The NeuroinflammatoryPotential of HIV-1 NefVariants in Modulating the Gene Expression Profile of Astrocytes.

HIV-1 mediated neurotoxicity is thought to be associated with HIV-1 viral proteins activating astrocytes and microglia by inducing inflammatory cytokines leading to the development of HIV-associated neurocognitive disorder (HAND). In the current study, we observe how HIV-1 Nef upregulates the levels of IL-6, IP-10, and TNF-α around 6.0fold in normal human astrocytes (NHAs) compared to cell and empty vector controls. Moderate downregulation in the expression profile of inflammatory cytokines was observed due to RNA interference. Furthermore, we determine the impact of inflammatory cytokines in the upregulation of kynurenine pathway metabolites, such as indoleamine 2,3-dioxygenase (IDO), and 3-hydroxyanthranilic acid oxygenase (HAAO) in NHA, and found the same to be 3.0- and 3.2-fold, respectively. Additionally, the variation in the level of nitric oxide before and after RNA interference was significant. The upregulated cytokines and pathway-specific metabolites could be linked with the neurotoxic potential of HIV-1 Nef. Thus, the downregulation in cytokines and kynurenine metabolites observed after siRNA-Nef interference indicates the possibility of combining the RNA interference approach with current antiretroviral therapy to prevent neurotoxicity development.

Re-sequencing of candidate genes FOXF1, HSPA6, HAAO, and KYNU in 522 individuals with VATER/VACTERL, VACTER/VACTERL-like association, and isolated anorectal malformation.

BACKGROUND: The acronym VATER/VACTERL association describes the combination of at least three component features (CFs): vertebral defects (V), anorectal malformations (ARM) (A), cardiac defects (C), tracheoesophageal fistula with or without esophageal atresia (TE), renal malformations (R), and limb defects (L). Individuals presenting two CFs have been termed VATER/VACTERL-like. Recently, FOXF1, HSPA6, HAAO, KYNU, TRAP1, and ZIC3 have been proposed as candidate genes for VATER/VACTERL, VATER/VACTERL-like, and ARM. Re-sequencing studies identified disease-causing variants in TRAP1 and ZIC3, the contribution of other genes was not independently investigated. One affected variant carrier in FOXF1 was previously identified. Here we re-sequenced FOXF1, HSPA6, HAAO, and KYNU in 522 affected individuals. METHODS: Using molecular inversion probe (MIP) technology, re-sequencing was performed in 63 individuals with VATER/VACTERL association, 313 with VATER/VACTERL-like association, and 146 with ARM. All individuals were of European ethnicity. Variant filtering considered variants with a minor allele frequency (MAF) ≤0.01 for putative recessive disease-genes HSPA6, HAAO, and KYNU. For the putative dominant disease-gene FOXF1 we considered variants with a MAF ≤0.0001. In silico prediction tools were used for further prioritization. RESULTS: Only two variants in FOXF1 in two independently affected individuals [c.443G>T, p.(Cys148Phe); c.850T>C, p.(Tyr284His)] passed our filter criteria. One individual presented with ARM, the second presented with TE and C comprising atrial and ventricular septal defects. Sanger sequencing confirmed both variants but also their inheritance from the healthy mother. CONCLUSION: Our analysis suggests that FOXF1, HSPA6, HAAO and KYNU do not play a major role in the formation of VACTER/VACTERL phenotypes or ARM.

Observing 3-hydroxyanthranilate-3,4-dioxygenase in action through a crystalline lens.

