage form ARSs Schematic Structure References MCS EPRS 1 1512 EPRSVariantsN1 Cleavage [17,18,35,36] MRS 1 900 T1-WRS [37–39] [6,13,20,22,27 MCSform WRS 1 471 mini-WRST1-WRS [6,13,20,22,27 WRSEPRS 1 1 471 1512 mini-WRSEPRS EPRSN1N1 [17T2-WRS,18[17,18,35,36],35,36 ] ,28,30–32] MCSform MRS 11 900 1512 [37–39] formMCS T2-WRS ,28,30–32] MRS 11 900 T1-WRS [[6,13,20,22,2737– [37–39]39] form WRSFreef 1 471 mini-WRS Freef HRS 1 509 HRSΔC T2-WRST1-WRS [6,13,20,22,27,28,30–32] [40–42] HRSMRSorm 1 1 509 900 HRS ΔC T1-WRS [6,13,20,22[40–42] [37–39],27,28, WRSWRS 11 471471 mini-WRS orm T2-WRST2-WRST1-WRS 30[6,13,20,22,27,28,30–32]–32] FreeFreef form WRS 1 471 mini-WRS HRSHRS 11 509509 HRS ∆ΔC[C T2-WRS 40,28,30–32]–[40–42]42] Freeform GRS 1 739 T1-WRS [6,13,20,22,27 [43,44] WRSGRS 1 471 mini-WRS [43,44] GRSHRS 1 11 509 739739 HRSΔC [43,44][40–42] Freeform T2-WRS ,28,30–32] HRS 1 509 HRSΔC [40–42] orm GRS GST1 CD 739 WHEP ERS PRS ABD [43,44] FreefGST CD WHEP ERS PRS ABD. HRS 1 509 HRSΔC [40–42] GRS 1 739 [43,44] orm 2. WRS as a Biomarker of Immune Response GST GRS CD1 WHEP 739 ERS PRS ABD. [43,44] 2.1. WRS Expression in Systemic Inflammatory Response 2. WRS as a Biomarker of Immune Response GST GRS CD1 2. DefiningWRS WHEP as a protein Biomarker739 expressionERS of Immune profilesPRS Response in various ABD. cell types in tissues and organs[43,44] is 2.1. WRS Expression in Systemic Inflammatory Response GST CD critical forWHEP understanding ERS their uniquePRS physiological characteristicsABD. in humans. Addressing 2.1. WRS Expression in Systemic Inflammatory Response this2. has WRS a major as a Biomarker impactDefining not of only Immune protein on efforts expression Response to identify profiles target in various protein cell functions, types in tissues but also and organs is crit- Defining protein expression profiles in various cell types in tissues and organs is crit- GST CD for appliedWHEP medical ical for research ERSunderstanding into diseasePRS their management, uniqueABD. physiological such as characteristics pharmaceutical in drughumans. Addressing ical2.1.2. WRS forWRS understanding as Expression a Biomarker in Systemictheir of Immune unique Inflammatory physiologicalResponse Response characteristics in humans. Addressing development andthis biomarker has a major discovery impact[ not45,46 only]. on efforts to identify target protein functions, but also for this2.2.1. 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WRS been In is characterizedaddition, more heavily microbial and expre relative infectionssed inratios intermediate and (%) IFN- of monocyteγ stimulation and non-classical subsets causes have mono-mono- been Classicalferentiation (CD14 fromcytes+CD16 monocytescompared−), intermediate toto themonocyte classical (CD14-derived subset+CD16 +(Figuremacrophages), and 1A).non-classical At (MDMs) the early (CD14 or stages dendritic−CD16 of+ )infection, FL- cytesdeterminedcytes, compared but not to B,aid to T, inthe or understanding classicalnatural killersubset the(NK) (Figure pathogenesis cells, 1A). to produceAt ofthe infectious early and stagessecrete and of otherWRS infection, inflamma-[19,24,25]. FL- monocytescells (DCs) have[48,49].WRS +been is In secreted characterizedaddition,− from microbial monocytesand relative infection+ and ratios directlyand+ (%) IFN- of binds monocyteγ stimulation to TLR4-MD2 subsets causes have complexes− mono- been+ on macro- WRStoryClassical disorders.is secreted (CD14 WRS fromCD16 is monocytes ),more intermediate heavily and expredirectly (CD14ssed bindsCD16 in intermediate to), TLR4-MD2and non-classical an dcomplexes non-classical (CD14 on macro- CD16mono-) determinedcytes, but not tophages B,aid T, in or tounderstanding naturalactivate killerphagocytosis the(NK) pathogenesis cells, [24]. to Initial produce of FL-WRSinfectious and secretesecretion and otherWRS is IFN- inflamma-[19,24,25].γ-independent and phagescytesmonocytes compared to activate have tobeen phagocytosis the characterized classical [24].subset and Initial (Figure relative FL-WRS 1A). ratios Atsecretion (%) the of early monocyte is IFN- stagesγ-independent subsets of infection, have been andFL- toryClassical disorders. (CD14acts WRS+ CD16as a is warning− ),more intermediate heavily to prime expre (CD14innatessed +immunity.CD16 in intermediate+), and Similar non-classical an tod thesenon-classical pro-inflammatory(CD14− CD16mono-+) functions actsWRSdetermined as is a secretedwarning to aid from to in prime understandingmonocytes innate immunity.and the directly pathogenesis Similar binds toto theseof TLR4-MD2 infectious pro-inflammatory complexesand other inflamma- onfunctions macro- cytesmonocytes compared have tobeen the characterized classical subset and (Figure relative 1A). ratios At (%) the of early monocyte stages subsets of infection, have been FL- phagestory disorders. to activate WRS phagocytosis is more heavily [24]. Initialexpressed FL-WRS in intermediate secretion is anIFN-d non-classicalγ-independent mono- and WRSdetermined is secreted to aid from in understandingmonocytes and the directly pathogenesis binds to ofTLR4-MD2 infectious complexesand other inflamma-on macro- actscytes as compared a warning to to the prime classical innate subset immunity. (Figure Similar 1A). toAt thesethe early pro-inflammatory stages of infection, functions FL- phagestory disorders. to activate WRS phagocytosis is more heavily [24]. Initialexpressed FL-WRS in intermediate secretion is anIFN-d non-classicalγ-independent mono- and WRS is secreted from monocytes and directly binds to TLR4-MD2 complexes on macro- actsphagescytes as compared a to warning activate to to phagocytosis the prime classical innate [24].subset immunity. Initial (Figure FL-WRS Similar 1A). toAtsecretion these the early pro-inflammatory is IFN- stagesγ-independent of infection, functions andFL- WRS is secreted from monocytes and directly binds to TLR4-MD2 complexes on macro- acts as a warning to prime innate immunity. Similar to these pro-inflammatory functions phages to activate phagocytosis [24]. Initial FL-WRS secretion is IFN-γ-independent and acts as a warning to prime innate immunity. Similar to these pro-inflammatory functions

