TRIM7 is a member of the TRIM (tripartite motif-containing) protein superfamily. This family has been implicated in many disorders such as genetic diseases, neurological diseases, and cancers. Little is known about the function of TRIM7 except that it interacts with glycogenin and may regulate glycogen biosynthesis. Recently, a yeast two-hybrid protein-protein interaction screen revealed the binding of TRIM7 to Sin1, a protein found in a complex with the mammalian target of rapamycin (mTOR) protein kinase. mTOR can form two complexes, mTORC1 and mTORC2, which are important for cell growth, differentiation, and survival. Sin1 is a core component of mTORC2 and is critical for mTORC2 stability and activity. It was confirmed by co-immunoprecipitation that TRIM7 associates with Sin1 and mTOR in cultured mammalian cells. Furthermore, it was demonstrated that TRIM7 is a phosphoprotein, although it was not directly targeted by mTOR in vitro. Similar to some other TRIM family proteins, it was demonstrated that TRIM7 has a ubiquitin E3 ligase function allowing it to autoubiquitinate both in vitro and in cells. The autoubiquitination of TRIM7 was dependent on its RING domain. Further characterization of TRIM7 indicated that it can both homo-oligomerise as well as hetero-oligomerise with other members of its sub-class of TRIM proteins and that it co-localises with them into discrete cytoplasmic loci. To determine the cellular function of TRIM7, a stable cell line expressing an shRNA directed against TRIM7 was generated. Successful knock down of TRIM7 was achieved and this led to an increase in the protein levels of components of the mTORC2 complex, including Sin1. This coincided with an increase in cell proliferation. In conclusion, this research identifies a novel role for TRIM7 as a ubiquitin ligase involved in regulating cell proliferation and provides a potential link between TRIM7 and the mTOR pathway, a major transducer of proliferative and cell survival signals.

京都大学 / 0048 / 新制・課程博士 / 博士(社会健康医学) / 甲第22749号 / 社医博第110号 / 新制||社医||11(附属図書館) / 京都大学大学院医学研究科社会健康医学系専攻 / (主査)教授 髙橋 良輔, 教授 髙折 晃史, 教授 中山 健夫 / 学位規則第4条第1項該当 / Doctor of Public Health / Kyoto University / DFAM

The ubiquitin-proteasome proteolysis pathway is responsible for the degradation of abnormal and short-lived proteins to regulate many important biochemical activities in eukaryotes. By employing affymetrix microarray analysis, we have identified a novel ubiquitin ligase E3 gene GhRING2 that is expressed differentially between two G. hirsutum lines - Texas Marker-1 and Chromosome Substitution Line CS-B25. The complete GhRING2 gene sequence was obtained by genomic and cDNA walking. The expression of GhRING2 in cotton fiber is developmentally regulated, suggesting that the ubiquitin-proteasome pathway may regulate cotton fiber growth and development. Using a yeast two-hybrid assay GhRING2 was found to interact with a PROTODERMAL FACTOR1 (GhPDF1) protein. GhPDF1 is expressed preferentially in immature ovules and fiber initials and the gene has been suggested to play a role in cell fate determination and fiber development. Pull down and plasmid swap assays were employed to confirm this interaction.

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A cotton fiber cDNA, GhRING1, and its corresponding gene have been cloned and characterized. The GhRING1 gene encodes a RING-type ubiquitin ligase (E3) containing 337 amino acids (aa). The GhRING1 protein contains a RING finger motif with conserved cysteine and histine residues at the C-terminus and is classified as a C3H2C3-type RING protein. Blast searches show that GhRING1 has the highest homology to At3g19950 from Arabidopsis. Real time RT-PCR analysis indicates that the GhRING1 gene is highly expressed in cotton fiber in a developmental manner. The transcript level of the GhRING1 gene reaches a maximum in elongating fibers at 15 DPA. In vitro auto-ubiquitination assays using wheat germ extract and a reconstitution system demonstrate that GhRING1 has the ubiquitin E3 ligase activity. A fiber specific lipid transfer protein 4 (FSltp4) is identified as the target substrate of GhRING1 by using a bacterial two-hybrid system. The binding of GhRING1 and FSltp4 is confirmed by using an in vitro pull down assay and a yeast two-hybrid system. The histochemical GUS assay was performed to analyze tissue specificity of the GhRING1 and At3g19950 promoters in transgenic Arabidopsis plants. The GUS assay shows that the promoter of At3g19950 is highly activated in leaves, roots, trichomes and also in anthers and stigma of flowers. In contrast, the GUS expression directed by the promoter of GhRING1 is only located at stipules and anthers and stigma of flowers. The GhRING1 is the first ubiquitin E3 gene isolated and studied from cotton. Based on the expression pattern of GhRING1, FSltp4, and GhUBC E2s and the identification of a fiber-specific target protein, FSltp4, we propose that protein ubiquitination occurs in fiber and the ubiquitin-proteasome pathway regulates fiber development.

