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Results for “spatial domain identification”

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PubMed2001

Integrin-mediated activation of transforming growth factor-beta(1) in pulmonary fibrosis.

The integrin alphavbeta6 is restricted to epithelial cells and is dramatically induced in response to injury and inflammation. Mice expressing a null mutation of this integrin develop exaggerated inflammation of the lungs and skin, but are dramatically protected from bleomycin-induced pulmonary fibrosis. This phenotype led to the identification of a unique role for this integrin in binding to and activating latent extracellular complexes of the anti-inflammatory, profibrotic cytokine, transforming growth factor-beta(1). This integrin-mediated activation is tightly spatially restricted and appears to require direct presentation of the activated cytokine to receptors on adjacent cells. The process also requires distinct regions of the beta6-subunit cytoplasmic domain and an intact actin cytoskeleton, suggesting the existence of additional cellular mechanisms to regulate this process. If this mechanism is found to be as important in humans as it is in mice, the integrin and as yet to be identified pathways for cellular regulation of this process could be exciting new targets for intervention in fibrotic diseases of the lung and other epithelial organs.

Animals↗
PubMed1992

Finding the gene(s) for Waardenburg syndrome(s).

Waardenburg syndrome is a recognizable disorder characterized by autosomal dominant inheritance of pigmentary abnormalities of the eyes, hair, and skin sometimes associated with congenital hearing impairment. At least two different types of Waardenburg syndrome have been described. The different types of Waardenburg syndrome are differentiated as follows: type I, with dystopia canthorum; type II, without dystopia canthorum; and type III, without dystopia, with unilateral eyelid ptosis and skeletal abnormalities. Results of pooled linkage analysis done by a consortium of investigators suggests that Waardenburg syndrome type I is genetically heterogeneous, meaning that there is probably more than one genotype that can be associated with the findings typical of the syndrome. A mutated gene that can cause Waardenburg syndrome type I has been discovered. Either a single base pair substitution or an 18 base pair deletion in exon 2 has been found in affected individuals but is not found in unaffected members. Most likely, the mutated gene causes a perturbation in DNA binding of a paired domain, in turn having an effect on the regulation of other genes so the development and spatial orientation of cells and structures derived from the neural crest are affected. Forty years after Waardenburg syndrome was first described, a mutant gene has been found that is known to cause the syndrome. The process of discovery that has transpired in the time from syndrome description to gene identification has been one of steady progress and interdisciplinary cooperation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1999

Identification of the ribosome binding sites of translation initiation factor IF3 by multidimensional heteronuclear NMR spectroscopy.

Titrations of Escherichia coli translation initiation factor IF3, isotopically labeled with 15N, with 30S ribosomal subunits were followed by NMR by recording two-dimensional (15N,1H)-HSQC spectra. In the titrations, intensity changes are observed for cross peaks belonging to amides of individual amino acids. At low concentrations of ribosomal subunits, only resonances belonging to amino acids of the C-domain of IF3 are affected, whereas all those attributed to the N-domain are still visible. Upon addition of a larger amount of 30S subunits cross peaks belonging to residues of the N-terminal domain of the protein are also selectively affected. Our results demonstrate that the two domains of IF3 are functionally independent, each interacting with a different affinity with the ribosomal subunits, thus allowing the identification of the individual residues of the two domains involved in this interaction. Overall, the C-domain interacts with the 30S subunits primarily through some of its loops and alpha-helices and the residues involved in ribosome binding are distributed rather symmetrically over a fairly large surface of the domain, while the N-domain interacts mainly via a small number of residues distributed asymmetrically in this domain. The spatial organization of the active sites of IF3, emerging through the comparison of the present data with the previous chemical modification and mutagenesis data, is discussed in light of the ribosomal localization of IF3 and of the mechanism of action of this factor.

Amino Acid Sequence↗
PubMed2002

The contribution of factor Xa to exosite-dependent substrate recognition by prothrombinase.

