G72 Protein Research Articles

Plasma G72 protein in schizophrenia: A comparative analysis of drug-naive schizophrenia patients, patients in acute exacerbation and healthy controls

ObjectivesSchizophrenia is a complex psychiatric disorder with an unclear etiopathogenesis. This study investigates the plasma G72 protein levels in drug-naive schizophrenia patients (DNS), those in acute psychotic episodes (AES), and healthy controls (HC). It also examines the correlation between the plasma G72 protein levels and Positive and Negative Syndrome Scale (PANSS) scores. MethodsThe study included 138 schizophrenia patients (84 DNS, 54 AES) and 83 HCs. Plasma G72 protein levels were measured by ELISA. Statistical analyses, including log-transformation and correlation analysis, were conducted. ResultsSchizophrenia patients had significantly lower plasma G72 levels than HCs (4.39 ± 5.38 vs. 8.06 ± 10.27 ng/mL, p < 0.001), while DNS and AES groups did not differ significantly. Log-transformed data confirmed these differences. Negative correlation was found between plasma G72 levels and age (r = −0.258, p = 0.02), PANSS-G (r = −0.249, p = 0.004), and total PANSS scores (r = −0.226, p = 0.008). ROC analysis showed poor discrimination between schizophrenia patients and controls (AUC: 0.587, p = 0.031). ConclusionsThis study's novel findings reveal that plasma G72 protein levels are significantly lower in schizophrenia patients and inversely correlated with age and symptom severity. However, the poor diagnostic accuracy observed in the ROC analysis suggests that G72 may not be a reliable biomarker for schizophrenia at this stage. These results underscore the need for further research to explore the potential clinical implications of these findings.

An Ensemble Approach to Predict Schizophrenia Using Protein Data in the N-methyl-D-Aspartate Receptor (NMDAR) and Tryptophan Catabolic Pathways.

In the wake of recent advances in artificial intelligence research, precision psychiatry using machine learning techniques represents a new paradigm. The D-amino acid oxidase (DAO) protein and its interaction partner, the D-amino acid oxidase activator (DAOA, also known as G72) protein, have been implicated as two key proteins in the N-methyl-D-aspartate receptor (NMDAR) pathway for schizophrenia. Another potential biomarker in regard to the etiology of schizophrenia is melatonin in the tryptophan catabolic pathway. To develop an ensemble boosting framework with random undersampling for determining disease status of schizophrenia, we established a prediction approach resulting from the analysis of genomic and demographic variables such as DAO levels, G72 levels, melatonin levels, age, and gender of 355 schizophrenia patients and 86 unrelated healthy individuals in the Taiwanese population. We compared our ensemble boosting framework with other state-of-the-art algorithms such as support vector machine, multilayer feedforward neural networks, logistic regression, random forests, naive Bayes, and C4.5 decision tree. The analysis revealed that the ensemble boosting model with random undersampling [area under the receiver operating characteristic curve (AUC) = 0.9242 ± 0.0652; sensitivity = 0.8580 ± 0.0770; specificity = 0.8594 ± 0.0760] performed maximally among predictive models to infer the complicated relationship between schizophrenia disease status and biomarkers. In addition, we identified a causal link between DAO and G72 protein levels in influencing schizophrenia disease status. The study indicates that the ensemble boosting framework with random undersampling may provide a suitable method to establish a tool for distinguishing schizophrenia patients from healthy controls using molecules in the NMDAR and tryptophan catabolic pathways.

Combination of G72 Genetic Variation and G72 Protein Level to Detect Schizophrenia: Machine Learning Approaches.

The D-amino acid oxidase activator (DAOA, also known as G72) gene is a strong schizophrenia susceptibility gene. Higher G72 protein levels have been implicated in patients with schizophrenia. The current study aimed to differentiate patients with schizophrenia from healthy individuals using G72 single nucleotide polymorphisms (SNPs) and G72 protein levels by leveraging computational artificial intelligence and machine learning tools. A total of 149 subjects with 89 patients with schizophrenia and 60 healthy controls were recruited. Two G72 genotypes (including rs1421292 and rs2391191) and G72 protein levels were measured with the peripheral blood. We utilized three machine learning algorithms (including logistic regression, naive Bayes, and C4.5 decision tree) to build the optimal predictive model for distinguishing schizophrenia patients from healthy controls. The naive Bayes model using two factors, including G72 rs1421292 and G72 protein, appeared to be the best model for disease susceptibility (sensitivity = 0.7969, specificity = 0.9372, area under the receiver operating characteristic curve (AUC) = 0.9356). However, a model integrating G72 rs1421292 only slightly increased the discriminative power than a model with G72 protein alone (sensitivity = 0.7941, specificity = 0.9503, AUC = 0.9324). Among the three models with G72 protein alone, the naive Bayes with G72 protein alone had the best specificity (0.9503), while logistic regression with G72 protein alone was the most sensitive (0.8765). The findings remained similar after adjusting for age and gender. This study suggests that G72 protein alone, without incorporating the two G72 SNPs, may have been suitable enough to identify schizophrenia patients. We also recommend applying both naive Bayes and logistic regression models for the best specificity and sensitivity, respectively. Larger-scale studies are warranted to confirm the findings.