The synthesis of quinolinic acid from tryptophan is a critical step in the de novo biosynthesis of nicotinamide adenine dinucleotide (NAD+) in mammals. Herein, the nonheme iron-based 3-hydroxyanthranilate-3,4-dioxygenase responsible for quinolinic acid production was studied by performing time-resolved in crystallo reactions monitored by UV-vis microspectroscopy, electron paramagnetic resonance (EPR) spectroscopy, and X-ray crystallography. Seven catalytic intermediates were kinetically and structurally resolved in the crystalline state, and each accompanies protein conformational changes at the active site. Among them, a monooxygenated, seven-membered lactone intermediate as a monodentate ligand of the iron center at 1.59-Å resolution was captured, which presumably corresponds to a substrate-based radical species observed by EPR using a slurry of small-sized single crystals. Other structural snapshots determined at around 2.0-Å resolution include monodentate and subsequently bidentate coordinated substrate, superoxo, alkylperoxo, and two metal-bound enol tautomers of the unstable dioxygenase product. These results reveal a detailed stepwise O-atom transfer dioxygenase mechanism along with potential isomerization activity that fine-tunes product profiling and affects the production of quinolinic acid at a junction of the metabolic pathway.

Dysregulation at multiple points of the kynurenine pathway is a ubiquitous feature of renal cancer: implications for tumour immune evasion.

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO), the first step in the kynurenine pathway (KP), is upregulated in some cancers and represents an attractive therapeutic target given its role in tumour immune evasion. However, the recent failure of an IDO inhibitor in a late phase trial raises questions about this strategy. METHODS: Matched renal cell carcinoma (RCC) and normal kidney tissues were subject to proteomic profiling. Tissue immunohistochemistry and gene expression data were used to validate findings. Phenotypic effects of loss/gain of expression were examined in vitro. RESULTS: Quinolate phosphoribosyltransferase (QPRT), the final and rate-limiting enzyme in the KP, was identified as being downregulated in RCC. Loss of QPRT expression led to increased potential for anchorage-independent growth. Gene expression, mass spectrometry (clear cell and chromophobe RCC) and tissue immunohistochemistry (clear cell, papillary and chromophobe), confirmed loss or decreased expression of QPRT and showed downregulation of other KP enzymes, including kynurenine 3-monoxygenase (KMO) and 3-hydroxyanthranilate-3,4-dioxygenase (HAAO), with a concomitant maintenance or upregulation of nicotinamide phosphoribosyltransferase (NAMPT), the key enzyme in the NAD+ salvage pathway. CONCLUSIONS: Widespread dysregulation of the KP is common in RCC and is likely to contribute to tumour immune evasion, carrying implications for effective therapeutic targeting of this critical pathway.

VPOT: A Customizable Variant Prioritization Ordering Tool for Annotated Variants.

Next-generation sequencing (NGS) technologies generate thousands to millions of genetic variants per sample. Identification of potential disease-causal variants is labor intensive as it relies on filtering using various annotation metrics and consideration of multiple pathogenicity prediction scores. We have developed VPOT (variant prioritization ordering tool), a python-based command line tool that allows researchers to create a single fully customizable pathogenicity ranking score from any number of annotation values, each with a user-defined weighting. The use of VPOT can be informative when analyzing entire cohorts, as variants in a cohort can be prioritized. VPOT also provides additional functions to allow variant filtering based on a candidate gene list or by affected status in a family pedigree. VPOT outperforms similar tools in terms of efficacy, flexibility, scalability, and computational performance. VPOT is freely available for public use at GitHub (https://github.com/VCCRI/VPOT/). Documentation for installation along with a user tutorial, a default parameter file, and test data are provided.

Single Nucleotide Polymorphisms of HAAO and IRX6 Genes as Risk Factors for Hypospadias.

