Evidence for the interaction of d-amino acid oxidase with pLG72 in a glial cell line

Sacchi, Silvia; Cappelletti, Pamela; Giovannardi, Stefano; Pollegioni, Loredano

doi:10.1016/j.mcn.2011.06.001

Cited by 53 publications

(111 citation statements)

References 26 publications

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“…The principle on which the proposed assay is based is that pLG72 binds to hDAAO, 5,7,8 alters hDAAO structure in a time-dependent manner, 7,22 and regulates hDAAO activity. This regulation has been reported as activation 5,23 by some researchers and inhibition by others.…”

Section: Plg72-directed Hdaao Activity Assay Htsmentioning

confidence: 99%

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

et al. 2015

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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 IC 20 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, 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.

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Section: Plg72-directed Hdaao Activity Assay Htsmentioning

confidence: 99%

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

et al. 2015

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“…Some findings indicated that LG72 binds to the D-serine degrading enzyme DAO (Chang et al 2013;Chumakov et al 2002;Sacchi et al 2008;Sacchi et al 2011). However, DAO and LG72 are localized in different subcellular compartments.…”

Section: Discussionmentioning

confidence: 99%

“…In contrast, several studies have shown a mitochondrial localization of LG72 (Kvajo et al 2008;Otte et al 2011;Sacchi et al 2011). In order to reconcile the disparate sub-cellular localization with a putative modulation of DAO activity by LG72, it has been suggested that the two proteins interact in the cytosol during the transport of DAO into peroxisomes (Sacchi et al 2011). Even if this were correct, we find it highly unlikely that modulation of DAO activity is the only function of LG72.…”

Section: Discussionmentioning

confidence: 99%

Identification of the Mitochondrial MSRB2 as a Binding Partner of LG72

et al. 2014

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Genetic studies have linked the evolutionary novel, anthropoid primate-specific gene locus G72/G30 in the etiology of schizophrenia and other psychiatric disorders.However, the function of the protein encoded by this locus, LG72, is currently controversially discussed. Some studies have suggested that LG72 binds to and regulates the activity of the peroxisomal enzyme D-amino-acid-oxidase, while others proposed an alternative role of this protein due to its mitochondrial location in vitro.Studies with transgenic mice expressing LG72 further suggested that high levels ofLG72 lead to an impairment of mitochondrial functions with a concomitant increase in reactive oxygen species (ROS) production. In the present study we now performed extensive interaction analyses and identified the mitochondrial methionine-Rsulfoxide reductase B2 (MSRB2) as a specific interaction partner of LG72. MSRB2 belongs to the MSR protein family and functions in mitochondrial oxidative stress defense. Based on our results we propose that LG72 is involved in the regulation of mitochondrial oxidative stress.

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“…All experiments were performed using a U87 human glioblastoma cell line stably expressing human DAAO [25]. Cellular amino acids were extracted as described previously [18]: 2.5 9 10 5 U87 cells were resuspended in 1 mL of ice-cold 5% trichloroacetic acid, sonicated and centrifuged for 45 min at 16 000 g. For the HPLC assay, the soluble fraction was extracted with water-saturated ether, neutralized and derivatized with o-phthaldialdehyde/N-acetyl-L-cysteine.…”

Section: Glycine/sarcosine Detection In Biological Samplesmentioning

confidence: 99%

“…The novel sensing assay shows a detection limit ≤ 0.5 lM (slightly lower than commercial assay kits) and was used to assay glycine and sarcosine in biological fluid, i.e. in a U87 glioblastoma cell line used to study the modulation of D-serine level by the catabolic enzyme D-amino acid oxidase [18,25]; D-serine and glycine are alternative ligands required, together with glutamate, to activate NMDARs [21,26]. The glycine concentration detected by the H244K GO-based assay was in good agreement with the value obtained by using the reference HPLC method (7.5 versus 6.7 lM, respectively) but in 1 s, compared with the 60 min required by chromatographic separation.…”

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confidence: 99%

Novel biosensors based on optimized glycine oxidase

et al. 2014

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Glycine is involved in several physiological functions, e.g. as a neurotransmitter in the central nervous system, and sarcosine has been identified as a differential metabolite greatly enhanced during prostate cancer progression and metastasis. Glycine oxidase from Bacillus subtilis (GO) was engineered with the final aim of producing specific analytical systems to detect these small achiral amino acids. Based on in silico analysis, site-saturation mutagenesis was independently performed at 11 positions: a total of 16 single-point GO variants were analyzed. Significantly improved kinetic parameters were observed on glycine for the A54R, H244K-N-Q-R, Y246W and M261R variants. The introduction of multiple mutations then identified the H244K/M261R variant showing a 5.4-fold increase in maximal activity on glycine. With sarcosine as substrate, a number of single-point variants showed increased maximal activity and/or affinity: the kinetic efficiency was increased 6-fold for the M49L variant. Two GO variants with a high substrate specificity ratio for glycine (versus sarcosine, i.e. H244K GO) or for sarcosine (versus glycine, i.e. M49L GO) combined with high substrate affinity were used to set up a simple fluorescence-based biosensor. This optical sensing assay represents a novel, inexpensive and fast tool to assay glycine or sarcosine concentrations in biological samples (detection limit ≤ 0.5 lM). IntroductionGlycine is the smallest and the only nonchiral natural a-amino acid used for protein biosynthesis. In addition to such a structural role, it possesses further physiological functions: it is an important biosynthetic precursor (i.e. it is used for de novo synthesis of porphyrins and purines) and it acts as a neurotransmitter in the central nervous system. Glycine is a coagonist, together with D-serine, of glutamate at glutamatergic N-methyl-Daspartate receptors (NMDARs) in the forebrain [1], thus serving both inhibitory and excitatory functions [2]. Due to this role, glycine is probably involved in the pathophysiology of schizophrenia and other neurological diseases. For example, a correlation exists between low levels of glycine and catatonia (a psychiatric disease), and the perturbation of the glycine deportation system is associated with ifosfamide encephalopathy and congenital nonketotic hyperglycinemia [2].Sarcosine, 2-(methylamino)acetic acid or N-methylglycine, is a natural, small, endogenous amino acid present in low concentrations in the blood and in urine. It has been proposed to play a role in the progression of prostate cancer, and assessing sarcosine concentrations in body fluids was suggested as a novel biomarker for prostate cancer [3]. With the final aim of developing a biosensor to assay glycine and sarcosine in biological fluids, we engineered glycine oxidase from Bacillus subtilis (GO) (EC 1.4.3.19) to improve its kinetic efficiency on these two compounds. GO is a homotetrameric flavoenzyme that contains one molecule of noncovalently bound FAD per 47 kDa protein monomer [4,5]. Purified recomb...

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Evidence for the interaction of d-amino acid oxidase with pLG72 in a glial cell line

Cited by 53 publications

References 26 publications

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

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

Identification of the Mitochondrial MSRB2 as a Binding Partner of LG72

Novel biosensors based on optimized glycine oxidase

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