No description available.

<p>We have provided the significance of Protein Methyltransferase 5 on skeletal muscle function and muscle development.</p>

infection is a leading cause of gastric cancer, which is the second-most common cancer-related death in the world. The chronic inflammatory environment in the gastric mucosal epithelia during infection stimulates intracellular signaling pathways, namely inflammatory signals, which may lead to the promotion and progression of cancer cells. We herein report two important signal transduction pathways, the LPS-TLR4 and CagA-MET pathways. Upon stimulation, lipopolysaccharide (LPS) binds to toll-like receptor 4 (TLR4) mainly on macrophages and gastric epithelial cells. This induces an inflammatory response in the gastric epithelia to upregulate transcription factors, such as NF-κB, AP-1, and IRFs, all of which contribute to the initiation and progression of gastric cancer cells. Compared with other bacterial LPSs, LPS has a unique function of inhibiting the mononuclear cell (MNC)-based production of IL-12 and IFN-γ. While this mechanism reduces the degree of inflammatory reaction of immune cells, it also promotes the survival of gastric cancer cells. The HGF/SF-MET signaling plays a major role in promoting cellular proliferation, motility, migration, survival, and angiogenesis, all of which are essential factors for cancer progression. infection may facilitate MET downstream signaling in gastric cancer cells through its CagA protein via phosphorylation-dependent and/or phosphorylation-independent pathways. Other signaling pathways involved in infection include EGFR, FAK, and Wnt/β-Catenin. These pathways function in the inflammatory process of gastric epithelial mucosa, as well as the progression of gastric cancer cells. Thus, infection-mediated chronic inflammation plays an important role in the development and progression of gastric cancer.

<p>  </p> <p>Pediatric hydrocephalus is a complex neurological condition associated with a pathological accumulation of cerebrospinal fluid (CSF), typically within the brain ventricular system. Pediatric hydrocephalus can be primary (due to genetic abnormalities or idiopathic causes), or secondary to injuries such as hemorrhage, trauma, or infection. The current permanent treatment paradigms for pediatric hydrocephalus are exclusively surgical and include the diversion of CSF via shunt or ventriculostomy. These surgical interventions are wrought with failures, burdening both the United States healthcare system and patients with repeat neurosurgical procedures. Thus, the development of nonsurgical interventions to treat hydrocephalus represents a clinically unmet need. To study hydrocephalus, we use a genetic rat model of primary neonatal hydrocephalus, the <em>Tmem67</em>P394L mutant. In several proof-of-concept studies, we identify antagonism of the transient receptor potential vanilloid 4 (TRPV4) channel and associated upstream regulatory kinase, serum-and-glucocorticoid-induced kinase 1 (SGK1) as therapeutics for the treatment of hydrocephalus. Using <em>in vitro</em> models of the choroid plexus epithelium, the tissue which produces CSF, we show compelling proof-of-mechanism for TRPV4 antagonism and SGK1 inhibition at preventing CSF production. Therefore, the studies in this dissertation provide substantive evidence on the role of TRPV4 in the choroid plexus in health and disease. </p>

<p dir="ltr">Protein phosphorylation is a ubiquitous post-translational modification controlled by the opposing activities of protein kinases and phosphatases, which regulate diverse biological processes in all kingdoms of life. One of the key challenges to a complete understanding of phosphoregulatory networks is the unambiguous identification of kinase and phosphatase substrates. Liquid chromatography-coupled mass spectrometry (LC-MS/MS) and associated phosphoproteomic tools enable global surveys of phosphoproteome changes in response to signaling events or perturbation of phosphoregulatory network components. Despite the power of LC-MS/MS, it is still challenging to directly link kinases and phosphatases to specific substrate phosphorylation sites in many experiments. Here we described two methods for the LC-MS/MS-based characterization of protein phosphatases and kinases. The first is an <i>in-vitro</i> method designed to probe the inherent substrate specificity of kinase or phosphatases. This method utilizes an enzyme reaction with synthetic peptides, serving served as substrate proxies, coupled with LC-MS/MS for rapid, accurate high-throughput quantification of the specificity constant (<i>k</i>_<em>cat</em><i>/K</i>_<em>M</em>) for each substrate in the reaction and amino acid preference in the enzyme active site, providing insight into their cellular roles. The second couple’s auxin-inducible degradation system (AID) with phosphoproteomics for protein functional characterization. AID is a surrogate for specific chemical inhibition, which minimizes non-specific effects associated with long-term target perturbation. Using this system, we demonstrate-PP2A in complex with its B-subunit Rox Three Suppressor 1 (PP2A^Rts1) contributes to the phosphoregulation of a conserved fungal-specific membrane protein complex called the eisosome. By maintaining eisosomes in their hypophosphorylated state, PP2A^Rts1 aids fungal cells in preserving metabolic homeostasis. This work demonstrates the power of mass spectrometry as a critical tool for protein functional characterization.</p>

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24665号 / 農博第2548号 / 新制||農||1098(附属図書館) / 学位論文||R5||N5446(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 阪井 康能, 教授 木岡 紀幸, 教授 井上 善晴 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

<p>  </p> <p>Calcium influx into pancreatic beta-cells is required for proper beta-cell growth and function. While the functional significance of calcium influx into the beta-cells is known, the significance of release of calcium from intracellular stores is less understood. Calcium-induced calcium release (CICR) is a process by which calcium influx into the cell through voltage-gated calcium channels activated release of calcium from intracellular stores. The functional significance of CICR is well understood in cardiac and vascular muscle cells in regard to excitation-contraction coupling. However, the functional significance of CICR in beta-cells in not well understood. </p> <p>To investigate the role of RyR2 in pancreatic beta-cell function, we utilized CRISPR-Cas9 gene editing to delete RyR2 from the rat insulinoma INS-1 cell line. we found that RyR2KO cells displayed an enhanced glucose-stimulated Ca2+ integral (area under the curve; AUC) which was sensitive to inhibition by the IP3R antagonist, xestospongin C. Loss of RyR2 also resulted in a reduction in IRBIT protein levels. Therefore, we deleted IRBIT from INS-1 cells (IRBITKO) and found that IRBITKO cells also displayed an increased Ca2+ AUC in response to glucose stimulation. RyR2 KO and IRBIT KO cells had reduced glucose-stimulated insulin secretion and insulin content. RT-qPCR revealed that <em>INS2</em> transcript levels were reduced in both RyR2KO and IRBITKO. Nuclear localization of AHCY were increase in both the RyR2KO and IRBITKO cells, corresponding with increased levels of insulin gene methylation. Proteomic analysis revealed that deletion of RyR2 or IRBIT resulted in differential regulation of 314 and 137 proteins, respectively, with 41 in common. Our results suggest that RyR2 and IRBIT activity regulate insulin content, insulin secretion, and regulate the proteome in INS-1 cells</p> <p>We next sought to assess the consequences on cellular Ca2+ handling in the absence of RyR2 and IRBIT in INS-1 cells. Store-operated Ca2+ entry (SOCE) stimulated with thapsigargin was reduced in RyR2KO cells compared to controls, but this was not different in IRBITKO cells. STIM1 protein levels were not different between the three cell lines. Basal and carbachol stimulated phospholipase C (PLC) activity was reduced specifically in RyR2KO cells and not IRBITKO cells. However, basal PIP2 levels were elevated in both RyR2KO and IRBITKO cells. Insulin secretion stimulated by tolbutamide was reduced in RyR2KO and IRBITKO cells compared to controls, but this was still potentiated by an EPAC-selective cAMP analog in all three cell lines. Cortical f-actin is known to regulate insulin secretion, and levels were markedly reduced in RyR2KO cells compared to control INS-1 cells. Whole-cell Cav channel current density was reduced in RyR2KO cells compared to controls, and Ba2+ current was significantly reduced by PIP2 depletion preferentially in RyR2KO cells over control INS-1 cells. Action potentials stimulated by 18 mM glucose were more frequent in RyR2KO cells compared to controls, and insensitive to the SK channel inhibitor apamin. Taken together, these results suggest that RyR2 plays a critical role in regulating PLC activity and PIP2 levels via regulation of SOCE. RyR2 also regulates beta-cell electrical activity by controlling Cav current density, via regulation of PIP2 levels, and SK channel activation.</p> <p>Lastly, we investigated the role of PDE subtypes cAMP in INS-1 cells and human islets. We utilized subtype selective inhibitors of PDE1, PDE3 and PDE8 to assess the potential of these PDEs as potential therapeutic targets. We found that PDE1 is the primary subtype in INS-1 cells, whereas PDE3 appears to be required in human pancreatic β-cells by cAMP measurements. PDE1 inhibition potentiated glucose-stimulated to the greatest extent in both INS-1 cells and human islets. PDE1 inhibition potentiated CREB phosphorylation to the greatest extent and was also capable of mitigating lipotoxicity in INS-1 cells. Collectivity, this work highlights the role of cAMP compartmentalized signaling in pancreatic β-cells, and this has drastic effects on pancreatic beta-cell function and survival.</p>