Kinetic studies support the concept that protein substrate recognition by the prothrombinase complex of coagulation is achieved by interactions at extended macromolecular recognition sites (exosites), distinct from the active site of factor Xa within the complex. We have used this formal kinetic model and a monoclonal antibody directed against Xa (alphaBFX-2b) to investigate the contributions of surfaces on the proteinase to exosite-mediated protein substrate recognition by prothrombinase. alphaBFX-2b bound reversibly to a fluorescent derivative of factor Xa (K(d) = 17.1 +/- 5.6 nm) but had no effect on active site function of factor Xa or factor Xa saturably assembled into prothrombinase. In contrast, alphaBFX-2b was a slow, tight binding inhibitor of the cleavage of either prethrombin 2 or meizothrombin des-fragment 1 by prothrombinase (K(i)(*) = 0.55 +/- 0.05 nm). Thus, alphaBFX-2b binding to factor Xa within prothrombinase selectively leads to the inhibition of protein substrate cleavage without interfering with active site function. Inhibition kinetics could adequately be accounted for by a kinetic model in which prethrombin 2 and alphaBFX-2b bind in a mutually exclusive way to prothrombinase. These are properties expected of an exosite-directed inhibitor. The site(s) on factor Xa responsible for antibody binding were evaluated by identification of immunoreactive fragments following chemical digestion of human and bovine Xa and were further confirmed with a series of recombinantly expressed fragments. These approaches suggest that residues 82-91 and 102-116 in the proteinase domain contribute to alphaBFX-2b binding. The data establish this antibody as a prototypic exosite-directed inhibitor of prothrombinase and suggest that the occlusion of a surface on factor Xa, spatially removed from the active site, is sufficient to block exosite-dependent recognition of the protein substrate by prothrombinase.

Amino Acid Sequence↗
PubMed1994

Molecular cloning of a novel member of the nuclear receptor superfamily related to the orphan receptor, TR2.

We have cloned a novel member (mTR2R1) of the nuclear receptor superfamily of transcription factors from a mouse brain cDNA library. The cDNA sequence predicts a protein primary structure of 629 amino acids with a calculated molecular weight of 68.8 kDa. The amino acid sequence of the protein contains three regions of consensus sequence that are conserved throughout the nuclear receptor superfamily. One of these regions encodes a type II zinc finger DNA binding domain that is characteristic of this family of transcription factors. Comparison of the amino acid sequence of mTR2R1 with other nuclear receptors indicates that it is most closely related to the orphan receptor, hTR2, and suggests that these proteins constitute a novel subfamily within the nuclear receptor superfamily. mTR2R1 is encoded by two mRNAs that show different but overlapping spatial patterns of expression in the adult mouse. The identification of a subfamily of TR2 receptors together with the existence of variant mRNAs for these receptors prompts a close examination of the tissue-specific regulatory role of this subfamily of nuclear receptors.

Amino Acid Sequence↗
PubMed1998

Validity of a cognitive computerized assessment system in brain-injured patients.

The primary objective of the study was to investigate the criterion validity of the Cognitive Drug Research (CDR) computerized system in assessing cognitive functioning among persons with brain injury or other organic illness. Understanding the cognitive effects of pharmacotherapy is important in improving long-term rehabilitation outcomes in persons with organic disorders. The CDR system evaluates cognitive skills of attention, short-term or working memory, long-term memory, and visuomotor and motor functioning. For validity testing, correlation coefficients were obtained from an analysis of CDR scores to IQ and Folstein Mini-Mental State Exam (MMSE) scores. Subtests of the CDR system that correlated with the MMSE were choice reaction time (R = -0.542, p = 0.04), spatial working memory (R = 0.938, p = 0.01), and word recognition (R = -0.949, p = 0.01). These tests primarily measured attention and working memory. Correlations between the CDR and IQ scores were not evident. In summary, the data suggest the CDR assessment system has high patient acceptability by persons with traumatic brain injury, and is a useful tool in assessing certain domains of cognition, specifically short-term memory, discrimination, and identification skills.

Brain Diseases↗
PubMed2004

Trichostatin A-induced histone acetylation causes decondensation of interphase chromatin.

The effect of trichostatin A (TSA)-induced histone acetylation on the interphase chromatin structure was visualized in vivo with a HeLa cell line stably expressing histone H2A, which was fused to enhanced yellow fluorescent protein. The globally increased histone acetylation caused a reversible decondensation of dense chromatin regions and led to a more homogeneous distribution. These structural changes were quantified by image correlation spectroscopy and by spatially resolved scaling analysis. The image analysis revealed that a chromatin reorganization on a length scale from 200 nm to >1 microm was induced consistent with the opening of condensed chromatin domains containing several Mb of DNA. The observed conformation changes could be assigned to the folding of chromatin during G1 phase by characterizing the effect of TSA on cell cycle progression and developing a protocol that allowed the identification of G1 phase cells on microscope coverslips. An analysis by flow cytometry showed that the addition of TSA led to a significant arrest of cells in S phase and induced apoptosis. The concentration dependence of both processes was studied.

Acetylation↗
PubMed1997

Site-directed spin-labeling study of the structure and subunit interactions along a conserved sequence in the alpha-crystallin domain of heat-shock protein 27. Evidence of a conserved subunit interface.

