Neuroblastoma (NB) is the most common extracranial solid tumour in childhood. NB is thought to arise from the failed differentiation of neural crest progenitor cells that would normally form tissues of the adrenal gland and sympathetic nervous system. These neural crest progenitors then uncontrollably proliferate forming a tumour. Despite aggressive surgery and chemotherapy, the cure rate of high-risk NB patients remains below 30%. Our laboratory has shown that human NB tumour specimens and high-risk patient derived cell lines express pannexin 1 (PANX1), and that treatment with the PANX1 channel blockers carbenoxolone or probenecid constitute reduce NB progression in vitro and in vivo. PANX1 is a glycoprotein that forms single membrane channels best known to serve as conduits for ATP release. Interestingly, while PANX1 was also detected in control neurons by western blotting, its banding pattern was strikingly different as a band at around 50 kDa was found in all NB cell lines, but not in neurons. Using shRNA targeting PANX1 and deglycosylation enzymes, I have shown that this band corresponds to a PANX1 glycosylated species. PANX1 has been reported to be phosphorylated in NB at amino acid Y10. PANX1 is also predicted to be glycosylated at N255. In order to study the role of these post-translational modifications, myc-tagged Y10F and N255A PANX1 mutants were engineered by site-directed mutagenesis. Immunolocalization and cell surface biotinylation assays suggest that the localization both mutants at the cell surface is reduced compared to that of myc-PANX1. Dye uptake assays revealed that myc-Y10F has significantly reduced channel activity. Expression of myc-Y10F and myc-N255A in NB cells inhibited cell proliferation and decreased metastatic potential in vitro. Further analysis of NB tumour specimens revealed that there is a missense mutation in PANX1 resulting in the formation of truncated peptide (amino acid 1-99). Interestingly, I have found that when co-expressed with myc-PANX1, PANX11-99, reduced PANX1 channel activity. Taken together, these findings indicate that phosphorylation on Y10 and glycosylation on N255 regulate PANX1 channel activity and exacerbate NB malignancy, while the expression of PANX11-99 in NB may be beneficial.

Phosphoenolpyruvate carboxylase (EC 4.1.1.31; PEPC) is one of the carbon dioxide- fixing enzymes, which yields oxaloacetate from phosphoenolpyruvate and bicarbonate. Regulation of PEPC activity occurs at many levels. In addition to pH and concentration of activators and inhibitors, it is phosphorylation as well. Phosphorylation of PEPC causes a change of kinetic parameters, such as maximal reaction rate, sensitivity to activation or inhibition. Considering that, there is still little information like this about C3 plants and that regulation is in various plant species different, I have dealt with monitoring of the kinetic parameters and regulation possibilities of PEPC isolated from C3 plant sources (Cannabis sativa L., Chenopodium quinoa, Pisum sativum L., Lens culinaris). While the activity of PEPC from leaves of Cannabis sativa L. was decreased by alkaline phosphatase, the activity of PEPC from seeds of Chenopodium quinoa, Pisum sativum L., Lens culinaris was not affected by alkaline phosphatase. The affinity of PEPC from seeds Chenopodium quinoa, Pisum sativum L., Lens culinaris to the substrate PEP was higher than in the case of PEPC from leaves of Cannabis sativa L.. For PEPC from Cannabis sativa L. was found that the apparent dephosphorylation leads to decrease of sensitivity to the...

Transport of proteins to the nucleus through a nuclear envelope is controlled mostly via nuclear localization signal (NLS). Nuclear localization signal is rich in positively charged amino acids arginine and lysine. It was observed that activity of this NLS could be regulated through a phosphorylation of serine in its close proximity. Either a phosphorylation of serine or phosphomimetic changes of these "presequences" could represent an important mechanism regulating a localization of protein in cells in relation to a cellular activation. In our laboratory was identified protein - Fragile X mental retardation syndrome 1 neighbor (Fmr1nb), whose cellular localization could be driven by this posttranslational modification.

