Construction and Characterization of Chimeric Enzyme Consisting of an Amino-Terminal Domain of Phenylalanine Dehydrogenase and a Carboxy-Terminal Domain of Leucine Dehydrogenase

1994 ◽  
Vol 116 (4) ◽  
pp. 931-936 ◽  
Author(s):  
Kunishige Kataoka ◽  
Harumi Takada ◽  
Katsuyuki Tanizawa ◽  
Tohru Yoshimura ◽  
Nobuyoshi Esaki ◽  
...  
Keyword(s):  
Chimeric Enzyme ◽  
Phenylalanine Dehydrogenase ◽  
Amino Terminal ◽  
Leucine Dehydrogenase ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

scholarly journals Ezrin contains cytoskeleton and membrane binding domains accounting for its proposed role as a membrane-cytoskeletal linker.

1993 ◽  
Vol 120 (1) ◽  
pp. 129-139 ◽  
Author(s):  
M Algrain ◽  
O Turunen ◽  
A Vaheri ◽  
D Louvard ◽  
M Arpin
Keyword(s):  
Plasma Membrane ◽  
Tyrosine Kinases ◽  
Actin Filaments ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Binding Domains ◽  
Carboxy Terminal Domain ◽  
Cell Surface Structures ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

Ezrin, a widespread protein present in actin-containing cell-surface structures, is a substrate of some protein tyrosine kinases. Based on its primary and secondary structure similarities with talin and band 4.1 it has been suggested that this protein could play a role in linking the cytoskeleton to the plasma membrane (Gould, K.L., A. Bretscher, F.S. Esch, and T. Hunter. 1989. EMBO (Eur. Mol. Biol. Organ.), J. 8:4133-4142; Turunen, O., R. Winqvist, R. Pakkanen, K.-H. Grzeschik, T. Wahlström, and A. Vaheri. 1989. J. Biol. Chem. 264:16727-16732). To test this hypothesis, we transiently expressed the complete human ezrin cDNA, or truncated cDNAs encoding the amino- and carboxy-terminal domains of the protein, in CV-1 cells. Protein epitope tagging was used to unambiguously determine the subcellular distribution of the protein encoded by the transfected cDNA. We show that this protein is concentrated underneath the dorsal plasma membrane in all actin-containing structures and is partially detergent insoluble. The amino-terminal domain displays the same localization but is readily extractable by nonionic detergent. The carboxy-terminal domain colocalizes with microvillar actin filaments as well as with stress fibers and remains associated with actin filaments after detergent extraction, and with disorganized actin structures after cytochalasin D treatment. Our results clearly demonstrate that ezrin interacts with membrane-associated components via its amino-terminal domain, and with the cytoskeleton via its carboxy-terminal domain. The amino-terminal domain could include the main determinant that restricts the entire protein to the cortical cytoskeleton in contact with the dorsal plasma membrane and its specialized microdomains such as microvilli, microspikes and lamellipodia.

scholarly journals Residues near the Amino Terminus of Rns Are Essential for Positive Autoregulation and DNA Binding

2008 ◽  
Vol 190 (7) ◽  
pp. 2279-2285 ◽  
Author(s):  
Georgeta N. Basturea ◽  
Maria D. Bodero ◽  
Mario E. Moreno ◽  
George P. Munson
Keyword(s):  
Dna Binding ◽  
Amino Terminus ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Amino Terminal Domain ◽  
Regulated Promoters

ABSTRACT Most members of the AraC/XylS family contain a conserved carboxy-terminal DNA binding domain and a less conserved amino-terminal domain involved in binding small-molecule effectors and dimerization. However, there is no evidence that Rns, a regulator of enterotoxigenic Escherichia coli virulence genes, responds to an effector ligand, and in this study we found that the amino-terminal domain of Rns does not form homodimers in vivo. Exposure of Rns to the chemical cross-linker glutaraldehyde revealed that the full-length protein is also a monomer in vitro. Nevertheless, deletion analysis of Rns demonstrated that the first 60 amino acids of the protein are essential for the activation and repression of Rns-regulated promoters in vivo. Amino-terminal truncation of Rns abolished DNA binding in vitro, and two randomly generated mutations, I14T and N16D, that independently abolished Rns autoregulation were isolated. Further analysis of these mutations revealed that they have disparate effects at other Rns-regulated promoters and suggest that they may be involved in an interaction with the carboxy-terminal domain of Rns. Thus, evolution may have preserved the amino terminus of Rns because it is essential for the regulator's activity even though it apparently lacks the two functions, dimerization and ligand binding, usually associated with the amino-terminal domains of AraC/XylS family members.