Int. J. Mol. Sci. 2021, 22, 4523 3 of 13

RNA and protein expression data generated by the Human Protein Atlas project indicates that WRS is abundantly expressed in monocytes (http://www.proteinatlas.org/ ENSG00000140105-WRS/blood, accessed on 17 December 2020). Monocytes are early responders to pathogens and maintain vascular homeostasis in acute infections [47]. Like proteins that specifically act on monocytes, WRS is abundantly expressed during differ- entiation from monocytes to monocyte-derived macrophages (MDMs) or dendritic cells (DCs) [48,49]. In addition, microbial infection and IFN-γ stimulation causes monocytes, but not B, T, or natural killer (NK) cells, to produce and secrete WRS [19,24,25]. Classical (CD14+CD16−), intermediate (CD14+CD16+), and non-classical (CD14−CD16+) monocytes have been characterized and relative ratios (%) of monocyte subsets have been determined to aid in understanding the pathogenesis of infectious and other inflammatory disorders. WRS is more heavily expressed in intermediate and non-classical monocytes compared to the classical subset (Figure1A). At the early stages of infection, FL-WRS is secreted from monocytes and directly binds to TLR4-MD2 complexes on macrophages to activate phagocytosis [24]. Initial FL-WRS secretion is IFN-γ-independent and acts as a warning to prime innate immunity. Similar to these pro-inflammatory functions of FL-WRS, classical monocytes also play a crucial role in phagocytosis during the initial inflammatory response [50].

Figure 1. WRS as a biomarker of immune response during infection. (A) Expression levels of WRS transcripts in various blood cell types. The expression of WRS is enhanced in intermediate and non-classical monocytes. (B) Kinetics of WRS and three monocyte subsets (classical, intermediate, and non-classical) in inﬂammatory responses caused by infection. The secretion of WRS increased consistently with an increase in the numbers of intermediate and non-classical monocytes.

Non-classical monocytes have been widely viewed as being anti-inflammatory in or- der to maintain vascular homeostasis. Intermediate and non-classical monocytes gradually increase and expand in infectious diseases such as sepsis, in which they constitute ~50% of all monocytes [47,51,52]. Pathological conditions such as sepsis and septic shock are characterized by the elevated expression of proteases such as fibrin, neutrophil elastase (NE), and matrix metalloproteinases (MMPs), which simultaneously produce the mini-WRS, T1-WRS, and T2-WRS truncated variants [22,26,53,54]. Anti-inflammatory pathways are concurrently activated at this time, leading to the release of anti-inflammatory cytokines that dampen and, ultimately, terminate the inflammatory response. With the gradual increase in intermediate and non-classical monocytes, a secondary increase in WRS expression is supposed to be occurred to maintain homeostasis (Figure1B). As the truncated variants have anti-inflammatory and antiangiogenic functions [20,26,30], it is critical to Int. J. Mol. Sci. 2021, 22, 4523 4 of 13

distinguish them from FL-WRS and to evaluate their kinetics during the inﬂammatory phase. Furthermore, understanding the pathological roles of the truncated variants in systemic inﬂammatory diseases might contribute to their potential as a new therapeutic target.

2.2. WRS as a Prognostic Biomarker in Sepsis Sepsis is characterized by an overwhelming systemic inflammatory reaction to microbial infection that can lead to severe sepsis and septic shock [55]. Early recognition of sepsis is critical for timely and effective intervention [56]. However, biomarkers that reflect the severity of infection in patients with sepsis have not yet been identified. Although C-reactive protein (CRP) is a traditional biomarker that is elevated in inflammatory states, it has low specificity for diagnosing sepsis [57]. Consistent with previous findings of abundant WRS secretion upon microbial infection in vitro and in vivo [24,25], high levels of WRS are detected in the serum of critically ill patients with sepsis. Choi et al. recently reported that WRS has clinical value for de- tecting sepsis and predicting mortality among critically ill patients with sepsis. The areas under the receiver operating characteristic curves (AUROCs) for sepsis discrimination with WRS, procalcitonin (PCT), CRP, and IL-6 are 0.864, 0.727, 0.625, and 0.651, respectively [34], indicating that WRS has better predictive value than other clinical factors of sepsis (Figure2A).