Site-directed spin-labeling (SDSL) was used to investigate the secondary structure, solvent accessibility, and tertiary and quaternary interactions along the sequence located between residues 133 and 144 in the alpha-crystallin domain of human heat-shock protein 27 (HSP 27). The sequence is conserved among mammalian sHSP and shows similarity to the region of highest homology between alpha A- and alpha B-crystallins. Eleven sequential single cysteine mutants were prepared and reacted with a sulfhydryl-specific spin-label. The accessibilities of attached nitroxide side chains to a paramagnetic probe in the aqueous solution were determined. Spectral line shapes were analyzed in terms of side-chain mobility and spatial proximity to nearby nitroxides. The sequence-specific mobilities and accessibilities varied with a period of 2, consistent with the presence of a beta-strand along the sequence. At even sites, the nitroxide environment is highly ordered with virtually no accessibility to the hydrophilic probe, indicating that one face of the strand is buried. Furthermore, spin-spin interactions between nitroxides in the oligomeric structure strongly suggest that equivalent strands from different subunits are in close spatial proximity. These structural characteristics are remarkably similar to those of the equivalent sequence in alpha A-crystallin [Berengian, A. R., Bova, M. P., and Mchaourab, H. S. (1997) Biochemistry 36, 9951-9957]. In both proteins, a beta-strand spans the sequence and is located at a subunit interface, indicating that one set of interactions between subunits, and its associated symmetry, is conserved. This is the first report of sequence-specific structural similarity between alpha A-crystallin and HSP 27 and the first identification of a conserved secondary structural element in the alpha-crystallin domain.

Amino Acid Sequence↗
PubMed2006

iMOTdb--a comprehensive collection of spatially interacting motifs in proteins.

Realization of conserved residues that represent a protein family is crucial for clearer understanding of biological function as well as for the better recognition of additional members in sequence databases. Functionally important residues are recognized well due to their high degree of conservation in closely related sequences and are annotated in functional motif databases. Structural motifs are central to the integrity of the fold and require careful analysis for their identification. We report the availability of a database of spatially interacting motifs in single protein structures as well as those among distantly related protein structures that belong to a superfamily. Spatial interactions amongst conserved motifs are automatically measured using sequence similarity scores and distance calculations. Interactions between pairs of conserved motifs are described in the form of pseudoenergies. iMOTdb database provides information for 854,488 motifs corresponding to 60,849 protein structural domains and 22,648 protein structural entries.

Amino Acid Motifs↗
PubMed2004

Novel retinal genes discovered by mining the mouse embryonic RetinalExpress database.

PURPOSE: Bioinformatics has emerged as a powerful tool for identifying novel genes and pathways associated with retinal biology and disease. The developing mouse retina expresses an exceedingly large and complex variety of genes. Many of these genes have not been characterized but nevertheless are likely to have important developmental or physiological functions. The purpose of this study was to use an in silico approach with a mouse embryonic retinal database of cDNAs/expressed sequence tags (ESTs) named RetinalExpress to identify previously uncharacterized genes that are represented in the developing retina. METHODS: cDNA clones unique to the RetinalExpress database were identified by comparing clones in the RetinalExpress database with those in other cDNA/EST databases. We used a hierarchical filtering procedure with high stringency criteria that included sequence quality, colinearity with hypothetical gene sequences, and absence of any substantial existing annotation to select clones that were likely to represent novel genes. Selected clones were located on mouse chromosomes using National Center for Biotechnology Informatics Map Viewer software and the database from the University of California at Santa Cruz Genome Bioinformatics Web browser. The expression of selected retinal transcripts was determined using reverse transcriptase (RT)-PCR. In situ hybridization of sectioned embryonic and postnatal retinas was performed to determine spatial expression patterns of selected transcripts. RESULTS: Of the 27,765 cDNA clones from RetinalExpress that we filtered through several public cDNA/EST databases, 26 cDNA/EST sequences were identified that, at the time of the analysis, were unique to RetinalExpress. Seventeen clones were selected for RT-PCR analysis, and retinal transcripts corresponding to previously uncharacterized genes were unambiguously detected for six clones. Three genes encoded open reading frames containing putative functional domains; one sequence contained an HMG DNA binding domain, another, an RFX DNA binding domain, and another, a phospholipase C catalytic domain X. Transcripts from the genes encoding DNA binding domains were expressed in embryonic and postnatal retinas with distinct spatial patterns. CONCLUSIONS: The characterization of 26 mouse genes whose partial nucleotide sequences were uniquely represented in the RetinalExpress cDNA/EST database demonstrated the feasibility of retinal gene discovery using in silico analysis. Two of these genes had distinctive spatial expression patterns in the retina and one was likely to function as a DNA binding protein in embryonic and postnatal retinas. The gene identification approach described here demonstrates the usefulness of establishing large cDNA/EST databases from highly specialized neuronal tissues such as the retina to find novel genes.

