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.

Tryptophan-Starved Human Cells Overexpressing Tryptophanyl-tRNA Synthetase Enhance High-Affinity Tryptophan Uptake via Enzymatic Production of Tryptophanyl-AMP.

Our previous study demonstrated that L-tryptophan (Trp)-depleted cells display a marked enhancement in Trp uptake facilitated by extracellular tryptophanyl-tRNA synthetase (TrpRS). Here, we show that Trp uptake into TrpRS-overexpressing cells is also markedly elevated upon Trp starvation. These findings indicate that a Trp-deficient condition is critical for Trp uptake, not only into cells to which TrpRS protein has been added but also into TrpRS-overexpressing cells. We also show that overexpression of TrpRS mutants, which cannot synthesize tryptophanyl-AMP, does not promote Trp uptake, and that inhibition of tryptophanyl-AMP synthesis suppresses this uptake. Overall, these data suggest that tryptophanyl-AMP production by TrpRS is critical for high-affinity Trp uptake.

Extracellular aaRSs drive autoimmune and inflammatory responses in rheumatoid arthritis via the release of cytokines and PAD4.

OBJECTIVES: Recent studies demonstrate that extracellular-released aminoacyl-tRNA synthetases (aaRSs) play unique roles in immune responses and diseases. This study aimed to understand the role of extracellular aaRSs in the pathogenesis of rheumatoid arthritis (RA). METHODS: Primary macrophages and fibroblast-like synoviocytes were cultured with aaRSs. aaRS-induced cytokine production including IL-6 and TNF-α was detected by ELISA. Transcriptomic features of aaRS-stimulated macrophages were examined using RNA-sequencing. Serum and synovial fluid (SF) aaRS levels in patients with RA were assessed using ELISA. Peptidyl arginine deiminase (PAD) 4 release from macrophages stimulated with aaRSs was detected by ELISA. Citrullination of aaRSs by themselves was examined by immunoprecipitation and western blotting. Furthermore, aaRS inhibitory peptides were used for inhibition of arthritis in two mouse RA models, collagen-induced arthritis and collagen antibody-induced arthritis. RESULTS: All 20 aaRSs functioned as alarmin; they induced pro-inflammatory cytokines through the CD14-MD2-TLR4 axis. Stimulation of macrophages with aaRSs displayed persistent innate inflammatory responses. Serum and SF levels of many aaRSs increased in patients with RA compared with control subjects. Furthermore, aaRSs released PAD4 from living macrophages, leading to their citrullination. We demonstrate that aaRS inhibitory peptides suppress cytokine production and PAD4 release by aaRSs and alleviate arthritic symptoms in a mouse RA model. CONCLUSIONS: Our findings uncovered the significant role of aaRSs as a novel alarmin in RA pathogenesis, indicating that their blocking agents are potent antirheumatic drugs.

Tryptophan Depletion Modulates Tryptophanyl-tRNA Synthetase-Mediated High-Affinity Tryptophan Uptake into Human Cells.

The novel high-affinity tryptophan (Trp)-selective transport system is present at elevated levels in human interferon-γ (IFN-γ)-treated and indoleamine 2,3-dioxygenase 1 (IDO1)-expressing cells. High-affinity Trp uptake into cells results in extracellular Trp depletion and immune suppression. We have previously shown that both IDO1 and tryptophanyl-tRNA synthetase (TrpRS), whose expression levels are increased by IFN-γ, have a crucial function in high-affinity Trp uptake into human cells. Here, we aimed to elucidate the relationship between TrpRS and IDO1 in high-affinity Trp uptake. We demonstrated that overexpression of IDO1 in HeLa cells drastically enhances high-affinity Trp uptake upon addition of purified TrpRS protein to uptake assay buffer. We also clarified that high-affinity Trp uptake by Trp-starved cells is significantly enhanced by the addition of TrpRS protein to the assay buffer. Moreover, we showed that high-affinity Trp uptake is also markedly elevated by the addition of TrpRS protein to the assay buffer of cells overexpressing another Trp-metabolizing enzyme, tryptophan 2,3-dioxygenase (TDO2). Taken together, we conclude that Trp deficiency is crucial for high-affinity Trp uptake mediated by extracellular TrpRS.

