Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant inherited neurodegenerative disease for which there is no cure. SCA7 belongs to the group of polyglutamine disorders, which are all caused by the expansion of a polyglutamine tract in different disease proteins. Common toxic mechanisms have been proposed for polyglutamine diseases; however the exact pathological mechanism(s) are still unclear. The aim of this thesis was to identify and characterize the molecular mechanisms by which polyglutamine expansion in the ATXN7 protein cause SCA7 and how this can be counteracted. We found that mutant ATXN7 can be degraded by the ubiquitin proteasome system (UPS) and autophagy, the two main cellular degradation pathways. However aggregation stabilized the protein against degradation. Moreover, we found that mutant ATXN7 blocked the induction of autophagy by interfering with p53 and the ULK1-ATG13-FIP200 complex. Pharmacological stimulation of autophagy ameliorated aggregation, as well as toxicity. We also found that oxidative stress plays an important role in mutant ATXN7 toxicity and that the oxidative stress is generated by activation of NADPH oxidase 1 (NOX1) complexes. Furthermore, we showed that the increased NOX1 activity, together with polyQ expanded ATXN7 mediated disruption of the transcription factor p53, results in metabolic alterations in SCA7 cells. The expression of key p53 regulated metabolic proteins like AIF, TIGAR and GLUT1 was altered in SCA7 cells and resulted in reduced mitochondrial respiration, a higher dependence on glycolysis and reduced ATP levels. In summary, our data indicate that mutant ATXN7 mediated dysregulation of p53, resulting in autophagic and metabolic alterations, could play a key role in SCA7 and possibly other polyglutamine diseases.

The type II tumour suppressor ING3 has been shown to modulate transcription, cell cycle control, and apoptosis. To investigate the putative role of ING3 in melanoma development, we examined the expression of ING3 in 58 dysplastic nevi, 114 primary melanomas, and 50 metastatic melanomas with tissue microarray and immunohistochemistry. Overall ING3 was reduced in metastatic melanomas compared with dyslastic nevi and primary melanomas. Reduced nuclear ING3 staining also correlated with melanoma progression, increased cytoplasmic ING3 level, tumour location at sun-exposed sites, and a poorer disease-specific 5-year survival of patients with primary melanoma. Multivariate analysis revealed that nuclear ING3 staining can independently predict patient outcome in primary melanomas. In melanoma cells, ING3 expression was rapidly induced by UV irradiation. Using stable clones of melanoma cells overexpressing ING3, we showed that ING3 significantly promoted UV-induced apoptosis. Unlike its homologues ING1b and ING2, ING3-enhanced apoptosis upon UV irradiation was independent of functional p53. Furthermore, ING3 did not affect the expression of mitochondrial proteins but increased the cleavage of Bid and caspases. Moreover, ING3 upregulated Fas expression and ING3-mediated apoptosis was blocked by inhibiting caspase-8 or Fas activation. Knockdown of ING3 expression decreased UV-induced apoptosis remarkably, suggesting that ING3 plays a crucial role in cellular response to UV radiation. To explore how ING3 is deregulated in advanced melanomas, we examined ING3 expression in metastatic melanoma cells and found that ING3 was downregulated due to a rapid protein turnover in these cells. Further studies demonstrated that ING3 undergoes degradation via the ubiquitin-proteasome pathway. We also demonstrate that ING3 interacts with the SCF (Skp1/Cul1/Roc1/Skp2) E3 ligase complex. Knockdown of Cul1 or Skp2 significantly stabilized ING3 in melanoma cells. In addition, lysine residue 96 is essential for ING3 ubiquitination as its mutation to arginine completely abrogated ING3 turnover and enhanced ING3-stimulatd apoptosis upon UV irradiation. Taken together, ING3 is deregulated in melanomas as a result of both nucleus-to-cytoplasm shift and rapid degradation. The level of ING3 in the nucleus may be an important marker for human melanoma progression and prognosis. Restoration of ING3 expression significantly sensitizes melanoma cells to UV radiation through the activation of Fas/caspase-8 pathway.

