微小管結合タンパク質の1つであるタウは、神経細胞の軸索に特異的に局在している。タウの軸索局在化分子機序を解明するために、外因性タウを神経細胞の発達初期に一時的に発現させ、軸索特異的に局在させる方法を構築した。この方法を用い、proline rich region 2 (PRR2)がタウの軸索局在化に重要であること、PRR2のリン酸化が軸索への移動に関与することを示唆した。またこの系の確立は局在や細胞内動態などの検討を行うことを可能にした。 / Microtubule-associated protein tau localizes specifically to neuronal axons. In order to elucidate the molecular mechanism of the axon localization of tau, we constructed an expression system for axon specific localization of exogenous tau in immature neurons in culture. Using this system, it suggested that the proline rich region 2 (PRR2) and phosphorylation of PRR2, which contains important phosphorylation sites, is critical for the localization. In the future, this experimental system will contribute greatly to the study of tau in normal and in the pathology of Alzheimer's disease. / 博士(理学) / Doctor of Philosophy in Science / 同志社大学 / Doshisha University

No description available.

L’hyperphosphorylation de la protéine Tau, à l’origine de la formation de dégénérescences neurofibrillaires et de la mort cellulaire observée, chez les patients atteints de la maladie d’Alzheimer, est causée par un déséquilibre entre l’activité des kinases et des phosphatases. Cette modification post traductionnelle touche de nombreux sites dans la Région Riche en Proline (RRP) de Tau. Or, des études RMN, réalisées au sein du laboratoire, ont montré que les polyphénols sont capables d’interagir avec des fragments de la protéine issues de la RRP avec des affinités de l’ordre de grandeur de celles décrites pour les kinases. Dans ce contexte, il a été envisagé de synthétiser une bibliothèque de polyphénols et de mettre au point une réaction de phosphorylation sur des peptides modèles afin d’étudier, par spectrométrie de masse, la capacité de ces composés à protéger Tau contre l’attaque des kinases. / Hyperphosphorylation of Tau protein, which leads to their abnormal aggregation into neurofibrillary tangles and to neuronal loss observed in patients with Alzheimer disease, is regulated by a disequilibrium between kinases and phosphatases activities. This post traductional modification affects several residues, in particular in the Proline Rich Region of Tau (PRR). But, NMR studies, realized in our laboratory, have shown that polyphenols are able to interact with peptide Tau models issued from the PRR and the affinity are in the same range that those described for the kinases. In this context, we have envisaged to synthetize several polyphenols and to develop a phosphorylation reaction of peptide models in order to study, by mass spectrometry, the ability of these compounds to protect Tau against kinase attack.

Hepatitis delta virus (HDV) is the smallest known mammalian RNA virus, containing a genome of ~ 1700 nt. Replication of HDV is extremely dependent on the host transcription machinery. Previous studies indicated that RNA polymerase II (RNAPII) directly binds to and forms an active preinitiation complex on the right terminal stem-loop fragment (R199G) of HDV genomic RNA, and that the polypyrimidine tract-binding protein-associated splicing factor (PSF) directly binds to the same region. Further studies demonstrated that PSF also binds to the carboxyl-terminal domain (CTD) of RNAP II. In my thesis, co-immunoprecipitation assays were performed to show that PSF stimulates the interaction of RNAPII with R199G. Results of co-immunoprecipitation experiments also suggest that both the RNA recognition motif 2 (RRM2) and N-terminal proline-rich region (PRR) of PSF are required for the interaction between PSF and RNAPII, while the two RNA recognition motifs (RRM1 and RRM2) might be required for the interaction of PSF with R199G. Furthermore, in vitro run-off transcription assays suggest that PSF facilitates the HDV RNA transcription from the R199G template. Together, the above experiments suggest that PSF might act as a transcription factor for the RNAPII transcription of HDV RNA by linking the CTD of RNAPII and the HDV RNA promoter. My experiments provide a better understanding of the mechanism of HDV RNA-dependent transcription by RNAP II.

No description available.

Hepatitis delta virus (HDV) is the smallest known mammalian RNA virus, containing a genome of ~ 1700 nt. Replication of HDV is extremely dependent on the host transcription machinery. Previous studies indicated that RNA polymerase II (RNAPII) directly binds to and forms an active preinitiation complex on the right terminal stem-loop fragment (R199G) of HDV genomic RNA, and that the polypyrimidine tract-binding protein-associated splicing factor (PSF) directly binds to the same region. Further studies demonstrated that PSF also binds to the carboxyl-terminal domain (CTD) of RNAP II. In my thesis, co-immunoprecipitation assays were performed to show that PSF stimulates the interaction of RNAPII with R199G. Results of co-immunoprecipitation experiments also suggest that both the RNA recognition motif 2 (RRM2) and N-terminal proline-rich region (PRR) of PSF are required for the interaction between PSF and RNAPII, while the two RNA recognition motifs (RRM1 and RRM2) might be required for the interaction of PSF with R199G. Furthermore, in vitro run-off transcription assays suggest that PSF facilitates the HDV RNA transcription from the R199G template. Together, the above experiments suggest that PSF might act as a transcription factor for the RNAPII transcription of HDV RNA by linking the CTD of RNAPII and the HDV RNA promoter. My experiments provide a better understanding of the mechanism of HDV RNA-dependent transcription by RNAP II.