To define the overall kinetic and chemical mechanism of adenosine 3',5'-monophosphate dependent protein kinase catalytic subunit, the mechanism in the direction of MgADP phosphorylation was determined, using studies of initial velocity in the absence and presence of dead-end inhibitors. The kinetic mechanism was determined as a function of uncomplexed Mg^2+ (Mg_f) at pH 7.2 and as a function of pH at low (0.5 mM) Mg_f. At pH 7.2 data are consistent with a random kinetic mechanism in the direction of MgADP phosphorylation with both pathways allowed: the pathway in which MgADP binds to enzyme prior to phosphorylated peptide (PSP) and that in which PSP binds before MgADP. One or the other pathway predominates, depending on Mg_f concentration. At 0.5 mM Mg_f, the mechanism is steady-state ordered with the pathway where PSP binds first preferred; at 10 mM Mg_f, the mechanism is equilibrium ordered, and the pathway in which MgADP binds first preferred. This change in mechanism to equilibrium ordered is due to an increase in affinity of enzyme for MgADP and a decrease in affinity for PSP. There is also a pH-dependent change in mechanism at 0.5 mM Mg_f. At pH 6 the mechanism is equilibrium ordered with the pathway where PSP binds first preferred. At pH 7.6 the mechanism is ordered with MgADP binding first. The log V/E_t vs. pH profile is pH-independent, suggesting only the correctly protonated form of each substrate binds to enzyme. The log V/K_MgADP vs. PH profile gives a pK of 7, likely that of a general acid, which must be protonated for activity. The pK_iPSP vs. pH profile gives a pK of 6.5, likely reflecting the peptide phosphoryl group, which must be unprotonated for activity.

Growing evidence supports the role of transcription factor activation in the pathophysiology of inflammatory disorders, sepsis, ARDS, SIRS, and shock. Kinase mediated phosphorylation of IκBα is a crucial step in the NFκB activation pathway. We investigated IκBα phosphorylation in murine liver and lung extracts after cecal ligation and puncture (CLP) in the presence and absence of a glucan ligand. ICR mice were subjected to CLP. Unoperated and sham-operated mice served as the controls. Glucan phosphate (50 mg/kg) was administered 1 h before or 15 min after CLP. CLP increased hepatic and pulmonary levels of phospho-IκBα by 48-192%. Pre-or post-treatment with glucan phosphate decreased (P < 0.05) tissue phospho-IκBα levels in CLP mice. Phospho-IκBα in the glucan-CLP group were not significantly different from the unoperated controls. To investigate mechanisms we examined IKKβ kinase activity, IκBα phosphorylation and degradation, and NFκB activity in a murine macrophage cell line, J774a.1, treated with LPS (1 μg/mL) and/or glucan phosphate (1 μg/mL) for up to 120 min. The glucan ligand blunted LPS-induced IKKβ kinase activity, phosphorylation and degradation of IκBα, and NFκB nuclear binding activity. The data indicate that one mechanism by which (1→3)-β-D-glucan may alter the response to endotoxin or polymicrobial sepsis involves modulation of IKKβ kinase activity with subsequent decreases in IκBα phosphorylation and NFκB activation.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23551号 / 生博第462号 / 新制||生||62(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 中野 雄司, 教授 見学 美根子, 教授 千坂 修 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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Indiana University-Purdue University Indianapolis (IUPUI) / Parkinson disease (PD) is the second most common neurodegenerative disease. It is characterized by loss of dopaminergic cells in the substantia nigra, which causes loss of dopaminergic synapses onto striatal medium spiny neurons (MSNs). Dendritic spines that are localized to these striatal MSNs receive synaptic inputs from both the nigral dopamine neurons and cortical glutamate neurons. Signaling downstream of excitatory, glutamatergic drive is modulated by dopamine. This tripartite connection: glutamate, dopamine, and MSN dendritic spine, is important for normal motor function. Glutamate released from presynaptic terminals binds to and activates two classes of inotropic glutamate receptors that are localized to dendritic spines on striatal MSNs: the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and the N-methyl-D-aspartate receptor (NMDAR). Once these receptors are activated, they allow for Ca2+ influx, which in turn activates Ca2+-dependent processes that underlie neural plasticity, including long-term potentiation (LTP) and long-term depression (LTD). Proper machinery in the pre- and post-synaptic neurons is required for normal signal transduction. Moreover, this signal transduction requires proper organization of synaptic proteins, which is achieved by specific protein-protein interactions. These protein-protein interactions are dynamic and can be modulated under various conditions, including pathological changes in the phosphorylation status of a specific protein. Catalytically active proteins called phosphatases and kinases specifically regulate the phosphorylation status of synaptic proteins. Pathologically, in PD there is increased autophosphorylation and activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). This increased phosphorylation may be due to changes in the activity of the serine/threonine protein phosphatase 1 (PP1), a highly conserved protein serine/threonine phosphatase that has a diverse set of functions in eukaryotes. Serine/threonine phosphatase substrate specificity is obtained via interactions with targeting and regulatory proteins. One such protein, spinophilin, is a scaffolding protein that targets PP1 to various synaptic substrates to regulate their phosphorylation. Interestingly, the association of PP1 with spinophilin is enhanced in a rat model of PD. The NMDAR is another protein that has altered phosphorylation in animal models of PD. We have found that there is a decrease in the NMDAR-spinophilin interaction in an animal model of PD. Here, we have found that spinophilin and the NMDAR interact in brain tissue and when overexpressed in a mammalian cell system. Moreover, we have identified novel mechanisms that regulate this interaction and have identified putative consequences of altering this association. These studies give us novel insight into mechanisms and consequences underlying pathological changes observed in an animal model of PD. Understanding these changes will inform novel therapeutic targets that may be useful in modulating striatal function.