PURPOSE: We evaluated the association of hypospadias and 17 susceptibility loci previously identified by a European genome-wide association study in a cohort of Japanese patients. We also examined the expression of candidate genes in male mouse embryos to determine the possible underlying mechanisms of this disease. MATERIALS AND METHODS: We enrolled 169 Japanese patients (mean age at surgery 3.7 years) who underwent repair of hypospadias. Genotyping of 17 single nucleotide polymorphisms was performed using a multiplex polymerase chain reaction invader assay. We also performed in situ hybridization to determine whether candidate genes were expressed in the male genital tubercle during embryonic development of the external genitalia in mice. RESULTS: Single nucleotide polymorphism rs3816183 of HAAO was significantly associated with susceptibility to hypospadias in general (p = 0.0019) and to anterior/middle hypospadias (p = 0.0283) and posterior hypospadias (p = 0.0226), while single nucleotide polymorphism rs6499755 of IRX6 showed an association with susceptibility to anterior/middle hypospadias (p = 0.0472). In mouse embryos there was no significant upregulation of Haao expression in the developing male external genitalia. Irx3 and Irx5, which are linked to Irx6 within the IrxB cluster, were expressed in the mesenchyme remote from the urethral plate epithelium during the critical embryonic period for masculinization. Irx6 was expressed in the ectodermal epithelium, demonstrating prominent dorsal ectodermal expression without expression in the ventral ectoderm adjacent to the urethral plate during the same period. CONCLUSIONS: Genetic variations of HAAO and IRX6 influence susceptibility to hypospadias in the Japanese population. Further research is needed to clarify the mechanism by which variations in these genes contribute to the pathogenesis of hypospadias.

Genetic alterations affecting the genes encoding the enzymes of the kynurenine pathway and their association with human diseases.

Tryptophan is metabolized primarily via the kynurenine pathway (KP), which involves several enzymes, including indoleamine 2,3-dioxygenase, tryptophan 2,3 dioxygenase (TDO), kynurenine aminotransferases (KATs), kynurenine monooxygenase (KMO) etc. The majority of metabolites are neuroactive: some of them, such as kynurenic acid, show neuroprotective effects, while others contribute to free radical production, leading to neurodegeneration. Imbalance of the pathway is assumed to contribute to the development of several neurodegenerative diseases, psychiatric disorders, migraine and multiple sclerosis. Our aim was to summarize published data on genetic alterations of enzymes involved in the KP leading to disturbances of the pathway that can be related to different diseases. To achieve this, a PubMed literature search was performed for publications on genetic alterations of the KP enzymes upto April 2017. Several genetic alterations of the KP have been identified and have been proposed to be associated with diseases. Here we must emphasize that despite the large number of recognized genetic alterations, the number of firmly established causal relations with specific diseases is still small. The realization of this by those interested in the field is very important and finding such connections should be a major focus of related research. Polymorphisms of the genes encoding the enzymes of the KP have been associated with autism, multiple sclerosis and schizophrenia, and were shown to affect the immune response of patients with bacterial meningitis, just to mention a few. To our knowledge, this is the first comprehensive review of the genetic alterations of the KP enzymes. We believe that the identification of genetic alterations underlying diseases has great value regarding both treatment and diagnostics in precision medicine, as this work can promote the understanding of pathological mechanisms, and might facilitate medicinal chemistry approaches to substitute missing components or correct the disturbed metabolite balance of KP.

NAD Deficiency, Congenital Malformations, and Niacin Supplementation.

BACKGROUND: Congenital malformations can be manifested as combinations of phenotypes that co-occur more often than expected by chance. In many such cases, it has proved difficult to identify a genetic cause. We sought the genetic cause of cardiac, vertebral, and renal defects, among others, in unrelated patients. METHODS: We used genomic sequencing to identify potentially pathogenic gene variants in families in which a person had multiple congenital malformations. We tested the function of the variant by using assays of in vitro enzyme activity and by quantifying metabolites in patient plasma. We engineered mouse models with similar variants using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system. RESULTS: Variants were identified in two genes that encode enzymes of the kynurenine pathway, 3-hydroxyanthranilic acid 3,4-dioxygenase (HAAO) and kynureninase (KYNU). Three patients carried homozygous variants predicting loss-of-function changes in the HAAO or KYNU proteins (HAAO p.D162*, HAAO p.W186*, or KYNU p.V57Efs*21). Another patient carried heterozygous KYNU variants (p.Y156* and p.F349Kfs*4). The mutant enzymes had greatly reduced activity in vitro. Nicotinamide adenine dinucleotide (NAD) is synthesized de novo from tryptophan through the kynurenine pathway. The patients had reduced levels of circulating NAD. Defects similar to those in the patients developed in the embryos of Haao-null or Kynu-null mice owing to NAD deficiency. In null mice, the prevention of NAD deficiency during gestation averted defects. CONCLUSIONS: Disruption of NAD synthesis caused a deficiency of NAD and congenital malformations in humans and mice. Niacin supplementation during gestation prevented the malformations in mice. (Funded by the National Health and Medical Research Council of Australia and others.).

