Author Correction: A novel homozygous mutation in GAD1 gene described in a schizophrenic patient impairs activity and dimerization of GAD67 enzyme.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Overexpression of sialidase NEU3 increases the cellular radioresistance potential of U87MG glioblastoma cells.

The plasma membrane-associated sialidase NEU3 is known to play important roles in different physiological and pathophysiological processes such as proliferation, cellular differentiation and tumorigenesis. Up-regulation of NEU3 has been associated to several tumors and recently it was demonstrated that its down-modulation in glioblastoma cells promotes cell invasiveness. To date, no information concerning the possible role played by NEU3 in relation to tumor radioresistance is available. Here we show that overexpression of NEU3 in glioblastoma U87MG cells activates PI3K/Akt signaling pathway resulting in an increased radioresistance capacity and in an improved efficiency of double strand DNA-repair mechanisms after irradiation. Our results demonstrate for the first time that NEU3 contributes to the radioresistance features of U87MG cells, bringing to evidence a novel rand peculiar role of the enzyme in cancer biology.

A novel homozygous mutation in GAD1 gene described in a schizophrenic patient impairs activity and dimerization of GAD67 enzyme.

Recently, by whole exome sequencing of schizophrenia (SCZ) patients, we identified a subject that was homozygous for a novel missense substitution (c.391 A > G) in the glutamate acid decarboxylase 1 (GAD1) gene. GAD1 encodes for GAD67 enzyme, catalyzing the production of gamma-aminobutyric acid (GABA) from L-glutamic acid. Here, we studied the impact of this mutation on GAD67 activity, dimerization and subcellular localization. Biochemical assay revealed that c.391 A > G reduces GAD67 enzymatic activity by ~30%, probably due to the impaired homodimerization of homozygous mutants as highlighted by proximity ligation assays. The mutational screening of 120 genes of the "GABAergic system" in a cohort of 4,225 SCZ cases and 5,834 controls (dbGaP: phs000473.v1.p2), did not identify other cases that were homozygous for ultra-rare variants in GAD1, but highlighted an increased frequency of cases that were homozygous for rare variants in genes of the GABA system (SCZ: 0.14% vs. Controls: 0.00%; p-value = 0.0055). In conclusion, this study demonstrates the functional impact of c.391 A > G variant and its biological effect makes it a good candidate as risk variant for SCZ. This study also supports an involvement of ultra-rare variants in GABAergic genes in the etiopathogenesis of SCZ.

Exploring the Role of Enzalutamide in Combination with Radiation Therapy: An In Vitro Study.

BACKGROUND/AIM: Androgen receptor plays a key role in prostate cancer development and is a causative agent of its radio-resistance. The present study investigated the potential radio-sensitizing effect of enzalutamide, a second-generation anti-androgen, in human prostate cancer cells. MATERIALS AND METHODS: The radio-sensitizing effect of enzalutamide was assessed in the androgen-dependent LNCaP cells and the androgen-independent PC3 cells by clonogenic assay and γ-H2AX assay. RESULTS: Enzalutamide-treated LNCaP cells showed a significant decrease of cell survival at all radiation doses tested. An increased number of γ-H2AX-positive nuclei was observed, suggesting a possible impairment of the DNA repair machinery. Conversely, enzalutamide did not exhibit a significant radio-sensitizing effect on PC3 cells. CONCLUSION: The combination of enzalutamide with ionizing radiation significantly improves radio-sensitivity of hormone-dependent LNCaP cells. Translated in the clinical practice, our results may help to find additional strategies to improve effectiveness of radiotherapy.

Exosomes Secreted by HeLa Cells Shuttle on Their Surface the Plasma Membrane-Associated Sialidase NEU3.

Sialidases are glycohydrolases that remove terminal sialic acid residues from oligosaccharides, glycolipids, and glycoproteins. The plasma membrane-associated sialidase NEU3 is involved in the fine-tuning of sialic acid-containing glycans directly on the cell surface and plays relevant roles in important biological phenomena such as cell differentiation, molecular recognition, and cancer transformation. Extracellular vesicles are membranous structures with a diameter of 0.03-1 µm released by cells and can be detected in blood, urine, and culture media. Among extracellular vesicles, exosomes play roles in intercellular communication and maintenance of several physiological and pathological conditions, including cancer, and could represent a useful diagnostic tool for personalized nanomedicine approaches. Using inducible expression of the murine form of NEU3 in HeLa cells, a study of the association of the enzyme with exosomes released in the culture media has been performed. Briefly, NEU3 is associated with highly purified exosomes and localizes on the external leaflet of these nanovesicles, as demonstrated by enzyme activity measurements, Western blot analysis, and dot blot analysis using specific protein markers. On the basis of these results, it is plausible that NEU3 activity on exosome glycans enhances the dynamic biological behavior of these small extracellular vesicles by modifying the negative charge and steric hindrance of their glycocalyx. The presence of NEU3 on the exosomal surface could represent a useful marker for the detection of these nanovesicles and a tool for improving our understanding of the biology of these important extracellular carriers in physiological and pathological conditions.