Animals↗
PubMed1996

Structure of the Ras-binding domain of c-Raf-1 as determined by NMR spectroscopy and identification of the region that interacts with Ras.

The structure of the Ras-binding domain of human c-Raf-1 (residues 55 to 132) as determined in solution by NMR spectroscopy is presented. It consists of a five-stranded beta-sheet, a twelve residue alpha-helix, and an additional one-turn helix. The fold belongs to a known family whose members include ubiquitin and protein G. The surface of Raf55-132 that interacts with Ras has been identified by resonance perturbation mapping. The binding site is a spatially contiguous patch comprised of the two-N-terminal beta-strands, the loop between them, and the C-terminal end of the alpha-helix. A model of the Raf-Ras complex is presented, which was derived by analogy to the complex between protein G and a Fab fragment of IgG. In the model, edge beta-strands of each protein align in an antiparallel orientation, forming a unified beta-sheet, and side chains from both proteins are able to participate in ionic and hydrophobic interactions at the interface.

Binding Sites↗
PubMed1999

An olfactory-specific glutathione-S-transferase in the sphinx moth Manduca sexta.

Insect antennae have a primary function of detecting odors including sex pheromones and plant volatiles. The assumption that genes uniquely expressed in these antennae have an olfactory role has led to the identification of several genes that are integral components of odorant transduction. In the present study, differential display polymerase chain reaction (ddPCR) was used to isolate 25 antennal-specific mRNAs from the male sphinx moth Manduca sexta. Northern blot analyses revealed that one clone, designated G7-9, was antennal-specific and was highly enriched in male antennae relative to female antennae. In situ hybridization indicated that G7-9 expression was restricted to a spatial domain of the olfactory epithelium occupied exclusively by sex-pheromone-sensitive olfactory sensilla. Amino acid homology and phylogenetic analyses identified G7-9 as a glutathione-S-transferase (GST); we have named the full-length clone GST-msolf1. GSTs are known to function primarily in the detoxification of noxious compounds. Spectrophotometric and chromatographic analyses of total GST activity indicate that the endogenous GSTs of male and female antennae can modify trans-2-hexenal, a plant-derived green leaf aldehyde known to stimulate the olfactory system of M. sexta. The restricted localization of GST-msolf1 to sex-pheromone-sensitive sensilla, the fact that the sex pheromone of M. sexta consists of a complex mixture of aldehyde components, and the observation that antennal GSTs can modify an aldehyde odorant suggest that GST-msolf1 may have a role in signal termination. In the light of the more commonly observed role of GSTs in xenobiotic metabolism, we propose that GST-msolf1 may play a dual role of protecting the olfactory system from harmful xenobiotics and inactivating aldehyde odorants, especially components of the M. sexta sex pheromone.

Amino Acid Sequence↗
PubMed2000

Identification of residues in the TPR domain of Ssn6 responsible for interaction with the Tup1 protein.

Ssn6, a yeast protein that comprises 10 tandem tetratricopeptide repeat (TPR) motifs, associates with Tup1 repressor protein and acts as a transcriptional corepressor. In this report we identify point mutations in the TPR1 of Ssn6 that disrupt Tup1 interaction. Furthermore, we construct a 3D model of the TPR domain of Ssn6, which is responsible for Tup1 binding, based on the known structure of protein phosphatase 5. According to this model all selected mutations reduce the ability of Ssn6 to interact with Tup1 by affecting the structural integrity of TPR1 and/or the correct spatial arrangement of TPR1 relative to TPR2 and TPR3.

Amino Acid Motifs↗
PubMed1989

Phylogenetic evidence for tertiary interactions in 16S-like ribosomal RNA.

Major efforts are underway to elucidate the spatial distribution of ribosomal RNAs (rRNAs) in the ribosome. An especially informative approach is the identification of likely base-base tertiary interactions within the RNA by phylogenetic comparison. Herein evidence is presented for three heretofore unrecognized candidate tertiary interactions, G506/C525, C779/G803 and A994/U1380 (1) in 16S-like rRNAs. This brings to eight the number of such interactions that are strongly supported by phylogenetic evidence. The three newly identified interactions further define the folding within domains II and III of 16S-like rRNA. No interactions have yet been found that would serve to orient the domains relative to one another.

