Isolation of organellar DNA from Codium fragile (Codiaceae, Codiales, Ulvophyceae)

Organellar DNA was isolated from Codium fragile (Suringar) Hariot (Codiaceae, Codiales, Ulvophyceae) by CsCI-buoyant density centrifugation in the presence of Hoechst dye 33258. Three bands were formed by ultracentrifugation and each fraction of DNA was identified by Southern hybridization. The uppermost fraction was identified as chloroplast DNA, the middle fraction was nuclear DNA and the bottom fraction was mitochondrial DNA. Nuclear rDNA was isolated in the same fraction as mitochondrial DNA. The estimated genome size of mitochondrial DNA by analysis with restriction endonucleases was more than 141.6 kb, which was larger than that of microalgae but smaller than land plants. Restriction endonuclease analysis of the chloroplast DNA showed no difference with that known of C. fragile in New York.     

Loss of Rab5 drives non-autonomous cell proliferation through TNF and Ras signaling in Drosophila

Deregulation of the endocytic machinery has been implicated in human cancers. However, the mechanism by which endocytic defects drive cancer development remains to be clarified. Here, we find through a genetic screen in Drosophila that loss of Rab5, a protein required for early endocytic trafficking, drives non-autonomous cell proliferation in imaginal epithelium. Our genetic data indicate that dysfunction of Rab5 leads to cell-autonomous accumulation of Eiger (a TNF homolog) and EGF receptor (EGFR), which causes activation of downstream JNK and Ras signaling, respectively. JNK signaling and its downstream component Cdc42 cooperate with Ras signaling to induce upregulation of a secreted growth factor Upd (an IL-6 homolog) through inactivation of the Hippo pathway. Such non-autonomous tissue growth triggered by Rab5 defect could contribute to epithelial homeostasis as well as cancer development within heterogeneous tumor microenvironment.     

Identification of the C-terminal portion of a protein by comparative peptide mapping

A method is presented for the simple identification of C-terminal fragment of proteins. The method consists of (i) C-terminal processing of a protein by carboxypeptidase and (ii) comparative peptide mapping of the intact and carboxypeptidase-excised protein after fragmentation by endoproteinase or by chemical cleavage. The peptide mapping was performed by means of high-performance reversed-phase chromatography, where the C-terminal fragment was identified as a peptide peak that was lost or decreased in the carboxypeptidase-excised protein. The C-terminal sequence of the protein could be then determined by sequential Edman degradation of the C-terminal fragment collected from the peptide mapping chromatography. The sensitivity of the method depends solely on the peptide detection and subsequent Edman degradation, currently available techniques of which require a nanomole to subnanomole quantity of protein. The present method can be coupled with conventional carboxypeptidase technology because it utilizes a protein portion remaining after carboxypeptidase digestion while released amino acids are needed in the conventional technique. The method would be particularly valuable in finding a gene probe site for a RNA message coding for the C-terminal portion of a molecule.     

Assessing photosynthetic efficiency in an experimental mangrove canopy using remote sensing and chlorophyll fluorescence

This study examined the ability of the photochemical reflectance index (PRI) to track changes in effective quantum yield (Δ F/F _m ′), non-photochemical quenching (NPQ), and the xanthophyll cycle de-epoxidation (DPS) in an experimental mangrove canopy. PRI was correlated with (Δ F/F _m ′) and NPQ over the 4-week measurement period and over the diurnal cycle. The normalised difference vegetation index (NDVI) was not correlated with any aspect of photochemical efficiency measured using chlorophyll fluorescence or xanthophyll pigments. This study demonstrated that photochemical adjustments were responsible for controlling the flow of energy through the photosynthetic apparatus in this mangrove forest canopy rather than canopy structural or chlorophyll adjustments.     