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Implication de ROQUIN dans la physiopathologie du lymphome T angio-immunoblastique. Le T-LAI est un lymphome T périphérique qui de part sa rareté bénéficie de peu d'études contrairement aux lymphomes B. En France, le T-LAI est le PTCL le plus fréquemment rencontré. Malgré une évolution clinique variable, le T-LAI reste une tumeur agressive dont la survie globale est inférieure à 3 ans. Un des objectifs de notre équipe est donc de mieux comprendre la physiopathologie de ce lymphome et d'identifier des évènements oncogéniques conduisant à son développement. Dans le cadre de ce projet, notre étude s'est portée sur le gène ROQUIN qui code une E3 ubiquitine ligase de la famille RING et dont la mutation est associée à l'apparition d'un syndrome proche du T-LAI chez la souris sanroque.Bien que nous n'ayons détecté aucune mutation dans la séquence codante de ROQUIN nous avons identifié un nouveau transcrit alternatif appelé ROQUIN ØE17. Celui-ci code une protéine qui, comme la forme sauvage, se localise dans les granules de stress et les corps P et interagit avec certains ARNm et micro-ARN. Néanmoins il est le seul à inhiber l'expression de la molécule de costimulation ICOS. ROQUIN ØE17, qui est présent en concentrations variables dans les différentes populations lymphocytaires T n'est quasiment pas exprimé dans les T-LAI. De ce fait, la perte du transcrit ROQUIN ØE17 pourrait participer à la genèse et/ou développement de ce lymphome. / Implication of ROQUIN in the physiopathology of angio-immunoblastic T cell lymphoma. AITL is a peripheral T cell lymphoma, poorly studied compared to B cell lymphomas due to its rarity. In France, AITL is the PTCL the most frequently encountered. Despite a variable clinical course, AITL is an aggressive tumor with an overall survival lower than 3 years. One of our goal is to better understand the physiopathology of this lymphoma and identify oncogenic events that lead to its development. In this project, our study was focused on ROQUIN gene that encodes a RING E3 ubiquitin ligase and whose mutation induces an AITL-like syndrom in sanroque mice.Although we did not detect any mutation in ROQUIN coding sequence, we identified a novel alternative transcript referred as ROQUIN ØE17. It encodes a protein that, like wild type protein, localizes to stress granules and P bodies and interacts with mRNAs and microRNAs. However, only ROQUIN ØE17 inhibits the expression of the costimulatory molecule ICOS. This transcript, whose expression varies between T cell populations, is hardly expressed in AITL. Consequently, the loss of ROQUIN ØE17 could be involved in the genesis and/or development of this lymphoma.

La Neuropathie à Axones Géants (NAG) est une maladie neurodégénérative rare et fatale caractérisée par une détérioration du système nerveux central et périphérique, impliquant les fonctions motrices et sensorielles. La détérioration massive du système nerveux est accompagnée d'une désorganisation générale des Filaments Intermédiaires ce qui la différencie de nombreuses maladies neurodégénératives où seuls les neurofilaments(NFs) sont affectés. La protéine déficiente, la gigaxonine, est la sous-unité d'une ubiquitine ligase E3, responsable de la reconnaissance spécifique des substrats MAP1B, MAP1S et TBCB, seuls connus à ce jour.Dans le but d'étudier le rôle de la gigaxonine sur la survie neuronale, la désorganisation du cytosquelette et d'avoir un modèle animal suffisamment fort pour envisager des tests thérapeutiques, j'ai caractérisé un modèle murin de NAG. Pour ce faire, j'ai réalisé une étude comportementale des fonctions motrices et sensorielles ainsi qu'une étude histopathologique. Les souris NAG (129/SvJ) développent un phénotype moteur modéré dès 60 semaines alors que les souris NAG (C57BL/6) présentent un phénotype sensoriel dès 60 semaines. Les données histopathologiques ne présentent pas de mort neuronale mais les NFs sont sévèrement altérés. Les NFs sont plus abondant, leur diamètre est augmenté et leur orientation hétérogène, comme c'est observé chez les patients NAG.Nos résultats montrent que l'absence de gigaxonine induit un phénotype moteur et sensoriel modéré mais par contre reproduit la désorganisation massive des NFs observée chez les patients. Ce modèle va nous permettred'étudier le rôle de la gigaxonine, une ligase E3, sur l'organisation des NFs et ainsi comprendre les processus pathologiques impliqués dans d'autres maladies neurodégénératives caractérisée par une accumulation des NFs et un dysfonctionnement du système ubiquitine-protéasome comme les maladies d'Azheimer, de Parkinson etd'huntington ou la sclérose latérale amyotrophique. / Giant Axonal Neuropathy (GAN) is a rare and fatale neurodegenerative disorder characterized by a deterioration of the peripheral and central nervous system. The broad deterioration of the nervous system is accompanied with a general disorganization of the Intermediate Filaments which makes it different from other neurodegenerative disorders wherein only neurofilaments (NFs) are affected. The defective protein, gigaxonin, is the substrate adaptator of an E3 ubiquitin ligase, in charge of the specific recognition of MAP1B, MAP1S and TBCB. In order to study the role of gigaxonin on neuronal survival, the cytoskeleton disorganization and to have a relevant GAN animal model to evaluate efficacy of GAN treatments, I have characterized a GAN mouse model. I did a motor and sensory behavioural study and an histopathologic study. The GAN mice (129/SvJ) shown mild motordeficits starting at 60 weeks of age while sensory deficits were evidenced in C57BL/6 GAN mice. No apparent neurodegeneration was evidenced in GAN mice, but dysregulation of NFs was massive. NFs were more abundant, they shown the abnormal increased diameter and misorientation that are characteristics of the human pathology. Our results show that gigaxonin depletion induces mild motor and sensory deficits but recapitulates the severe NFs dysregulation seen in patients. Our model will allow us to study the role of the gigaxonin-E3 ligase in organizing NFs and understand the pathological processes engaged in other neurodegenerative disorders characterized by accumulation of NFs and dysfunction of the Ubiquitin Proteasome System, such as Amyotrophic Lateral Sclerosis, Huntington's, Alzheimer's and Parkinson's diseases.

No description available.