Figure 2. Non-canonical physiopathological roles of WRS. (A) Secreted WRS as a sepsis biomarker. Secreted WRS is a potential biomarker not only for the early detection of sepsis but also for predicting 28-day mortality. (B) Different angiostatic activities of each WRS variant in angiogenesis. Mini-, T1-, and T2-WRS have antiangiogenic effects that may be related to metastasis in tumor microenvironments. (C) Intracellular oligomerized WRS with NFTs and extracellular plaque-like WRS, along with Aβ plaques, are detected in AD. Int. J. Mol. Sci. 2021, 22, 4523 5 of 13

Moreover, existing diagnostic markers of sepsis were only able to detect bacterial sepsis. Among several markers of inflammation and sepsis, PCT is used to identify bacterial infections, but also has the disadvantage of not identifying viral infections. [58,59]. Besides higher sensitivity towards sepsis discrimination of WRS, another advantage of WRS is that it can be used to diagnose sepsis regardless of whether the causal infection is bacterial, fungal, or viral. Furthermore, biomarkers are needed that can accurately predict mortality due to severe sepsis and septic shock. PCT and CRP levels do not provide statistically significant prediction of 28-day mortality in sepsis patients; however, WRS is significantly associated with 28-day overall mortality among patients with sepsis in intensive care units [34]. Based on these clinical results, WRS secretion at early infection stages is crucial in the recognition and phagocytosis of microbes, which are the main roles of classical monocytes. Sepsis gradually worsens and WRS, which is synthesized de novo and secreted upon IFN-γ activation, is likely to function similarly to non-classical monocytes that are involved in pathophysiology associated with anti-inflammatory response and blood vessel remodeling. Secreted WRS is a sensitive and accurate biomarker not only for discriminating sepsis but also for predicting mortality among critically ill patients.

3. WRS as a Therapeutic Target in Cancer 3.1. WRS as a Target for Anti-Angiogenic Therapy Several ARSs have been identified as secretory cytokines that control angiogenesis and immune responses in the tumor microenvironment. Fragments of the closely related mammalian tyrosyl-tRNA synthetase (YRS) and WRS are known to regulate angiogenesis [60]. Native FL-WRS does not affect angiogenic signaling; however, mini-WRS, T1-WRS, and T2-WRS, are all angiostatic factors [26,30,32,60,61]. Expression of the truncated WRS variants, like that of many angiostatic factors, is induced by IFN-γ. In particular, T2-WRS has proven potently antiangiogenic in several cell-based assays of vascular endothelial growth factor (VEGF)-induced Matrigel angiogenesis in vitro and in vivo. T2-WRS is a potent inhibitor of retinal angiogenesis in neonatal mice, where it localizes to retinal blood vessels [30]. Moreover, the inhibitory activity of T2-WRS in endothelial cells (ECs) in vitro abrogates cellular responses to flow-induced fluid shear stress, including endothelial nitric-oxide synthase (eNOS), extracellular signal-regulated kinase (ERK1/2), and Akt activation [31]. The eight-residue D382-TIEEHR-Q389 sequence, which is located within the tRNA anticodon-binding (TAB) domain, plays a crucial role in the angiostatic activity of the truncated variants. This implies that WRS uses the same domain for both antiangiogenic receptor binding and classical protein synthesis [2,61]. Vascular endothelial (VE)-cadherin is a calcium-dependent adhesion molecule that is selectively expressed at EC intercellular junctions and is essential for normal vascular development. T2-WRS binds at EC intercellular junctions and VE-cadherin has been identified as a receptor for T2-WRS. The binding of T2-WRS to human umbilical vein ECs (HUVECs) inhibits VEGF-induced ERK activation and EC migration [32]. Adam et al. sug- gested that human WRS manifests antiangiogenic activity only when a eukaryote-specific N-terminal extension is removed, due to steric hindrance that blocks Trp2 and Trp4 of VE-cadherin from accessing the active site pocket [62]. T2-WRS contains a Rossmann fold nucleotide-binding domain that is conserved throughout all prokaryotic and eukaryotic WRSs. FL-WRS, mini-WRS, and T1-WRS variants retain aminoacylation activity, whereas T2-WRS does not [30]. Thus, T2-WRS may apparently be produced only to inhibit angiogenesis without contributing to aminoacylation (Figure2B and Table2). Proteases including fibrin, NE, and MMPs are abundant in tumor microenvironments or at sites of infection, where they produce T2-WRS by cleaving the N-terminus. This could explain why WRS secreted by IFN-γ activation is truncated to T2-WRS, and its role in inhibiting angiogenesis is of great importance in clinical approaches targeting WRS. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 of 14 Int. J. Mol. Sci. 2021, 22 , x FOR PEER REVIEW 7 of 14

