区分同位体標識を用いたβ2アドレナリン受容体のリン酸化によるシグナル制御機構の解明

学位の種別: 課程博士審査委員会委員 : （主査）東京大学教授 嶋田 一夫, 東京大学教授 船津 高志, 東京大学准教授 花岡 健二郎, 東京大学特任准教授 加藤 大, 東京大学講師 大戸 梅治University of Tokyo(東京大学

Phosphorylation-induced conformation of beta(2)-adrenoceptor related to arrestin recruitment revealed by NMR

The C-terminal region of G-protein-coupled receptors (GPCRs), stimulated by agonist binding, is phosphorylated by GPCR kinases, and the phosphorylated GPCRs bind to arrestin, leading to the cellular responses. To understand the mechanism underlying the formation of the phosphorylated GPCR-arrestin complex, we performed NMR analyses of the phosphorylated beta(2)-adrenoceptor (beta(2)AR) and the phosphorylated beta(2)AR-beta-arrestin 1 complex, in the lipid bilayers of nanodisc. Here we show that the phosphorylated C-terminal region adheres to either the intracellular side of the transmembrane region or lipids, and that the phosphorylation of the C-terminal region allosterically alters the conformation around M215(5.54) and M279(6.41), located on transemembrane helices 5 and 6, respectively. In addition, we found that the conformation induced by the phosphorylation is similar to that corresponding to the beta-arrestin-bound state. The phosphorylation-induced structures revealed in this study propose a conserved structural motif of GPCRs that enables beta-arrestin to recognize dozens of GPCRs.Peer reviewe

Dramatic Transcriptional Changes in an Intracellular Parasite Enable Host Switching between Plant and Insect

Phytoplasmas are bacterial plant pathogens that have devastating effects on the yields of crops and plants worldwide. They are intracellular parasites of both plants and insects, and are spread among plants by insects. How phytoplasmas can adapt to two diverse environments is of considerable interest; however, the mechanisms enabling the “host switching” between plant and insect hosts are poorly understood. Here, we report that phytoplasmas dramatically alter their gene expression in response to “host switching” between plant and insect. We performed a detailed characterization of the dramatic change that occurs in the gene expression profile of Candidatus Phytoplasma asteris OY-M strain (approximately 33% of the genes change) upon host switching between plant and insect. The phytoplasma may use transporters, secreted proteins, and metabolic enzymes in a host-specific manner. As phytoplasmas reside within the host cell, the proteins secreted from phytoplasmas are thought to play crucial roles in the interplay between phytoplasmas and host cells. Our microarray analysis revealed that the expression of the gene encoding the secreted protein PAM486 was highly upregulated in the plant host, which is also observed by immunohistochemical analysis, suggesting that this protein functions mainly when the phytoplasma grows in the plant host. Additionally, phytoplasma growth in planta was partially suppressed by an inhibitor of the MscL osmotic channel that is highly expressed in the plant host, suggesting that the osmotic channel might play an important role in survival in the plant host. These results also suggest that the elucidation of “host switching” mechanism may contribute to the development of novel pest controls

Degradation of ionic liquids by a UV/H2O2 process and CMCase from novel ionic liquid-tolerant alkaliphilic Nocardiopsis sp. SSC4

We demonstrated the degradation of two ionic liquids (1-butyl-3-methylimidazolium chloride, [BMIM]Cl, and 1-ethylpyridinium bromide, [EtPy]Br) that are useful for the solubilization of wood components. [BMIM]+ and [EtPy]+ were detected by thin-layer chromatography (TLC) and electrospray ionization–mass spectrometry (ESI-MS). [BMIM]+ was harder to degrade than [EtPy]+. Ultraviolet (UV) irradiation with 0.2% (v/v) H2O2 for 16 h degraded 1 mmol/L [BMIM]+, whereas UV irradiation alone degraded 1 mmol/L [EtPy]+. Additionally, we isolated an ionic liquid-tolerant alkaliphilic actinomycete, Nocardiopsis sp. SSC4. Strain SSC4 produced carboxymethylcellulase (CMCase) in the presence of 1.0% (v/v, 48.1 mmol/L) 1-ethyl-3-methylimidazolium trifluoromethanesulphonate ([EMIM]CF3SO3), which is useful for the extraction of cellulose-rich materials from wood. In the case of strain SSC4, CMCase was inducibly synthesized by more than 0.5% CMC. The addition of 0%–1.0% tryptone or 0%–2.0% yeast extract decreased the CMCase activity in a concentration-dependent manner. After cultivation of strain SSC4 with 1.0% (w/v) CMC medium (pH 9.0) for 48 h at 37 °C, the culture supernatant exhibited CMCase activity at 0.03 U/mg. The optimum reaction temperature of CMCase was 45 °C. CMCase was stable up to 37 °C for 20 h incubation. The degradation characteristics of [BMIM]+ and [EtPy]+ and the activity of CMCase in the presence of [EMIM]CF3SO3 may be useful for the development of a bioconversion system for biomass resources

Function-Related Dynamics in Multi-Spanning Helical Membrane Proteins Revealed by Solution NMR

A primary biological function of multi-spanning membrane proteins is to transfer information and/or materials through a membrane by changing their conformations. Therefore, particular dynamics of the membrane proteins are tightly associated with their function. The semi-atomic resolution dynamics information revealed by NMR is able to discriminate function-related dynamics from random fluctuations. This review will discuss several studies in which quantitative dynamics information by solution NMR has contributed to revealing the structural basis of the function of multi-spanning membrane proteins, such as ion channels, GPCRs, and transporters

Effects of Helicobacter pylori Infection on Mucin Phenotype of Early Differentiated Gastric Carcinoma; Study with Gastric Mucosal Specimens Obtained by Endoscopic Mucosal Resection

We investigated the effect of Helicobacter pylori (H. pylori) infection on development of early differentiated gastric cancer with regard to mucin phenotypic expression and cancer’s surrounding mucosa. The H. pylori antibody (HP-Ab) positive rate of patients with gastric cancer was significantly higher than that of control. Furthermore, we classified the gastric cancer group according to mucin histochemistry and immunohistochemistry into 3 types, namely gastric type, intestinal type and gastric-intestinal type whose occurrence rates were 34%, 35% and 31%, respectively. The surrounding mucosa of the intestinal phenotype cancer showed severe atrophy and high degree of intestinal metaplasia but its H. pylori density was very low. On the contrary, the surrounding mucosa of the gastric phenotype cancer revealed mild atrophy and mild intestinal metaplasia whereas the H. pylori density was relatively high. However, no statistically significant difference was observed in HP-Ab positive rate between these cancer types. Moreover, we found that the tumor diameter was inversely correlated with the incidence of the gastric phenotype cancer. These results indicate that intestinal phenotype cancer is related to atrophy and intestinal metaplasia of surrounding mucosa caused by H. pylori infection. Furthermore, we conclude that gastric phenotype cancer is associated with the early stage of H. pylori infection but not atrophy nor intestinal metaplasia