Reconstitution of blue-green reversible photoconversion of a cyanobacterial photoreceptor, PixJ1, in phycocyanobilin-producing Escherichia coli

PixJ1, a photoreceptor in the unicellular cyanobacterium Synechocystis sp. PCC 6803, mediates positive phototactic motility and contains two GAF domains, the latter of which binds a bilin chromophore. Full-length PixJ1 expressed and purified from Synechocystis showed unique reversible photoconversion between a blue light-absorbing (Pb) form and a green light-absorbing (Pg) form (1) in contrast to the reversible phototransformation between the red light-absorbing form and far-red light-absorbing form of the other GAF-containing photoreceptors such as plant or bacterial phytochromes. To clarify the origin of the blue-shifted photoconversion, we tried to reconstitute this blue-green reversible phototransformation by synthesizing the second GAF domain in Escherichia coli transformed with genes for biosynthesis of four different bilins, biliverdin (BV), bilirubin (BR), phycocyanobilin (PCB), and phycocyanorubin (PCR), as final products. The three expression systems, the BR system being the exception, produced a GAF polypeptide with a covalently bound bilin. The GAF polypeptide from the BV-synthesizing system exhibited an irreversible photoconversion, while that from the PCB-synthesizing system revealed photoconversion between Pb and Pg almost identical to that of the full-length PixJ1, indicating that PCB is responsible for the blue-green reversible photoconversion. Furthermore, the GAF polypeptide from the PCR-producing system exhibited almost the same reversible spectral change, possibly coming from the PCB accumulated in the PCR-biosynthetic pathway. Mass spectrometry (MS) of the main tryptic chromopeptide revealed that the chromophore binds to a 21-amino acid peptide that contains a cysteine-histidine motif for phytochrome chromophore binding and that an ion signal can be assigned to desorbed PCB. The absorption spectra of the denatured GAF polypeptide suggested that PCB is attached to the protein moiety in a twisted conformation that disrupts the pi-electron conjugation between the A and B rings, possibly being held in position through a second covalent linkage.     

Phase-separated protein droplets of amyotrophic lateral sclerosis-associated p62/SQSTM1 mutants show reduced inner fluidity

Several amyotrophic lateral sclerosis (ALS)-related proteins such as FUS, TDP-43, and hnRNPA1 demonstrate liquid–liquid phase separation, and their disease-related mutations correlate with a transition of their liquid droplet form into aggregates. Missense mutations in SQSTM1/p62, which have been identified throughout the gene, are associated with ALS, frontotemporal degeneration (FTD), and Paget’s disease of bone. SQSTM1/p62 protein forms liquid droplets through interaction with ubiquitinated proteins, and these droplets serve as a platform for autophagosome formation and the antioxidative stress response via the LC3-interacting region (LIR) and KEAP1-interacting region (KIR) of p62, respectively. However, it remains unclear whether ALS/FTD-related p62 mutations in the LIR and KIR disrupt liquid droplet formation leading to defects in autophagy, the stress response, or both. To evaluate the effects of ALS/FTD-related p62 mutations in the LIR and KIR on a major oxidative stress system, the Keap1-Nrf2 pathway, as well as on autophagic turnover, we developed systems to monitor each of these with high sensitivity. These methods such as intracellular protein–protein interaction assay, doxycycline-inducible gene expression system, and gene expression into primary cultured cells with recombinant adenovirus revealed that some mutants, but not all, caused reduced NRF2 activation and delayed autophagic cargo turnover. In contrast, while all p62 mutants demonstrated sufficient ability to form liquid droplets, all of these droplets also exhibited reduced inner fluidity. These results indicate that like other ALS-related mutant proteins, p62 missense mutations result in a primary defect in ALS/FTD via a qualitative change in p62 liquid droplet fluidity.     

E6AP ubiquitin ligase mediates ubiquitylation and degradation of hepatitis C virus core protein

Hepatitis C virus (HCV) core protein is a major component of viral nucleocapsid and a multifunctional protein involved in viral pathogenesis and hepatocarcinogenesis. We previously showed that the HCV core protein is degraded through the ubiquitin-proteasome pathway. However, the molecular machinery for core ubiquitylation is unknown. Using tandem affinity purification, we identified the ubiquitin ligase E6AP as an HCV core-binding protein. E6AP was found to bind to the core protein in vitro and in vivo and promote its degradation in hepatic and nonhepatic cells. Knockdown of endogenous E6AP by RNA interference increased the HCV core protein level. In vitro and in vivo ubiquitylation assays showed that E6AP promotes ubiquitylation of the core protein. Exogenous expression of E6AP decreased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected Huh-7 cells. Furthermore, knockdown of endogenous E6AP by RNA interference increased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected cells. Taken together, our results provide evidence that E6AP mediates ubiquitylation and degradation of HCV core protein. We propose that the E6AP-mediated ubiquitin-proteasome pathway may affect the production of HCV particles through controlling the amounts of viral nucleocapsid protein.