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 of 14

Table 2. Schematic structure and angiostatic activity of human WRS variants. The Rossmann fold (RF) and anticodon Int. J. Mol.Table Sci. 2. 2021 Schematic, 22, x FOR structure PEER REVIEW and angiostatic activity of human WRS variants. The Rossmann fold (RF) and anticodon7 of 14 binding domain (ABD) are well conserved in all WRS variants. The eukaryotic-specific extension (ESE) is common in binding domain (ABD)eukaryotic are well WRS. conserved Human full-lengthin all WRS (FL)-WRSvariants. Thehas aeukaryotic-specific vertebrate-specific exte extensionnsion (ESE) (VSE), is also common known in as WHEP domain. Int. J. Mol.Table Sci. 2. 2021 Schematic, 22, x FOR structure PEER REVIEW and angiostatic activity of human WRS variants. The Rossmann fold (RF) and anticodon7 of 14 eukaryotic WRS. HumanThe aminoacylation full-length (FL)-WRS or angios hastatic a activitiesvertebrate-specific of WRS va extensionriants are (VSE),indicated also in known +/−. as WHEP domain. bindingThe aminoacylation domain (ABD) or angios are welltatic conserved activities ofin WRSall WRS variants variants are indicated. The eukaryotic-specific in +/−. extension (ESE) is common in Table 2. Schematic structure and angiostatic activity of human WRS variants. The Rossmann fold (RF) and anticodon Int. J. Mol. Sci.eukaryotic2021, 22, 4523 WRS. Human full-length (FL)-WRS has a vertebrate-specific extension (VSE), also known as WHEP domain.6 of 13 bindingThe aminoacylation domain (ABD)WRS or angios are welltatic conserved activities ofin WRSall WRS variants variants are indicated. The eukaryotic-specific in +/−. extensionAminoacylation (ESE) is common in Angiostatic TableWRS 2. Schematic structure and angiostatic activitySchematic of huma nStructure WRS variants. Aminoacylation The Rossmann fold (RF)Angiostatic and anticodon eukaryotic WRS. VariantsHuman full-length (FL)-WRS has a vertebrate-specific extension (VSE), also knownActivity as WHEP domain. Activity bindingThe aminoacylation domain (ABD) or angios are welltaticSchematic conserved activities ofinStructure WRSall WRS variants variants are .indicated The eukaryotic-specific in +/−. extension (ESE) is common in VariantseukaryoticWRS WRS. Human full-length (FL)-WRS has a vertebrate-specific extensionAminoacylationActivity (VSE), also known as WHEPAngiostaticActivity domain. Table 2. Schematic structure and angiostatic activity of human WRS variants. The Rossmann fold (RF) and anticodon The aminoacylation or angiostaticSchematic activities of Structure WRS variants are indicated in +/−. bindingVariantsWRS domain (ABD)FL-WRS are well conserved in1 all WRS variants. The eukaryotic-speciﬁc Aminoacylation 471Activity extension (ESE) is common+Angiostatic Activity in − FL-WRS 1 Schematic Structure 471 + − eukaryoticVariantsWRS WRS. Human full-length (FL)-WRS has a vertebrate-speciﬁc extension (VSE),Aminoacylation alsoActivity known as WHEP domain.AngiostaticActivity The aminoacylation or angiostatic1 activities Schematic of WRS variants Structure are indicated 471 in +/−. VariantsFL-WRS Activity+ Activity− mini-WRS 48 471 + − WRSmini-WRSFL-WRS Variants 1 48 Schematic Structure 471471 Aminoacylation Activity+ Angiostatic Activity− FL-WRS 1 471 + − mini-WRSFL-WRS 1 48 471471 + − T1-WRS 71 471 + − mini-WRS 48 471 + + mini-WRST1-WRS 4871 471471 + − mini-WRST1-WRS 48 71 471 ++ + − T1-WRS 71 471 + − T2-WRS 94 471 − ++ T2-WRST1-WRS 71 471 − + ++ − T2-WRS 9494 471471 − ++ T1-WRST2-WRS 71 94 471 +− ++− WHEP ESE Rossmann fold ABD. T2-WRSWHEP ESE 94 Rossmann fold 471 ABD. − ++

WHEP ESE Rossmann fold ABD. T2-WRS 3.2. Implications94 of Angiostaic WRS in471 Cancer Metastasis − ++

