The high-affinity tryptophan uptake transport system in human cells

The L-tryptophan (Trp) transport system is highly selective for Trp with affinity in the nanomolar range. This transport system is augmented in human interferon (IFN)-γ-treated and indoleamine 2,3-dioxygenase 1 (IDO1)-expressing cells. Up-regulated cellular uptake of Trp causes a reduction in extracellular Trp and initiates immune suppression. Recent studies demonstrate that both IDO1 and tryptophanyl-tRNA synthetase (TrpRS), whose expression levels are up-regulated by IFN-γ, play a pivotal role in high-affinity Trp uptake into human cells. Furthermore, overexpression of tryptophan 2,3-dioxygenase (TDO2) elicits a similar effect as IDO1 on TrpRS-mediated high-affinity Trp uptake. In this review, we summarize recent findings regarding this Trp uptake system and put forward a possible molecular mechanism based on Trp deficiency induced by IDO1 or TDO2 and tryptophanyl-AMP production by TrpRS.

More recently, a distinct amino acid transport system with high affinity and selectivity for Trp was identified in interferon (IFN)-γ-treated and indoleamine 2,3-dioxygenase 1 (IDO1)-expressing cells [5][6][7][8].Trp uptake into cells such as monocyte-derived macrophages (MDMs) and those with high IDO1 expression levels is characterized by a high degree of specificity and affinity for Trp (K m for Trp is 0.1-0.3μM), with no observed uptake of other neutral amino acids (e.g.Tyr or Phe) (Figure 1B) [5,8].Furthermore, this novel Trp uptake mechanism, which does not require a transmembrane Na + gradient, is not inhibited by other amino acids or by BCH [5].These observations indicate that the transporter operates via a mechanism independent of system L.
Here, we review new insights regarding the underlying mechanism of high-affinity Trp uptake.In particular, we introduce the crucial roles of tryptophanyl-tRNA synthetase (TrpRS) and IDO1 on high-affinity Trp uptake.Finally, we discuss a possible molecular mechanism for TrpRS-mediated high-affinity Trp uptake into human cells.
IDO1 performs numerous biological roles including controlling T cell propagation, regulating antitumor immunity, and maintaining maternal tolerance towards the allogeneic fetus [12][13][14][18][19][20][21][22][23].For example, IDO1 expression is up-regulated in the placenta leading to Trp depletion at the maternal-fetal interface, which aids in protecting the fetus from rejection by the maternal immune system [12,27].Human IDO is expressed in MDMs and specific monocyte-derived dendritic cells (DCs) [13,14].The diminished extracellular levels of Trp resulting from IDO1-mediated Trp breakdown in MDMs and DCs blocks T cell proliferation, leading to immunosuppression [12][13][14]20,22,23].The basal expression level of the high-affinity Trp-specific transport system is low in monocytes but undergoes selective induction upon the differentiation of MDMs [5].This highaffinity transport system allows MDMs to efficiently take up Trp even at low substrate levels.A lack of IDO1 expression and/or activity has been shown to augment various autoimmune disorders [30].Furthermore, IDO1 expression levels are significantly up-regulated in certain types of cancer and IFN-γ-stimulated cells [7,8,[12][13][14][18][19][20][21][22][23]25,26].Some cancers also take advantage of the Trp metabolism-mediated immunosuppressive effect to block host immune rejection [18,20,[23][24][25][26]28], although other cancers are reported to require Trp for survival (i.e.similar to T cells) [31].For example, it has been demonstrated that up-regulated levels of IDO1 in immunogenic mouse tumor cells block their rejection in preimmunized mice [18,20].Furthermore, constitutive expression of IDO1 prevents tumor cells from being rejected by T cells [18,20].Intriguingly, expression of the high-affinity Trp transport system in mouse and human tumor cells is induced by IDO1 [6].As such, the highaffinity Trp uptake system could be a promising target for anticancer therapeutics.Another example of an enzyme that breaks down Trp is tryptophan 2,3-dioxygenase (TDO2).TDO2, which is mainly expressed in the liver and contains a heme prosthetic group, converts Trp to Kyn.In addition, TDO2 is known to be expressed in cancer cells where it induces immune resistance [24,32].
Additionally, high-affinity Trp uptake is suppressed by siRNA-mediated down-regulation of human TrpRS or IDO1 expression [8].In contrast, overexpression of human TrpRS or IDO1 enhances high-affinity Trp uptake [8].These results suggest that high-affinity Trp uptake relies on both IDO1 and TrpRS.