The human body consists of about 40 % skeletal muscles which control the body’s movement, ability to stand up, force generation, locomotion, heat production and are also the body’s protein reservoir. Muscle mass is controlled by the relationship between protein synthesis and protein degradation. Atrophy, a decrease in muscle mass, can be trigged by disuse, immobilization, inflammation and cancer. Hypertrophy, an increase in muscle mass, can occur after increased mechanical load, high usage and/or anabolic stimulation. The aim of this thesis was to investigate changes in expression and post translational modifications of some factors involved in the regulation of protein synthesis and protein degradation in 6-days denervated atrophic hind-limb muscles (anterior tibial and pooled gastrocnemius and soleus muscles) and in 6-days denervated hypertrophic hemidiaphragm muscle in mice. Protein expression and post translational modifications were studied semi-quantitatively using Western blots with whole muscle homogenates and separated nuclear and cytosolic fractions from both innervated and denervated muscles.  An increase in protein synthesis after denervation in both atrophic and hypertrophic muscles was suggested after studies of factors downstream of mTOR (paper I).  Other results suggest that FoxO1 and MuRF1 (paper II) participate in the tissue remodeling that occurs after denervation. A differential response of MK2 phosphorylation in denervated hypertrophic and atrophic muscles was confirmed (paper III). An increase in phosphorylation of the MK2 substrate Hsp 25 in all denervated muscles studied (paper III) indicates that other factors than MK2 are involved in regulating this phosphorylation. eIF4G phosphorylation at S1108 was investigated (paper IV) and a decrease was observed in atrophic muscle but an increase in hypertrophic muscle. The results in this thesis suggest that there are several factors that control protein degradation and protein synthesis in denervated atrophic and hypertrophic skeletal muscles. This is an intricate labyrinth with many different cell signaling factors, the function of which are still far from fully understood.

The eukaryotic endoplasmic reticulum (ER) is a multifunctional organelle, primarily responsible for the folding and maturation of secretory proteins, as well as lipid metabolism, calcium homeostasis, ubiquitin-dependent signalling and cell fate decisions. ER-associated degradation (ERAD) oversees protein folding and delivers misfolded proteins for degradation by the proteasome via ubiquitin conjugation mediated by RING-type E3 ubiquitin ligases. An intact ERAD is crucial to cellular homeostasis, as unresolved protein imbalances cause ER stress that ultimately lead to apoptosis. The human ER accommodates at least 25 E3s, however our understanding is mostly limited to Hrd1 and AMFR/gp78, both of which have a defined function in ERAD. To understand the contribution of ER E3s to cellular and organelle homeostasis, this study used mass spectrometry of purified E3 complexes to identify cofactors and build interaction networks of ER-resident E3s. These findings will form the foundation for investigating the biological roles of these ubiquitin ligases. Transcriptional analysis highlighted the centrality of Hrd1 among all ER-resident E3s in response to protein misfolding in the ER. Additionally, the contribution of individual Hrd1 complex components to resolving proteotoxic stress was assessed using a misfolded antibody subunit (IgM heavy chain), rather than conventional pharmacological treatments. The ERAD components essential for substrate degradation and survival under proteotoxic stress were identified, highlighting the pivotal role of Hrd1, its cofactor SEL1L and the Derlin family members. Finally, it was demonstrated that autophagy induction in response to proteasome inhibition is key to relieve the burden of protein misfolding in the ER, as it sustained the survival of cells defective for ERAD. Importantly, this study proposes a potential involvement of Hrd1 in signalling from the ER to autophagy, suggesting potential crosstalk between the ERAD and autophagic pathways.

The type II tumour suppressor ING3 has been shown to modulate transcription, cell cycle control, and apoptosis. To investigate the putative role of ING3 in melanoma development, we examined the expression of ING3 in 58 dysplastic nevi, 114 primary melanomas, and 50 metastatic melanomas with tissue microarray and immunohistochemistry. Overall ING3 was reduced in metastatic melanomas compared with dyslastic nevi and primary melanomas. Reduced nuclear ING3 staining also correlated with melanoma progression, increased cytoplasmic ING3 level, tumour location at sun-exposed sites, and a poorer disease-specific 5-year survival of patients with primary melanoma. Multivariate analysis revealed that nuclear ING3 staining can independently predict patient outcome in primary melanomas. In melanoma cells, ING3 expression was rapidly induced by UV irradiation. Using stable clones of melanoma cells overexpressing ING3, we showed that ING3 significantly promoted UV-induced apoptosis. Unlike its homologues ING1b and ING2, ING3-enhanced apoptosis upon UV irradiation was independent of functional p53. Furthermore, ING3 did not affect the expression of mitochondrial proteins but increased the cleavage of Bid and caspases. Moreover, ING3 upregulated Fas expression and ING3-mediated apoptosis was blocked by inhibiting caspase-8 or Fas activation. Knockdown of ING3 expression decreased UV-induced apoptosis remarkably, suggesting that ING3 plays a crucial role in cellular response to UV radiation. To explore how ING3 is deregulated in advanced melanomas, we examined ING3 expression in metastatic melanoma cells and found that ING3 was downregulated due to a rapid protein turnover in these cells. Further studies demonstrated that ING3 undergoes degradation via the ubiquitin-proteasome pathway. We also demonstrate that ING3 interacts with the SCF (Skp1/Cul1/Roc1/Skp2) E3 ligase complex. Knockdown of Cul1 or Skp2 significantly stabilized ING3 in melanoma cells. In addition, lysine residue 96 is essential for ING3 ubiquitination as its mutation to arginine completely abrogated ING3 turnover and enhanced ING3-stimulatd apoptosis upon UV irradiation. Taken together, ING3 is deregulated in melanomas as a result of both nucleus-to-cytoplasm shift and rapid degradation. The level of ING3 in the nucleus may be an important marker for human melanoma progression and prognosis. Restoration of ING3 expression significantly sensitizes melanoma cells to UV radiation through the activation of Fas/caspase-8 pathway. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第22633号 / 理博第4622号 / 新制||理||1664(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 森 和俊, 教授 平野 丈夫, 教授 川口 真也 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21012号 / 医博第4358号 / 新制||医||1028(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 戸口田 淳也, 教授 鈴木 茂彦, 教授 開 祐司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Target of Myb 1 (TOM1) is an endosomal adaptor protein that plays a role in cargo membrane trafficking for degradation by serving as an alternative endosomal sorting complex required for transport component. TOM1 has also been shown to serve as a novel phosphatidylinositol 5-phosphate (PtdIns5P) effector at signaling endosomes through its VHS domain, delaying cargo degradation in a bacterial infection model. The aim of this thesis is to clarify the structural and functional basis of the autoregulation mechanism of TOM1 to switch from endosomal protein trafficking to the bacterial survival signaling pathway. Our thermal denaturation and spectroscopic studies demonstrate that PtdIns5P reduced thermostability, interhelical contacts, and conformational compaction of TOM1 VHS. The thermodynamic studies indicate that TOM1 VHS endothermically binds to PtdIns5P through two potential noncooperative binding sites, with its acyl chains playing a relevant role in the interaction. These findings suggest that, under Shigella flexneri infection, TOM1 may interact with downstream effectors in a different VHS domain conformational state, thus involving the protein in bacterial survival signaling pathways. In order to obtain molecular details for the interaction of the TOM1 VHS domain for PtdIns5P and Ubiquitin (Ub), the backbone assignment information was obtained by performing NMR experiments, which assigned backbone 1H, 13C, and 15N resonances of the TOM1 VHS domain. With this structural information, our heteronuclear single quantum coherence and molecular dynamics simulations data revealed that TOM1 VHS interacts with PtdIns5P following a fast-exchange regime, with the PtdIns5P binding site predicted to be at a region spanning α-helices 6 to 8. Further mutagenesis and lipid-protein overlay assay studies indicated that K147 plays a critical role in the binding of TOM1 VHS domain to PtdIns5P. TOM1, unexpectedly, did not bind PtdIns5P. Using truncated forms of TOM1 protein, we discovered that neither TOM1 GAT domain nor the C-terminal domain modulated TOM1 VHS's PtdIns5P binding; however, surprisingly, a linker sequence between the TOM1 VHS and GAT domains exhibited an autoinhibition role for TOM1 binding to PtdIns5P. This linker region was observed to induce local conformational changes on the structure of TOM1 VHS domain, especially around α-helices 6 and 8, which are proposed to build up the binding pocket for PtdIns5P. In order to investigate whether the linker region between TOM1 VHS and GAT domain can also regulate the Ub association of TOM1 VHS domain, the binding properties of TOM1 and its domains to Ub were explored. Unexpectedly, the binding affinity of TOM1 VHS-linker for Ub was increased about 10-fold when compared with that for the TOM1 VHS domain, suggesting that the linker enhances the avidity of TOM1 for ubiquitinated cargo. Structural analysis indicated that the linker region may cap the conventional Ub-binding site of TOM1 VHS, thus forming a more compact structure. In summary, this study uncovered a novel intramolecular modulatory mechanism in TOM1 that regulates ligand recognition by its VHS domain. By providing the molecular basis of the TOM1 interactions, we may provide cargo sorting mechanistic insights, create functionally specific mutations, and precisely manipulate TOM1 function under bacterial infection conditions, and other yet-to-be-discovered PtdIns5P-dependent signaling pathways. / Doctor of Philosophy / Membrane trafficking is a delivery network established in a cell to transport proteins (cargoes) from one intracellular place to another one to control their activity. TOM1 is a protein involved in this process, which plays a role in transporting cargoes for degradation. Defects in this trafficking pathway lead to human diseases, such as immunodeficiency and neurodegeneration diseases. TOM1 has also been shown to be beneficial for bacterial survival in human cells. However, how TOM1 switches its role form protein trafficking to bacterial pathogenesis is still unclear. In our study, we discovered an internal region of TOM1 may serve as a switch to shift the role of TOM1 in human cells. In an "on" status, TOM1 favors to transport cargoes, while in an "off" status, TOM1 is used for bacteria survival. This study provides insights in the function of TOM1 which is beneficial for the design of novel therapeutic strategies against TOM1, which will prevent the progress of bacterial infections.

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