Dynamics of D-amino acid oxidase ain kidney epithelial cells under amino acid starvation.

D-amino acid oxidase (DAO) is a flavoenzyme catalyzing the oxidation of D-amino acid (AA)s. In the kidney, its expression is detected in proximal tubules, and DAO is considered to play a role in the conversion of D-form AAs to α-keto acids. LLC-PK1 cells, a pig renal proximal tubule cell line, were used to elucidate the regulation of DAO protein synthesis and degradation. In this study, we showed that trypsinization of LLC-PK1 cells in culture system rapidly reduced the intracellular DAO protein level to ∼33.9% of that before treatment, even within 30 min. Furthermore, we observed that the DAO protein level was decreased when LLC-PK1 cells were subjected to AA starvation. To determine the degradation pathway, we treated the cells with chloroquine and MG132. DAO degradation was found to be inhibited by chloroquine, but not by MG132 treatment. We next examined whether or not DAO was degraded by autophagy. We found that AA starvation led to an increased accumulation of LC3-II, suggesting that DAO protein is degraded by autophagy due to AA starvation conditions. Furthermore, treatment with cycloheximide inhibited DAO protein degradation. Taken together, DAO protein is degraded by autophagy under starvation. The present study revealed the potential dynamics of DAO correlated with renal pathophysiology.

Novel oseltamivir-resistant mutations distant from the active site of influenza B neuraminidase.

We performed a neuraminidase sequence analysis of thirty-two pediatric patients with influenza B who visited Teikyo University Hospital from January 2016 to March 2017, and found oseltamivir-resistant samples belonging to the Yamagata and Victoria lineages. Comparison with the neuraminidase sequence of oseltamivir-susceptible B/Brisbane/60/2008 revealed 5 common amino acid substitutions in many of these samples. According to the binding free energy calculation, the N340D and E358K substitutions reduced the affinity of oseltamivir to neuraminidase. Unexpectedly, these substitutions were located distant from the oseltamivir-binding site in neuraminidase. According to the molecular dynamics simulations, the N340D substitution rearranged complicated hydrogen bond networks in an extensive surface region of neuraminidase. The E358K substitution extensively altered the electrostatic potential map of the overall neuraminidase structure. Through these novel mechanisms, the N340D and E358K substitutions indirectly influenced the affinity reduction. These results may be useful for designing drugs for the treatment of oseltamivir-resistant virus infections.Communicated by Ramaswamy H. Sarma.

P219L substitution in human D-amino acid oxidase impacts the ligand binding and catalytic efficiency.

Human D-amino acid oxidase (DAO) is a flavoenzyme that is implicated in neurodegenerative diseases. We investigated the impact of replacement of proline with leucine at Position 219 (P219L) in the active site lid of human DAO on the structural and enzymatic properties, because porcine DAO contains leucine at the corresponding position. The turnover numbers (kcat) of P219L were unchanged, but its Km values decreased compared with wild-type, leading to an increase in the catalytic efficiency (kcat/Km). Moreover, benzoate inhibits P219L with lower Ki value (0.7-0.9 µM) compared with wild-type (1.2-2.0 µM). Crystal structure of P219L in complex with flavin adenine dinucleotide (FAD) and benzoate at 2.25 Å resolution displayed conformational changes of the active site and lid. The distances between the H-bond-forming atoms of arginine 283 and benzoate and the relative position between the aromatic rings of tyrosine 224 and benzoate were changed in the P219L complex. Taken together, the P219L substitution leads to an increase in the catalytic efficiency and binding affinity for substrates/inhibitors due to these structural changes. Furthermore, an acetic acid was located near the adenine ring of FAD in the P219L complex. This study provides new insights into the structure-function relationship of human DAO.

Age- and gender-dependent D-amino acid oxidase activity in mouse brain and peripheral tissues: implication for aging and neurodegeneration.

