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

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

OBJECTIVETo evaluate the impact of surface modification on the DNA-binding ability of nano-hydroxyapatite (nHA).

METHODSChemical co-precipitation-hydrothermal synthesis was utilized to prepare the nHA particles, and polyethylenimine (PEI) was used for surface modification of the nHA. Transmission electron microscopic (TEM) observation and zeta potential detection of the nHA were carried out before and after surface modification. The abilities of the nanoparticles, at different pH values and different concentrations, for DNA-binding and DNA protection against nuclease digestion were assessed before and after surface modification by electrophoresis.

RESULTSTEM observation showed a short rod-like morphology of PEI-modified nHA with uniform particle size and good dispersion; the nHA without the modification tended to aggregate with poor dispersion. With a positive zeta potential, the PEI-modified nHA showed an obviously enhanced ability of DNA binding at different pH values and concentrations, with strong capacity to protect the DNA against Dnase I digestion. At the concentration of 250 µg/ml and a pH value of 7.0, the nHA-PEI showed an optimal efficiency of DNA-binding and DNA protection.

CONCLUSIONnHA with surface modification by PEI can serve as an effective vector for DNA binding and transfer.

Amino Acid Motifs ; DNA ; chemistry ; Durapatite ; chemistry ; Gene Transfer Techniques ; Genetic Vectors ; Nanoparticles ; chemistry ; Polyethyleneimine ; chemistry

As a protein originally found in plant pathogenic bacteria, transcription activator-like effectors (TALEs) can be fused with the cleaving domain of restriction endonuclease (For example Fok I) to form artificial nucleases named TALENs. These proteins are dependent on variable numbers of tandem Repeats of TALEs to recognize and bind DNA sequences. Each of these repeats consists of a set of approximately 34 amino acids, composed of about 32 conserved amino acids and 2 highly variable amino acids called repeat variant di-residues (RVDs). RVDs distinguish one TALE from another and can make TALEs have a simple cipher for the one-to-one recognition for proteins and DNA bases. Based on this, in theory, artificially constructed TALENs could recognize and break DNA sites specifically and arbitrarily to perform gene knockout, insertion or modification. We reviewed the development of this technology in multi-level and multi species, and its advantages and disadvantages compared with ZFNs and CRISPR/Cas technology. We also address its special advantages in industrial microbe breeding, vector construction, targeting precision, high efficiency of editing and biological safety.
Amino Acid Motifs ; Biotechnology ; DNA ; chemistry ; Endonucleases ; chemistry ; Tandem Repeat Sequences ; Trans-Activators ; chemistry

Amino Acid Motifs ; DEAD-box RNA Helicases ; chemistry ; Humans ; Protein Domains ; Structure-Activity Relationship

The WD40 domain exhibits a β-propeller architecture, often comprising seven blades. The WD40 domain is one of the most abundant domains and also among the top interacting domains in eukaryotic genomes. In this review, we will discuss the identification, definition and architecture of the WD40 domains. WD40 domain proteins are involved in a large variety of cellular processes, in which WD40 domains function as a protein-protein or protein-DNA interaction platform. WD40 domain mediates molecular recognition events mainly through the smaller top surface, but also through the bottom surface and sides. So far, no WD40 domain has been found to display enzymatic activity. We will also discuss the different binding modes exhibited by the large versatile family of WD40 domain proteins. In the last part of this review, we will discuss how post-translational modifications are recognized by WD40 domain proteins.
Amino Acid Motifs ; Amino Acid Sequence ; Animals ; Humans ; Molecular Sequence Data ; Protein Processing, Post-Translational ; Protein Structure, Tertiary ; Proteins ; chemistry ; metabolism ; Repetitive Sequences, Amino Acid

Types of cofactor independency for newly found oxidoreductases sequences are usually determined by experimental analysis. These experimental methods are both time-consuming and costly. With the explosion of oxidoreductases sequences entering into the databanks, it is highly desirable to explore the feasibility of selectively classifying newly found oxidoreductases into their respective cofactor independency classes by means of an automated method. In this study, we proposed a modified Chou's pseudo-amino acid composition method to extract features from sequences and the k-nearest neighbor was used as the classifier, and the results were very encouraging. When lambda = 48, w = 0.1, the areas under the ROC curve of k-nearest neighbor in 10-fold cross-validation was 0.9536; and the success rate was 92.0%, which was 3.5% higher than that of pseudo-amino acid composition. It was also better than all the other 7 feature extraction methods. Our results showed that predicting the cofactors of oxidoreductases was feasible and the modified pseudo-amino acid composition method may be a useful method for extracting features from protein sequences.
Amino Acid Motifs ; Amino Acids ; analysis ; Coenzymes ; chemistry ; Computational Biology ; Models, Chemical ; Oxidoreductases ; chemistry ; Predictive Value of Tests