Conditions of establishment for the Salix community at lower-than-normal water levels along a dam reservoir shoreline

In limited-water-level reservoirs, areas along the reservoir shoreline are often exposed within the drawdown zone when water levels are reduced from normal to limited levels during the flood season. To prevent erosion and conserve the landscape, test plantings and landscaping along the shoreline have been implemented at some dam reservoirs. The establishment of vegetation within the drawdown zone contributes to both landscape and wildlife habitat conservation. Distribution, habitat, and seed-dispersal period of willows such as Salix subfragilis were investigated at Miharu Dam reservoir in northeastern Japan. Eight willow species were found around the dam, but S. subfragilis dominated within the drawdown zone. Field survey results beginning in 1995 indicate that the size of the S. subfragilis community increased after initial impoundment. Although many other Salix species disperse seeds prior to the drawdown period, S. subfragilis disperses during and after drawdown in the spring; thus, when water levels are lower than normal, its seeds are supplied to the newly exposed zones that provide suitable habitat for Salix seedling establishment. Our results suggest that S. subfragilis dominated because of water-resistant properties and timing of the seed dispersal period.     

Three-Step Photoconversion of Only Three Subunits of the Water-Soluble Chlorophyll-Binding Protein Tetramer from Chenopodium album

Water-soluble chlorophyll (Chl)-binding proteins (WSCPs) have been found in various plants. WSCPs are categorized into two classes based on their photoconvertibility: Class I (photoconvertible) and Class II (non-photoconvertible). Based on their absorption peaks, which occur in the red wavelengths, the pre- and post-photoconverted forms of Chenopodium album WSCP (CaWSCP) are called CP668 and CP742, respectively. Although various biochemical and biophysical properties of CaWSCP have already been characterized, questions remain regarding the structural dynamics of the photoconversion from CP668 to CP742, and the relationship between the photoconversion activity and incident light wavelength. To address how the wavelength of incident light affects the photoconversion, we performed time-course analyses of CaWSCP photoconversion by using light-emitting diodes that emit either white light, or at the discrete wavelengths 670, 645, 525, 470, or 430 nm. The most efficient photoconversion was observed under irradiation at 430 nm. Less efficient photoconversion was observed under irradiation with 670, 645, 470, or 525 nm light, in that order. The relationship between photoconversion activity and wavelength corresponded with the absorption peak intensities of Chls in the CaWSCP complex. The observed time dependence of the A₇₄₂/A₆₆₈ ratio during photoconversion of the CaWSCP complex indicated that the photoconversion from CP668 to CP742 occurs in a three-step reaction, and that only three subunits in the complex could be photoconverted.     

The CHLI1 subunit of Arabidopsis thaliana magnesium chelatase is a target protein of the chloroplast thioredoxin

Insertion of magnesium into protoporphyrin IX by magnesium chelatase is a key step in the chlorophyll biosynthetic pathway, which takes place in plant chloroplasts. ATP hydrolysis by the CHLI subunit of magnesium chelatase is an essential component of this reaction, and the activity of this enzyme is a primary determinant of the rate of magnesium insertion into the chlorophyll molecule (tetrapyrrole ring). Higher plant CHLI contains highly conserved cysteine residues and was recently identified as a candidate protein in a proteomic screen of thioredoxin target proteins (Balmer, Y., Koller, A., del Val, G., Manieri, W., Schurmann, P., and Buchanan, B. B. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 370-375). To study the thioredoxin-dependent regulation of magnesium chelatase, we first investigated the effect of thioredoxin on the ATPase activity of CHLI1, a major isoform of CHLI in Arabidopsis thaliana. The ATPase activity of recombinant CHLI1 was found to be fully inactivated by oxidation and easily recovered by thioredoxin-assisted reduction, suggesting that CHLI1 is a target protein of thioredoxin. Moreover, we identified one crucial disulfide bond located in the C-terminal helical domain of CHLI1 protein, which may regulate the binding of the nucleotide to the N-terminal catalytic domain. The redox state of CHLI was also found to alter in a light-dependent manner in vivo. Moreover, we successfully observed stimulation of the magnesium chelatase activity in isolated chloroplasts by reduction. Our findings strongly suggest that chlorophyll biosynthesis is subject to chloroplast biogenesis regulation networks to coordinate them with the photosynthetic pathways in chloroplasts.     

