Keisuke Wakasugi

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.

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.

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.

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 (SOD1^G93A mice). Intracerebroventricular administration of CysC during the early symptomatic SOD1^G93A 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. image

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.

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.

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 G_i/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 Gα_i1. Moreover, we show that Lys46, Lys70, Arg208, Lys209, and Lys210 residues of Gα_i1 are important for binding to human Ngb. We propose a molecular docking model of the complex between human Ngb and Gα_i1.