Expression in Escherichia coli and in vitro refolding of the human protein pLG72

Molla, Gianluca; Bernasconi, Mariagrazia; Sacchi, Silvia; Pilone, Mirella S.; Pollegioni, Loredano

doi:10.1016/j.pep.2005.08.003

Cited by 37 publications

(52 citation statements)

References 9 publications

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“…The final enzyme preparation was stored in 20 mM Tris-HCl buffer, pH 8.0, 100 mM NaCl, 5% glycerol, 5 mM 2-mercaptoethanol, and 40 M FAD. The G72 gene was also expressed in E. coli cells; the protein was expressed in the insoluble fraction and then solubilized and purified according to the procedure reported in Molla et al (18); see Fig. 1A.…”

Section: Methodsmentioning

confidence: 99%

“…To provide biochemical evidence supporting the involvement of the identified genes in this pathologic process, we recently reported on the overexpression of pLG72 and human DAAO (hDAAO) in Escherichia coli and biochemically characterized hDAAO (18,19). In the present study we have investigated the interaction of pLG72 with hDAAO in vitro and the effect of pLG72 binding on the functional and structural properties of the flavoenzyme as well as their effect on the cellular concentration of D-serine.…”

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confidence: 96%

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pLG72 Modulates Intracellular D-Serine Levels through Its Interaction with D-Amino Acid Oxidase

Sacchi

Bernasconi

Martineau

et al. 2008

Journal of Biological Chemistry

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Human genes coding for pLG72 and D-amino acid oxidase have recently been linked to the onset of schizophrenia. pLG72 was proposed as an activator of the human FAD-containing flavoprotein D-amino acid oxidase (hDAAO). In the brain this oxidizes D-serine, a potent activator of N-methyl-D-aspartate receptor. We have investigated the mechanistic regulation of hDAAO by pLG72. Immunohistochemical analyses revealed that hDAAO and pLG72 are both expressed in astrocytes of the human cortex, where they most likely interact, considering their partial overlapping subcellular distribution and their coimmunoprecipitation. We demonstrated that the specific in vitro interaction of the two proteins yields a complex composed of 2 hDAAO homodimers and 2 pLG72 molecules. Binding of pLG72 did not affect the kinetic properties and FAD binding ability of hDAAO; instead, a time-dependent loss of hDAAO activity in the presence of an excess of pLG72 was found. The binding affects the tertiary structure of hDAAO, altering the amount of the active form. We finally demonstrated that overexpression of hDAAO in glioblastoma cells decreases the levels of D-serine, an effect that is null when pLG72 is coexpressed. These data indicate that pLG72 acts as a negative effector of hDAAO. Therefore, a decrease in the synaptic concentration of D-serine as the result of an anomalous increase in hDAAO activity related to hypoexpression of pLG72 may represent a molecular mechanism by which hDAAO and pLG72 are involved in schizophrenia susceptibility.Schizophrenia is one of the most widely spread psychiatric disorders; it is a complex disease or, more likely, a group of related illnesses to which an individual has a strong genetic predisposition (1). Among the identified schizophrenia susceptibility genes (2), the gene G72 encodes for several splicing isoforms; pLG72 represents the longest open reading frame (153 amino acids), which is mainly expressed in brain (3). G72 is present only in primates: there are no homologues of this gene in databases nor has sequence analysis of the putative open reading frame revealed any likely function (2, 3). Yeast twohybrid experiments using pLG72 as bait identified D-amino acid oxidase (EC 1.4.3.3, DAAO 4 ) on 12q24 as a putative interacting partner, and preliminary functional measurements showed that pLG72 should function as an in vitro activator of pig kidney DAAO (pkDAAO) (3). DAAO is a FAD-containing flavoenzyme that catalyzes the oxidative deamination of D-amino acids to the corresponding ␣-keto acids, hydrogen peroxide and ammonia (4, 5).Based on current findings we can hypothesize that in brain, the physiological role of DAAO is to modulate the levels of D-serine, an important glial-derived messenger that acts as the endogenous allosteric modulator of the glutamatergic NMDA receptor subtype (6 -8). D-and L-serine can be reversibly isomerized in astrocytic glia, which unsheathes synapses, by serine racemase. Compelling evidence has indicated that glutamate neurotransmission hypofunction is associated with symptoms...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 96%

pLG72 Modulates Intracellular D-Serine Levels through Its Interaction with D-Amino Acid Oxidase

Sacchi

Bernasconi

Martineau

et al. 2008

Journal of Biological Chemistry

Self Cite

139

277

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“…7,8 The EGFP-hDAAO protein form (see later) was also expressed in E. coli cells by using the pET20 plasmid: the recombinant enzyme was purified with a 35% yield and >85% purity as reported in. 7 The final hDAAO preparation was in 20 mM Tris-HCl buffer, pH 8.0, 100 mM NaCl, 5% glycerol, 5 mM 2-mercaptoethanol, and 40 lM FAD and diluted in plain buffer without FAD before use.…”

Section: Enzymes and Activity Assaymentioning

confidence: 99%

“…6 We recently demonstrated that in vitro hDAAO specifically interacts with pLG72, yielding a % 200-kDa complex constituted by 2 hDAAO homodimers and 2 pLG72 monomers. [7][8][9] This interaction results in a time-dependent loss of hDAAO activity which is mainly due to alteration of the tertiary structure of hDAAO. Furthermore, we confirmed in vivo the hDAAO-pLG72 interaction and demonstrated that the cellular concentration of D-serine decreases in U87 glioblastoma cells transfected with a plasmid encoding for hDAAO but is not modified in those simultaneously transfected with cDNAs encoding both pLG72 and hDAAO.…”

Section: Introductionmentioning

confidence: 99%

Effect of ligand binding on human D‐amino acid oxidase: Implications for the development of new drugs for schizophrenia treatment

et al. 2010

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In human brain the flavoprotein D-amino acid oxidase (hDAAO) is responsible for the degradation of the neuromodulator D-serine, an important effector of NMDA-receptor mediated neurotransmission. Experimental evidence supports the concept that D-serine concentration increase by hDAAO inhibition may represent a valuable therapeutic approach to improve the symptoms in schizophrenia patients. This study investigated the effects on hDAAO conformation and stability of the substrate D-serine (or of the pseudo-substrate trifluoro-D-alanine), the FAD cofactor, and two inhibitors (benzoate, a classical substrate-competitive inhibitor and the drug chlorpromazine (CPZ), which competes with the cofactor). We demonstrated that all these compounds do not alter the interaction of hDAAO with its physiological partner pLG72. The ligands used affect the tertiary structure of hDAAO differently: benzoate or trifluoro-D-alanine binding increases the amount of the holoenzyme form in solution and stabilizes the flavoprotein, while CPZ binding favors a protein conformation resembling that of the apoprotein, which is more sensitive to degradation. Interestingly, the apoprotein form of hDAAO binds the substrate D-serine: this interaction increases FAD binding thus increasing the amount of active holoenzyme in solution. Benzoate and CPZ similarly modify the short-term cellular D-serine concentration but affect the cellular concentration of hDAAO differently. In conclusion, the different alteration of hDAAO conformation and stability by the ligands used represents a further parameter to take into consideration during the development of new drugs to cope schizophrenia.

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“…3 G72 is a novel, primate-specific gene encoding for a protein with no recognizable motifs and a high alpha-helix content suggestive of a membrane localization. 4 Notably, G72 represents a rare case of a primate-specific gene with a rapidly evolving protein structure. 3,5 The gene is located within a genomic region enriched in repeat elements, and a part of its coding sequence and 5 0 flanking region have been derived from long terminal repeat elements (http://genome.ucsc.edu).…”

Section: Introductionmentioning

confidence: 99%

Evidence implicating the candidate schizophrenia/bipolar disorder susceptibility gene G72 in mitochondrial function

et al. 2007

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G72 is a strong candidate susceptibility gene for schizophrenia and bipolar disorder, whose function remains enigmatic. Here we show that one splicing isoform of the gene (LG72 ) encodes for a mitochondrial protein. We also provide convergent lines of evidence that increase of endogenous or exogenous G72 levels promotes robust mitochondrial fragmentation in mammalian cell lines and primary neurons, which proceeds in a manner that does not depend on induction of apoptosis or alteration in mitochondrial transmembrane potential. Finally, we show that increase in G72 levels in immature primary neurons is accompanied by a marked increase in dendritic arborization. By contrast, we failed to confirm the originally proposed functional interaction between G72 and D-amino acid oxidase (DAO) in two tested cell lines. Our results suggest an alternative role for G72 in modulating mitochondrial function.

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Expression in Escherichia coli and in vitro refolding of the human protein pLG72

Cited by 37 publications

References 9 publications

pLG72 Modulates Intracellular D-Serine Levels through Its Interaction with D-Amino Acid Oxidase

pLG72 Modulates Intracellular D-Serine Levels through Its Interaction with D-Amino Acid Oxidase

Effect of ligand binding on human D‐amino acid oxidase: Implications for the development of new drugs for schizophrenia treatment

Evidence implicating the candidate schizophrenia/bipolar disorder susceptibility gene G72 in mitochondrial function

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