D-amino acid oxidase (DAO) is a flavoenzyme, catalysing oxidative deamination of D-amino acids to produce corresponding α-keto acids, ammonia and hydrogen peroxide. In our search for DAO activity among various tissues, we developed a sensitive assay based on hydrogen peroxide production involving enzyme-coupled colorimetric assay with peroxidase. We first optimized buffer components to extract DAO protein from mouse tissues. Here we show that DAO activity was detected in kidney, cerebellum, medulla oblongata, midbrain and spinal cord, but not in liver. In addition, we observed that DAO activity and expression were decreased in thoracic and lumbar regions of spinal cord in aged mice when compared with young mice, indicating that decreased DAO is involved in motoneuron degeneration during senescence. We also found gender difference in DAO activity in the kidney, suggesting that DAO activity is influenced by sexual dimorphism. We newly detected DAO activity in the epididymis, although undetected in testis. Furthermore, DAO activity was significantly higher in the caput region than corpus and cauda regions of epididymis, indicating that D-amino acids present in the testis are eliminated in epididymis. Taken together, age- and gender-dependent DAO activity in each organ may underlie the human pathophysiology regulated by D-amino acid metabolism.

Structural basis for potent inhibition of d-amino acid oxidase by thiophene carboxylic acids.

A series of thiophene-2-carboxylic acids and thiophene-3-carboxylic acids were identified as a new class of DAO inhibitors. Structure-activity relationship (SAR) studies revealed that small substituents are well-tolerated on the thiophene ring of both the 2-carboxylic acid and 3-carboxylic acid scaffolds. Crystal structures of human DAO in complex with potent thiophene carboxylic acids revealed that Tyr224 was tightly stacked with the thiophene ring of the inhibitors, resulting in the disappearance of the secondary pocket observed with other DAO inhibitors. Molecular dynamics simulations of the complex revealed that Tyr224 preferred the stacked conformation irrespective of whether Tyr224 was stacked or not in the initial state of the simulations. MM/GBSA indicated a substantial hydrophobic interaction between Tyr244 and the thiophene-based inhibitor. In addition, the active site was tightly closed with an extensive network of hydrogen bonds including those from Tyr224 in the stacked conformation. The introduction of a large branched side chain to the thiophene ring markedly decreased potency. These results are in marked contrast to other DAO inhibitors that can gain potency with a branched side chain extending to the secondary pocket due to Tyr224 repositioning. These insights should be of particular importance in future efforts to optimize DAO inhibitors with novel scaffolds.

A ternary complex model of Sirtuin4-NAD+-Glutamate dehydrogenase.

Sirtuin4 (Sirt4) is one of the mammalian homologues of Silent information regulator 2 (Sir2), which promotes the longevity of yeast, C. elegans, fruit flies and mice. Sirt4 is localized in the mitochondria, where it contributes to preventing the development of cancers and ischemic heart disease through regulating energy metabolism. The ADP-ribosylation of glutamate dehydrogenase (GDH), which is catalyzed by Sirt4, downregulates the TCA cycle. However, this reaction mechanism is obscure, because the structure of Sirt4 is unknown. We here constructed structural models of Sirt4 by homology modeling and threading, and docked nicotinamide adenine dinucleotide+ (NAD+) to Sirt4. In addition, a partial GDH structure was docked to the Sirt4-NAD+ complex model. In the ternary complex model of Sirt4-NAD+-GDH, the acetylated lysine 171 of GDH is located close to NAD+. This suggests a possible mechanism underlying the ADP-ribosylation at cysteine 172, which may occur through a transient intermediate with ADP-ribosylation at the acetylated lysine 171. These results may be useful in designing drugs for the treatment of cancers and ischemic heart disease.

De novo non-synonymous TBL1XR1 mutation alters Wnt signaling activity.

Here we report de novo non-synonymous single-nucleotide variants (SNVs) by conducting whole exome sequencing of 18 trios consisting of Japanese patients with sporadic schizophrenia and their parents. Among nine SNVs, we explored the functional impact of the de novo mutation in TBL1XR1 [c.30 C > G (p.Phe10Leu)], a gene previously found to be associated with autism spectrum disorder and epilepsy. Protein structural analysis revealed that Phe10Leu mutation may decrease the structural stability of the TBL1XR1 protein. We demonstrate that Phe10Leu mutation alters the interaction of TBL1XR1 with N-CoR and ß-catenin, which play critical roles in regulation of Wnt-mediated transcriptional activity. Consistently, TBL1XR1-mediated activation of Wnt signaling was up-regulated by Phe10Leu mutation. These results suggest that a de novo TBL1XR1 point mutation could alter Wnt/ß-catenin signaling activity. Further studies are required to clarify the involvement of TBL1XR1 mutations in neuropsychiatric conditions.