WHEP ESE Rossmann3.2. Implicationsfold of Angiosta ABD. ic WRS in Cancer Metastasis ARS3.2. Implications expression of profiles Angiosta canic WRS be a in useful Cancer prognostic Metastasis tool for cancers, as they correlate with overall patient survivalARS expression in each cancer profiles type. can WRSbe a useful is dysregulated prognostic intool different for cancers, as they correlate WHEP ESE ARS Rossmannexpression foldprofiles can be a ABD.useful prognostic tool for cancers, as they correlate cancers,3.2. includingImplications ovarian, ofwith Angiosta overall cervical,ic WRS patient colorectal, in Cancersurvival Metastasis oral in each squamous cancer ty cellpe. carcinoma WRS is dysregulated (OSCC), in different cancers, with overall patient survival in each cancer type. WRS is dysregulated in different cancers, uveal melanomaARS expression (UM)including and profiles pancreatic ovarian, can be cancers cervical,a useful [63 colorectal,progno–68]. Instic ovarian oraltool squamousfor cancer, cancers, WRScell as carcinomathey protein correlate (OSCC), uveal mel- 3.2.including Implications ovarian, of Angiosta cervical,ic colorectal,WRS in Cancer oral Metastasissquamous cell carcinoma (OSCC), uveal mel- levelswith were overall up-regulated patientanoma survival in highly (UM) in malignantand each pancreatic cancer clear type. cancers cell WRS adenocarcinoma [63–68].is dysregulated In ovarian when in different cancer, compared WRScancers, protein levels were anomaARS (UM) expression and pancreatic profiles cancerscan be a [63–68]. useful prognoIn ovarianstic toolcancer, for cancers,WRS protein as they levels correlate were with3.2. mucinousincluding Implications ovarianovarian, ofup-regulated Angiosta adenocarcinomacervical,ic colorectal,WRS in highly in [ 63Cancer ].oral malignant The Metastasissquamous expression clear cell cell level carcinoma adenocarcinoma of WRS is(OSCC), increased when uveal in compared mel- with mucin- withup-regulated overall patient in highly survival malignant in each clear cancer cell tyadenocarcinomape. WRS is dysregulated when compared in different with cancers, mucin- cervicalanoma carcinomaARS (UM) expression and tissueous pancreatic whenprofilesovarian compared cancerscanadenocarcinoma be a [63–68]. withuseful normal progno In [63] ovarian tissue. sticThe tool [cancer,expression64]. for WRS cancers,WRS is level overexpressedprotein as of they WRS levels correlate is were increased in cervical includingous ovarian ovarian, adenocarcinoma cervical, colorectal, [63]. The oral expression squamous level cell of carcinoma WRS is increased (OSCC), uvealin cervical mel- in OSCCwithup-regulated tissuesoverall when patient in highlycarcinoma compared survival malignant tissue in with each clearwhen adjacent cancer cell compared ty normaladenocarcinomape. WRS with tissues. is dysregulatednormal Importantly, when tissue compared in[64]. different WRS WRS with levels is cancers, mucin-overexpressed in OSCC anomacarcinoma (UM) tissue and when pancreatic compared cancers with [63–68]. normal In tissue ovarian [64]. cancer, WRS is WRS overexpressed protein levels in OSCC were are significantlyincludingous ovarian ovarian, higher adenocarcinomatissues in cervical, tumor when cellscolorectal, [63]compared from. The metastatic oral expression with squamous adjacent lymph level cell nodesnormal of carcinoma WRS than tissues. is in increased primary(OSCC), Importantly,tumor uvealin cervical WRSmel- levels are signif- up-regulatedtissues when comparedin highly malignant with adjacent clear normal cell adenocarcinoma tissues. Importantly, when compared WRS levels with are mucin- signif- sites [anomacarcinoma65,69]. (UM) Bioinformatics tissue and icantlywhen pancreatic analysiscompared higher cancers basedin withtumor [63–68]. on normal Genecells In Expressionfromtissue ovarian metastatic [64]. cancer, WRS Omnibus lymph isWRS overexpressed (GEO) protein nodes database thanlevels in inOSCC were primary tumor sites ousicantly ovarian higher adenocarcinoma in tumor cells from[63]. Themetastatic expression lymph level nodes of WRSthan isin increasedprimary tumor in cervical sites showedup-regulatedtissues that when WRS expressionincompared highly[65,69]. malignant inwithBioinformatics UM adjacent metastatic clear normal cell analysis cancer adenocarcinoma tissues. was based significantly Importantly, on whenGene higher compared ExpressionWRS thanlevels with that areOmnibus inmucin- signif- (GEO) database carcinoma[65,69]. Bioinformatics tissue when comparedanalysis based with normal on Gene tissue Expression [64]. WRS Omnibus is overexpressed (GEO) databasein OSCC non-metastaticousicantly ovarian higher group. adenocarcinoma inshowed Kaplan-Meiertumor cellsthat fromWRS[63] analysis. Theexpressionmetastatic expression based onlymphin UM The level Cancernodesmetastatic of WRS than Genome cancer isin increasedprimary Atlas was (TCGA)significantly tumorin cervical sites higher than that tissuesshowed when that WRScompared expression with adjacent in UM metastatic normal tissues. cancer Importantly, was significantly WRS levelshigher are than signif- that databasecarcinoma[65,69]. showed Bioinformatics tissue that in highwhen non-metastatic WRS comparedanalysis expression basedwith group. wasnormal on associatedKaplan-MeierGene tissue Expression [64]. with WRSan loweralysis Omnibus is survivaloverexpressed based (GEO) [66on]. The Most indatabase OSCCCancer Genome Atlas icantlyin non-metastatic higher in tumor group. cells Kaplan-Meier from metastatic analysis lymph based nodes on thanThe inCancer primary Genome tumor Atlas sites cancer-associatedtissuesshowed when that WRS mortalitycompared(TCGA) expression iswith database due adjacent in to UM tumor showed metastatic normal cell that metastasis tissues. highcancer WRSImportantly, was to otherexpr significantlyession organs. WRS was levelshigher WRS associated isare than ex- signif- that with lower survival [65,69].(TCGA) Bioinformaticsdatabase showed analysis that high based WRS on expr Geneession Expression was associated Omnibus with (GEO) lower databasesurvival pressedicantlyin abundantlynon-metastatic higher in [66]. patientstumor group. Most cells who Kaplan-Meiercancer-associated from did notmetastatic relapse analysis mortality afterlymph based surgery nodes is dueon for than toThe triple-negative tumor inCancer primary cell metastasisGenome tumor breast Atlas sitesto other organs. WRS showed[66]. Most that cancer-associated WRS expression mortality in UM metastatic is due to tumor cancer cell was metastasis significantly to other higher organs. than WRS that cancer[65,69].(TCGA) (TNBC), Bioinformaticsdatabase whenis compared showed expressed analysis that with abundantly high thosebased WRS who on inexpr didGene patientsession [70 Expression]. Moreover, whowas didassociated Omnibusnot low relapse WRS with (GEO) expression afterlower surgerydatabase survival for triple-negative inis expressednon-metastatic abundantly group. in Kaplan-Meier patients who didanalysis not relapse based afteron The surgery Cancer for Genometriple-negative Atlas is associatedshowed[66]. Most withthat cancer-associated anWRS increasedbreast expression cancer risk mortality of in(TNBC), lymphUM metastatic is nodewhen due to metastasis compared tumor cancer cell was and with metastasis significantly reduced those who to survival other higherdid organs.