| Abstract |
A major task of the Human Genome Project is the detailed structural and functional analysis of novel genes. Within the chromosomal region 10q23.3-25.1 several, yet unidentified, disease-related gene loci have been localized. One of them is NEURL, the human homologue of Drosophila neuralized (neur) gene. NEURL haw been mapped within 10q25.1, a region most frequently deleted in malignant astrocytomas. Drosophila neuralized belongs to the neurogenic genes also including Notch (N), Delta (Dl), Enhancer of split [E(spl)], big brain (bib), mastermind (mam) and almondex (AMX). It is expressed in various tissues during development and participates in Notch receptordependent intracellular communication. Because of its significant sequence similarity with the Drosophila protein, and its defects determined in astrocytoma cell lines, it has been hypothesized that NEURL may be involved in cell fate determination of the mammalian nervous system, and that its inactivation is associated with the progression of malignant neuroectodermal tumors. In order to contribute to the elucidation of the functional role of neuralized in mammals, we isolated and analyzed the structure and the expression of the mouse ortholog, Neurl. Furthermore, we decided to analyze in parallel, both the insect and the mouse molecules using the well-studied Drosophila experimental model system. This approach would offer us the opportunity to obtain particularly useful information regarding the biological role of the insect polypeptide and at the same time, extrapolate this information to the mammalian homolog. Mammalian and insect neuralized proteins share extensive structural similarities through-out the entire molecule. The most recognizable similarities are two neuralized-specific sequence repeats, NEUZ1 and 2, of unknown function and a carboxy-terminal RING Zinc finger, characterizing proteins with E3 ubiquitin ligase activity. Expression profile determination in embryo and adult mouse tissues, suggested that Neurl is active from the earliest embryonal developmental stages in neural and skeletal system, in the developing limbs and in epithelial cell layers of sense and internal organs. Transient expression in mammalian cell lines of GFP-tagged neuralized polypeptides (using either full length or truncated molecules) as well as targeted over-expression of either Neur or Neurl in Drosophila tissues, showed that both proteins are localized in the cytoplasmic side of the plasma membrane and possibly, in endocytotic vesicles. Biochemical experiments and immunofluorescence microscopy observations suggested that the association of neuralized protein or with other molecules of the cytoplasmic periphery. Futhermore, it was shown that the amino-terminal part of the neuralized protein extended up to the first NEUZ domain, is necessary and sufficient for its subcellular localization. The functional similarities of the insect and the mammalian Neuralized were further corroborated with extensive functional experimentation in the Drosophila model system. Targeted over-expression of a neur or Neurl transgenes in Drosophila embryos resulted in similar phenotypic consequences, i.e.: loss of Notch function in adults as manifested by cut wings and vein thickening. Down-regulation of E(spl)m8, a transcription target gene of the Notch signaling pathway, at the wing margin was also observed. Possibly due to the evolutionary distance between the two homologs, the phenotypic effects of the mammalian transgene were weaker than those observed with the Drosophila neur. Detailed functional analysis of the Drosophila gene was undertaken in order to elucidate the exact function of Neuralized protein in Notch signaling and to get as much information as possible concerning the role of the murine polypeptide. Thus, it was shown that Neur plays a significant role in enhancing the ability of Delta ligand to activate Notch receptor, as well as in subsequent endocytosis, in cis, and posttranslational degradation of the former. Moreover, Delta endocytosis is followed by Notch extracellular region co-internalization. Furthermore, the Neur RING domain is required for DI internalization but is dispensable for DI signaling activity. The potent modulatory effect of Neur on DI activity makes Neur a good candidate for establishing signaling asymmetries and thus, different developmental fates within cellular equivalence groups.
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Δομική και λειτουργική ανάλυση των γονιδίων neuralized του ποντικού και της Drosophila και διερεύνηση, in vivo, του ρόλου τους στη σηματοδότηση Notch
