Project description:Analysis of genes and biological processes influenced by Neuronal calcium sensor 1 (NCS1) based on whole transcriptome analyiss of Ncs1+/+ (wildtype) and Ncs1-/- (knockout) mouse brain tissues, i.d. the frontal cortex and the hippocampus.
Project description:Phosphatidylinositol 4 kinase III (PI4KIII/PI4KA) is an essential lipid kinase that is critical for regulating plasma membrane identity. PI4KA is primarily recruited to the plasma membrane through targeted recruitment by the proteins EFR3A and EFR3B, with these binding to the PI4KA accessory proteins TTC7 (TTC7A and TTC7B) and FAM126 (FAM126A and FAM126B). Here we characterised how both EFR3 isoforms interact with all possible TTC7 and FAM126 combinations and developed a nanobody that specifically blocks EFR3 mediated PI4KA recruitment in TTC7B containing complexes. Using a yeast display approach, we generated a nanobody that is selective for TTC7B and blocks EFR3 binding. Cryo-electron microscopy and hydrogen deuterium exchange mass spectrometry reveal that the nanobody sterically blocks EFR3 recruitment and shows an extended interface with both PI4KA and TTC7B. Overall, this work provides insight into PI4KA regulation and is a useful tool for manipulating unique complexes of PI4KA that may be valuable for future therapeutic targeting of PI4KA.
Project description:Metastases in the bone marrow (BM) are grim prognostic factors in patients with neuroblastoma (NB). In spite of extensive analysis of primary tumor cells from high- and low-risk NB patients, a characterization of freshly isolated BM-infiltrating metastatic NB cells is still lacking. Our aim was to identify proteins specifically expressed by metastatic NB cells, that may be relevant for prognostic and therapeutic purposes. Metastatic NB cells were freshly isolated from patients’ BM by positive immunomagnetic bead manipulation using anti-GD2 monoclonal antibody. Unselected BM samples from patients with metastatic NB were also included. Gene expression profiles were compared with those obtained from archived NB primary tumors from patients with 5y-follow-up. After validation by RT-qPCR, expression/secretion of the proteins encoded by the up-regulated genes in the BM-infiltrating NB cells was evaluated by flow cytometry and ELISA. Compared to primary tumor cells, BM-infiltrating NB cells down-modulated the expression of CX3CL1, AGT, ATP1A2 mRNAs, whereas they up-regulated several genes commonly expressed by various lineages of BM resident cells. BM-infiltrating NB cells expressed indeed the proteins encoded by the top-ranked genes, S100A8 and A9 (calprotectin), CD177 and CD3, and secreted the CXCL7 chemokine. BM-infiltrating NB cells also expressed CD271 and HLA-G. We have identified proteins specifically expressed by BM-infiltrating NB cells. Among them, calprotectin, a potent inflammatory protein, and HLA-G, endowed with tolerogenic properties facilitating tumor escape from host immune response, may represent novel biomarkers and/or targets for therapeutic intervention in high-risk NB patients.