[70]. among than Moreover, WRS that low WRS ex- patients(TCGA)breast with cancer colondatabase (TNBC), cancer showed [ 68when]. that compared high WRS with expr thoseession who was did associated [70]. Moreover, with lower low WRSsurvival ex- inis expressednon-metastatic abundantlypression group. in isKaplan-Meier patientsassociated who with didan alysisan not increased relapse based afteronrisk The ofsurgery lymph Cancer for node Genometriple-negative metast Atlasasis and reduced sur- Tumor[66].pression Most cells is cancer-associated metastasizeassociated with via lymphatic an mortality increased or is hematogenousdue risk to of tumor lymph cell dissemination.node metastasis metastasis to Although other and reducedorgans. lym- WRS sur- (TCGA)breast cancer database (TNBC),vival showed amongwhen that compared patients high WRS with with expr colon thoseession cancer who was did [68]. associated [70]. Moreover, with lower low WRSsurvival ex- phaticisvival metastasis expressed amongcan abundantlypatients occur with via in extant colon patients vessels,cancer who [68]. evidence did not supportsrelapse after the notionsurgery that for metastasistriple-negative [66].pression Most is cancer-associated associatedTumor with an mortality cells increased metastasize is due risk to of tumorvia lymph lymphatic cell node metastasis metast or hematogenousasis to other and organs.reduced disse WRS sur-mination. Although is significantlybreastTumor cancer improved cells (TNBC), metastasize when when lymphatic compared via lymphatic vessel with thoseor density hematogenous who increases did [70]. duedisse Moreover, tomination. lymphangio- low Although WRS ex- isvival expressed among abundantlypatientslymphatic with in colon patientsmetastasis cancer who can [68]. did occur not viarelapse extant after vessels, surgery evidence for triple-negative supports the notion that me- genesis.pressionlymphatic For example, is metastasisassociated VEGF-C canwith occur stimulatesan increased via extant the ri growthskve ssels,of lymph ofevidence lymphatic node supportsmetast endotheliumasis the and notion reduced and that is sur-me- breastTumor cancer cells (TNBC),tastasis metastasize when is significantly compared via lymphatic with improved thoseor hematogenous whowhen did lymphatic [70]. disse Moreover, vesselmination. densitylow Although WRS increases ex- due to lym- overexpressedvivaltastasis among is insignificantly breastpatients cancer with improved colon cells. cancer Stimulation when [68]. lymphatic by VEGF-A vessel anddensity -Cis increases also associated due to lym- pressionlymphatic is metastasisassociatedphangiogenesis. canwith occur an increased via For extant example, ri skve ssels,of VEGF-Clymph evidence node stimul supportsmetastates asisthe the growthand notion reduced of that lymphatic sur-me- endothelium with lymphaticphangiogenesis.Tumor vessel cells For formationmetastasize example, and viaVEGF-C sentinel lymphatic stimul node oratesmetastasis hematogenous the growth [71– 73of disse ].lymphatic WRSmination. are endothelium overex- Although vivaltastasis among is significantly patientsand iswith overexpressedimproved colon cancer when in [68]. lymphaticbreast cancer vessel cells. density Stimulation increases by VEGF-A due to lym-and -C is also associ- pressedlymphaticand at is stages overexpressed metastasis of cancer canin metastasis breast occur cancer via and extant T2-WRScells. ve Stssels,imulation has evidence a potent by VEGF-A inhibitorysupports and the effect -C notion is against also that associ- me- phangiogenesis.Tumor cells ated Formetastasize example, with lymphatic viaVEGF-C lymphatic vessel stimul formation orates hematogenous the andgrowth sentinel of disse lymphatic nodemination. metastasis endothelium Although [71–73]. WRS are over- VEGF-inducedtastasisated with is lymphatic vesselsignificantly formation. vessel improved formation However, when and whether lymphatic sentinel the node angiostaticvessel metastasis density activity [71–73].increases of T2-WRS WRS due are to is over-lym- lymphaticand is overexpressed metastasisexpressed canin breast occur at stages cancer via extant of cells. cancer ve Stssels, imulationmetastasis evidence by and VEGF-A supports T2-WRS and thehas -C notiona potentis also that inhibitoryassoci- me- effect against associatedphangiogenesis.expressed with at lymphangiogenesis stages For of example, cancer metastasis VEGF-C during metastatic stimuland T2-WRSates progression the has growth a potent remains of lymphaticinhibitory unknown. effectendothelium against tastasisated with is lymphaticsignificantlyVEGF-induced vessel improved formation vessel when and formation. lymphatic sentinel However,node vessel metastasis density whether [71–73].increases the angiostatic WRS due are to over- lym-activity of T2-WRS is TheandVEGF-induced truncatedis overexpressed variants vessel information. are breast antagonistic cancer However, cells. in the Stwhetimulation controlher the of by angiostatic angiogenesis VEGF-A activityand and -C immune isof also T2-WRS associ- is phangiogenesis.expressed at stages Forassociated of example, cancer with metastasis VEGF-C lymphangiogenesis stimuland T2-WRSates the duringhas growth a potent metastatic of lymphaticinhibitory progression effectendothelium against remains unknown. stimulationatedassociated with and lymphatic with has lymphangiogenesis potential vessel as formation a therapeutic during and sentinel targetmetastatic node for cancer.progression metastasis In contrast, [71–73].remains WRS FL-WRSunknown. are over- andVEGF-induced is overexpressed vessel in formation. breast cancer However, cells. Stwhetimulationher the by angiostatic VEGF-A andactivity -C is of also T2-WRS associ- is showsexpressed the opposite at stages function of cancer compared metastasis to the and truncated T2-WRS variants, has a potent suggesting inhibitory that effect caution against atedassociated with lymphatic with lymphangiogenesis vessel formation during and sentinel metastatic node progression metastasis [71–73].remains WRS unknown. are over- is neededVEGF-induced when interpreting vessel formation. the relationship However, between whether FL-WRS the angiostatic and its truncated activity variants.of T2-WRS is expressed at stages of cancer metastasis and T2-WRS has a potent inhibitory effect against Furthermore,associated the with targets lymphangiogenesis of each truncated during WRS metastatic variant need progression to be validated remains and unknown. their effectivenessVEGF-induced as an immuno-oncology vessel formation. targetHowever, should whet beher confirmed. the angiostatic activity of T2-WRS is associated with lymphangiogenesis during metastatic progression remains unknown. 4. Pathological Role of WRS in Alzheimer’s Disease Alzheimer’s disease (AD) is an age-dependent neurodegenerative disorder and the