Non-canonical functions of human cytoplasmic tyrosyl-, tryptophanyl- and other aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases catalyze the aminoacylation of their cognate tRNAs. Here we review the accumulated knowledge of non-canonical functions of human cytoplasmic aminoacyl-tRNA synthetases, especially tyrosyl- (TyrRS) and tryptophanyl-tRNA synthetase (TrpRS). Human TyrRS and TrpRS have an extra domain. Two distinct cytokines, i.e., the core catalytic "mini TyrRS" and the extra C-domain, are generated from human TyrRS by proteolytic cleavage. Moreover, the core catalytic domains of human TyrRS and TrpRS function as angiogenic and angiostatic factors, respectively, whereas the full-length forms are inactive for this function. It is also known that many synthetases change their localization in response to a specific signal and subsequently exhibit alternative functions. Furthermore, some synthetases function as sensors for amino acids by changing their protein interactions in an amino acid-dependent manner. Further studies will be necessary to elucidate regulatory mechanisms of non-canonical functions of aminoacyl-tRNA synthetases in particular, by analyzing the effect of their post-translational modifications.

Tryptophanyl-tRNA synthetase mediates high-affinity tryptophan uptake into human cells.

The tryptophan (Trp) transport system has a high affinity and selectivity toward Trp, and has been reported to exist in both human and mouse macrophages. Although this system is highly expressed in interferon-γ (IFN-γ)-treated cells and indoleamine 2,3-dioxygenase 1 (IDO1)-expressing cells, its identity remains incompletely understood. Tryptophanyl-tRNA synthetase (TrpRS) is also highly expressed in IFN-γ-treated cells and also has high affinity and selectivity for Trp. Here, we investigated the effects of human TrpRS expression on Trp uptake into IFN-γ-treated human THP-1 monocytes or HeLa cells. Inhibition of human TrpRS expression by TrpRS-specific siRNAs decreased and overexpression of TrpRS increased Trp uptake into the cells. Of note, the TrpRS-mediated uptake system had more than hundred-fold higher affinity for Trp than the known System L amino acid transporter, promoted uptake of low Trp concentrations, and had very high Trp selectivity. Moreover, site-directed mutagenesis experiments indicated that Trp- and ATP-binding sites, but not tRNA-binding sites, in TrpRS are essential for TrpRS-mediated Trp uptake into the human cells. We further demonstrate that the addition of purified TrpRS to cell culture medium increases Trp uptake into cells. Taken together, our results reveal that TrpRS plays an important role in high-affinity Trp uptake into human cells.

Intracerebroventricular administration of Cystatin C ameliorates disease in SOD1-linked amyotrophic lateral sclerosis mice.

Cystatin C (CysC) is a major protein component of Bunina bodies, which are a pathological hallmark observed in the remaining motor neurons of patients with amyotrophic lateral sclerosis (ALS). Dominant mutations in the SOD1 gene, encoding Cu/Zn superoxide dismutase (SOD1), are causative for a subset of inherited ALS cases. Our previous study showed that CysC exerts a neuroprotective effect against mutant SOD1-mediated toxicity in vitro; however, in vivo evidence of the beneficial effects mediated by CysC remains obscure. Here we examined the therapeutic potential of recombinant human CysC in vivo using a mouse model of ALS in which the ALS-linked mutated SOD1 gene is expressed (SOD1G93A mice). Intracerebroventricular administration of CysC during the early symptomatic SOD1G93A mice extended their survival times. Administered CysC was predominantly distributed in ventral horn neurons including motor neurons, and induced autophagy through AMP-activated kinase activation to reduce the amount of insoluble mutant SOD1 species. Moreover, PGC-1α, a disease modifier of ALS, was restored by CysC through AMP-activated kinase activation. Finally, the administration of CysC also promoted aggregation of CysC in motor neurons, which is similar to Bunina bodies. Taken together, our findings suggest that CysC represents a promising therapeutic candidate for ALS.

An alternative conformation of human TrpRS suggests a role of zinc in activating non-enzymatic function.

Tryptophanyl-tRNA synthetase (TrpRS) in vertebrates contains a N-terminal extension in front of the catalytic core. Proteolytic removal of the N-terminal 93 amino acids gives rise to T2-TrpRS, which has potent anti-angiogenic activity mediated through its extracellular interaction with VE-cadherin. Zinc has been shown to have anti-angiogenic effects and can bind to human TrpRS. However, the connection between zinc and the anti-angiogenic function of TrpRS has not been explored. Here we report that zinc binding can induce structural relaxation in human TrpRS to facilitate the proteolytic generation of a T2-TrpRS-like fragment. The zinc-binding site is likely to be contained within T2-TrpRS, and the zinc-bound conformation of T2-TrpRS is mimicked by mutation H130R. We determined the crystal structure of H130R T2-TrpRS at 2.8 Å resolution, which reveals drastically different conformation from that of wild-type (WT) T2-TrpRS. The conformational change creates larger binding surfaces for VE-cadherin as suggested by molecular dynamic simulations. Surface plasmon resonance analysis indicates more than 50-fold increase in binding affinity of H130R T2-TrpRS for VE-cadherin, compared to WT T2-TrpRS. The enhanced interaction is also confirmed by a cell-based binding analysis. These results suggest that zinc plays an important role in activating TrpRS for angiogenesis regulation.

Chimeric ZHHH neuroglobin acts as a cell membrane-penetrating inducer of neurite outgrowth.

Neuroglobin (Ngb) is a heme protein expressed in the vertebrate brain. We previously engineered a chimeric Ngb protein, in which module M1 of human Ngb is replaced by that of zebrafish Ngb, and showed that the chimeric ZHHH Ngb has a cell membrane-penetrating activity similar to that of zebrafish Ngb and also rescues cells from death caused by hypoxia/reoxygenation as does human Ngb. Recently, it was reported that overexpression of mammalian Ngb in neuronal cells induces neurite outgrowth. In this study, we performed neurite outgrowth assays of chimeric Ngb using rat pheochromocytoma PC12 cells. Addition of chimeric Ngb, but not human or zebrafish Ngb, exogenously to the cell medium induces neurite outgrowth. On the other hand, the K7A/K9Q chimeric Ngb double mutant, which cannot translocate into cells, did not induce neurite outgrowth, suggesting that the cell membrane-penetrating activity of the chimeric Ngb is crucial for its neurite outgrowth-promoting activity. We also prepared several site-directed chimeric Ngb mutants and demonstrated that residues crucial for neurite outgrowth-inducing activity of the chimeric Ngb are not exactly the same as those for its neuroprotective activity.

A novel function of neuroglobin for neuroregeneration in mice after optic nerve injury.

Neuroglobin (Ngb) is a recently discovered heme protein in the vertebrate brain that can bind to oxygen molecules. Mammalian Ngb plays a crucial role in neuroprotection under conditions of oxidative stress. To investigate other potential functions of Ngb, we investigated the mouse retinal Ngb system following optic nerve injury. In the retina of control mice, Ngb immunoreactivity was limited to the retinal ganglion cell (RGC) layer, and this immunoreactivity rapidly decreased to less than 50% of the control level 5 days after optic nerve injury. On the basis of this decrease, we designed in vivo experiments with enhanced expression of Ngb using adult mouse retina. The enhanced expression of Ngb was achieved by injecting chimeric human Ngb protein, which included the cell membrane-penetrating module of fish Ngb. One-day pretreatment with chimeric Ngb increased immunoreactivity levels of Ngb two-fold in mouse RGCs and increased the number of surviving RGCs three-fold by 14 days after optic nerve injury compared with vehicle controls. Furthermore, in the mouse retinas showing enhanced Ngb expression, several regenerating central optic axons exhibited outgrowth and were found to pass through the nerve crush site 14 days after nerve injury. No such regenerating optic axons were observed in the control mouse optic nerve during the same time frame. The data obtained from in vivo experiments strongly indicate that mammalian Ngb has neuroprotective and neuroregenerative properties.

Identification of a residue crucial for the angiostatic activity of human mini tryptophanyl-tRNA synthetase by focusing on its molecular evolution.

Human tryptophanyl-tRNA synthetase (TrpRS) exists in two forms: a full-length TrpRS and a mini TrpRS. We previously found that human mini, but not full-length, TrpRS is an angiostatic factor. Moreover, it was shown that the interaction between mini TrpRS and the extracellular domain of vascular endothelial (VE)-cadherin is crucial for its angiostatic activity. However, the molecular mechanism of the angiostatic activity of human mini TrpRS is only partly understood. In the present study, we investigated the effects of truncated (mini) form of TrpRS proteins from human, bovine, or zebrafish on vascular endothelial growth factor (VEGF)-stimulated chemotaxis of human umbilical vein endothelial cells (HUVECs). We show that both human and bovine mini TrpRSs inhibited VEGF-induced endothelial migration, whereas zebrafish mini TrpRS did not. Next, to identify residues crucial for the angiostatic activity of human mini TrpRS, we prepared several site-directed mutants based on amino acid sequence alignments among TrpRSs from various species and demonstrated that a human mini K153Q TrpRS mutant cannot inhibit VEGF-stimulated HUVEC migration and cannot bind to the extracellular domain of VE-cadherin. Taken together, we conclude that the Lys153 residue of human mini TrpRS is a VE-cadherin binding site and is therefore crucial for its angiostatic activity.

Identification of residues crucial for the interaction between human neuroglobin and the α-subunit of heterotrimeric Gi protein.

Mammalian neuroglobin (Ngb) protects neuronal cells under conditions of oxidative stress. We previously showed that human Ngb acts as a guanine nucleotide dissociation inhibitor (GDI) for the α-subunits of heterotrimeric Gi/o proteins and inhibits the decrease in cAMP concentration, leading to protection against cell death. In the present study, we used an eukaryotic expression vector driving high-level expression of human wild-type Ngb or Ngb mutants that either exhibit or lack GDI activities in human cells. We demonstrate that the GDI activity of human Ngb is tightly correlated with its neuroprotective activity. We further demonstrate that Glu53, Glu60, and Glu118 of human Ngb are crucial for both the neuroprotective activity and interaction with Gαi1. Moreover, we show that Lys46, Lys70, Arg208, Lys209, and Lys210 residues of Gαi1 are important for binding to human Ngb. We propose a molecular docking model of the complex between human Ngb and Gαi1.

A Possible Role of Neuroglobin in the Retina After Optic Nerve Injury: A Comparative Study of Zebrafish and Mouse Retina.

Neuroglobin (Ngb) is a new member of the family of heme proteins and is specifically expressed in neurons of the central and peripheral nervous systems in all vertebrates. In particular, the retina has a 100-fold higher concentration of Ngb than do other nervous tissues. The role of Ngb in the retina is yet to be clarified. Therefore, to understand the functional role of Ngb in the retina after optic nerve injury (ONI), we used two types of retina, from zebrafish and mice, which have permissible and non-permissible capacity for nerve regeneration after ONI, respectively. After ONI, the Ngb protein in zebrafish was upregulated in the amacrine cells within 3 days, whereas in the mouse retina, Ngb was downregulated in the retinal ganglion cells (RGCs) within 3 days. Zebrafish Ngb (z-Ngb) significantly enhanced neurite outgrowth in retinal explant culture. According to these results, we designed an overexpression experiment with the mouse Ngb (m-Ngb) gene in RGC-5 cells (retinal precursor cells). The excess of m-Ngb actually rescued RGC-5 cells under hypoxic conditions and significantly enhanced neurite outgrowth in cell culture. These data suggest that mammalian Ngb has positive neuroprotective and neuritogenic effects that induce nerve regeneration after ONI.

Expression of the rodent-specific alternative splice variant of tryptophanyl-tRNA synthetase in murine tissues and cells.

