Chemical Tools for Exploring Metabolite Interactions with Nuclear Receptors and Beyond

The identification and functional characterization of specific metabolite–protein interactions remains a major challenge in chemical biology and drug development. The functionalization of native metabolites and synthetic small molecules with bio-orthogonal detection tags (alkyne, azide, and others) has afforded chemical reporters for the biochemical analysis of metabolically labelled proteins, whereas functionalization with photo-crosslinkers enables non-covalent metabolite–protein interactions to be captured. While we and many others have employed this approach to different metabolites and synthetic ligands, the installation of bio-orthogonal detection tags and photo-crosslinkers to more complex and diverse small molecules can be challenging and limiting. In this regard, activity-based protein profiling (ABPP) using active site–directed probes in combination with small molecule competition studies allows the discovery of potential substrates and inhibitors of selective enzyme families. Moreover, the development of proteome-wide labelling of select amino acid (cysteine and lysine) has revealed new sites in proteins for function modulation. However, some key protein classes and specific ligand-binding domains have been inaccessible by these methods. New activity-based probes and targeted chemical reporters of specific protein families are thus needed to characterize their endogenous and exogenous ligands for fundamental biology and drug discovery. In this thesis, we describe two projects aimed at developing chemical probes to assist in small molecule target engagement and target deconvolution studies. In Chapter 1, we provide an overview of the origin and evolution of ABPP and related chemical reporter–based chemical proteomics methods. We also summarise recent developments in label-free methods that work with underivatized molecules. In Chapter 2, we describe the development of a ligand-directed probe (alk-GW9662) for a subset of nuclear receptors (NRs). NRs are a family of ligand-activated transcription factors that regulate diverse physiological processes in animals and are key targets for therapeutic development. Recent studies implicated NRs in host–microbiota interactions and suggested that various microbiota-derived metabolites may serve as ligands for NRs. We demonstrate alk-GW9662’s utility to assess target engagement of candidate ligands of peroxisome proliferator–activated receptor (PPAR) γ. We also explore repurposing alk-GW9662. We profile its target proteins using chemical proteomics and demonstrate that the probe can be used in target engagement study of other proteins. Overall, our results suggest that alk-GW9662 may be useful for target engagement analysis of candidate PPAR ligands and offer a starting scaffold in developing probes for other proteins. In Chapter 3, we describe efforts towards the combined use of proteolysis-targeting chimeras (PROTACs) and chemical proteomics in target deconvolution studies. We describe the design and synthesis of PROTACs that recruit putative E3 ligases. Overall, the projects described herein underline the utility of chemical tools such as ligand-directed probes and PROTACs in target engagement and target deconvolution studies

Role of adsorption kinetics in the low-temperature Si growth by gas-source molecular beam epitaxy: In situ observations and detailed modeling of the growth

科研費報告書収録論文(課題番号:12650025・基盤研究(C)(2)・H12～H13/研究代表者:末光, 眞希/有機ケイ素を用いたSiCエピタキシャル成長の表面化学

Periodic alternation between intake and exhaust of air in dynamic insulation

Dynamic insulation (DI) can recover heat lost in conduction by drawing cold outdoor air into indoor through an insulation wall in winter. A “breathing DI” system we proposed in the past has functions both as an insulated envelope and as a highly efficient heat exchanger for ventilation. It is alternated periodically that the outdoor air is drawn through half of walls made of breathable inorganic concrete (BIC) and the indoor air is exhausted through the other half of the BIC walls. In order to put the breathing DI system into practice in housing construction, this paper presents some studies from various points of view in addition to the past studies on heat and moisture transport based on laboratory experiments and numerical simulations. We first experimentally studied the filtering efficiency and clogging of a BIC panel. This showed that approximately 30 % of the atmospheric dust can be captured by a BIC panel and no clogging would occur for at least 10 years. We also measured the sorption and desorption of formaldehyde by a BIC panel to confirm the effectiveness of a BIC wall to sorb gaseous state formaldehyde. We furthermore constructed a new test house at Ibaraki, Japan, to confirm the thermal performance of the breathing DI system based on full scale experiments

On the Problems in Design and Performance of Earth Work for the General Utilization Rice Paddy Field in the Peaty Soil.

論文(Article)journal articl

Activation of plant immunity by exposure to dinitrogen pentoxide gas generated from air using plasma technology

Reactive nitrogen species (RNS) play an important role in plant immunity as signaling factors. We previously developed a plasma technology to partially convert air molecules into dinitrogen pentoxide (N(2)O(5)), an RNS whose physiological action is poorly understood. To reveal the function of N(2)O(5) gas in plant immunity, Arabidopsis thaliana was exposed to plasma-generated N(2)O(5) gas once (20 s) per day for 3 days, and inoculated with Botrytis cinerea, Pseudomonas syringae pv. tomato DC3000 (Pst), or cucumber mosaic virus strain yellow (CMV(Y)) at 24 h after the final N(2)O(5) gas exposure. Lesion size with B. cinerea infection was significantly (P < 0.05) reduced by exposure to N(2)O(5) gas. Propagation of CMV(Y) was suppressed in plants exposed to N(2)O(5) gas compared with plants exposed to the air control. However, proliferation of Pst in the N(2)O(5)-gas-exposed plants was almost the same as in the air control plants. These results suggested that N(2)O(5) gas exposure could control plant disease depending on the type of pathogen. Furthermore, changes in gene expression at 24 h after the final N(2)O(5) gas exposure were analyzed by RNA-Seq. Based on the gene ontology analysis, jasmonic acid and ethylene signaling pathways were activated by exposure of Arabidopsis plants to N(2)O(5) gas. A time course experiment with qRT-PCR revealed that the mRNA expression of the transcription factor genes, WRKY25, WRKY26, WRKY33, and genes for tryptophan metabolic enzymes, CYP71A12, CYP71A13, PEN2, and PAD3, was transiently induced by exposure to N(2)O(5) gas once for 20 s peaking at 1–3 h post-exposure. However, the expression of PDF1.2 was enhanced beginning from 6 h after exposure and its high expression was maintained until 24–48 h later. Thus, enhanced tryptophan metabolism leading to the synthesis of antimicrobial substances such as camalexin and antimicrobial peptides might have contributed to the N(2)O(5)-gas-induced disease resistance