most common cause of dementia [74]. It is characterized by neuroﬁbrillary tangles (NFTs)

Int. J. Mol. Sci. 2021, 22, 4523 7 of 13

composed of paired helical filaments and extracellular senile plaques containing aggre- gated amyloid fibrils and non-amyloid components in the brain [71]. Neither an effective treatment nor a biochemical drug for AD is currently available. Neuronal cell death triggers the release of cytoplasmic proteins, some of which interact with Amyloid-β (Aβ). Specifically, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been shown to interact with neurodegenerative disease-related proteins including β- amyloid precursor protein (AβPP). Aβ induces disulfide bond formation and aggregation of GAPDH, possibly due to the formation of neurotoxic aggregates in AD [75–78]. The amount of GAPDH disulfide bonding is increased in detergent-insoluble extracts from patients with AD when compared with age-matched controls [79]. Interestingly, bovine WRS forms complexes with GAPDH. This complex formation between WRS and GAPDH i) does not influence aminoacylation activity and ii) predominantly involves the dispensable N-terminal domain of WRS [72]. The tendency of mammalian WRS to self-aggregate has been observed by electron microscopy, small-angle X-ray scattering, and biochemical experiments [10,73,74]. Oligomeric WRS is found in cytotoxic fibrils and extracellular plaque-like WRS aggregates detected in AD have features similar to extracellular senile plaques and NFTs (Figure2C). Electron microscopy has shown intensive fibril formation with a synthetic N-terminal peptide of WRS, corresponding to residues Ser32 to Tyr50, whereas fewer and less organized fibrils were formed by the C-terminal peptide (Glu414 to Val437) [11]. Tryptamine is an inhibitor that competes with Trp for binding to the WRS active site [75]. The expression of WRS is decreased in the cytoplasm and elevated in the detergent- insoluble “cytoskeleton” fraction of tryptamine-treated human neuronal cells. Moreover, about three-fold higher WRS immunogold reactivity was associated with NFTs than with the cytoplasm of tryptamine-treated human neuronal cells [74]. Paley et al. reported in tryptamine-treated cells that WRS is secreted into the extracellular space as either a free protein or within vesicles extending from the cytoplasm and then pinched off from the plasma membrane. Extracellular vesicles fuse in congophilic WRS+ plaques in tryptamine-treated cultures and in the brain in AD. Prominent WRS immunoreactivity is associated with plasma and the vesicle membranes of satellites and degenerated neurons in the brain with AD [76]. Biochemically purified bovine WRS is highly susceptible to aggregation [10] and recombinant human WRS and N-terminal synthetic peptides self- assemble in fibrils [11]. These findings provide a new perspective on the versatility of WRS functions and indicate that WRS is involved in the pathology of AD. Therefore, further studies are needed to clarify the relationship between human WRS and AD and to investigate whether WRS could be a novel therapeutic target, in particular by focusing on anti-WRS therapy to prevent or hinder the clinical progression of AD.

5. Immunological Role of WRS in Trp Metabolism 5.1. Increased Trp Production by WRS in Cancer Cell Tryptophan (Trp) is an essential amino acid that is important for cell survival and proliferation. Indoleamin-2,3-dioxygenase (IDO) is the initial and rate-limiting enzyme for Trp degradation through the kynurenine pathway (KP). Trp catabolism suppresses antitumor immune responses in pathological states such as cancer [80,81]. IFN-γ enhances expression of IDO as well as WRS. Both IDO and WRS are involved in regulating the immune response by modulating Trp metabolism. IFN-γ induces WRS to increase Trp uptake into cells through a process that has high afﬁnity and selectivity for Trp [82]. Consistent with this, Adam et al. reported that Trp depletion mediated by IDO1 and tryptophan-2,3-dioxygenase (TDO2) upregulates WRS expression to increase Trp production in cancer cells. Due to WRS mediated-Trp synthesis, cancer cells are thought to maintain protein synthesis and proliferate despite low Trp levels [83]. The depletion of Trp induced by IDO up-regulation results in speciﬁcally inhibited T cell proliferation, thus facilitating tumor escape from immune surveillance. Additionally, increased WRS expression and WRS-mediated Trp production directly facilitate cancer cell proliferation and survival, implying that both Int. J. Mol. Sci. 2021, 22, 4523 8 of 13

IDO and WRS are associated with immune evasion by cancer cells. A new approach to controlling Trp production by WRS might therefore resolve the redundancy of Trp metabo- lites in cancer immunotherapy. However, rather than pathological role of WRS acting on cancer itself, the involvement and regulation of WRS in tolerogenic immune response and autoinﬂammatory disease are prominent topics of interest in the context of therapeutics.

5.2. Implications of Increased WRS in Tolerogenic Immune Response Under healthy conditions, Trp-degrading activity is abundant in the lungs, intestines, and particularly in placental tissues [84]. Notably, WRS transcripts are also abundantly expressed in placenta and lung tissues, mostly known as immune privileged sites (https: //www.proteinatlas.org/ENSG00000140105-WARS/tissue, accessed on 17 December 2020). The importance of Trp depletion in the placenta is emphasized in maintaining immune privilege to create an environment that suppresses T cell activity and defends itself against rejection [85,86]. The expression of IDO1 and TDO2 is positively correlated with WRS, which in turn is significantly correlated with the expression of T cell markers. Recombinant soluble cytotoxic T lymphocyte antigen-4 (CTLA-4-Fc) is clinically effective for suppressing T cell activation in autoimmune diseases such as rheumatoid arthritis (RA). The expression of IDO and WRS in immune cells, such as DCs and CD4+ T cells is increased by CTLA-4-Fc. Notably, CD8+ T cells from CTLA-4-Fc-treated peripheral blood mononuclear cells (PBMCs) express increased levels of WRS but not IDO, suggesting that WRS-mediated regulation and IDO are involved in the immune tolerance mechanism of CTLA-4 [87]. Overexpression of WRS in T cells from patients with RA is apparently related to the pathogenesis of this disease. Although IDO is overexpressed in DCs in the synovial joints of patients with RA, IDO+ DCs do not have sufficient immunosuppressive ability when WRS is overexpressed in T cells [88–90]. The activity of IDO is decreased, whereas WRS expression is increased in T cells from patients with immune thrombocytopenia (ITP) [91] and Graves’ disease [92], which are autoimmune disorders that result in platelet destruction and hyperthyroidism, respectively. The question remains as to whether WRS, which is specifically overexpressed in T cells in patients with autoimmune diseases, indirectly affects Trp metabolism or whether WRS is a cause of autoimmune diseases as a result of its direct involvement in the immune tolerance of T cells. Ongoing clinical trials have shown that IDO inhibitors are well tolerated in cancer, systemic diseases, and central nervous system disorders targeting the pathogenic KP. Nonetheless, results using monotherapy have been somewhat limited and need an additional emerging target to overcome IDO redundancy. Increased WRS expression is likely to act as a pathological factor, not only in the tumor microenvironment, but also in Trp metabolism in autoimmune diseases such as RA. The identification of WRS as a biomarker and the development of selective WRS inhibitors might help improve therapeutic outcomes when combined with IDO inhibitors.

6. Conclusions and Perspectives The various features that distinguish eukaryotic WRS from prokaryotic WRS or other ARSs suggest that WRS has evolutionarily acquired diverse functions that are closely associated with human disease. Human WRS (hWRS) shows great potential as a suitable drug target, having non-canonical functions that underlie its physiopathological roles in human diseases, including infection, cancer, and neurodegenerative diseases (Figure2). Upon infection, hWRS is promptly secreted from monocytes for the priming of innate immunity [24]. Secretory hFL-WRS acted as a therapeutic immune-stimulatory agent and showed a promising potential as a novel biomarker for diagnosis of sepsis [34]. Moreover, alternatively spliced and truncated variants, including mini-WRS, T1-WRS, and T2-WRS, exhibit anti-angiogenic and anti-inﬂammatory properties. Each truncated WRS variant is likely to exert its own speciﬁc biochemical effect for the maintenance of homeostasis in pathological states. hWRS, together with oligomerized plaques, contributed to the pathogenesis of Alzheimer’s disease. Int. J. Mol. Sci. 2021, 22, 4523 9 of 13

The hWRS research field has extended from basic research to antibody and diagnostic development. However, the development of hWRS-targeted therapeutics remains a chal- lenge. Above all, sensitive analytical methods are required to distinguish each WRS variant, owing to their different mechanism of action and pathological roles. Subsequently, the therapeutic effects acting on the hWRS target sites can be easily and accurately monitored. Achieving this goal will lead a progressive development in the field of hWRS-targeted therapeutics. Currently, JW Bioscience, a company, is actively developing WRS diagnostic kits for sepsis and the products are undergoing clinical trials. Although significant progress has been made in the research on hWRS as a therapeutic target, a qualitative study is required to determine the unknown features of ARS variants in various diseases.

Author Contributions: Y.H.A. and S.-C.O. wrote the paper. S.Z. and T.-D.K. reviewed and edited the manuscript before submission. All authors have read and agreed to the published version of the manuscript. Funding: This work supported by “R&D Convergence Program” of the National Research Council of Science and Technology (NST) grant (CAP-18-02-KRIBB) and KRIBB Research Initiative Program by the Korea Government. Acknowledgments: I sincerely thank Je-In Youn and Sang Jun Ha for their valuable advices in preparing this review. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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