Id (Inhibitor of Differentiation) proteins belong to a family of transcriptional modulators that are characterized by a helix loop helix (HLH) region but lack the basic amino acid domain. Id proteins are known to interact with basic helix-loop-helix (bHLH) transcription factors and function as their negative regulators. The negative role of Id proteins has been well demonstrated in muscle development and some in neuronal cells. In this study, we investigated the effect of Id on the function of BETA2/NeuroD, a bHLH transcription factor responsible for neuron and endocrine cell specific gene expression. cDNAs of several Id isoforms were isolated by yeast two-hybrid system using the bHLH domain of E47, a ubiquitous bHLH partner as a bait. Id proteins expressed in COS M6 cells, were found in both cytosolic and nuclear fractions. Electrophoretic mobility shift assay showed that coexpression of Id2 proteins inhibited BETA2/ NeuroD binding to its target sequence, E-box. Id2 inhibited E-box mediated gene expression in a dose dependent manner in BETA2/NeuroD expressing HIT cells. Id coexpressed with BETA2/NeuroD in HeLa cells, inhibited the stimulatory activity of BETA2/NeuroD. These results suggest that Id proteins may negatively regulate tissue specific gene expression induced by BETA2/NeuroD in neuroendocrine cells and the inhibitory role of Id proteins during differentiation may be conserved in various tissues.
Amino Acid Sequence ; Animals ; Base Sequence ; Binding Sites ; Cells, Cultured ; DNA-Binding Proteins/genetics/*metabolism ; E-Box Elements ; Gene Expression Regulation/physiology ; Helix-Loop-Helix Motifs ; Human ; Islets of Langerhans/cytology/metabolism ; Mice ; Molecular Sequence Data ; Nerve Tissue Proteins/genetics/*metabolism ; Neurons/cytology/metabolism ; Organ Specificity ; Transcription Factors/genetics/*metabolism ; Two-Hybrid System Techniques

Entero virus 71 (EV71) causes hand, foot, and mouth disease (HFMD) and occasionally leads to severe neurological complications and even death. Scavenger receptor class B member 2 (SCARB2) is a functional receptor for EV71, that mediates viral attachment, internalization, and uncoating. However, the exact binding site of EV71 on SCARB2 is unknown. In this study, we generated a monoclonal antibody (mAb) that binds to human but not mouse SCARB2. It is named JL2, and it can effectively inhibit EV71 infection of target cells. Using a set of chimeras of human and mouse SCARB2, we identified that the region containing residues 77-113 of human SCARB2 contributes significantly to JL2 binding. The structure of the SCARB2-JL2 complex revealed that JL2 binds to the apical region of SCARB2 involving α-helices 2, 5, and 14. Our results provide new insights into the potential binding sites for EV71 on SCARB2 and the molecular mechanism of EV71 entry.
Amino Acid Sequence ; Animals ; Antibodies, Monoclonal ; chemistry ; genetics ; metabolism ; Binding Sites ; Cell Line ; Crystallography, X-Ray ; Enterovirus A, Human ; drug effects ; genetics ; growth & development ; immunology ; Fibroblasts ; drug effects ; virology ; Gene Expression ; HEK293 Cells ; Humans ; Immunoglobulin Fab Fragments ; chemistry ; genetics ; metabolism ; Lysosome-Associated Membrane Glycoproteins ; chemistry ; genetics ; immunology ; Mice ; Models, Molecular ; Protein Binding ; Protein Conformation, alpha-Helical ; Protein Conformation, beta-Strand ; Protein Interaction Domains and Motifs ; Receptors, Scavenger ; chemistry ; genetics ; immunology ; Receptors, Virus ; chemistry ; genetics ; immunology ; Recombinant Fusion Proteins ; chemistry ; genetics ; immunology ; Sequence Alignment ; Sequence Homology, Amino Acid ; Sf9 Cells ; Spodoptera ; Thermodynamics

KM-HN-1 is a C-terminal coiled-coil domain containing protein previously referred to as image clone MGC33607. This protein has been previously identified as a cancer/testis antigen and reported as nuclear and chromatin localizing protein. We raised polyclonal antisera with the GST fusion protein and identified them as a 105 kDa protein. Motif analysis showed that this protein harbors the leucine zipper motif in internal 1/3 region and the coiled-coil domain in the C-terminal region. Using the full length and various deletion mutants, we determined the motif that governs the subcellular localization of KM-HN-1. Immunofluorescence staining of the endogenous KM-HN-1 and various kinds of GFP-tagged KM-HN-1 revealed that KM-HN-1 localizes to the centrosomes as well as nucleus. The centrosomal localization-determining region of this protein is C-terminal coiled-coil domain in which the leucine zipper motif and the nuclear export signal (NES) harbor.
Amino Acid Motifs/physiology ; Amino Acid Sequence ; Antigens, Neoplasm/chemistry/*metabolism ; Cells, Cultured ; Centrosome/*metabolism ; Fluorescent Antibody Technique ; Humans ; Leucine Zippers/*physiology ; Molecular Sequence Data ; Mutation ; Nuclear Proteins/chemistry/*metabolism ; Protein Conformation ; Protein Structure, Tertiary ; Sequence Analysis, Protein

