Centaurin-α2 interacts with ß-tubulin and stabilizes microtubules.

Centaurin-α2 is a GTPase-activating protein for ARF (ARFGAP) showing a diffuse cytoplasmic localization capable to translocate to membrane, where it binds phosphatidylinositols. Taking into account that Centaurin-α2 can localize in cytoplasm and that its cytoplasmatic function is not well defined, we searched for further interactors by yeast two-hybrid assay to investigate its biological function. We identified a further Centaurin-α2 interacting protein, ß-Tubulin, by yeast two-hybrid assay. The interaction, involving the C-terminal region of ß-Tubulin, has been confirmed by coimmunoprecipitation experiments. After Centaurin-α2 overexpression in HeLa cells and extraction of soluble (αß dimers) and insoluble (microtubules) fractions of Tubulin, we observed that Centaurin-α2 mainly interacts with the polymerized Tubulin fraction, besides colocalizing with microtubules (MTs) in cytoplasm accordingly. Even following the depolimerizing Tubulin treatments Centaurin-α2 remains mainly associated to nocodazole- and cold-resistant MTs. We found an increase of MT stability in transfected HeLa cells, evaluating as marker of stability the level of MT acetylation. In vitro assays using purified Centaurin-α2 and tubulin confirmed that Centaurin-α2 promotes tubulin assembly and increases microtubule stability. The biological effect of Centaurin-α2 overexpression, assessed through the detection of an increased number of mitotic HeLa cells with bipolar spindles and with the correct number of centrosomes in both dividing and not dividing cells, is consistent with the Centaurin-α2 role on MT stabilization. Centaurin-α2 interacts with ß-Tubulin and it mainly associates to MTs, resistant to destabilizing agents, in vitro and in cell. We propose Centaurin-α2 as a new microtubule-associated protein (MAP) increasing MT stability.

Breakpoint characterization of a novel NF1 multiexonic deletion: a case showing expression of the mutated allele.

Neurofibromatosis type 1 (NF1) is a common genetic disease caused by haploinsufficiency of the NF1 tumor-suppressor gene. Different pathogenetic mechanisms have been identified, with the majority (95%) causing intragenic lesions. Single or multiexon NF1 copy number changes occur in about 2% of patients, but little is known about the molecular mechanisms behind these intragenic deletions. We report here on the molecular characterization of a novel NF1 multiexonic deletion. The application of a multidisciplinary approach including multiplex ligation-dependent probe amplification, allelic segregation analysis, and fluorescent in situ hybridization allowed us to map the breakpoints in IVS27b and IVS48. Furthermore, the breakpoint junction was characterized by sequencing. Using bioinformatic analysis, we identified some recombinogenic motifs in close proximity to the centromeric and telomeric breakpoints and predicted the presence of a mutated messenger ribonucleic acid, which was deleted between exons 28 and 48 and encodes a neurofibromin that lacks some domains essential for its function. Through reverse transcriptase-polymerase chain reaction, the expression of the mutated allele was verified, showing the junction between exons 27b and 49 and, as expected, was not subjected to nonsense-mediated decay. Multiexonic deletions represent 2% of NF1 mutations, and until now, the breakpoint has been identified in only a few cases. The fine characterization of multiexonic deletions broadens the mutational repertoire of the NF1 gene, allowing for the identification of different pathogenetic mechanisms causing NF1.

Evaluation of 1p36 markers and clinical outcome in a skull base chordoma study.

Chordomas are rare embryogenetic tumors, arising from remnants of the notochord, characterized by local invasiveness and variable tendency for recurrence. No molecular markers are currently used in a clinical setting to distinguish chordomas with an indolent or an aggressive pattern. Among the genetic lesions observed in this tumor, one of the most commonly detected is 1p loss. In a previous study we observed 1p36 loss of heterozygosity (LOH) in 85% of the analyzed chordomas. We studied a group of 16 homogeneously treated skull base chordomas (SBCs), reporting 1p36 LOH in 75% of them and determining the expression pattern of eight apoptotic genes mapped at 1p36. No tumors shared a common expression profile with nucleus pulposus, which is considered the only adult normal tissue deriving from notochord. In particular, tumor necrosis factor receptor superfamily genes TNFRSF8, TNFRSF9, and TNFRSF14 were differently expressed compared with control in a higher percentage of tumors (40%-53%) than were the remaining analyzed genes, suggesting that the deregulation of these three genes might have a role in chordoma tumorigenesis. The presence/absence of LOH and the expression/nonexpression of each apoptotic gene were studied in a survival analysis. Our results suggest that the lack of 1p36 LOH or the presence of TNFRSF8 expression might be associated with a better prognosis in patients with SBCs.