Indiana University-Purdue University Indianapolis (IUPUI) / Voltage-gated sodium channels (Navs) undergo remarkably complex modes of modulation to fine tune membrane excitability and neuronal firing properties. In neurons, the isoform Nav1.6 is highly enriched at the axon initial segment and nodes, making it critical for the initiation and propagation of neuronal impulses. Thus, Nav1.6 modulation and dysfunction may profoundly impact the input-output properties of neurons in normal and pathological conditions. Phosphorylation is a powerful and reversible mechanism that exquisitely modulates ion channels. To this end, the multifunctional calcium/calmodulin-dependent protein kinase II (CaMKII) can transduce neuronal activity through phosphorylation of diverse substrates to serve as a master regulator of neuronal function. Because Nav1.6 and CaMKII are independently linked to excitability disorders, I sought to investigate modulation of Nav1.6 function by CaMKII signaling to reveal an important mechanism underlying neuronal excitability. Multiple biochemical approaches show Nav1.6 is a novel substrate for CaMKII and reveal multi-site phosphorylation within the L1 domain; a hotspot for post-translational regulation in other Nav isoforms. Consistent with these findings, pharmacological inhibition of CaMKII reduces transient and persistent sodium currents in Purkinje neurons. Because Nav1.6 is the predominant sodium current observed in Purkinje neurons, these data suggest that Nav1.6 may be modulated through CaMKII signaling. In support of this, my studies demonstrate that CaMKII inhibition significantly attenuates Nav1.6 transient and persistent sodium currents and shifts the voltage-dependence of activation to more depolarizing potentials in heterologous cells. Interestingly, I show that these functional effects are likely mediated by CaMKII phosphorylation of Nav1.6 at S561 and T642, and that each phosphorylation site regulates distinct biophysical characteristics of the channel. These findings are further extended to investigate CaMKII modulation of disease-linked mutant Nav1.6 channels. I show that different Nav1.6 mutants display distinct responses to CaMKII modulation and reveal that acute CaMKII inhibition attenuates gain-of-function effects produced by mutant channels. Importantly, computational simulations modeling the effects of CaMKII inhibition on WT and mutant Nav1.6 channels demonstrate dramatic reductions in neuronal excitability in Purkinje and cortical pyramidal cell models. Together, these findings suggest that CaMKII modulation of Nav1.6 may be a powerful mechanism to regulate physiological and pathological neuronal excitability. / 2022-02-02

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20568号 / 医博第4253号 / 新制||医||1022(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 原田 浩, 教授 武藤 学, 教授 戸井 雅和 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM