The D-amino acid oxidase-carbon nanotubes: evaluation of cytotoxicity and biocompatibility of a potential anticancer nanosystem

The ‘enzyme prodrug therapy’ represents a promising strategy to overcome limitations of current cancer treatments by the systemic administration of prodrugs, converted by a foreign enzyme into an active anticancer compound directly in tumor sites. One example is D-amino acid oxidase (DAAO), a dimeric favoenzyme able to catalyze the oxidative deamination of D-amino acids with production of hydrogen peroxide, a reactive oxygen species (ROS), able to favor cancer cells death. A DAAO variant containing fve aminoacidic substitutions (mDAAO) was demonstrated to possess a better therapeutic efcacy under low O2 concentration than wild-type DAAO (wtDAAO). Recently, aiming to design promising nanocarriers for DAAO, multi-walled carbon nanotubes (MWCNTs) were functionalized with polyethylene glycol (PEG) to reduce their tendency to aggregation and to improve their biocompatibility. Here, wtDAAO and mDAAO were adsorbed on PEGylated MWCNTs and their activity and cytotoxicity were tested. While PEG-MWCNTs-DAAOs have shown a higher activity than pristine MWCNTs-DAAO (independently on the DAAO variant used), PEG-MWCNTs-mDAAO showed a higher cytotoxicity than PEG-MWCNTs-wtDAAO at low O2 concentration. In order to evaluate the nanocarriers’ biocompatibility, PEG-MWCNTsDAAOs were incubated in human serum and the composition of protein corona was investigated via nLC-MS/MS, aiming to characterize both soft and hard coronas. The mDAAO variant has infuenced the bio-corona composition in both number of proteins and presence of opsonins and dysopsonins: notably, the soft corona of PEG-MWCNTs-mDAAO contained less proteins and was more enriched in proteins able to inhibit the immune response than PEG-MWCNTs-wtDAAO. Considering the obtained results, the PEGylated MWCNTs conjugated with the mDAAO variant seems a promising candidate for a selective antitumor oxidative therapy: under anoxic-like conditions, this novel drug delivery system showed a remarkable cytotoxic efect controlled by the substrate addition, against diferent tumor cell lines, and a bio-corona composition devoted to prolong its blood circulation time, thus improving the drug’s biodistribution

Biochemical and cellular studies of three human 3‐phosphoglycerate dehydrogenase variants responsible for pathological reduced L‐serine levels

In the brain, the non-essential amino acid L-serine is produced through the phosphorylated pathway (PP) starting from the glycolytic intermediate 3-phosphoglycerate: among the different roles played by this amino acid, it can be converted into D-serine and glycine, the two main co-agonists of NMDA receptors. In humans, the enzymes of the PP, namely phosphoglycerate dehydrogenase (hPHGDH, which catalyzes the first and rate-limiting step of this pathway), 3-phosphoserine aminotransferase, and 3-phosphoserine phosphatase are likely organized in the cytosol as a metabolic assembly (a "serinosome"). The hPHGDH deficiency is a pathological condition biochemically characterized by reduced levels of L-serine in plasma and cerebrospinal fluid and clinically identified by severe neurological impairment. Here, three single-point variants responsible for hPHGDH deficiency and Neu-Laxova syndrome have been studied. Their biochemical characterization shows that V261M, V425M, and V490M substitutions alter either the kinetic (both maximal activity and Km for 3-phosphoglycerate in the physiological direction) and the structural properties (secondary, tertiary, and quaternary structure, favoring aggregation) of hPHGDH. All the three variants have been successfully ectopically expressed in U251 cells, thus the pathological effect is not due to hindered expression level. At the cellular level, mistargeting and aggregation phenomena have been observed in cells transiently expressing the pathological protein variants, as well as a reduced L-serine cellular level. Previous studies demonstrated that the pharmacological supplementation of L-serine in hPHGDH deficiencies could ameliorate some of the related symptoms: our results now suggest the use of additional and alternative therapeutic approaches

Assays of D-Amino Acid Oxidase Activity

D-amino acid oxidase (DAAO) is a well-known flavoenzyme that catalyzes the oxidative FAD-dependent deamination of D-amino acids. As a result of the absolute stereoselectivity and broad substrate specificity, microbial DAAOs have been employed as industrial biocatalysts in the production of semi-synthetic cephalosporins and enantiomerically pure amino acids. Moreover, in mammals, DAAO is present in specific brain areas and degrades D-serine, an endogenous coagonist of the N-methyl-D-aspartate receptors (NMDARs). Dysregulation of D-serine metabolism due to an altered DAAO functionality is related to pathological NMDARs dysfunctions such as in amyotrophic lateral sclerosis and schizophrenia. In this protocol paper, we describe a variety of direct assays based on the determination of molecular oxygen consumption, reduction of alternative electron acceptors, or α-keto acid production, of coupled assays to detect the hydrogen peroxide or the ammonium production, and an indirect assay of the α-keto acid production based on a chemical derivatization. These analytical assays allow the determination of DAAO activity both on recombinant enzyme preparations, in cells, and in tissue samples

D-amino acid oxidase-nanoparticle system: a potential novel approach for cancer enzymatic therapy.