Structure and function of human TrpRS
TrpRS is an aminoacyl-tRNA synthetase that mediates the ligation of Trp to its corresponding tRNA (tRNA Trp ) in the cytosol [47,48].This reaction entails the formation of tryptophanyl-AMP from Trp and ATP with the concurrent release of inorganic pyrophosphate (PPi) [47].Transfer of the aminoacyl moiety of tryptophanyl-AMP to tRNA Trp then yields tryptophanyl-tRNA Trp and AMP [47].The aminoacylation activity of mammalian TrpRS is regulated by its interaction with either Zn 2+ or heme [49][50][51][52][53].
Two types of TrpRS are found in human cells.The predominant type is the full-length polypeptide (a.a.1-471) along with a minor truncated version (mini TrpRS) (a.a.where the first 47 residues from the NH 2 -terminal domain of TrpRS are missing as a result of alternative splicing (Figure 2A) [54,55].IFN-γ markedly up-regulate the expression level of both types of TrpRSs in human cells [33][34][35][36][37]. Human mini TrpRS display almost the same aminoacylation activity as human full-length TrpRS [56].

Human TrpRS mediates high-affinity Trp uptake
Akin to the high-affinity Trp uptake system, the expression of TrpRS is markedly increased in IFN-γ-treated cells and TrpRS has a strong selectivity and affinity for Trp [66,67].TrpRS-specific siRNA has been shown to inhibit the expression of human TrpRS, which then decreases high-affinity Trp uptake [8].In contrast, highaffinity Trp uptake is enhanced by up-regulated expression of human TrpRS [8].Moreover, the addition of purified TrpRS into assay buffer including living cells increased the rate of high-affinity Trp uptake [8].Both the full-length and mini form of human TrpRS stimulate high-affinity Trp uptake (Table 1) [8].These observations demonstrate that human TrpRS plays a central role in regulating high-affinity Trp uptake into human cells.

Trp depletion, induced by Trp-metabolizing enzymes, enhances TrpRS-mediated high-affinity Trp uptake
Cellular expression of IDO1 leads to Trp starvation, resulting in increased amounts of activating transcription factor 4 (ATF4) protein [68][69][70].ATF4 is a stress-induced transcription factor [71].Its expression is known to be enhanced by amino acid starvation [72][73][74].Up-regulation of IDO1 significantly increases high-affinity Trp uptake upon addition of purified TrpRS protein into the assay buffer [70].In contrast, overexpression of a mutant form of IDO1 (H346A), which cannot bind heme or convert Trp to Kyn [10], has no discernable influence on TrpRS-mediated Trp uptake into cells [70].Furthermore, overexpression of TDO2 also stimulates ATF4 expression, which markedly increases extracellular TrpRS-mediated high-affinity Trp uptake [70].Despite catalyzing the same reaction, IDO1 and TDO2 display discrete enzymatic properties [17,[75][76][77].Amino acid sequence similarity between IDO1 and TDO2 is low and although IDO1 is a monomer TDO2 exists as a homotetrameric form.Moreover, the overall tertiary architecture and relative 3D orientation of the small domains are  dissimilar in human IDO1 and TDO2 [11,78,79].The evident disparity between IDO1 and TDO2 suggests that high-affinity Trp uptake does not rely on protein-protein interactions involving these two enzymes.Addition of TrpRS protein to Trp-starved cells incubated in Trp-free medium results in a marked enhancement in high-affinity Trp uptake, akin to cells expressing IDO1or TDO2 [70].In contrast, Kyn has no effect on high-affinity Trp uptake brought about by extracellular TrpRS [70].Moreover, high-affinity Trp uptake into human TrpRS-overexpressing cells is also significantly enhanced upon Trp starvation [80].In conclusion, Trp starvation is crucial for high-affinity Trp uptake not only into cells to which TrpRS protein has been added but also into cells that are overexpressing TrpRS.