Tryptophanyl-tRNA synthetase (TrpRS) catalyzes the aminoacylation of tRNA(Trp). mRNA of a rodent-specific alternative splice variant of TrpRS (SV-TrpRS), which results in the inclusion of an additional hexapeptide at the C-terminus of full-length TrpRS (FL-TrpRS), has been identified in murine embryonic stem (ES) cells. In the present study, we evaluated the expression of mouse TrpRS mRNA by real-time reverse transcription PCR. We show that SV-TrpRS and FL-TrpRS mRNAs are highly expressed in murine ES cells, embryo, spleen, lung, liver and uterus, and that the relative expression of SV-TrpRS compared to FL-TrpRS is significantly less in the brain. Moreover, we found that interferon-γ increases the expression of TrpRS in a mouse cell line. These results provide the first evidence for tissue-specific expression and alternative splicing of mouse TrpRS.

Crucial roles of Glu60 in human neuroglobin as a guanine nucleotide dissociation inhibitor and neuroprotective agent.

Mammalian neuroglobin (Ngb) protects neuronal cells under conditions of oxidative stress. We previously showed that human Ngb acts as a guanine nucleotide dissociation inhibitor (GDI) for the α-subunits of heterotrimeric Gi/o proteins and inhibits reductions in cAMP concentration, leading to protection against cell death. In the present study, we created human E60Q Ngb mutant and clarified that Glu60 of human Ngb is a crucial residue for its GDI and neuroprotective activities. Moreover, we investigated structural and functional properties of several human Ngb mutants and demonstrated that the neuroprotective effect of human Ngb is due to its GDI activity and not due to its scavenging activity against reactive oxygen species.

Functional characterization of fish neuroglobin: zebrafish neuroglobin is highly expressed in amacrine cells after optic nerve injury and can translocate into ZF4 cells.

Neuroglobin (Ngb) is a recently discovered vertebrate heme protein that is expressed in the brain and can reversibly bind oxygen. Mammalian Ngb is involved in neuroprotection under conditions of oxidative stress, such as ischemia and reperfusion. We previously found that zebrafish Ngb can penetrate the mammalian cell membrane. In the present study, we investigated the functional characteristics of fish Ngb by using the zebrafish cell line ZF4 and zebrafish retina. We found that zebrafish Ngb translocates into ZF4 cells, but cannot protect ZF4 cells against cell death induced by hydrogen peroxide. Furthermore, we demonstrated that a chimeric ZHHH Ngb protein, in which module M1 of human Ngb is replaced by that of zebrafish, is a cell-membrane-penetrating protein that can protect ZF4 cells against hydrogen peroxide exposure. Moreover, we investigated the localization of Ngb mRNA and protein in zebrafish retina and found that Ngb mRNA is expressed in amacrine cells in the inner nuclear layer and is significantly increased in amacrine cells 3days after optic nerve injury. Immunohistochemical studies clarified that Ngb protein levels were increased in both amacrine cells and presynaptic regions in the inner plexiform layer after nerve injury. Taken together, we hypothesize that fish Ngb, whose expression is upregulated in amacrine cells after optic nerve injury, might be released from amacrine cells, translocate into neighboring ganglion cells, and function in the early stage of optic nerve regeneration. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Human neuroglobin functions as an oxidative stress-responsive sensor for neuroprotection.

Mammalian neuroglobin (Ngb) protects neuronal cells under conditions of oxidative stress. The mechanism underlying this function is only partly understood. Here, we report that human Ngb exists in lipid rafts only during oxidative stress and that lipid rafts are crucial for neuroprotection by Ngb. The ferrous oxygen-bound form of Ngb, which exists under normoxia, is converted to the ferric bis-His conformation during oxidative stress, inducing large tertiary structural changes. We clarified that ferric bis-His Ngb, but not ferrous ligand-bound Ngb, specifically binds to flotillin-1, a lipid raft microdomain-associated protein, as well as to α-subunits of heterotrimeric G proteins (Gα(i/o)). Moreover, we found that human ferric bis-His Ngb acts as a guanine nucleotide dissociation inhibitor for Gα(i/o) that has been modified by oxidative stress. In addition, our data shows that Ngb inhibits the decrease in cAMP concentration that occurs under oxidative stress, leading to protection against cell death. Furthermore, by using a mutated Ngb protein that cannot form the bis-His conformation, we demonstrate that the oxidative stress-induced structural changes of human Ngb are essential for its neuroprotective activity.