Characterization of a Mode of Specific Binding of Fibrin Monomer through its Amino-Terminal Domain by Macrophages and Macrophage Cell-Lines

1990 ◽  
Vol 63 (02) ◽  
pp. 193-203 ◽  
Author(s):  
John R Shainoff ◽  
Deborah J Stearns ◽  
Patricia M DiBello ◽  
Youko Hishikawa-Itoh
Keyword(s):  
Peritoneal Macrophages ◽  
Affinity Binding ◽  
Macrophage Cell ◽  
High Affinity Binding ◽  
Amino Terminal ◽  
High Affinity ◽  
Terminal Domain ◽  
Weak Binding ◽  
Amino Terminal Domain

SummaryThe studies reported here probe the existence of a receptor-mediated mode of fibrin-binding by macrophages that is associated with the chemical change underlying the fibrinogen-fibrin conversion (the release of fibrinopeptides from the amino-terminal domain) without depending on fibrin-aggregation. The question is pursued by 1) characterization of binding in relation to fibrinopeptide content of both the intact protein and the CNBr-fragment comprising the amino-terminal domain known as the NDSK of the protein, 2) tests of competition for binding sites, and 3) photo-affinity labeling of macrophage surface proteins. The binding of intact monomers of types lacking either fibrinopeptide A alone (α-fibrin) or both fibrinopeptides A and B (αβ-fibrin) by peritoneal macrophages is characterized as proceeding through both a fibrin-specific low density/high affinity (BMAX ≃ 200–800 molecules/cell, KD ≃ 10−12 M) interaction that is not duplicated with fibrinogen, and a non-specific high density/low affinity (BMAX ≥ 105 molecules/cell, KD ≥ 10−6 M) interaction equivalent to the weak binding of fibrinogen. Similar binding characteristics are displayed by monocyte/macrophage cell lines (J774A.1 and U937) as well as peritoneal macrophages towards the NDSK preparations of these proteins, except for a slightly weaker (KD ≃ 10−10 M) high-affinity binding. The high affinity binding of intact monomer is inhibitable by fibrin-NDSK, but not fibrinogen-NDSK. This binding appears principally dependent on release of fibrinopeptide-A, because a species of fibrin (β-fibrin) lacking fibrinopeptide-B alone undergoes only weak binding similar to that of fibrinogen. Synthetic Gly-Pro-Arg and Gly-His-Arg-Pro corresponding to the N-termini of to the α- and the β-chains of fibrin both inhibit the high affinity binding of the fibrin-NDSKs, and the cell-adhesion peptide Arg-Gly-Asp does not. Photoaffinity-labeling experiments indicate that polypeptides with elec-trophoretically estimated masses of 124 and 187 kDa are the principal membrane components associated with specifically bound fibrin-NDSK. The binding could not be up-regulated with either phorbol myristyl acetate, interferon gamma or ADP, but was abolished by EDTA and by lipopolysaccharide. Because of the low BMAX, it is suggested that the high-affinity mode of binding characterized here would be too limited to function by itself in scavenging much fibrin, but may act cooperatively with other, less limited modes of fibrin binding.

scholarly journals Characterization of the amino-terminal domain of Mx2/MxB-dependent interaction with the HIV-1 capsid

2014 ◽  
Vol 5 (12) ◽  
pp. 954-957 ◽  
Author(s):  
Jia Kong ◽  
Bo Xu ◽  
Wei Wei ◽  
Xin Wang ◽  
Wei Xie ◽  
...  
Keyword(s):  
Amino Terminal ◽  
Terminal Domain ◽  
Amino Terminal Domain ◽  
Hiv 1 ◽  
Dependent Interaction

Disruption of dynamic cell surface architecture of NIH3T3 fibroblasts by the N-terminal domains of moesin and ezrin: in vivo imaging with GFP fusion proteins

1999 ◽  
Vol 112 (1) ◽  
pp. 111-125 ◽  
Author(s):  
M.R. Amieva ◽  
P. Litman ◽  
L. Huang ◽  
E. Ichimaru ◽  
H. Furthmayr
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Full Length ◽  
Cell Behavior ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal

Lamellipodia, filopodia, microspikes and retraction fibers are characteristic features of a dynamic and continuously changing cell surface architecture and moesin, ezrin and radixin are thought to function in these microextensions as reversible links between plasma membrane proteins and actin microfilaments. Full-length and truncated domains of the three proteins were fused to green fluorescent protein (GFP), expressed in NIH3T3 cells, and distribution and behaviour of cells were analysed by using digitally enhanced differential interference contrast (DIC) and fluorescence video microscopy. The amino-terminal (N-)domains of all three proteins localize to the plasma membrane and fluorescence recordings parallel the dynamic changes in cell surface morphology observed by DIC microscopy of cultured cells. Expression of this domain, however, significantly affects cell surface architecture by the formation of abnormally long and fragile filopodia that poorly attach and retract abnormally. Even more striking are abundant irregular, branched and motionless membraneous structures that accumulate during retraction of lamellipodia. These are devoid of actin, endogenous moesin, ezrin and radixin, but contain the GFP-labeled domain. While a large proportion of endogenous proteins can be extracted with non-ionic detergents as in untransfected control cells, >90% of N-moesin and >60% of N-ezrin and N-radixin remain insoluble. The minimal size of the domain of moesin required for membrane localization and change in behavior includes residues 1–320. Deletions of amino acid residues from either end result in diffuse intracellular distribution, but also in normal cell behavior. Expression of GFP-fusions of full-length moesin or its carboxy-terminal domain has no effect on cell behavior during the observation period of 6–8 hours. The data suggest that, in the absence of the carboxy-terminal domain, N-moesin, -ezrin and -radixin interact tightly with the plasma membrane and interfere with normal functions of endogeneous proteins mainly during retraction.

scholarly journals Both Carboxy- and Amino-Terminal Domains of the Vaccinia Virus Interferon Resistance Gene, E3L, Are Required for Pathogenesis in a Mouse Model

2001 ◽  
Vol 75 (2) ◽  
pp. 850-856 ◽  
Author(s):  
Teresa A. Brandt ◽  
Bertram L. Jacobs
Keyword(s):  
Cell Culture ◽  
Weight Loss ◽  
Mouse Model ◽  
Vaccinia Virus ◽  
Host Range ◽  
Broad Host Range ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

ABSTRACT The vaccinia virus (VV) E3L gene is responsible for providing interferon (IFN) resistance and a broad host range to VV in cell culture. The E3L gene product contains two distinct domains. A conserved carboxy-terminal domain, which is required for the IFN resistance and broad host range of the virus, has been shown to bind double-stranded RNA (dsRNA) and inhibit the antiviral dsRNA-dependent protein kinase, PKR. The amino-terminal domain, while conserved among orthopoxviruses, is dispensable in cell culture. To study the role of E3L in whole-animal infections, WR strain VV recombinants either lacking E3L (VVΔE3L) or expressing an amino-terminal (VVE3LΔ83N) or carboxy-terminal (VVE3LΔ26C) truncation of E3L were constructed. Whereas wild-type VV had a 50% lethal dose of approximately 104 PFU after intranasal infection, and elicited severe weight loss and morbidity, VVΔE3L was apathogenic, leading to no death, weight loss, or morbidity. VVΔE3L was also apathogenic after intracranial injection. Although the amino-terminal domain of E3L is dispensable for infection of cells in culture, both the amino- and carboxy-terminal domains of E3L were required for full pathogenesis in intranasal infections. These results demonstrate that the entire E3L gene is required for pathogenesis in the mouse model.

scholarly journals Fimbrin is a homologue of the cytoplasmic phosphoprotein plastin and has domains homologous with calmodulin and actin gelation proteins.

1990 ◽  
Vol 111 (3) ◽  
pp. 1069-1079 ◽  
Author(s):  
M V de Arruda ◽  
S Watson ◽  
C S Lin ◽  
J Leavitt ◽  
P Matsudaira
Keyword(s):  
Calcium Binding ◽  
Actin Binding ◽  
Cdna Clones ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Actin Binding Domain ◽  
Solid Tissues ◽  
Complete Protein ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

Fimbrin is an actin-bundling protein found in intestinal microvilli, hair cell stereocilia, and fibroblast filopodia. The complete protein sequence (630 residues) of chicken intestine fimbrin has been determined from two full-length cDNA clones. The sequence encodes a small amino-terminal domain (115 residues) that is homologous with two calcium-binding sites of calmodulin and a large carboxy-terminal domain (500 residues) consisting of a fourfold-repeated 125-residue sequence. This repeat is homologous with the actin-binding domain of alpha-actinin and the amino-terminal domains of dystrophin, actin-gelation protein, and beta-spectrin. The presence of this duplicated domain in fimbrin links actin bundling proteins and gelation proteins into a common family of actin cross-linking proteins. Fimbrin is also homologous in sequence with human L-plastin and T-plastin. L-plastin is found in only normal or transformed leukocytes where it becomes phosphorylated in response to IL 1 or phorbol myristate acetate. T-plastin is found in cells of solid tissues where it does not become phosphorylated. Neoplastic cells derived from solid tissues express both isoforms. The differences in expression, sequence, and phosphorylation suggest possible functional differences between fimbrin isoforms.

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