Development of human hepatocellular carcinoma cell-targeted protein cages

We described herein a human hepatocellular carcinoma (HCC) cell-targeted protein cage for which the HCC-binding peptide termed SP94 was modified at the surface of a naturally occurred heat shock protein (Hsp) cage. Six types of HCC-targeted Hsp cages were chemically synthesized using two types of heterobifunctional linker (SM(PEG)(n)) with different lengths and two types of SP94 peptide, which contained a unique Cys residue at the N- or C-terminus of the peptide. These Hsp cages were characterized using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF MS) analyses, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses, and dynamic light scattering (DLS) measurement. Fluorescence microscopic observations revealed that all the engineered protein cages bind selectively to HCC cells but not to the other cell lines tested (including normal liver cell). Moreover, the number of SP94 peptides on Hsp cages, conjugation site of SP94 peptide, and linker length between a Hsp cage and a SP94 peptide had important effects upon the binding of engineered Hsp cages to HCC cells. An engineered Hsp cage conjugated to the N-terminus of SP94 peptide via a longer linker molecule and containing high SP94 peptide levels showed greater binding toward HCC cells. Surprisingly, through optimization of these three factors, up to 10-fold greater affinity toward HCC cells was achieved. These results are critically important not only for the development of HCC cell-targeting devices using SP94 peptide, but also to create other cell-targeting materials that utilize other peptide ligands.     

Phospholipid Flippase Activities and Substrate Specificities of Human Type IV P-type ATPases Localized to the Plasma Membrane

Type IV P-type ATPases (P4-ATPases) are believed to translocate aminophospholipids from the exoplasmic to the cytoplasmic leaflets of cellular membranes. The yeast P4-ATPases, Drs2p and Dnf1p/Dnf2p, flip nitrobenzoxadiazole-labeled phosphatidylserine at the Golgi complex and nitrobenzoxadiazole-labeled phosphatidylcholine (PC) at the plasma membrane, respectively. However, the flippase activities and substrate specificities of mammalian P4-ATPases remain incompletely characterized. In this study, we established an assay for phospholipid flippase activities of plasma membrane-localized P4-ATPases using human cell lines stably expressing ATP8B1, ATP8B2, ATP11A, and ATP11C. We found that ATP11A and ATP11C have flippase activities toward phosphatidylserine and phosphatidylethanolamine but not PC or sphingomyelin. By contrast, ATPase-deficient mutants of ATP11A and ATP11C did not exhibit any flippase activity, indicating that these enzymes catalyze flipping in an ATPase-dependent manner. Furthermore, ATP8B1 and ATP8B2 exhibited preferential flippase activities toward PC. Some ATP8B1 mutants found in patients of progressive familial intrahepatic cholestasis type 1 (PFIC1), a severe liver disease caused by impaired bile flow, failed to translocate PC despite their delivery to the plasma membrane. Moreover, incorporation of PC mediated by ATP8B1 can be reversed by simultaneous expression of ABCB4, a PC floppase mutated in PFIC3 patients. Our findings elucidate the flippase activities and substrate specificities of plasma membrane-localized human P4-ATPases and suggest that phenotypes of some PFIC1 patients result from impairment of the PC flippase activity of ATP8B1.     

Discovery of highly reactive peptide tag by ELISA-type screening for specific cysteine conjugation

We report the discovery of a highly reactive peptide tag for the specific cysteine conjugation of proteins. Screening of cysteine-containing peptides using ELISA-type screening yielded a 19-amino acid tag (DCPPPDDAADDAADDAADD), named DCP3 tag, which enabled the rapid and selective labeling of the tag-fused protein with a synthetic zinc complex on the surface of living cells.     

Genome-wide screening identified SEC61A1 as an essential factor for mycolactone-dependent apoptosis in human premonocytic THP-1 cells

Buruli ulcer is a chronic skin disease caused by a toxic lipid mycolactone produced by Mycobacterium ulcerans, which induces local skin tissue destruction and analgesia. However, the cytotoxicity pathway induced by mycolactone remains largely unknown. Here we investigated the mycolactone-induced cell death pathway by screening host factors using a genome-scale lenti-CRISPR mutagenesis assay in human premonocytic THP-1 cells. As a result, 884 genes were identified as candidates causing mycolactone-induced cell death, among which SEC61A1, the α-subunit of the Sec61 translocon complex, was the highest scoring. CRISPR/Cas9 genome editing of SEC61A1 in THP-1 cells suppressed mycolactone-induced endoplasmic reticulum stress, especially eIF2α phosphorylation, and caspase-dependent apoptosis. Although previous studies have reported that mycolactone targets SEC61A1 based on mutation screening and structural analysis in several cell lines, we have reconfirmed that SEC61A1 is a mycolactone target by genome-wide screening in THP-1 cells. These results shed light on the cytotoxicity of mycolactone and suggest that the inhibition of mycolactone activity or SEC61A1 downstream cascades will be a novel therapeutic modality to eliminate the harmful effects of mycolactone in addition to the 8-week antibiotic regimen of rifampicin and clarithromycin.