Ectopic overexpression of LAPTM5 results in lysosomal targeting and induces Mcl-1 down-regulation, Bak activation, and mitochondria-dependent apoptosis in human HeLa cells.

Human lysosomal-associated protein multispanning membrane 5 (LAPTM5) was identified by an ordered differential display-polymerase chain reaction (ODD-PCR) as an up-regulated cDNA fragment during 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced differentiation of U937 cells into monocytes/macrophages. After TPA-treatment, the levels of LAPTM5 mRNA and protein increased and reached a maximum at 18-36 h. In healthy human tissues, LAPTM5 mRNA was expressed at high levels in hematopoietic cells and tissues, at low levels in the lung and fetal liver, and was not detected in other non-hematopoietic tissues. LAPTM5 mRNA was detected in immature malignant cells of myeloid lineage, such as K562, HL-60, U937, and THP-1 cells, and in unstimulated peripheral T cells, but was absent or barely detectable in lymphoid malignant or non-hematopoietic malignant cells. The LAPTM5 level in HL-60 cells increased more significantly during TPA-induced monocyte/macrophage differentiation than during DMSO-induced granulocyte differentiation. Ectopic expression of GFP-LAPTM5 or LAPTM5 in HeLa cells exhibited the localization of LAPTM5 to the lysosome. In HeLa cells overexpressing LAPTM5, the Mcl-1 and Bid levels declined markedly and apoptosis was induced via Bak activation, Δψm loss, activation of caspase-9, -8 and -3, and PARP degradation without accompanying necrosis. However, these LAPTM5-induced apoptotic events except for the decline of Bid level were completely abrogated by concomitant overexpression of Mcl-1. The pan-caspase inhibitor (z-VAD-fmk) could suppress the LAPTM5-induced apoptotic sub-G1 peak by ~40% but failed to block the induced Δψm loss, whereas the broad-range inhibitor of cathepsins (Cathepsin Inhibitor I) could suppress the LAPTM5-induced apoptotic sub-G1 peak and Δψm loss, by ~22% and ~23%, respectively, suggesting that the LAPTM5-mediated Δψm loss was exerted at least in part in a cathepsin-dependent manner. Together, these results demonstrate that ectopic overexpression of LAPTM5 in HeLa cells induced apoptosis via cleavage of Mcl-1 and Bid by a LAPTM5-associated lysosomal pathway, and subsequent activation of the mitochondria-dependent caspase cascade.

Elucidation of inhibitor-binding pockets of d-amino acid oxidase using docking simulation and N-sulfanylethylanilide-based labeling technology.

Because of the relevance of d-serine (d-Ser) to schizophrenia, inhibitors of d-amino acid oxidase (DAO), which catalyzes degradation of d-Ser in the presence of flavin adenine dinucleotide (FAD), are expected to be anti-schizophrenia therapeutics. In this study, binding pockets of DAO to its inhibitor 4-bromo-3-nitrobenzoic acid were searched by combining in silico docking simulation and labeling experiments employing an N-sulfanylethylanilide-based labeling technology that we have developed. The results clearly demonstrated that there are two binding pockets: one is shared with d-Ser and FAD, and the other is an unexpected cleft between the subunits of a DAO dimer. These findings will provide insight to aid the development of new DAO inhibitors. In addition, it was also proved that our labeling technology could be applicable to elucidate the binding pockets of proteins.

Structure models of G72, the product of a susceptibility gene to schizophrenia.

The G72 gene is one of the most susceptible genes to schizophrenia and is contained exclusively in the genomes of primates. The product of the G72 gene modulates the activity of D-amino acid oxidase (DAO) and is a small protein prone to aggregate, which hampers its structural studies. In addition, lack of a known structure of a homologue makes it difficult to use the homology modelling method for the prediction of the structure. Thus, we first developed a hybrid ab initio approach for small proteins prior to the prediction of the structure of G72. The approach uses three known ab initio algorithms. To evaluate the hybrid approach, we tested our prediction of the structure of the amino acid sequences whose structures were already solved and compared the predicted structures with the experimentally solved structures. Based on these comparisons, the average accuracy of our approach was calculated to be ∼5 Å. We then applied the approach to the sequence of G72 and successfully predicted the structures of the N- and C-terminal domains (ND and CD, respectively) of G72. The predicted structures of ND and CD were similar to membrane-bound proteins and adaptor proteins, respectively.