Antiviral Effect of IDO in Mouse Fibroblast Cells During Influenza Virus Infection.

While upregulation of 2,3-dioxygenase (IDO) accompanied by degradation of tryptophan along the kynurenine pathway have been reported to exert antimicrobial effects against a wide range of infectious agents, its role in the replication of influenza A virus remains uncertain. We performed experiments using influenza A/WSN/33 virus infection of mouse fibroblast cell-line (NIH-3T3) to study the effects of IDO on viral replication. Influenza infection resulted in prominent elevations of transcripts encoding IDO, interferon (IFN)-ß, and segment 8 of the virus in NIH-3T3 cells. Introduction of siRNA targeted against IDO followed by infection resulted in further increased levels of viral RNA without altering IFN-ß expression. Inhibition of IDO during the infection also resulted in reduction of virus-driven upregulation of 3-hydroxyanthranilate 3,4-dioxygenase (HAAO), but not kynurenine 3-monooxygenase (KMO), which are enzymes downstream in the kynurenine pathway. Thus, induction of IDO appears to contribute to limiting replication of the WSN/33 strain of influenza A virus in murine NIH-3T3 cells.

Expression of tryptophan 2,3-dioxygenase and production of kynurenine pathway metabolites in triple transgenic mice and human Alzheimer's disease brain.

To assess the role of the kynurenine pathway in the pathology of Alzheimer's disease (AD), the expression and localization of key components of the kynurenine pathway including the key regulatory enzyme tryptophan 2,3 dioxygenase (TDO), and the metabolites tryptophan, kynurenine, kynurenic acid, quinolinic acid and picolinic acid were assessed in different brain regions of triple transgenic AD mice. The expression and cell distribution of TDO and quinolinic acid, and their co-localization with neurofibrillary tangles and senile ß amyloid deposition were also determined in hippocampal sections from human AD brains. The expression of TDO mRNA was significantly increased in the cerebellum of AD mouse brain. Immunohistochemistry demonstrated that the density of TDO immuno-positive cells was significantly higher in the AD mice. The production of the excitotoxin quinolinic acid strongly increased in the hippocampus in a progressive and age-dependent manner in AD mice. Significantly higher TDO and indoleamine 2,3 dioxygenase 1 immunoreactivity was observed in the hippocampus of AD patients. Furthermore, TDO co-localizes with quinolinic acid, neurofibrillary tangles-tau and amyloid deposits in the hippocampus of AD. These results show that the kynurenine pathway is over-activated in AD mice. This is the first report demonstrating that TDO is highly expressed in the brains of AD mice and in AD patients, suggesting that TDO-mediated activation of the kynurenine pathway could be involved in neurofibrillary tangles formation and associated with senile plaque. Our study adds to the evidence that the kynurenine pathway may play important roles in the neurodegenerative processes of AD.

An exploratory study of the interplay between decreased concentration of tryptophan, accumulation of kynurenines, and inflammatory arthritis.

