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Ras acts as a molecular switch to control intracellular signaling on the plasma membrane (PM). Elucidating how Ras associates with PM in the native cellular environment is crucial for understanding its control mechanism. Here, we used in-cell nuclear magnetic resonance (NMR) spectroscopy combined with site-specific 19 F-labeling to explore the membrane-associated states of H-Ras in living cells. The site-specific incorporation of p -trifluoromethoxyphenylalanine (OCF 3 Phe) at three different sites of H-Ras, i.e., Tyr32 in switch I, Tyr96 interacting with switch II, and Tyr157 on helix α5, allowed the characterization of their conformational states depending on the nucleotide-bound states and an oncogenic mutational state. Exogenously delivered 19 F-labeled H-Ras protein containing a C-terminal hypervariable region was assimilated via endogenous membrane-trafficking, enabling proper association with the cell membrane compartments. Despite poor sensitivity of the in-cell NMR spectra of membrane-associated H-Ras, the Bayesian spectral deconvolution identified distinct signal components on three 19 F-labeled sites, thus offering the conformational multiplicity of H-Ras on the PM. Our study may be helpful in elucidating the atomic-scale picture of membrane-associated proteins in living cells., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Ficifolidione, a natural insecticidal compound isolated from the essential oils of Myetaceae species, is a spiro phloroglucinol with an isobutyl group at the C-4 position. We found that ficifolidione showed cytotoxicity against cancer cells via apoptosis. Replacement of the isobutyl group by n -propyl group did not influence the potency, but the effect of the replacement of this group by a shorter or longer alkyl group on the biological activity remains unknown. In this study, ficifolidione derivatives with alkyl groups such as methyl, n -pentyl, and n -heptyl group-instead of the isobutyl group at the C-4 position-were synthesized to evaluate their cytotoxicity against the human promyelocytic leukaemia cell line HL60 and their insecticidal activity against mosquito larvae. The biological activities of their corresponding 4-epimers were also evaluated. As a result, the conversion of the isobutyl group to another alkyl group did not significantly influence the cytotoxicity or insecticidal activity. In HL60 cells treated with the n -heptyl-ficifolidione derivative, the activation of caspase 3/7 and the early stages of apoptosis were detected by using immunofluorescence and flow cytometric techniques, respectively, suggesting that the cytotoxicity should be induced by apoptosis even though the alkyl group was changed.

To obtain biocontrol agents for suppression of food-deteriorating fungi during storage of agricultural products, bacteria producing volatile organic compounds (VOCs) with strong antifungal activity were screened and isolated from various environmental samples. Among 136 bacterial isolates, strain TM-R showed the strongest and broadest antifungal activity. Based on physiological and genetical characterization, the bacterium was identified as Bacillus pumilus . The effects of VOCs produced by the bacterium, which was grown on four types of agar media (nutrient, Trypto-Soya, Luria-Bertani, and TM Enterprise), were examined against six species of fungi ( Alternaria alternata , Aspergillus niger, Cladosporium cladosporioides , Curvularia lunata , Fusarium oxysporum , and Penicillium italicum) in both small- and large-scale tests (plate and 12-L tests, respectively). In the plate test, the bacterium markedly suppressed the mycelial growth of five fungi ( Alternaria alternata , Cladosporium cladosporioides , Curvularia lunata , F. oxysporum , and P. italicum ) but promoted the growth of Aspergillus niger . In the 12-L test, the degree of growth inhibitiondecreased; however, the bacterium grown on TMEA still exhibited the strongest inhibition, especially against P. italicum (growth inhibition rate of 93%). Surprisingly, the growth of Aspergillus niger was promoted even more strongly (-36%) by the bacterium on TMEA than in the plate test (-9%). Twenty-two of 32 VOCs detected by GC-MS were identified using three databases (NIST 2011, AromaOffice, and AroChemBase). The species and concentration of detected VOCs differed greatly among growth media. To identify causative antifungal VOCs, we estimated the correlation between growth inhibition of P. italicum by the bacterium grown on each of the four media vs. the relative abundance of individual VOCs. As a result, four VOCs (methyl isobutyl ketone, ethanol, 5-methyl-2-heptanone, and S-(-)-2-methylbutylamine) were determined to be the predominant antifungal VOCs. To the best of our knowledge, this study is the first to specify causative antifungal VOCs using such an approach.

SWIRM is an evolutionarily conserved domain involved in several chromatin-modifying complexes. Recently, the LSD1 protein, which bears a SWIRM domain, was found to be a demethylase for Lys4-methylated histone H3. Here, we report a solution structure of the SWIRM domain of human LSD1. It forms a compact fold composed of 6 alpha helices, in which a 20 amino acid long helix (alpha4) is surrounded by 5 other short helices. The SWIRM domain structure could be divided into the N-terminal part (alpha1-alpha3) and the C-terminal part (alpha4-alpha6), which are connected to each other by a salt bridge. While the N-terminal part forms a SWIRM-specific structure, the C-terminal part adopts a helix-turn-helix (HTH)-related fold. We discuss a model in which the SWIRM domain acts as an anchor site for a histone tail.

Among the many PWWP-containing proteins, the largest group of homologous proteins is related to hepatoma-derived growth factor (HDGF). Within a well-conserved region at the extreme N-terminus, HDGF and five HDGF-related proteins (HRPs) always have a PWWP domain, which is a module found in many chromatin-associated proteins. In this study, we determined the solution structure of the PWWP domain of HDGF-related protein-3 (HRP-3) by NMR spectroscopy. The structure consists of a five-stranded beta-barrel with a PWWP-specific long loop connecting beta2 and beta3 (PR-loop), followed by a helical region including two alpha-helices. Its structure was found to have a characteristic solvent-exposed hydrophobic cavity, which is composed of an abundance of aromatic residues in the beta1/beta2 loop (beta-beta arch) and the beta3/beta4 loop. A similar ligand binding cavity occurs at the corresponding position in the Tudor, chromo, and MBT domains, which have structural and probable evolutionary relationships with PWWP domains. These findings suggest that the PWWP domains of the HDGF family bind to some component of chromatin via the cavity.