Mechanism of a Mutation in Non-Structural Protein 1 Inducing High Pathogenicity of Avian Influenza Virus H5N1.

Avian influenza H5N1 has shown high mortality rate in human. Non-structural protein 1 (NS1) is a virulence factor of H5N1. Mutation at the 42nd residue within the RNA-binding domain (RBD) of NS1 dramatically changes the degree of pathogenicity of H5N1 in mice. We here studied the impact of this mutation on the function of RBD, and found that RBD with serine at the 42th residue binds double-stranded RNA (dsRNA), whereas that with proline at the 42th residue does not. Analysis of structural models of the RBD proteins with S42 and P42 suggested remarkable difference in the structure of the dsRNA-binding interface, whereas structural analysis by analytical gel filtration and CD measurements did not indicate difference between those RBD proteins. Our results suggest that the single amino acid replacement induces a minor, but global structural change leading to the loss of function of NS1 thereby the change in the degree of pathogenicity.

In Situ Click Chemistry for the Identification of a Potent D-Amino Acid Oxidase Inhibitor.

In situ click chemistry is a target-guided synthesis approach for discovering novel lead compounds by assembling organic azides and alkynes into triazoles inside the affinity site of target biogenic molecules such as proteins. We report in situ click chemistry screening with human D-amino acid oxidase (hDAO), which led to the identification of a more potent hDAO inhibitor. The hDAO inhibitors have chemotherapeutic potential as antipsychotic agents. The new inhibitor displayed competitive inhibition of hDAO and showed significantly increased inhibitory activity against hDAO compared with that of an anchor molecule of in situ click chemistry.

Nucling, a novel apoptosis-associated protein, controls mammary gland involution by regulating NF-κB and STAT3.

Postpartum mammary gland involution is the physiological process by which the lactating gland returns to its pre-pregnant state. In rodent models, the microenvironment of mammary gland involution is sufficient to induce enhanced tumor cell growth, local invasion, and metastasis. Therefore, a deeper understanding of the physiological regulation of involution may provide in-depth information on breast cancer therapy. We herein identified Nucling as an important regulator of involution of the mammary gland. A knock-out mouse model was generated and revealed that postpartum involution were impaired in mice lacking Nucling. Involution is normally associated with an increase in the activation of NF-κB and STAT3, which is required for the organized regulation of involution, and was observed in WT glands, but not in the absence of Nucling. Furthermore, the loss of Nucling led to the suppression of Calpain-1, IL-6, and C/EBPδ factors, which are known to be essential for normal involution. The number of M2 macrophages, which are crucial for epithelial cell death and adipocyte repopulation after weaning, was also reduced in Nucling-KO glands. Taken together, the results of the present study demonstrated that Nucling played an important role in mammary gland involution by regulating NF-κB and STAT3 signaling pathways.

Identification of DNA-binding proteins that interact with the 5'-flanking region of the human D-amino acid oxidase gene by pull-down assay coupled with two-dimensional gel electrophoresis and mass spectrometry.

D-Amino acid oxidase (DAO) is a flavoenzyme that metabolizes D-amino acids and is expected to be a promising therapeutic target of schizophrenia and glioblastoma. The study of DNA-binding proteins has yielded much information in the regulation of transcription and other biological processes. However, proteins interacting with DAO gene have not been elucidated. Our assessment of human DAO promoter activity using luciferase reporter system indicated the 5'-flanking region of this gene (-4289 bp from transcription initiation site) has a regulatory sequence for gene expression, which is regulated by multi-protein complexes interacting with this region. By using pull-down assay coupled with two-dimensional gel electrophoresis and mass spectrometry, we identified six proteins binding to the 5'-flanking region of the human DAO gene (zinc finger C2HC domain-containing protein 1A; histidine-tRNA ligase, cytoplasmic; molybdenum cofactor biosynthesis protein; 60S ribosomal protein L37; calponin-1; calmodulin binding protein and heterogeneous nuclear ribonucleoprotein A2/B1). These preliminary results will contribute to the advance in the understanding of the potential factors associated with the regulatory mechanism of DAO expression.

