標的タンパク質のリガンドを必要としないプロテインノックダウン法

学位の種別: 課程博士審査委員会委員 : （主査）東京大学教授 橋本 祐一, 東京大学教授 井上 将行, 東京大学教授 船津 高志, 東京大学教授 富田 泰輔, 東京大学准教授 花岡 健二郎University of Tokyo(東京大学

Penetration through Outer Membrane and Efflux Potential in <i>Pseudomonas aeruginosa</i> of Bulgecin A as an Adjuvant to β-Lactam Antibiotics

The treatment of infections by Gram-negative bacteria remains a difficult clinical challenge. In the light of the dearth of discovery of novel antibiotics, one strategy that is being explored is the use of adjuvants to enhance antibacterial activities of existing antibiotics. One such adjuvant is bulgecin A, which allows for the lowering of minimal-inhibitory concentrations for β-lactam antibiotics. We have shown that bulgecin A inhibits three of the pseudomonal lytic transglycosylases in its mode of action, yet high concentrations are needed for potentiation activity. Herein, we document that bulgecin A is not a substrate for pseudomonal efflux pumps, whose functions could have been a culprit in the need for high concentrations. We present evidence that the penetration barrier into the periplasm is at the root of the need for high concentrations of bulgecin A in its potentiation of β-lactam antibiotics

Penetration through Outer Membrane and Efflux Potential in Pseudomonas aeruginosa of Bulgecin A as an Adjuvant to &beta;-Lactam Antibiotics

The treatment of infections by Gram-negative bacteria remains a difficult clinical challenge. In the light of the dearth of discovery of novel antibiotics, one strategy that is being explored is the use of adjuvants to enhance antibacterial activities of existing antibiotics. One such adjuvant is bulgecin A, which allows for the lowering of minimal-inhibitory concentrations for &beta;-lactam antibiotics. We have shown that bulgecin A inhibits three of the pseudomonal lytic transglycosylases in its mode of action, yet high concentrations are needed for potentiation activity. Herein, we document that bulgecin A is not a substrate for pseudomonal efflux pumps, whose functions could have been a culprit in the need for high concentrations. We present evidence that the penetration barrier into the periplasm is at the root of the need for high concentrations of bulgecin A in its potentiation of &beta;-lactam antibiotics

Near-Infrared-Light-Activatable Proximity Labeling of Bead-Binding Proteins

Photocatalytic proximity labeling has recently undergone significant advances as a valuable tool for understanding protein–protein and cell–cell interactions. This paper reports the first photocatalytic protein-labeling approach in which the reaction can be controlled using near-infrared (NIR) light (810 nm). Magnetic affinity beads with encapsulated sulfur-substituted silicon (IV) phthalocyanine, which produces singlet oxygen upon NIR irradiation, were prepared. We have developed a method in which the histidine residues of proteins bound to the ligands on the beads are selectively oxidized and labeled by the nucleophilic labeling reagent while minimizing nonspecific adsorption to the dye. Beads with aryl sulfamide, lactose, or CZC-8004 ligands immobilized on their surface can be used to label proteins that bind these ligands, as well as their protein–protein interaction partners

Switching of Photocatalytic Tyrosine/Histidine Labeling and Application to Photocatalytic Proximity Labeling

Weak and transient protein interactions are involved in dynamic biological responses and are an important research subject; however, methods to elucidate such interactions are lacking. Proximity labeling is a promising technique for labeling transient ligand&ndash;binding proteins and protein&ndash;protein interaction partners of analytes via an irreversible covalent bond. Expanding chemical tools for proximity labeling is required to analyze the interactome. We developed several photocatalytic proximity-labeling reactions mediated by two different mechanisms. We found that numerous dye molecules can function as catalysts for protein labeling. We also identified catalysts that selectively modify tyrosine and histidine residues and evaluated their mechanisms. Model experiments using HaloTag were performed to demonstrate photocatalytic proximity labeling. We found that both ATTO465, which catalyzes labeling by a single electron transfer, and BODIPY, which catalyzes labeling by singlet oxygen, catalyze proximity labeling in cells

Total Syntheses of Bulgecins A, B, and C and Their Bactericidal Potentiation of the β‑Lactam Antibiotics

The bulgecins are iminosaccharide secondary metabolites of the Gram-negative bacterium <i>Paraburkholderia acidophila</i> and inhibitors of lytic transglycosylases of bacterial cell-wall biosynthesis and remodeling. The activities of the bulgecins are intimately intertwined with the mechanism of a cobiosynthesized β-lactam antibiotic. β-Lactams inhibit the penicillin-binding proteins, enzymes also critical to cell-wall biosynthesis. The simultaneous loss of the lytic transglycosylase (by bulgecin) and penicillin-binding protein (by β-lactams) activities results in deformation of the septal cell wall, observed microscopically as a bulge preceding bacterial cell lysis. We describe a practical synthesis of the three naturally occurring bulgecin iminosaccharides and their mechanistic evaluation in a series of microbiological studies. These studies identify potentiation by the bulgecin at subminimum inhibitory concentrations of the β-lactam against three pathogenic Gram-negative bacteria and establish for the first time that this potentiation results in a significant increase in the bactericidal efficacy of a clinical β-lactam