Genomic and biochemical characterization of sialic acid acetylesterase (siae) in zebrafish.

Sialic acid acetylesterase (SIAE) removes acetyl moieties from the carbon 9 and 4 hydroxyl groups of sialic acid and recently a debate has been opened on its association to autoimmunity. Trying to get new insights on this intriguing enzyme we have studied siae in zebrafish (Danio rerio). In this teleost siae encodes for a polypeptide with a high degree of sequence identity to human and mouse counterparts. Zebrafish Siae behavior upon transient expression in COS7 cells is comparable to human enzyme concerning pH optimum of enzyme activity, subcellular localization and glycosylation. In addition, and as already observed in case of human SIAE, the glycosylated form of the enzyme from zebrafish is released into the culture media. During embryogenesis, in situ hybridization experiments demonstrate that siae transcript is always detectable during development, with a more specific expression in the central nervous system, in pronephric ducts and liver in the more advanced stages of the embryo development. In adult fish an increasing amount of siae mRNA is detectable in heart, eye, muscle, liver, brain, kidney and ovary. These results provide novel information about Siae and point out zebrafish as animal model to better understand the biological role(s) of this rather puzzling enzyme in vertebrates, regarding immune system function and the development of central nervous system.

Human sialic acid acetyl esterase: Towards a better understanding of a puzzling enzyme.

Sialic acid acetyl esterase (SIAE) removes acetyl moieties from the hydroxyl groups in position 9 and 4 of sialic acid. Recently, a dispute has been opened on its association to autoimmunity. In order to get new insights on human SIAE biology and to clarify its seemingly contradictory molecular properties, we combined in silico characterization, phylogenetic analysis and homology modeling with cellular studies in COS7 cells. Genomic and phylogenetic analysis revealed that in most tissues only the "long" isoform, originally referred to lysosomal sialic acid esterase, is detected. Using the homology modeling approach, we predicted a model of SIAE 3D structure, which fulfills the topological features of SGNH-hydrolase family. In addition, the model and site-directed mutagenesis experiments allowed the definition of the residues involved in catalysis. SIAE transient expression revealed that the protein is glycosylated and is active in vitro as an esterase with a pH optimum corresponding to 8.4-8.5. Moreover, glycosylation influences the biological activity of the enzyme and is essential for release of SIAE into the culture medium. According to these findings, co-localization experiments demonstrated the presence of SIAE in membranous structures corresponding to endoplasmic reticulum and Golgi complex. Thus, at least in COS7 cells, SIAE behaves as a typical secreted enzyme, subjected to glycosylation and located along the classical secretory route or in the extracellular space. In these environments, the enzyme could act on 9-O-acetylated sialic acid residues, contributing to the fine-tuning of the various functions played by this acidic sugar.

Sialidase NEU3 dynamically associates to different membrane domains specifically modifying their ganglioside pattern and triggering Akt phosphorylation.

Lipid rafts are known to regulate several membrane functions such as signaling, trafficking and cellular adhesion. The local enrichment in sphingolipids and cholesterol together with the low protein content allows their separation by density gradient flotation after extraction with non-ionic detergent at low temperature. These structures are also referred to as detergent resistant membranes (DRM). Among sphingolipids, gangliosides play important roles in different biological events, including signal transduction and tumorigenesis. Sialidase NEU3 shows high enzymatic specificity toward gangliosides. Moreover, the enzyme is present both at the cell surface and in endosomal structures and cofractionates with caveolin. Although changes in the expression level of NEU3 have been correlated to different tumors, little is known about the precise distribution of the protein and its ability in modifying the ganglioside composition of DRM and non-DRM, thus regulating intracellular events. By means of inducible expression cell system we found that i) newly synthesized NEU3 is initially associated to non-DRM; ii) at steady state the protein is equally distributed between the two membrane subcompartments, i.e., DRM and non-DRM; iii) NEU3 is degraded via the proteasomal pathway; iv) the enzyme specifically modifies the ganglioside composition of the membrane areas where it resides; and v) NEU3 triggers phosphorylation of Akt, even in absence of exogenously administered EGF. Taken together our data demonstrate that NEU3 regulates the DRM ganglioside content and it can be considered as a modulator of Akt phosphorylation, further supporting the role of this enzyme in cancer and tumorigenesis.