Plasma and cerebrospinal fluid G72 protein levels in schizophrenia and major depressive disorder

G72 is a modulator of D-amino acid oxidase, the enzyme that degrades D-serine, an amino acid that plays a critical role in glutamate neurotransmission, and has been implicated in psychiatric disorders. The aim of this study was to examine whether plasma or cerebrospinal fluid (CSF) G72 protein levels were altered in either schizophrenia or major depressive disorder (MDD) and whether any correlation between G72 levels and disease severity existed. Initially, 27 schizophrenic patients, 26 MDD patients, and 27 healthy controls matched for age, sex, and ethnicity were enrolled. Compared to those of controls, plasma or CSF G72 levels were not significantly different in patients with schizophrenia or MDD. Although we found a significant positive correlation between plasma G72 levels and a positive symptoms score on the positive and negative syndrome scale (PANSS), this was not replicated in the second study (40 schizophrenic patients). CSF G72 levels showed no significant correlation with PANSS scores. In MDD, neither plasma nor CSF G72 levels correlated significantly with depression severity. Since severity of our patients were relatively mild, further investigations in a large number of subjects including drug-free patients, younger patients, and more severely affected patients are warranted.

Increased serum G72 protein levels in patients with schizophrenia: a potential candidate biomarker.

The product of the G72 gene is an activator of d-amino acid oxidase and has been suggested to play a role in the pathogenesis of schizophrenia. Increased G72 protein levels may be associated with disturbed glutamatergic transmission and increased reactive oxygen species. Only one pilot study by Lin et al. has investigated the potential role of serum G72 protein levels as a biomarker for schizophrenia. In this study, we aimed to compare serum G72 protein levels between patients with schizophrenia and healthy controls, and to retest the results of the previous pilot study. Materials and methods In total, 107 patients with a diagnosis of schizophrenia according to the inclusion and exclusion criteria and 60 age-sex-matched healthy controls were included in the study. The groups were compared regarding serum G72 protein levels. The mean serum G72 protein values were 495.90±152.03 pg/ml in the schizophrenia group and 346.10±102.08 pg/ml in the healthy control group. The mean serum G72 protein level was significantly increased in the schizophrenia group compared with the healthy control group (t=-3.89, p<0.001). A receiver operating characteristics analysis was performed to compare the schizophrenia and healthy control groups. It was determined that the cut-off value was 141.51 pg/ml with a sensitivity of 0.991 and a specificity of 0.821. We suggest that serum G72 protein levels may represent a candidate biomarker for schizophrenia and have confirmed the results of the previous preliminary study. Additional studies with larger sample sizes and the inclusion of first episode schizophrenia patients are required to clarify the reliability and validity of serum G72 protein levels as a biomarker for schizophrenia.

PM539. Evaluation of plasma and cerebrospinal fluid G72 protein levels and their correlations with psychiatric symptoms in schizophrenia and major depression

PM539. Evaluation of plasma and cerebrospinal fluid G72 protein levels and their correlations with psychiatric symptoms in schizophrenia and major depression

High-Throughput Screening Strategy Identifies Allosteric, Covalent Human D-Amino Acid Oxidase Inhibitor

Genome-wide association studies have linked polymorphisms in the gene G72 to schizophrenia risk in several human populations. Although controversial, biochemical experiments have suggested that the mechanistic link of G72 to schizophrenia is due to the G72 protein product, pLG72, exerting a regulatory effect on human D-amino acid oxidase (hDAAO) activity. In an effort to identify hDAAO inhibitors of novel mechanism of action, we designed a pLG72-directed hDAAO activity assay suitable for high-throughput screening (HTS). During assay development, we confirmed that pLG72 was an inhibitor of hDAAO. Thus, our assay employed an IC20 pLG72 concentration that was high enough to allow dynamic pLG72-hDAAO complexes to form but with sufficient remaining hDAAO activity to measure during an HTS. After conducting an approximately 150,000-compound HTS, we further characterized a class of compound hits that were less potent hDAAO inhibitors when pLG72 was present. Focusing primarily on compound 2 [2-(2,5-dimethylphenyl)-6-fluorobenzo[d]isothiazol-3(2H)-on], we demonstrated that these compounds inhibited hDAAO via an allosteric, covalent mechanism. Although there is significant interest in the therapeutic potential of compound 2 and its analogues, their sensitivity to reducing agents and their capacity to bind cysteines covalently would need to be addressed during therapeutic drug development.

Lipidomics reveals dysfunctional glycosynapses in schizophrenia and the G72/G30 transgenic mouse

BackgroundAbnormal structural/functional connectivity has been proposed to underlie the pathophysiology of schizophrenia. However, the biochemical basis of abnormal connectivity remains undefined. MethodsWe undertook a shotgun lipidomic analysis of over 700 lipids across 26 lipid subclasses in the frontal cortex of schizophrenia subjects and hippocampus of G72/G30 transgenic mice. ResultsWe demonstrate that glycosphingolipids and choline plasmalogens, structural lipid pools in myelin, are significantly elevated in the frontal cortex obtained from patients suffering from schizophrenia and the hippocampus of G72/G30 transgenic mice. ConclusionsOur data suggest that structural lipid alterations in oligodendrocyte glycosynapses are responsible for dysconnectivity in schizophrenia and that increased expression of G72 protein may play a role in the development of abnormal glycosynapses.

Distinctively higher plasma G72 protein levels in patients with schizophrenia than in healthy individuals

Distinctively higher plasma G72 protein levels in patients with schizophrenia than in healthy individuals

Psychosis and relapse in bipolar disorder are related to GRM3, DAOA, and GRIN2B genotype

Dysfunction in glutamate signalling is thought to play a role in the pathophysiology of bipolar disorder (BD). There is evidence of associations between single nucleotide polymorphisms (SNPs) in GRM3, GRIN2B, and DAOA genes and the diagnosis of BD. In this pilot study, we investigated the frequency of SNP variants in these 3 genes within South African population groups, and assessed interactions between genes and phenotypes of BD disease severity. Multiplex SNaPshotTM PCR was used to genotype 191 case and 188 control samples. Cases comprised of 191 individuals in a South African cohort of mixed ancestry and Caucasians, with BD Type 1. Phenotypes of BD disease severity were: age of onset, number of illness episodes, number of hospitalisations for depression or mania and history of psychotic symptoms. There were no significant difference in SNP allele frequencies between cases and controls. In the case-only analysis, the GRM3 rs6465084 heterozygote was associated with a 4-fold increased risk of lifetime history of psychotic symptoms, and the specific variants within the gene pair, DAOA and GRIN2B, had a significant interaction with the number of hospitalisations for mania, with lowest admission rates associated with both pairs of ancestral alleles. In BD, variations in glutamatergic genes may influence phenotypes related to the severity of illness. Speculatively, newly derived genes associated with various evolutionary advantages, may also increase the risk for more severe BD. These preliminary findings deserve validation in a larger cohort.

Investigation of G72 (DAOA) expression in the human brain.

BackgroundPolymorphisms at the G72/G30 locus on chromosome 13q have been associated with schizophrenia or bipolar disorder in more than ten independent studies. Even though the genetic findings are very robust, the physiological role of the predicted G72 protein has thus far not been resolved. Initial reports suggested G72 as an activator of D-amino acid oxidase (DAO), supporting the glutamate dysfunction hypothesis of schizophrenia. However, these findings have subsequently not been reproduced and reports of endogenous human G72 mRNA and protein expression are extremely limited. In order to better understand the function of this putative schizophrenia susceptibility gene, we attempted to demonstrate G72 mRNA and protein expression in relevant human brain regions.MethodsThe expression of G72 mRNA was studied by northern blotting and semi-quantitative SYBR-Green and Taqman RT-PCR. Protein expression in human tissue lysates was investigated by western blotting using two custom-made specific anti-G72 peptide antibodies. An in-depth in silico analysis of the G72/G30 locus was performed in order to try and identify motifs or regulatory elements that provide insight to G72 mRNA expression and transcript stability.ResultsDespite using highly sensitive techniques, we failed to identify significant levels of G72 mRNA in a variety of human tissues (e.g. adult brain, amygdala, caudate nucleus, fetal brain, spinal cord and testis) human cell lines or schizophrenia/control post mortem BA10 samples. Furthermore, using western blotting in combination with sensitive detection methods, we were also unable to detect G72 protein in a number of human brain regions (including cerebellum and amygdala), spinal cord or testis. A detailed in silico analysis provides several lines of evidence that support the apparent low or absent expression of G72.ConclusionOur results suggest that native G72 protein is not normally present in the tissues that we analysed in this study. We also conclude that the lack of demonstrable G72 expression in relevant brain regions does not support a role for G72 in modulation of DAO activity and the pathology of schizophrenia via a DAO-mediated mechanism. In silico analysis suggests that G72 is not robustly expressed and that the transcript is potentially labile. Further studies are required to understand the significance of the G72/30 locus to schizophrenia.

Human D‐amino acid oxidase: an update and review

The flavoprotein D-amino acid oxidase (DAO) degrades the gliotransmitter D-Ser, a potent activator of N-methyl-D-aspartate-type glutamate receptors. A body of evidence suggests that DAO, together with its activator, G72 protein, may play a key role in the pathophysiology of schizophrenia. It has also been suggested that 3,4-dihydroxy-D-phenylalanine (D-DOPA), the stereoisomer of 3,4-dihydroxy-L-phenylalanine (L-DOPA), is oxidized by DAO and converted to dopamine via an alternative biosynthetic pathway. We determined the crystal structures of human DAO in complex with the reaction products of two clinically important substrates, D-Ser and D-DOPA. Kinetic data show that the maximum velocity is much greater for D-DOPA than that for D-Ser, which strongly supports the proposed alternative pathway for dopamine biosynthesis in the treatment of Parkinson's disease. In addition, biochemical characterization of human DAO indicates that it binds FAD more weakly than does porcine D-amino acid oxidase (pDAO) and exists as a stable homodimer, even in the apoprotein form. Determination of the structures of human DAO in various states reveals that, in contrast to pDAO, the hydrophobic-Val-Ala-Ala-Gly-Leu (VAAGL) stretch (residues 47-51, structurally ambivalent peptide) located at the si-face of the flavin ring assumes a uniquely stable conformation, which provides a structural basis for the unique kinetic features of human DAO.

Crystal structure of human D‐amino acid oxidase: Context‐dependent variability of the backbone conformation of the VAAGL hydrophobic stretch located at the si‐face of the flavin ring

In the brain, the extensively studied FAD-dependent enzyme D-amino acid oxidase (DAO) degrades the gliotransmitter D-serine, a potent activator of N-methyl-D-aspartate type glutamate receptors, and evidence suggests that DAO, together with its activator G72 protein, may play a key role in the pathophysiology of schizophrenia. Indeed, its potential clinical importance highlights the need for structural and functional analyses of human DAO. We recently succeeded in purifying human DAO, and found that it weakly binds FAD and shows a significant slower rate of flavin reduction compared with porcine DAO. However, the molecular basis for the different kinetic features remains unclear because the active site of human DAO was considered to be virtually identical to that of porcine DAO, as would be expected from the 85% sequence identity. To address this issue, we determined the crystal structure of human DAO in complex with a competitive inhibitor benzoate, at a resolution of 2.5 Angstrom. The overall dimeric structure of human DAO is similar to porcine DAO, and the catalytic residues are fully conserved at the re-face of the flavin ring. However, at the si-face of the flavin ring, despite the strict sequence identity, a hydrophobic stretch (residues 47-51, VAAGL) exists in a significantly different conformation compared with both of the independently determined porcine DAO-benzoate structures. This suggests that a context-dependent conformational variability of the hydrophobic stretch accounts for the low affinity for FAD as well as the slower rate of flavin reduction, thus highlighting the unique features of the human enzyme.

Genetic and physiological data implicating the new human gene G72 and the gene for D-amino acid oxidase in schizophrenia.

A map of 191 single-nucleotide polymorphism (SNPs) was built across a 5-Mb segment from chromosome 13q34 that has been genetically linked to schizophrenia. DNA from 213 schizophrenic patients and 241 normal individuals from Canada were genotyped with this marker set. Two 1,400- and 65-kb regions contained markers associated with the disease. Two markers from the 65-kb region were also found to be associated to schizophrenia in a Russian sample. Two overlapping genes G72 and G30 transcribed in brain were experimentally annotated in this 65-kb region. Transfection experiments point to the existence of a 153-aa protein coded by the G72 gene. This protein is rapidly evolving in primates, is localized to endoplasmic reticulum/Golgi in transfected cells, is able to form multimers and specifically binds to carbohydrates. Yeast two-hybrid experiments with the G72 protein identified the enzyme d-amino acid oxidase (DAAO) as an interacting partner. DAAO is expressed in human brain where it oxidizes d-serine, a potent activator of N-methyl-D-aspartate type glutamate receptor. The interaction between G72 and DAAO was confirmed in vitro and resulted in activation of DAAO. Four SNP markers from DAAO were found to be associated with schizophrenia in the Canadian samples. Logistic regression revealed genetic interaction between associated SNPs in vicinity of two genes. The association of both DAAO and a new gene G72 from 13q34 with schizophrenia together with activation of DAAO activity by a G72 protein product points to the involvement of this N-methyl-d-aspartate receptor regulation pathway in schizophrenia.