Tryptophan is an essential amino acid which influences a wide range of physiological processes, including mood, cognition, and immunity. In the autoimmune diseases, such as rheumatoid arthritis (RA), the induction of tryptophan catabolism may help to diminish exacerbated immune responses. In this study, using collagen-induced arthritis (CIA) in DBA/1 mice which is an animal model of RA, the endogenous activity of the kynurenine pathway in the immune system was monitored before and after onset of the disease. An increased rate of the initiation of tryptophan catabolism via the kynurenine pathway throughout CIA has been observed. However, decreased tryptophan concentration in the lymph nodes from pre-arthritic mice was not enough to prevent development of CIA. In contrast, resolution of inflammation coincided with the decreased concentration of tryptophan and accumulation of its catabolites: kynurenine, anthranilic acid, and 3-hydroxyanthranilic acid in lymph nodes but not in the spleen. In addition, the lack of the accumulation of kynurenine and its downstream metabolites in the pre-arthritic lymph nodes coincided with increased mRNA expression for genes involved in the catabolism of kynurenine (Kynureninase, kynurenine 3-monooxygenase, and 3-hydroxyanthranilate 3,4 dioxygenase). However, in the lymph nodes from mice with established CIA, mRNA expression for these genes was normalized. Hence, keeping in mind an exploratory character of the results, it can be postulated that an anti-inflammatory role of the kynurenine pathway reaches its full potential only when decreased concentration of tryptophan coincides with accumulation of kynurenines driven by metabolic regulation of gene expression on the kynurenine pathway.

Promoter hypermethylation of CIDEA, HAAO and RXFP3 associated with microsatellite instability in endometrial carcinomas.

OBJECTIVE: DNA promoter methylation is an epigenetic phenomenon for long-term gene silencing during tumorigenesis. The purpose of this study is to identify novel hypermethylated loci associated with clinicopathologic variables in endometrioid endometrial carcinomas. METHODS: To find hypermethylated promoter loci, we used differential methylation hybridization coupling with microarray and further validated by combined bisulfite restriction analysis and MassARRAY assay. Methylation levels of candidate loci were corrected with clinicopathologic factors of endometrial carcinomas. RESULTS: Increased promoter methylation of CIDE, HAAO and RXFP3 was detected in endometrial carcinomas compared with adjacent normal tissues, and was associated with decreased gene expression of all three genes. In a clinical cohort, promoter hypermethylation on CIDEA, HAAO and RXFP3 was detected in 85, 63 and 71% of endometrial carcinomas, respectively (n=118, P<0.001) compared with uninvolved normal endometrium. Methylation status of CIDEA, HAAO and RXFP3 had significant association with microsatellite instability in tumors (P<0.001). Furthermore, methylation levels of HAAO were further found to relate to disease-free survivals (P=0.034). CONCLUSIONS: Hypermethylation of CIDEA, HAAO and RXFP3 promoter regions appears to be a frequent event in endometrial carcinomas. Hypermethylation at these loci is strongly associated with microsatellite instability status. Moreover, HAAO methylation predicts disease-free survival in this cohort of patients with endometrioid endometrial cancer.

Identification of candidate epigenetic biomarkers for ovarian cancer detection.

Ovarian cancer ranks the most lethal among gynecologic neoplasms in women. To develop potential biomarkers for diagnosis, we have identified five novel genes (CYP39A1, GTF2A1, FOXD4L4, EBP, and HAAO) that are hypermethylated in ovarian tumors, compared with the non-malignant normal ovarian surface epithelia, using the quantitative methylation-specific polymerase chain reactions. Interestingly enough, multivariate Cox regression analysis has identified hypermethylation of CYP39A1 correlated with an increase rate of relapsing (P=0.032, hazard ratio >1). Concordant hypermethylation in at least three loci was observed in 50 out of 55 (91%) of ovarian tumors examined. The test sensitivity and specificity were assessed to be 96 and 67% for CYP39A1; 95 and 88% for GTF2A1; 93 and 67% for FOXD4L4; 81 and 67% for EBP; 89 and 82% for HAAO, respectively. Our data have identified, for the first time, GTF2A1 alone, or GTF2A1 plus HAAO are excellent candidate biomarkers for detecting this disease. Moreover, the known functions of these gene products further implicate dysregulated transcriptional control, cholesterol metabolism, or synthesis of quinolinic acids, may play important roles in attributing to ovarian neoplasm. Molecular therapies, by reversing the aberrant epigenomes using inhibitory agents or by abrogating the upstream signaling pathways that convey the epigenomic perturbations, may be developed into promising treatment regimens.