Identification of two promoters for human D-amino acid oxidase gene: implication for the differential promoter regulation mediated by PAX5/PAX2.

D-amino acid oxidase (DAO) is a flavoenzyme that metabolizes d-amino acids. Until now, the DAO expression mechanism is still unclear. Our assessment of human DAO (hDAO) promoter activity using luciferase reporter system indicated the proximal upstream region of exon1 (-237/+1) has promoter activity (P1). Interestingly, we identified an alternative promoter in the proximal upstream region of exon2 (+4,126/+4,929) (P2). This alternative promoter has stronger activity than that of P1. Our results also revealed a negative regulatory segment (+1,163/+1,940) in intron1; that would act in concert with P1 and P2. Bioinformatics analyses elucidated the conservation of transcription factor PAX5 family binding sites among species. These sites (-60/-31) and (+4,464/+4,493), locate in P1 and P2 of hDAO, respectively. Gel shift assays demonstrated P1 contains a site (-60/-31) for PAX5 binding while P2 has three sites for both paired box gene 2 (PAX2) and paired box gene 5 (PAX5) binding. The dual roles of PAX5 family in regulating hDAO transcription by modulating promoter activity of P1 and activating promoter activity of P2 were implicated based on the site-directed mutagenesis experiment. Altogether, our data suggested the differential regulation of hDAO expression by two promoters whose activities may be modulated by the binding of PAX2 and PAX5.

Evaluation of human D-amino acid oxidase inhibition by anti-psychotic drugs in vitro.

It is of importance to determine whether antipsychotic drugs currently prescribed for schizophrenia exert D-amino acid oxidase (DAO)-inhibitory effects. We first investigated whether human (h)DAO can metabolize D-kynurenine (D-KYN) to produce the fluorescent compound kynurenic acid (KYNA) by using high-performance liquid chromatography with mass spectrometry, and fluorescence spectrometry. After confirmation of KYNA production from D-KYN by hDAO, 8 first- and second-generation antipsychotic drugs, and 6 drugs often prescribed concomitantly, were assayed for hDAO-inhibitory effects by using in vitro fluorometric methods with D-KYN as the substrate. DAO inhibitors 3-methylpyrazole-5-carboxylic acid and 4H-thieno[3,2-b]pyrrole-5-carboxylic acid inhibited KYNA production in a dose-dependent manner. Similarly, the second-generation antipsychotics blonanserin and risperidone were found to possess relatively strong hDAO-inhibitory effects in vitro (5.29 ± 0.47 µM and 4.70 ± 0.17 µM, respectively). With regard to blonanserin and risperidone, DAO-inhibitory effects should be taken into consideration in the context of their in vivo pharmacotherapeutic efficacy.

Modulation of extracellular d-serine content by calcium permeable AMPA receptors in rat medial prefrontal cortex as revealed by in vivo microdialysis.

In mammalian brains, d-serine has been shown to be required for the regulation of glutamate neurotransmission as an endogenous co-agonist for the N-methyl-d-aspartate type glutamate receptor that is essential for the expression of higher-order brain functions. The exact control mechanisms for the extracellular d-serine dynamics, however, await further elucidation. To obtain an insight into this issue, we have characterized the effects of agents acting at the α-amino-3-hydroxy-5-methyl-4-isoxazolepropioinic acid (AMPA) type glutamate receptor on the extracellular d-serine contents in the medial prefrontal cortex of freely moving rats by an in vivo microdialysis technique in combination with high-performance liquid chromatography with fluorometric detection. In vivo experiments are needed in terms of a crucial role of d-serine in the neuron-glia communications despite the previous in vitro studies on AMPA receptor-d-serine interactions using the separated preparations of neurons or glial cells. Here, we show that the intra-cortical infusion of (S)-AMPA, an active enantiomer at the AMPA receptor, causes a significant and concentration-dependent reduction in the prefrontal extracellular contents of d-serine, which is reversed by an AMPA/kainate receptor antagonist, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt, and a calcium permeable AMPA receptor antagonist, 1-naphthyl acetyl spermine. The d-serine reducing effects of (S)-AMPA are augmented by co-infusion of cyclothiazide that prevents AMPA receptor desensitization. Our data support the view that a calcium permeable AMPA receptor subtype may exert a phasic inhibitory control on the extracellular d-serine release in the mammalian prefrontal cortex in vivo.

