シンカク セイブツ ニ オケル D - アミノサン タイシャ ニ カンスル コウソガクテキ ケンキュウ

京都大学0048新制・課程博士博士(農学)甲第8986号農博第1168号新制||農||819(附属図書館)学位論文||H13||N3505(農学部図書室)UT51-2001-F316京都大学大学院農学研究科応用生命科学専攻(主査)教授 江﨑 信芳, 教授 清水 昌, 教授 天地 輝夫学位規則第4条第1項該当Doctor of Agricultural ScienceKyoto UniversityDA

Histone Deacetylase Inhibitors Prevent p53-Dependent and p53-Independent Bax-Mediated Neuronal Apoptosis Through Two Distinct Mechanisms

Pharmacological manipulation of protein acetylation levels by histone deacetylase (HDAC) inhibitors represents a novel therapeutic strategy to treat neurodegeneration as well as cancer. However, the molecular mechanisms that determine how HDAC inhibition exerts a protective effect in neurons as opposed to a cytotoxic action in tumor cells has not been elucidated. We addressed this issue in cultured postnatal mouse cortical neurons whose p53-dependent and p53-independent intrinsic apoptotic programs require the proapoptotic multidomain protein, Bax. Despite promoting nuclear p53 accumulation, Class I/II HDAC inhibitors (HDACIs) protected neurons from p53-dependent cell death induced by camptothecin, etoposide, heterologous p53 expression or the MDM2 inhibitor, nutlin-3a. HDACIs suppressed p53-dependent PUMA expression, a critical signaling intermediate linking p53 to Bax activation, thus preventing postmitochondrial events including cleavage of caspase-9 and caspase-3. In human SH-SY5Y neuroblastoma cells, however, HDACIs were not able to prevent p53-dependent cell death. Moreover, HDACIs also prevented caspase-3 cleavage in postnatal cortical neurons treated with staurosporine, 3-nitropropionic acid and a Bcl-2 inhibitor, all of which require the presence of Bax but not p53 to promote apoptosis. Although these three toxic agents displayed a requirement for Bax, they did not promote PUMA induction. These results demonstrate that HDACIs block Bax-dependent cell death by two distinct mechanisms to prevent neuronal apoptosis, thus identifying for the first time a defined molecular target for their neuroprotective actions

Druggable Metabolic Vulnerabilities Are Exposed and Masked during Progression to Castration Resistant Prostate Cancer

There is an urgent need for exploring new actionable targets other than androgen receptor to improve outcome from lethal castration-resistant prostate cancer. Tumor metabolism has reemerged as a hallmark of cancer that drives and supports oncogenesis. In this regard, it is important to understand the relationship between distinctive metabolic features, androgen receptor signaling, genetic drivers in prostate cancer, and the tumor microenvironment (symbiotic and competitive metabolic interactions) to identify metabolic vulnerabilities. We explore the links between metabolism and gene regulation, and thus the unique metabolic signatures that define the malignant phenotypes at given stages of prostate tumor progression. We also provide an overview of current metabolism-based pharmacological strategies to be developed or repurposed for metabolism-based therapeutics for castration-resistant prostate cancer

Evaluation of the Acid-Cleavable Isotope-Coded Affinity Tag Reagents: Application to Camptothecin-Treated Cortical Neurons

The new generation of isotope-coded affinity tag (ICAT) reagents have been evaluated by labeling an equimolar amount of bovine serum albumin (BSA) with ICAT-12C9 and ICAT-13C9, combining the mixtures, digesting them with trypsin and analyzing the digestate both by muRPLC-tandem MS and by matrix-assisted laser desorption ionization (MALDI) TOF/TOF MS. The use of 13C in place of 2H resulted in both of the labeled peptides having identical elution characteristics in a reversed-phase separation. This similarity in elution allows ICAT-labeled peptides to be effectively analyzed using a muRPLC-MALDI-MS strategy as well. All of the cysteinyl-containing tryptic peptides from BSA were identified with only a 10% variation in the relative abundance measurements between the light and heavy versions of each peptide. A facile method for the removal of contaminants that arise from the cleaved biotin moiety that otherwise interfere with downstream separations and MS analysis has also been developed. The new ICAT reagents were then applied to the analysis of a cortical neuron proteome sample to identify proteins regulated by the antitumor drug, camptothecin

Evaluation of Liquid Chromatography–Mass Spectrometry for Routine Proteome Analyses

Many different separation strategies have been developed for conducting proteomic studies, but microcapillary reversed‐phase liquid chromatography (µLC) coupled online to mass spectrometry (MS) has played an undeniably central role. We have conducted a study to evaluate two different common column configurations, along with two common stationary phase materials, for their ability to identify peptides from a complex proteome digest. A 10 cm long × 75 µm inner diameter (id) capillary column with an integrated electrospray tip, and a 30 cm long × 75 µm id capillary column in which the electrospray tip is separated from the separation capillary via a stainless steel union, both packed in‐house with reversed phase C18, 5 µm, 300 Å pore size particles, were evaluated for their ability to effectively resolve a complex mixture of tryptic peptides from a mouse cortical neuron proteome sample for online tandem MS (MS/MS) analysis. The results demonstrate that the continuous 10 cm long × 75 µm id capillary column with the integrated electrospray tip, enables the identification of a comparable number of peptides in a single µLC‐MS/MS analysis of a proteome tryptic digestate. We further evaluated the relationship between the numbers of peptides identified vs. the amount of sample loaded onto a 10 cm long × 50 µm id column packed with 3 µm, 100 Å, C18 reversed phase particles. The results show that more than 100 peptides can be identified from as little as 5 ng of tryptic peptides loaded, and that the number of identified peptides did not significantly increase beyond the loading of 50 ng

The Proteomics of Neurodegeneration

The continuing improvement and refinement of proteomic and bioinformatic tools has made it possible to obtain increasing amounts of structural and functional information about proteins on a global scale. The emerging field of neuroproteomics promises to provide powerful strategies for further characterizing neuronal dysfunction and cell loss associated with neurodegenerative diseases. Neuroproteomic studies have thus far revealed relatively comprehensive quantitative changes and post-translational modifications (mostly oxidative damage) of high abundance proteins, confirming deficits in energy production, protein degradation, antioxidant protein function, and cytoskeletal regulation associated with neurodegenerative diseases such as Alzheimer and Parkinson disease. The identification of changes in low-abundance proteins and characterization of their functions based on protein-protein interactions still await further development of proteomic methodologies and more dedicated application of these technologies by neuroscientists. Once accomplished, however, the resulting information will certainly provide a truly comprehensive view of neurodegeneration-associated changes in protein expression, facilitating the identification of novel biomarkers for the early detection of neurodegenerative diseases and new targets for therapeutic intervention

Proteomic Analysis in the Neurosciences

Proteomics is a field of study directed toward providing a comprehensive view of the characteristics and activity of every cellular protein. Rapid innovations in the core technologies required to characterize proteins on a global scale are poised to bring about a comprehensive understanding of how dynamic changes in protein expression, post-translational modification, and function affect complex signaling and regulatory networks. These advances have significant implications for understanding the multitude of pathways that govern behavior and cognition and the response of the nervous system to injury and disease