Crystal structure of bovine 3-hydroxyanthranilate 3,4-dioxygenase.

3-Hydroxyanthranilate 3,4-dioxygenase, the enzyme that catalyzes the conversion of 3-hydroxyanthranilate to quinolinic acid, has been extracted and purified from bovine kidney, crystallized and its structure determined at 2.5 A resolution. The enzyme, which crystallizes in the triclinic P1 space group, is a monomer, characterized by the so-called cupin fold. The monomer of the bovine enzyme mimics the dimer present in lower species, such as bacteria and yeast, since it is composed of two domains: one of them is equivalent to one monomer, whilst the second domain corresponds to only a portion of it. The active site consists of an iron ion coordinated by two histidine residues, one glutamate and an external ligand, which has been interpreted as a solvent molecule. It is contained in the N-terminal domain, whilst the function of the C-terminal domain is possibly structural. The catalytic mechanism very likely has been conserved through all species, since the positions of all residues considered relevant for the reaction are present from bacteria to humans.

Regulation of gene transcription by the histone H2A N-terminal domain.

Histone N-terminal domains play critical roles in regulating chromatin structure and gene transcription. Relatively little is known, however, about the role of the histone H2A N-terminal domain in transcription regulation. We have used DNA microarrays to characterize the changes in genome-wide expression caused by mutations in the N-terminal domain of histone H2A. Our results indicate that the N-terminal domain of histone H2A functions primarily to repress the transcription of a large subset of the Saccharomyces cerevisiae genome and that most of the H2A-repressed genes are also repressed by the histone H2B N-terminal domain. Using the histone H2A microarray data, we selected three reporter genes (BNA1, BNA2, and GCY1), which we subsequently used to map regions in the H2A N-terminal domain responsible for this transcriptional repression. These studies revealed that a small subdomain in the H2A N-terminal tail, comprised of residues 16 to 20, is required for the transcriptional repression of these reporter genes. Deletion of either the entire histone H2A N-terminal domain or just this small subdomain imparts sensitivity to UV irradiation. Finally, we show that two residues in this H2A subdomain, serine-17 and arginine-18, are specifically required for the transcriptional repression of the BNA2 reporter gene.

Crystal structure of 3-hydroxyanthranilic acid 3,4-dioxygenase from Saccharomyces cerevisiae: a special subgroup of the type III extradiol dioxygenases.

3-Hydroxyanthranilic acid 3,4-dioxygenase (3HAO) is a non-heme ferrous extradiol dioxygenase in the kynurenine pathway from tryptophan. It catalyzes the conversion of 3-hydroxyanthranilate (HAA) to quinolinic acid (QUIN), an endogenous neurotoxin, via the activation of N-methyl-D-aspartate (NMDA) receptors and the precursor of NAD(+) biosynthesis. The crystal structure of 3HAO from S. cerevisiae at 2.4 A resolution shows it to be a member of the functionally diverse cupin superfamily. The structure represents the first eukaryotic 3HAO to be resolved. The enzyme forms homodimers, with two nickel binding sites per molecule. One of the bound nickel atoms occupies the proposed ferrous-coordinated active site, which is located in a conserved double-strand beta-helix domain. Examination of the structure reveals the participation of a series of residues in catalysis different from other extradiol dioxygenases. Together with two iron-binding residues (His49 and Glu55), Asp120, Asn51, Glu111, and Arg114 form a hydrogen-bonding network; this hydrogen-bond network is key to the catalysis of 3HAO. Residues Arg101, Gln59, and the substrate-binding hydrophobic pocket are crucial for substrate specificity. Structure comparison with 3HAO from Ralstonia metallidurans reveals similarities at the active site and suggests the same catalytic mechanism in prokaryotic and eukaryotic 3HAO. Based on sequence comparison, we suggest that bicupin of human 3HAO is the first example of evolution from a monocupin dimer to bicupin monomer in the diverse cupin superfamilies. Based on the model of the substrate HAA at the active site of Y3HAO, we propose a mechanism of catalysis for 3HAO.