A novel pathway for the production of hydrogen sulfide from D-cysteine in mammalian cells.

In eukaryotes, hydrogen sulphide acts as a signalling molecule and cytoprotectant. Hydrogen sulphide is known to be produced from L-cysteine by cystathionine ß-synthase, cystathionine γ-lyase and 3-mercaptopyruvate sulfurtransferase coupled with cysteine aminotransferase. Here we report an additional biosynthetic pathway for the production of hydrogen sulphide from D-cysteine involving 3-mercaptopyruvate sulfurtransferase and D-amino acid oxidase. Unlike the L-cysteine pathway, this D-cysteine-dependent pathway operates predominantly in the cerebellum and the kidney. Our study reveals that administration of D-cysteine protects primary cultures of cerebellar neurons from oxidative stress induced by hydrogen peroxide and attenuates ischaemia-reperfusion injury in the kidney more than L-cysteine. This study presents a novel pathway of hydrogen sulphide production and provides a new therapeutic approach to deliver hydrogen sulphide to specific tissues.

Nucling, a novel protein associated with NF-κB, regulates endotoxin-induced apoptosis in vivo.

Nucling is a proapoptotic protein that regulates the apoptosome and nuclear factor-kappa B (NF-κB) signalling pathways. Strong stimuli, such as Gram-negative bacterial lipopolysaccharide (LPS), induce the simultaneous secretion of cytokines following the activation of NF-κB. Proinflammatory cytokines can induce liver damage through several mechanisms such as increases in oxidative stress and apoptotic reactions leading to tissue necrosis. Herein, we show that Nucling-knockout (KO) mice are resistant to LPS that consistently caused mortality in wild-type (WT) counterparts. Although serum levels of cytokines such as tumour necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-6 did not differ significantly between WT and Nucling-KO mice after the LPS challenge, hepatocytes of Nucling-KO mice were refractory to LPS- or TNF-α-induced cell death. These results were consistent with the decreased expression of proapoptotic proteins including apoptosis-inducing factor and cleaved form of poly (ADP-ribose) polymerase and terminal deoxynucleotidyl transferase dUTP nick end-labelling positive cells in the liver of Nucling-KO mice after the administration of a lethal dose of LPS. Moreover, the upregulation of NF-κB-regulated anti-apoptotic molecules including cellular inhibitor of apoptosis (cIAP) 1 and cIAP2 was observed in the liver of Nucling-KO mice after LPS treatment. These findings indicate that the Nucling deficiency leads to resistance to apoptosis in liver. We propose that Nucling is important for the induction of apoptosis in cells damaged by cytotoxic stressors through the NF-κB signalling pathway.

The role of Kupffer cells in carbon tetrachloride intoxication in mice.

Carbon tetrachloride (CCl(4))-induced acute hepatitis is assumed to involve two phases. The initial phase, initiated within 2 h after CCl(4) administration, involves the generation of reactive oxygen species. The second phase is assumed to start about 8 h subsequent to CCl(4) administration and involves the oxidant-induced activation of Kupffer cells, which release various pro-inflammatory mediators such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). We investigated the role of Kupffer cells during CCl(4) intoxication using Nucling-knockout mice (the KO group), in which the number of Kupffer cells is largely reduced. Plasma alanine transaminase and aspartate transaminase levels demonstrated that the liver necrosis during the second phase was significantly alleviated in the KO group compared with that in the wild-type mice (the WT group). Plasma TNF-α concentrations in the WT group significantly increased 24 h after CCl(4) intoxication, whereas those in the KO group did not significantly increase. Plasma IL-6 levels also significantly increased in the WT group 24 h after CCl(4) administration, but those in the KO group did not increase at any time point. These results indicated that excess reactions of Kupffer cells, once primed by oxidants, were involved in the exacerbation of oxidative stress and liver damage during the second phase of CCl(4) intoxication.