A hydrophobic amino acid cluster inserted into the C-terminus of a recycling cell surface receptor functions as an endosomal sorting signal

Cell surface receptors ubiquitylated after ligand stimulation are internalized and delivered to the lysosomal pathway for degradation. Ubiquitylated receptors are captured by ESCRT protein complexes that sort them to the lysosomal pathway. Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a component of endosomal sorting complexes required for transport (ESCRT)-0 that recognizes ubiquitin attached to receptors, indicating that it functions as a key molecule for ubiquitin-dependent endosomal sorting. In a previous study on interleukin (IL)-2 receptor β (IL-2Rβ) and IL-4 receptor α (IL-4Rα), which are constitutively internalized without ligand stimulation, we revealed that Hrs bound to IL-2Rβ and IL-4Rα in a ubiquitin-independent manner, and identified a hydrophobic amino acid cluster in the cytoplasmic region of IL-2Rβ and IL-4Rα as the Hrs-interacting domain. However, a chimeric receptor containing the hydrophobic amino acid cluster inserted into the C-terminal of IL-2Rα was not delivered to late endosomes, but recycled back to the plasma membrane. In the present study, we explored the functional domain related to endosomal sorting in IL-2Rβ together with the hydrophobic amino acid cluster, and discovered the importance of an approximately 30-amino acid stretch following the C-terminus of the hydrophobic amino acid cluster in IL-2Rβ. Even though the amino acid stretch following the hydrophobic amino acid cluster was composed of arbitrary amino acids, such a stretch was also permissive for the sorting ability, suggesting that the hydrophobic amino acid cluster functions as an endosomal sorting signal. These findings clarify part of the molecular mechanism underlying the ubiquitin-independent endosomal sorting of cytokine receptors that are constitutively internalized without ligand stimulation.     

Asymmetric structure and domain binding interfaces of human tyrosyl‐tRNA synthetase studied by molecular dynamics simulations

Human tyrosyl‐tRNA synthetase (HsTyrRS) is composed of two structural modules: N‐terminal catalytic core and an EMAP II‐like C‐terminal domain. The structures of these modules are known, but no crystal structure of the full‐length HsTyrRS is currently available. An all‐atom model of the full‐length HsTyrRS was developed in this work. The structure, dynamics, and domain binding interfaces of HsTyrRS were investigated by extensive molecular dynamics (MD) simulations. Our data suggest that HsTyrRS in solution consists of a number of compact asymmetric conformations, which differ significantly by their rigidity, internal mobility, orientation of C‐terminal modules, and the strength of interdomain binding. Interfaces of domain binding obtained in MD simulations are in perfect agreement with our previous coarse‐grained hierarchical rotations technique simulations. Formation of the hydrogen bonds between R93 residue of the ELR cytokine motif and the residues A340 and E479 in the C‐module was observed. This observation supports the idea that the lack of cytokine activity in the full‐length HsTyrRS is explained by interactions between N‐modules and C‐modules, which block the ELR motif. Copyright © 2013 John Wiley & Sons, Ltd.     

Chitin Coated Cellulose as an Adsorbent of lysozyme like Enzymes Preparation and Properties

As a new adsorbent of lysozyme-like enzymes, chitin coated (CC-)cellulose was prepared. CC-cellulose was stable and had good flow properties for use in column chromatography. Affinity chromatography with CC-cellulose showed that 3~5 mg of lysozyme/ml resin was adsorbed specifically and desorbed quantitatively under mild conditions. The utilities of the method of affinity chromatography with CC-cellulose are discussed.     

Isolation and Properties of New Photosynthetic Bacteria Isolated from Seawater

Three strains of purple nonsulfur photosynthetic bacteria were isolated from seawater. They showed the characteristic properties of Rhodobacter species. Their biochemical, physiological, and chemotaxonomical properties were different from those of previously known species.     

Dexamethasone Inhibits Mitogen-dependent Synthesis of Histamine Due to Histidine Decarboxylase Induced in Cultured Mouse Spleen Cells

Schizosaccharomyces pombe has four α-amylase homologs (Aah1p-Aah4p) with a glycosylphosphatidylinositol (GPI) modification site at the C-terminal end. Disruption mutants of aah genes were tested for mislocalization of vacuolar carboxypeptidase Y (CPY), and aah3Δ was found to secrete CPY. The conversion rate from pro- to mature CPY was greatly impaired in aah3Δ, and fluorescence microscopy inidicated that a sorting receptor for CPY, Vps10p, mislocalized to the vacuolar membrane. These results indicate that aah3Δ had a defect in the retrograde transport of Vps10p, and that Aah3p is the first S. pombe specific protein required for vacuolar protein sorting.     

Photoaffinity electrophoretic mobility shift assay using photoreactive DNA bearing 3-trifluoromethyl-3-phenyldiazirine in its phosphate backbone

Photoaffinity cross-linking enables the analysis of interactions between DNA and proteins even under denaturing conditions. We present a photoaffinity electrophoretic mobility shift assay (EMSA) in which two heterogeneous techniques―photoaffinity cross-linking using DNA bearing 3-trifluoromethyl-3-phenyldiazirine and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) analysis—are combined. To prepare the photoreactive DNA, which is an essential tool for photoaffinity EMSA, we first determined the optimal conditions for the integration of 4-(3-trifluoromethyl-3H-diazirin-3-yl)benzyl bromide to the specific site of oligonucleotide where phosphodiester linkage was replaced with phosphorothioate linkage. The photoaffinity EMSA was developed using the POU (initial letters of three genes: Pit-l, Oct-1,2, and unc-86) domain region of Oct-1 protein, which specifically bound to octamer DNA motif (ATGCAAAT). The affinity-purified recombinant POU domain proteins conjugated with glutathione-S-transferase (GST) contained three distinct proteins with molecular weights of 34, 36, and 45 kDa. The photoaffinity EMSA could clearly distinguish the individual binding abilities of three proteins on a single lane and showed that the whole POU domain protein specifically bound to octamer DNA motif by competition experiments. Using the nuclear extract of HeLa cells, the photoaffinity EMSA revealed that at least five specific proteins could bind to the octamer DNA motif. These results show that photoaffinity EMSA using 3-trifluoromethyl-3-phenyldiazirine can provide high-performance analysis of DNA-binding proteins.     

The New Face of the Lipid Droplet: Lipid Droplet Proteins

The lipid droplet (LD) is an organelle with vital functions found in nearly all organisms. LD proteomic research has provided fundamentally important insights into this organelle's functions. The review provides a summary of LD proteomic studies conducted across diverse organisms and cell and tissue types. The accumulated proteomic data are reviewed for evidence of a protein targeting mechanism for the organelle. The hypotheses for several specific localization mechanisms based on what is known about targeting mechanisms for other organelles and vesicles are provided. Although the nature of the mechanism is not known, the functional data demonstrate that the targeting mechanism and, indeed, the organelle itself, is conserved from prokaryotes to eukaryotes. It is hoped that the review will help inspire further research leading to novel discoveries in the field.