Aim: The authors propose a new magnetic nanoparticle–enzyme system for cancer therapy capable of targeting the enzyme and consequently decreasing the adverse effects, meanwhile improving the patient's life quality. Materials & methods: The authors have functionalized Fe3O4 nanoparticles with 3-amino-propyltriethoxysilane (APTES) and conjugated it to yeast D-amino acid oxidase (DAAO) by coupling this with glutaraldehyde. Results & conclusion: The authors have tested the Fe3O4-APTES–DAAO system on three tumor cell lines. Exposed cells show, at the electron microscope level, nanoparticles on the surface of the plasma membrane and inside endocytic vesicles. Fe3O4-APTES–DAAO caused a substantial decrease of cell viability greatly augmented when D-alanine, a DAAO substrate, was added. Fe3O4-APTES–DAAO was demonstrated to be more effective than free DAAO, confirming the validity of the system in cancer therapy. Original submitted 27 March 2012; Revised submitted 20 September 2012; Published online 5 February 201

Human d-amino acid oxidase: The inactive G183R variant

In the brain, the enzyme d-amino acid oxidase (DAAO) catalyzes the oxidative deamination of d-serine, a main positive modulator of the N-methyl-d-aspartate subtype of glutamate receptors (NMDAR). Dysregulation in d-serine signaling is implicated in the NMDAR dysfunctions observed in various brain diseases, such as amyotrophic lateral sclerosis, Alzheimer's disease, schizophrenia. A strain of ddY mice lacking DAAO activity due to the G181R substitution (DAAOG181Rmice) and exhibiting increased d-serine concentration as compared to wild-type mice shows altered pain response, improved adaptative learning and cognitive functions, and larger hippocampal long-term potentiation. In past years, this mice line has been used to shed light on physiological and pathological brain functions related to NMDAR. Here, we decided to introduce the corresponding substitution in human DAAO (hDAAO). The recombinant G183R hDAAO is produced as an inactive apoprotein: the substitution alters the protein conformation that negatively affects the ability to bind the flavin cofactor in the orientation required for hydride-transfer during catalysis. At the cellular level, the overexpressed G183R hDAAO is not fully targeted to peroxisomes, forms protein aggregates showing a strong colocalization with ubiquitin, and significantly (7-fold) increases both the d-serine cellular concentration and the D/(D+L)-serine ratio. Taken together, our investigation warrants caution in using DAAOG181Rmice: the abolition of enzymatic activity is coupled to DAAO aggregation, a central process in different pathological conditions. The effect due to G181R substitution in DAAO could be misleading: the effects due to impairment of d-serine degradation overlap with those related to aggregates accumulation

Extrasynaptic NMDARs activation by co-agonist glycine controls the occurrence of bursts in nigral dopamine neurons

peer reviewe

Relevance of weak flavin binding in human d-amino acid oxidase

In the brain, the human flavoprotein d-amino acid oxidase (hDAAO) is involved in the degradation of the gliotransmitter d-serine, an important modulator of NMDA-receptor-mediated neurotransmission; an increase in hDAAO activity (that yields a decrease in d-serine concentration) was recently proposed to be among the molecular mechanisms leading to the onset of schizophrenia susceptibility. This human flavoenzyme is a stable homodimer (even in the apoprotein form) that distinguishes from known d-amino acid oxidases because it shows the weakest interaction with the flavin cofactor in the free form. Instead, cofactor binding is significantly tighter in the presence of an active site ligand. In order to understand how hDAAO activity is modulated, we investigated the FAD binding process to the apoprotein moiety and compared the folding and stability properties of the holoenzyme and the apoprotein forms. The apoprotein of hDAAO can be distinguished from the holoenzyme form by the more “open” tertiary structure, higher protein fluorescence, larger exposure of hydrophobic surfaces, and higher sensitivity to proteolysis. Interestingly, the FAD binding only slightly increases the stability of hDAAO to denaturation by urea or temperature. Taken together, these results indicate that the weak cofactor binding is not related to protein (de)stabilization or oligomerization (as instead observed for the homologous enzyme from yeast) but rather should represent a means of modulating the activity of hDAAO. We propose that the absence in vivo of an active site ligand/substrate weakens the cofactor binding, yielding the inactive apoprotein form and thus avoiding excessive d-serine degradation