TrpRS increases high-affinity Trp uptake via the biosynthesis of tryptophanyl-AMP
Tertiary structural position of amino acid residues crucial for Trp-, ATP-and tRNA-binding in human fulllength TrpRS is shown in Figure 2B.Overexpression of a double mutant of TrpRS (Y159A/Q194A), which does not bind Trp, or a single mutant of TrpRS (A310W), in which the ATP-binding pocket is blocked [58], did not enhance Trp uptake into either normal or Trp-starved cells [8,80].Importantly, both these TrpRS mutants cannot generate tryptophanyl-AMP.A truncated mutant of TrpRS (Δ382-389), in which amino acid residues 382-389 are missing, is capable of generating tryptophanyl-AMP but unable to aminoacylate tRNA because it cannot bind tRNA [81].Overexpression of TrpRS (Δ382-389) increases Trp uptake into both normal and Trp-starved cells at levels equivalent to that observed in cells overexpressing wild-type TrpRS [8,80].These findings indicate that unlike the ability of human TrpRS to bind tRNA, its ability to bind Trp and ATP are an absolute requirement for high-affinity Trp uptake into Trp-starved cells.
Trp uptake into human cells overexpressing TrpRS that were starved of Trp was significantly inhibited by the addition of PPi [80].The presence of free PPi can disturb the equilibrium of the initial reaction in favor of breaking down aminoacyl-AMP, thereby blocking transfer of the amino acid to the tRNA.The presence of extracellular human TrpRS enhances Trp uptake into Trp-starved cells, but this uptake is inhibited by addition of PPi into the assay buffer [80].These findings suggest that TrpRS orchestrates high-affinity Trp uptake via the generation of tryptophanyl-AMP.

Proposed molecular mechanism for high-affinity Trp uptake induced by IFN-γ
The proposed model for TrpRS-mediated high-affinity Trp uptake into human cells elicited by IFN-γ is depicted in Figure 3. IFN-γ brings about increased expression of both IDO1 and TrpRS [8,15,17,[33][34][35][36][37].Up-regulated levels of IDO1 enhances the metabolism of Trp, leading to Trp deficiency, which in turn induces up-regulation of ATF4.Additional work is required to identify molecules at the cell surface that interact with extracellular TrpRS and determine whether these molecules are produced as a result of intracellular Trp depletion.Moreover, it has been reported that some TrpRS is secreted [82] and that extracellular TrpRS is present in the cell culture medium [40,42,43].A recent study reported that human TrpRS is secreted from cells after infection with a pathogen as part of a defense mechanism [42].Additional investigations are required to investigate whether cellular secretion of TrpRS is increased upon Trp starvation.It is proposed that secreted TrpRS will bind to Trp in the extracellular environment, which may lead to selective transportation of Trp into the cell via the TrpRS-interacting cell-surface molecules.Further studies are necessary to establish whether the novel highaffinity Trp transport system comprises TrpRS itself or an unidentified transporter modulated by TrpRS.