The EF-hand superfamily is a large group of proteins which contain EF-hand motif formed by helix-loop-helix. These proteins always have the ability of binding metal ions or forming dimmers. Troponin C, known as having ability of binding Ca2+, is one member of the EF-hand superfamily. Troponin C interacts with troponin I and troponin T, forming a troponin complex which takes part in regulating muscle contraction. It is interesting that troponin C was also found in non-muscular tissue, and its function was proved to be different from that of troponin C found in muscular tissue. To date, a lot of researches about troponin C have been carried out widely. However, most of them focused on vertebrate, seldom were done on invertebrate. Our group carried out a research on troponin C from silkworm, a model organism of insects, aiming to clarify the structure and function of silkworm troponin C. Here, we mainly discuss the characters of the EF-hand superfamily and the classification, structure and function of troponin C . We also introduced our work about silkworm troponin C briefly, hoping of making a little contribution to the research of invertebrate troponin C.
Amino Acid Sequence ; Animals ; Binding Sites ; genetics ; Bombyx ; genetics ; metabolism ; Calcium ; metabolism ; EF Hand Motifs ; Molecular Sequence Data ; Phylogeny ; Protein Binding ; Troponin C ; classification ; genetics ; metabolism

The important and diverse regulatory roles of Ca(2+) in eukaryotes are conveyed by the EF-hand containing calmodulin superfamily. However, the calcium-regulatory proteins in prokaryotes are still poorly understood. In this study, we report the three-dimensional structure of the calcium-binding protein from Streptomyces coelicolor, named CabD, which shares low sequence homology with other known helix-loop-helix EF-hand proteins. The CabD structure should provide insights into the biological role of the prokaryotic calcium-binding proteins. The unusual structural features of CabD compared with prokaryotic EF-hand proteins and eukaryotic sarcoplasmic calcium-binding proteins, including the bending conformation of the first C-terminal α-helix, unpaired ligand-binding EF-hands and the lack of the extreme C-terminal loop region, suggest it may have a distinct and significant function in calcium-mediated bacterial physiological processes, and provide a structural basis for potential calcium-mediated regulatory roles in prokaryotes.
Amino Acid Sequence ; Binding Sites ; Calcium ; physiology ; Calcium-Binding Proteins ; chemistry ; Crystallography, X-Ray ; EF Hand Motifs ; Molecular Sequence Data ; Protein Binding ; Protein Structure, Tertiary ; Sequence Alignment ; Sequence Homology, Amino Acid ; Streptomyces coelicolor ; Structural Homology, Protein ; Surface Properties

Actin binding proteins play key roles in cell structure and movement particularly as regulators of the assembly, stability and localization of actin filaments in the cytoplasm. In the present study, a cDNA clone encoding an actin bundling protein named as AhABP was isolated from Acanthamoeba healyi, a causative agent of granulomatous amebic encephalitis. This clone exhibited high similarity with genes of Physarum polycephalum and Dictyostelium discoideum, which encode actin bundling proteins. Domain search analysis revealed the presence of essential conserved regions, i.e., an active actin binding site and 2 putative calcium binding EF-hands. Transfected amoeba cells demonstrated that AhABP is primarily localized in phagocytic cups, peripheral edges, pseudopods, and in cortical cytoplasm where actins are most abundant. Moreover, AhABP after the deletion of essential regions formed ellipsoidal inclusions within transfected cells. High-speed co-sedimentation assays revealed that AhABP directly interacted with actin in the presence of up to 10 micrometer of calcium. Under the electron microscope, thick parallel bundles were formed by full length AhABP, in contrast to the thin actin bundles formed by constructs with deletion sites. In the light of these results, we conclude that AhABP is a novel actin bundling protein that is importantly associated with actin filaments in the cytoplasm.
Transfection ; Sequence Analysis, DNA ; Sequence Alignment ; Microscopy, Electron, Transmission ; Microfilament Proteins/*chemistry/genetics/*metabolism ; EF Hand Motifs ; DNA, Complementary ; Culture Media ; Cloning, Molecular ; Animals ; Amino Acid Sequence ; Actins/*metabolism ; Acanthamoeba/genetics/growth & development/*metabolism

Twenty-seven ERF transcription factor family genes were isolated from Arnebia euchroma, with an average size of 1,010 bp, each gene encoded a 212 amino acids on average. The gene structure and expression of physicochemical properties, subcellular localization, signal peptides, senior structural domains and conservative forecasting, and analysis of A. euchroma were studied comparing with ERF gene gi261363612 of Lithospermum erythrorhizon, and phylogenetic analysis of A. euchroma ERF family was carried out. The results showed the existence of three conserved domains in this family, the senior structure based on random coil and it clustered into CBF/DREB and ERF subfamilies.
Amino Acid Motifs ; Amino Acid Sequence ; Boraginaceae ; genetics ; Cloning, Molecular ; Computational Biology ; Genome, Plant ; genetics ; High-Throughput Nucleotide Sequencing ; Medicine, Chinese Traditional ; Molecular Sequence Data ; Multigene Family ; Phylogeny ; Plant Proteins ; chemistry ; genetics ; Plants, Medicinal ; Protein Structure, Secondary ; Protein Structure, Tertiary ; Sequence Analysis, DNA ; Transcription Factors ; chemistry ; genetics ; Transcriptome

The PE_PGRS family of proteins unique to mycobacteria is demonstrated to contain multiple calcium-binding and glycine-rich sequence motifs GGXGXD/NXUX. This sequence repeat constitutes a calcium-binding parallel beta-roll or parallel beta-helix structure and is found in RTX toxins secreted by many Gram-negative bacteria. It is predicted that the highly homologous PE PGRS proteins containing multiple copies of the nona-peptide motif could fold into similar calcium-binding structures. The implication of the predicted calcium-binding property of PE PGRS proteins in the light of macrophage-pathogen interaction and pathogenesis is presented.
Amino Acid Motifs ; Amino Acid Sequence ; Antigens, Bacterial ; chemistry ; genetics ; metabolism ; Bacterial Proteins ; chemistry ; genetics ; metabolism ; Base Sequence ; Binding Sites ; genetics ; Calcium ; metabolism ; DNA, Bacterial ; genetics ; Membrane Proteins ; chemistry ; genetics ; metabolism ; Models, Molecular ; Molecular Sequence Data ; Mycobacterium tuberculosis ; genetics ; metabolism ; Protein Structure, Secondary

OBJECTIVETo observe the effects of thermal stress on proliferation of human vascular endothelial cells (VECs) and explore its significance.

METHODSChanges of VECs proliferation were investigated with (3)H-TdR incorporation method after ECV304 was treated at 43 degrees for 2 hours, while expressions of intercellular adhesion molecule-1 (ICAM-1), inhibitor of differentiation-1 (ID1), and P16 and P21 proteins were determined by Western Blotting.

RESULTSThe effect of inhibition of VECs growth after thermal stress was detected by (3)H-TdR incorporation experiment. Western blotting showed ICAM-1, a marker of activated endothelial cells, was increased markedly after thermal stress. Expression of ID1 protein declined gradually with increasing expressions of its downstream genes, P16 and P21 following the thermal stress.

CONCLUSIONSThermal stress could strongly activate VECs and inhibit proliferation of VECs through ID1, thus down regulating cyclin-dependent kinase inhibitors, P16 and P21, which might be an essential pathway for recovery of VECs after thermal stress.

Blotting, Western ; Cells, Cultured ; Endothelium, Vascular ; cytology ; Helix-Loop-Helix Motifs ; physiology ; Humans ; Inhibitor of Differentiation Protein 1 ; Intercellular Adhesion Molecule-1 ; metabolism ; Repressor Proteins ; Temperature ; Transcription Factors ; metabolism ; Umbilical Veins ; cytology

This study was conducted to construct eukaryotic recombinant vector of LipL32-HlyX fusion gene from Leptospira serovar Lai and express it in mammalian cell. Both of LipL32 gene and HlyX gene were amplified from Leptospira strain O17 genomic DNA by PCR. Then with the two genes as template, LipL32-HlyX fusion gene was obtained by SOE PCR (gene splicing by overlap extension PCR). The fusion gene was then cloned into pcDNA3.1 by restriction nuclease digestion. Having been transformed into E. coli DH5alpha, the recombiant plasmid was identified by restriction nuclease digestion, PCR analysis and sequencing. The recombinant plasmid was then transfected into COS7 cell whose expression was detected by RT-PCR and Western blotting analysis. RT-PCR amplified a fragment about 2000 bp and Western blotting analysis found a specific band about 75 KD which was consistent with the expected fusion protein size. In conclusion, the successful construction of eukaryotic recombinant vector containing LipL32-HlyX fusion gene and the effective expression in mammalian have laid a foundation for the application of Leptospira DNA vaccine.
Animals ; Bacterial Outer Membrane Proteins ; genetics ; metabolism ; COS Cells ; Cercopithecus aethiops ; Gene Fusion ; Genetic Vectors ; Helix-Loop-Helix Motifs ; genetics ; Leptospira ; genetics ; Lipoproteins ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism