Enzymatic activity of glycosyltransferase GLT8D1 promotes human glioblastoma cell migration.

peer reviewedGlioblastoma (GBM) is the most aggressive primary brain tumor characterized by infiltrative growth of malignant glioma cells into the surrounding brain parenchyma. In this study, our analysis of GBM patient cohorts revealed a significantly higher expression of Glycosyltransferase 8 domain containing 1 (GLT8D1) compared to normal brain tissue and could be associated with impaired patient survival. Increased in vitro expression of GLT8D1 significantly enhanced migration of two different sphere-forming GBM cell lines. By in silico analysis we predicted the 3D-structure as well as the active site residues of GLT8D1. The introduction of point mutations in the predicted active site reduced its glycosyltransferase activity in vitro and consequently impaired GBM tumor cell migration. Examination of GLT8D1 interaction partners by LC-MS/MS implied proteins associated with cytoskeleton and intracellular transport as potential substrates. In conclusion, we demonstrated that the enzymatic activity of glycosyltransferase GLT8D1 promotes GBM cell migration

Marqueurs non-invasifs de la compétence ovocytaire au développement dans les cellules de cumulus chez l'humain

Prédire la capacité de développement et d'implantation des embryons reste un enjeu majeur pour l’Assistance Médicale à la Procréation (AMP). L’AMP doit répondre au désir du couple d’avoir un enfant en limitant les risques encourus par la mère et l’enfant en cas de grossesse multiple. Tous les laboratoires d’AMP utilisent des critères morphologiques pour évaluer la compétence au développement des embryons en dépit de la faible valeur prédictive de cette analyse. L'interaction ovocyte-cumulus participe à l’acquisition par l'ovocyte de sa compétence au développement. Cette interaction met en jeu l’expression de gènes spécifiques dans les cellules de cumulus (CCs). Notre objectif était d'identifier des marqueurs non invasifs de la compétence ovocytaire au développement. Ainsi nous avons recherché au niveau des CCs des gènes et des protéines exprimés en fonction de l’aptitude de l’ovocyte fécondé à atteindre le stade de blastocyste. L'expression des gènes des CCs a été étudiée par puce à ADN et qPCR haut débit. Après avoir tenu compte de la variabilité des patientes, nous avons identifié les gènes RGS2, POLR3K et CUL4B comme biomarqueurs. L'expression des protéines des CCs a été étudiée par puce à protéines et après validation des anticorps ciblant les protéines d'intérêt, les protéines RGS2, POLR3K et MERTK ont été identifiées comme biomarqueurs de la compétence au développement de l'ovocyte. Ces résultats permettent d’envisager la création d’un modèle prédictif multicritère incluant la morphologie de l’embryon à J2, les gènes et protéines marqueurs.The ability to predict the developmental and implantation ability of embryos remains a major goal in human assisted reproductive technology (ART).ART should allow couple to become parents while limiting the risks to the mother and the child in case of multiple pregnancy. ART laboratories use morphological criteria to evaluate the oocyte competence despite the poor predictive value of this analysis. The oocyte-cumulus interaction helps the oocyte to acquire its developmental competence partly through the expression of specific genes at the cumulus level. Therefore our aim was to identify at the level of cumulus cells (CCs) genes and proteins related to oocyte developmental competence as non-invasive marker. Gene expression of CCs was studied using microarray and high throughput qPCR according to the developmental competence of the oocyte (ability to reach the blastocyst stage after fertilization). While taking into account the patient variability we identified RGS2, POLR3K and CUL4B as biomarkers at RNA level. Then protein expression of CCs was studied using Reverse Phase Protein Array. After validation of the antibodies targeting the proteins of interests, RGS2, POLR3K and MERTK were identified as protein biomarkers of the developmental competence of the oocyte. These results lead us to consider a multi variables predictive model including the morphology of the embryo at J2, genes and protein markers

Oocyte developmental competence: an improved predictive model combining morphological criteria with transcripts or proteins biomarkers

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Biomarkers of oocyte quality in cumulus cells using Reverse Phase Protein Array (RPPA)

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Marqueurs protéiques de la qualité de l'ovocyte identifiés dans les cellules de cumulus par la spectrométrie de masse MALDI-TOF (ICM-MS). Une aide à la décision pour le transfert mono-embryonnaire en Assistance Médicale à la Procréation ?

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Reverse phase protein array (RPPA): a new approach for the identification of cumulus cells biomarkers of oocyte quality

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Analyse protéomique du cumulus humain : une approche indirecte de la viabilité de l’embryon

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Protéomique par Intact Cells Maldi-Tof Mass Spectrometry sur cumulus humain. Une aide à la décision pour le transfert mono-embryonnaire en Assistance Médicale à la Procréation ?

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Semi-quantitative measurement of specific proteins in human cumulus cells using reverse phase protein array

The ability to predict the developmental and implantation ability of embryos remains a major goal in human assisted-reproductive technology (ART) and most ART laboratories use morphological criteria to evaluate the oocyte competence despite the poor predictive value of this analysis. Transcriptomic and proteomic approaches on somatic cells surrounding the oocyte (granulosa cells, cumulus cells [CCs]) have been proposed for the identification of biomarkers of oocyte competence. We propose to use a Reverse Phase Protein Array (RPPA) approach to investigate new potential biomarkers of oocyte competence in human CCs at the protein level, an approach that is already used in cancer research to identify biomarkers in clinical diagnostics. Antibodies targeting proteins of interest were validated for their utilisation in RPPA by measuring siRNA-mediated knockdown efficiency in HEK293 cells in parallel with Western blotting (WB) and RPPA from the same lysates. The proteins of interests were measured by RPPA across 13 individual human CCs from four patients undergoing intracytoplasmic sperm injection procedure. The knockdown efficiency of VCL, RGS2 and SRC were measured in HEK293 cells by WB and by RPPA and were acceptable for VCL and SRC proteins. The antibodies targeting these proteins were used for their detection in human CCs by RPPA. The detection of protein VCL, SRC and ERK2 (by using an antibody already validated for RPPA) was then carried out on individual CCs and signals were detected for each individual sample. After normalisation by VCL, we showed that the level of expression of ERK2 was almost the same across the 13 individual CCs while the level of expression of SRC was different between the 13 individual CCs of the four patients and between the CCs from one individual patient. The exquisite sensitivity of RPPA allowed detection of specific proteins in individual CCs. Although the validation of antibodies for RPPA is labour intensive, RRPA is a sensitive and quantitative technique allowing the detection of specific proteins from very small quantities of biological samples. RPPA may be of great interest in clinical diagnostics to predict the oocyte competence prior to transfer of the embryo using robust protein biomarkers expressed by CCs