Animals↗
PubMed2004

Propagation of terahertz pulses in random media.

We describe measurements of single-cycle terahertz pulse propagation in a random medium. The unique capabilities of terahertz time-domain spectroscopy permit the characterization of a multiply scattered field with unprecedented spatial and temporal resolution. With these results, we can develop a framework for understanding the statistics of broadband laser speckle. Also, the ability to extract information on the phase of the field opens up new possibilities for characterizing multiply scattered waves. We illustrate this with a simple example, which involves computing a time-windowed temporal correlation between fields measured at different spatial locations. This enables the identification of individual scattering events, and could lead to a new method for imaging in random media.

Journal Article↗
PubMed2005

Common and specific amino acid residues in the prokaryotic polypeptide release factors RF1 and RF2: possible functional implications.

Termination of protein synthesis is promoted in ribosomes by proper stop codon discrimination by class 1 polypeptide release factors (RFs). A large set of prokaryotic RFs differing in stop codon specificity, RF1 for UAG and UAA, and RF2 for UGA and UAA, was analyzed by means of a recently developed computational method allowing identification of the specificity-determining positions (SDPs) in families composed of proteins with similar but not identical function. Fifteen SDPs were identified within the RF1/2 superdomain II/IV known to be implicated in stop codon decoding. Three of these SDPs had particularly high scores. Five residues invariant for RF1 and RF2 [invariant amino acid residues (IRs)] were spatially clustered with the highest-scoring SDPs that in turn were located in two zones within the SDP/IR area. Zone 1 (domain II) included PxT and SPF motifs identified earlier by others as 'discriminator tripeptides'. We suggest that IRs in this zone take part in the recognition of U, the first base of all stop codons. Zone 2 (domain IV) possessed two SDPs with the highest scores not identified earlier. Presumably, they also take part in stop codon binding and discrimination. Elucidation of potential functional role(s) of the newly identified SDP/IR zones requires further experiments.

Algorithms↗
PubMed2006

Functional interactions between Dlx2 and lymphoid enhancer factor regulate Msx2.

Dlx2, Lymphoid Enhancer Factor (Lef-1) and Msx2 transcription factors are required for several developmental processes. To understand the control of gene expression by these factors, chromatin immunoprecipitation (ChIP) assays identified Msx2 as a downstream target of Dlx2 and Lef-1. Dlx2 activates the Msx2 promoter in several cell lines and binds DNA as a monomer and dimer. A Lef-1 beta-catenin-dependent isoform minimally activates the Msx2 promoter and a Lef-1 beta-catenin-independent isoform is inactive, however co-expression of Dlx2 and both Lef-1 isoforms synergistically activate the Msx2 promoter. Co-immunoprecipitation and protein pull-down experiments demonstrate Lef-1 physically interacts with Dlx2. Deletion analyses of the Lef-1 protein reveal specific regions required for synergism with Dlx2. The Lef-1 beta-catenin binding domain (betaDB) is not required for its interaction with Dlx2. Msx2 can auto-regulate its promoter and repress Dlx2 activation. Msx2 repression of Dlx2 activation is dose-specific and both bind a common DNA-binding element. These transcriptional mechanisms correlate with the temporal and spatial expression of these factors and may provide a mechanism for the control of several developmental processes. We demonstrate new transcriptional activities for Dlx2, Msx2 and Lef-1 through protein interactions and identification of downstream targets.

Animals↗
PubMed1996

Identification and localization of polycystin, the PKD1 gene product.

Polycystin, the product of autosomal dominant polycystic kidney disease (ADPKD) 1 gene (PKD1) is the cardinal member of a novel class of proteins. As a first step towards elucidating the function of polycystin and the pathogenesis of ADPKD, three types of information were collected in the current study: the subcellular localization of polycystin, the spatial and temporal distribution of the protein within normal tissues and the effects of ADPKD mutations on the pattern of expression in affected tissues. Antisera directed against a synthetic peptide and two recombinant proteins of different domains of polycystin revealed the presence of an approximately 400-kD protein (polycystin) in the membrane fractions of normal fetal, adult, and ADPKD kidneys. Immunohistological studies localized polycystin to renal tubular epithelia, hepatic bile ductules, and pancreatic ducts, all sites of cystic changes in ADPKD, as well as in tissues such as skin that are not known to be affected in ADPKD. By electron microscopy, polycystin was predominantly associated with plasma membranes. Polycystin was significantly less abundant in adult than in fetal epithelia. In contrast, polycystin was overexpressed in most, but not all, cysts in ADPKD kidneys.

Antibodies↗