Future directions
The biosynthesis of tryptophanyl-AMP has been shown to be crucial for TrpRS-mediated high-affinity Trp uptake [80].Nonetheless, the precise mechanism by which tryptophanyl-AMP mediates Trp uptake remains to be established.It was recently reported that aminoacyl-tRNA synthetases produce aminoacyl-AMP and act as aminoacyl transferases to modify the ɛ-amino group of L-lysine (Lys) residues in proteins [83][84][85].This posttranslational modification can subsequently be eliminated by NAD + -dependent protein deacetylases (e.g.sirtuins) [83].Given that the biosynthesis of tryptophanyl-AMP is required for high-affinity Trp uptake [80], TrpRS could facilitate this Trp uptake by protein tryptophanylation.For instance, extracellular TrpRS could tryptophanylate specific cell surface proteins that then initiate high-affinity Trp uptake.Additional studies are needed to determine whether protein tryptophanylation mediated by TrpRS is involved in cellular Trp uptake.
Our previous work demonstrated that Kyn, a physiological agonist of the aryl hydrocarbon receptor (AhR) [86], did not play a major role in high-affinity Trp uptake facilitated by extracellular TrpRS [70].Nonetheless, some cell types are reported to display enhanced high-affinity Trp uptake when treated with Kyn [7].A recent study shows low levels of Trp sensitizes the AhR pathway by up-regulating AhR expression [87].Thus, additional investigations are required to establish whether the AhR pathway is implicated in the molecular mechanism of high-affinity Trp uptake using various cell types.
Starvation of most amino acids is reported to induce ATF4 expression and enhance inactivation of a mechanistic target of rapamycin complex 1 (mTORC1), leading to induction of autophagy [88,89].Trp depletion also stimulates ATF4 expression [68][69][70].However, high-affinity Trp uptake, which is enhanced under Trp-starved conditions, into TrpRS-overexpressing cells is markedly reduced by incubation of the cells with Torin-1 [80], a powerful inhibitor of mTORC1/2 and an effective inducer of autophagy [90].These findings imply that Trp depletion does not enhance the inactivation of mTORC1/2.This mechanism is unique to Trp.It was recently demonstrated that ribosomes in Trp-depleted cells bypass Trp codons of mRNA and Trp-to-Phe codon reassignment occurs [90][91][92].Intringuingly, Torin-1 was found to suppress ribosomal frameshifting in Trp-starved cells [90].Thus, suppression of frameshifting and Trp-to-Phe codon reassignment by Torin-1 may have decreased high-affinity Trp uptake into Trp-starved cells.Given that these changes are specific to conditions of Trp starvation, they may be associated with high-affinity Trp uptake.

Concluding remarks
Both TrpRS and IDO1, which are up-regulated by IFN-γ, are crucial for high-affinity Trp uptake into cells.Overexpression of IDO1 or TDO2 increases this TrpRS-mediated Trp uptake.Moreover, a deficiency in Trp induced by IDO1 or TDO2 is critical for TrpRS-mediated high-affinity Trp uptake into cells.It should be emphasized that TrpRS facilitates high-affinity uptake via the generation of tryptophanyl-AMP.Additional data is needed to reveal the precise molecular mechanism of high-affinity Trp uptake via extracellular TrpRS, particularly under Trp-depleted conditions.Trp uptake is also utilized by certain types of cancer to prevent immune rejection by T cells.Thus, proteins related to high-affinity Trp uptake represent potential targets for new anticancer agents.

Perspectives
• High-affinity Trp uptake into human cells leads to extracellular Trp depletion, thereby eliciting an immunosuppressive action.
• Both IDO1 and TrpRS, which are up-regulated by IFN-γ, play a central role in high-affinity Trp uptake.Trp deficiency induced by IDO1 is critical for the Trp uptake and TrpRS facilitates the Trp uptake via the biosynthesis of tryptophanyl-AMP.
• Proteins related to high-affinity Trp uptake could be a promising target for anticancer agents.

Figure 1 .
Figure 1.Comparison between normal Trp uptake mediated by System L transporter and high-affinity Trp uptake into cells.(A) Normal Trp uptake mediated by System L transporter.(B) High-affinity Trp uptake mediated by a novel Trp transporter system.

Figure 2 .
Figure 2. Structure of human TrpRS.(A) Schematic representation of human full-length and mini TrpRSs.The extra NH 2 -terminal domain of human TrpRS is shown in black.Numbers on the left and right correspond to the NH 2 -and COOH-terminal residues relative to human full-length TrpRS, respectively.(B) Tertiary structure of the dimeric human full-length TrpRS in complex with tryptophanyl-AMP (Protein Data Bank entry: 1R6T).The catalytic domain of human TrpRS is shown in yellow.Tryptophanyl-AMP is indicated in red.The extra NH 2 -terminal domain of human TrpRS is shown in black.Y159, Q194, and A310 residues are shown as space-filling models and colored in blue.

Figure 3 .
Figure 3.A schematic model of the regulatory mechanism of TrpRS-mediated high-affinity Trp uptake upon IFN-γ treatment of human cells.IFN-γ stimulates expression of both IDO1 and TrpRS.Up-regulated levels of IDO1 leads to Trp depletion, which in turn induces up-regulation of ATF4 expression.Secreted TrpRS will bind to Trp in the extracellular environment and regulate Trp uptake into human cells.

Table 1 .
Examples of the function of human full-length and mini TrpRSs