Species-specific functional evolution of neuroglobin.

Neuroglobin (Ngb) is a recently discovered vertebrate heme protein that is expressed in the brain and can reversibly bind oxygen. Human Ngb is involved in neuroprotection under oxidative stress conditions such as ischemia and reperfusion. We previously demonstrated that, on the one hand, human ferric Ngb binds to the α-subunit of heterotrimeric G proteins (Gα(i)) and acts as a guanine nucleotide dissociation inhibitor (GDI) for Gα(i). On the other hand, zebrafish Ngb does not exhibit GDI activity. By using wild-type and Ngb mutants, we demonstrated that the GDI activity of human Ngb is tightly correlated with its neuroprotective activity. The crucial residues for both GDI and neuroprotective activity, corresponding to Glu53, Arg97, Glu118, and Glu151 of human Ngb, are conserved among boreotheria of mammalia. Recently, we found that zebrafish, but not human, Ngb can translocate into cells and clarified that module M1 of zebrafish Ngb is important for protein transduction. By performing site-directed mutagenesis, we showed that Lys7, Lys9, Lys21, and Lys23 of zebrafish Ngb are crucial for protein transduction activity. Because these residues are conserved among fishes, but not among mammals, birds, reptilians, or amphibians, the ability to penetrate cell membranes may be a unique characteristic of fish Ngb proteins. Moreover, we clarified that zebrafish Ngb interacts with negatively charged cell-surface glycosaminoglycan. Taken together, these results suggest that the function of Ngb proteins has been changing dynamically throughout the evolution of life.

Module M1 of zebrafish neuroglobin acts as a structural and functional protein building block for a cell-membrane-penetrating activity.

Neuroglobin (Ngb) is a recently discovered vertebrate globin that is expressed in the brain and can reversibly bind oxygen. Mammalian Ngb is involved in neuroprotection during oxidative stress that occurs, for example, during ischemia and reperfusion. Recently, we found that zebrafish, but not human, Ngb can translocate into cells. Moreover, we demonstrated that a chimeric ZHHH Ngb protein, in which the module M1 of human Ngb is replaced by the corresponding region of zebrafish Ngb, can penetrate cell membranes and protect cells against oxidative stress-induced cell death, suggesting that module M1 of zebrafish Ngb is important for protein transduction. Furthermore, we recently showed that Lys7, Lys9, Lys21, and Lys23 in module M1 of zebrafish Ngb are crucial for protein transduction activity. In the present study, we have investigated whether module M1 of zebrafish Ngb can be used as a building block to create novel cell-membrane-penetrating folded proteins. First, we engineered a chimeric myoglobin (Mb), in which module M1 of zebrafish Ngb was fused to the N-terminus of full-length human Mb, and investigated its functional and structural properties. Our results showed that this chimeric Mb protein is stable and forms almost the same heme environment and α-helical structure as human wild-type Mb. In addition, we demonstrated that chimeric Mb has a cell-membrane-penetrating activity similar to zebrafish Ngb. Moreover, we found that glycosaminoglycan is crucial for the cell-membrane-penetrating activity of chimeric Mb as well as that of zebrafish Ngb. These results enable us to conclude that such module substitutions will facilitate the design and production of novel functional proteins.

Identification of residues critical for the cell-membrane-penetrating activity of zebrafish neuroglobin.

Neuroglobin (Ngb) is a globin found in the vertebrate brain. Recently, we found that zebrafish Ngb can translocate into cells and clarified that module M1 of zebrafish Ngb is important for protein transduction. In the present study, we used site-directed mutagenesis to identify residues of module M1 that are important for protein transduction. We show that Lys7, Lys9, Lys21, and Lys23 of zebrafish Ngb are crucial for its activity. Since these residues are conserved among fishes, but not among mammals, birds, or amphibians, the ability to penetrate cell membranes may be a unique characteristic of fish Ngb proteins.