<p>Regulators of G protein signaling (RGS) proteins are key negative regulators of Gα signaling, a branch of G-protein-coupled receptor (GPCR)-mediated signal transduction. Approximately 35% of drugs approved by the Food and Drug Administration (FDA) target GPCRs, so it is not surprising that the discovery of RGS proteins has triggered an interest in them as new drug targets. Even though many studies have been shown the involvement of RGS proteins in cancers, there is still a knowledge gap in understanding function and regulation of RGS proteins in these diseases. Consequently, in this thesis, I explored roles of two RGS proteins that have been implicated in cancers.</p> <p>RGS2 is proposed to act as a tumor suppressor in many different cancers, such as breast cancer, bladder, and ovarian cancer. Here, we investigated if RGS2 also plays a tumor suppressor role in UM, whose growth is driven by overactivated Gαq/11 signaling. We found that increased expression levels of RGS2 inhibit cell growth of UM 92.1 and Mel-202 cells. Mechanistically, this cell growth inhibition is dependent on the association between RGS2 and Gαq, but independent of its canonical GTPase-accelerating protein (GAP) activity. Furthermore, RGS2 inhibited the Mitogen-activated protein kinases (MAPK) signaling, downstream of Gαq, while leaving Yes-associated protein 1/Transcriptional coactivator with PDZ-binding motif (YAP/TAZ) activation unaffected. These data indicate a tumor suppressor role for RGS2 in UM and proposes RGS2 stabilization as a potential therapeutic targeting strategy. </p> <p>In contrast to RGS2, RGS20 contributes to cancer progression, particularly in breast cancer. However, how RGS20 is regulated is understudied. Palmitoylation, a reversible post-translational modification, regulates functions of other RGS proteins, and RGS20 is predicted to be palmitoylated. We provided direct evidence of RGS20 palmitoylation in cells and validated the palmitoylation site as the conserved cysteine (Cys148) in the RGS domain. Our results showed that palmitoylation on this site does not affect its GAP activity and subcellular localization, but it affects the association between RGS20 and active Gαo, and inhibition of Gαo-mediated signaling. This study serves as a foundation for future studies in furthering understating the role of palmitoylation in RGS20 function and its possible implications in cancer biology. </p>

Previous research discovered the existence of sedimentation-resistant mutants of E. coli. Genomic studies revealed that these mutants resisted sedimentation due to independent modifications to genes that influenced the Rcs signal transduction pathway, causing increased secretion of an exopolysaccharide capsule comprised primarily of colanic acid. The Rcs system is responsible for detecting envelope stressors; consequently, ampicillin and osmotic stress were used to perturb the cellular envelope and study the response of the mutants compared to wild-type cells. It was found that the overproduction of colanic acid in the mutants confers some resistance to envelope stress; however, the mutants still behaved similarly to wild-type cells. The doubling times of the strains grown in sodium chloride solutions were calculated. A wavelength scan from 400 nm to 800 nm was performed on strains grown in different salt concentrations to determine if there were significant differences in light scattering between the wild-type and mutant cells. Further analysis was performed that, along with the doubling time data, suggested that wild-type cells may have turned on genes for capsule production in response to being grown in high salt concentrations. Additional research could be conducted to test this hypothesis, perhaps through the quantification of colanic acid through a methyl pentose assay for wild-type cultures grown with high salt concentrations. The idea that wild-type cells could digest colanic acid as a carbon source when lacking resources was also investigated with different preparations of colanic acid. One preparation of colanic acid showed promising results, which could indicate that bacteria are able to digest their capsule in a novel method to produce energy when starved. Again, additional investigation should be conducted to confirm these results. Other future experiments could study the metabolome of these mutants to determine if they have increased quantities of alarmones related to biofilm formation.

B cell development in chicken takes place in a specific primary lymphoid organ, the bursa of Fabricius. The bursa is considered to provide a microenvironment that promotes B lymphocyte survival and maturation. The most important maturation step in the bursa is the immunoglobulin (Ig) gene conversion, a process that is responsible for immunoglobulin repertoire in avian species. The Ig-gene conversion is strictly regulated, and only progenitors that are able to initiate the process will develop into fully functional B lymphocytes. In this study the late embryonic B lymphocyte stages are investigated, the bursal stem cell stage and the onset of Ig-gene conversion stage. Previous studies identified functional and phenotypic differences between the two stages, showing high rate of proliferation at both, but a significant increase in apoptotic activity at the onset of gene conversion stage. The molecular basis behind the initiation of Ig-gene conversion is not well understood. Here two approaches are presented to provide information on the B lymphocyte developmental process. In chapter II proteomic analysis of the two cell stages was performed. The proteins were sorted into functional groups and signal transductions pathways were identified that are associated with proliferation, differentiation, cell adhesion and apoptosis. The project identified differences in protein profiles that might explain the changes in B lymphocyte physiology and bursal microenvironment at the initiation of Ig-gene conversion. In chapter III the antigen recognized by a bursal secretory dendritic cell specific monoclonal antibody, GIIF3 was identified and cloned. The antigen was shown to be expressed by bursal secretory dendritic cells only during the late embryonic period. The antigen was identified as smooth muscle gamma actin. Futher work will investigate what role the gene plays in dendritic cell funtion.