scholarly journals Primary structure and domain organization of human alpha and beta adducin.

1991 ◽  
Vol 115 (3) ◽  
pp. 665-675 ◽  
Author(s):  
R Joshi ◽  
D M Gilligan ◽  
E Otto ◽  
T McLaughlin ◽  
V Bennett
Keyword(s):  
Amino Acids ◽  
Sequence Similarity ◽  
Complete Sequence ◽  
Amino Acid Sequences ◽  
Actin Binding ◽  
Cell Contact ◽  
Binding Motif ◽  
Rat Tissues ◽  
Erythroleukemia Cells ◽  
Skeletal Protein

Adducin is a membrane-skeletal protein which is a candidate to promote assembly of a spectrin-actin network in erythrocytes and at sites of cell-cell contact in epithelial tissues. The complete sequence of both subunits of human adducin, alpha (737 amino acids), and beta (726 amino acids) has been deduced by analysis of the cDNAs. The two subunits have strikingly conserved amino acid sequences with 49% identity and 66% similarity, suggesting evolution by gene duplication. Each adducin subunit has three distinct domains: a 39-kD NH2-terminal globular protease-resistant domain, connected by a 9-kD domain to a 33-kD COOH-terminal protease-sensitive tail comprised almost entirely of hydrophilic amino acids. The tail is responsible for the high frictional ratio of adducin noted previously, and was visualized by EM. The head domains of both adducin subunits exhibit a limited sequence similarity with the NH2-terminal actin-binding motif present in members of the spectrin superfamily and actin gelation proteins. The COOH-termini of both subunits contain an identical, highly basic stretch of 22 amino acids with sequence similarity to the MARCKS protein. Predicted sites of phosphorylation by protein kinase C include the COOH-terminus and sites at the junction of the head and tail. Northern blot analysis of mRNA from rat tissues, K562 erythroleukemia cells and reticulocytes has shown that alpha adducin is expressed in all the tissues tested as a single message size of 4 kb. In contrast, beta adducin shows tissue specific variability in size of mRNA and level of expression. A striking divergence between alpha and beta mRNAs was noted in reticulocytes, where alpha adducin mRNA is present in at least 20-fold higher levels than that of beta adducin. The beta subunit thus is a candidate to perform a limiting role in assembly of functional adducin molecules.

Hereditary elliptocytosis due to both qualitative and quantitative defects in membrane skeletal protein 4.1

Blood ◽  
1991 ◽  
Vol 78 (9) ◽  
pp. 2438-2443 ◽  
Author(s):  
JG Conboy ◽  
R Shitamoto ◽  
M Parra ◽  
R Winardi ◽  
A Kabra ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mechanical Stability ◽  
Membrane Skeleton ◽  
Actin Binding ◽  
Erythrocyte Morphology ◽  
Protein 4.1 ◽  
Hereditary Elliptocytosis ◽  
Exon Expression ◽  
Amino Acid Peptide ◽  
Skeletal Protein

Abstract Protein 4.1 is an important structural component of the membrane skeleton that helps determine erythrocyte morphology and membrane mechanical properties. In a previous study we identified a case of human hereditary elliptocytosis (HE) in which decreased membrane mechanical stability was due to deletion of 80 amino acids encompassing the entire 10-Kd spectrin-actin binding domain. A portion of this domain (21 amino acids) is encoded by an alternatively spliced exon that is expressed in late but not early erythroid cells. We now report a case of canine HE in which the abnormal phenotype is caused by failure to express this alternative peptide in the mature red blood cell (RBC) membrane skeleton, in conjunction with quantitative deficiency of protein 4.1. Western blotting of RBC membranes from a dog with HE showed a truncated protein 4.1 that did not react with antibodies directed against the alternative peptide. In addition, sequencing of cloned reticulocyte protein 4.1 cDNA showed a precise deletion of 63 nucleotides comprising this exon. Normal dog reticulocytes did express this exon. Expression of this 21 amino acid peptide during erythroid maturation is therefore essential for proper assembly of a mechanically competent membrane skeleton, because RBCs lacking this peptide have unstable membranes.

scholarly journals Supervillin (p205): A Novel Membrane-associated, F-Actin–binding Protein in the Villin/Gelsolin Superfamily

1997 ◽  
Vol 139 (5) ◽  
pp. 1255-1269 ◽  
Author(s):  
Kersi N. Pestonjamasp ◽  
Robert K. Pope ◽  
Julia D. Wulfkuhle ◽  
Elizabeth J. Luna
Keyword(s):  
Plasma Membranes ◽  
Adherens Junctions ◽  
Intracellular Localization ◽  
Amino Acid Sequences ◽  
Actin Binding ◽  
Striking Similarity ◽  
Cell Contact ◽  
Sequence Information ◽  
Nuclear Targeting ◽  
E Cadherin

Actin-binding membrane proteins are involved in both adhesive interactions and motile processes. We report here the purification and initial characterization of p205, a 205-kD protein from bovine neutrophil plasma membranes that binds to the sides of actin filaments in blot overlays. p205 is a tightly bound peripheral membrane protein that cosediments with endogenous actin in sucrose gradients and immunoprecipitates. Amino acid sequences were obtained from SDS-PAGE–purified p205 and used to generate antipeptide antibodies, immunolocalization data, and cDNA sequence information. The intracellular localization of p205 in MDBK cells is a function of cell density and adherence state. In subconfluent cells, p205 is found in punctate spots along the plasma membrane and in the cytoplasm and nucleus; in adherent cells, p205 concentrates with E-cadherin at sites of lateral cell–cell contact. Upon EGTA-mediated cell dissociation, p205 is internalized with E-cadherin and F-actin as a component of adherens junctions “rings.” At later times, p205 is observed in cytoplasmic punctae. The high abundance of p205 in neutrophils and suspension-grown HeLa cells, which lack adherens junctions, further suggests that this protein may play multiple roles during cell growth, adhesion, and motility. Molecular cloning of p205 cDNA reveals a bipartite structure. The COOH terminus exhibits a striking similarity to villin and gelsolin, particularly in regions known to bind F-actin. The NH2 terminus is novel, but contains four potential nuclear targeting signals. Because p205 is now the largest known member of the villin/gelsolin superfamily, we propose the name, “supervillin.” We suggest that supervillin may be involved in actin filament assembly at adherens junctions and that it may play additional roles in other cellular compartments.

scholarly journals Loss of cargo binding in the human myosin VI deafness mutant (R1166X) leads to increased actin filament binding

2016 ◽  
Vol 473 (19) ◽  
pp. 3307-3319 ◽  
Author(s):  
Susan D. Arden ◽  
David A. Tumbarello ◽  
Tariq Butt ◽  
John Kendrick-Jones ◽  
Folma Buss
Keyword(s):  
Amino Acids ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Actin Binding ◽  
Binding Motif ◽  
Myosin Vi ◽  
Binding Ability ◽  
Activated State ◽  
Cargo Binding

Mutations in myosin VI have been associated with autosomal-recessive (DFNB37) and autosomal-dominant (DFNA22) deafness in humans. Here, we characterise an myosin VI nonsense mutation (R1166X) that was identified in a family with hereditary hearing loss in Pakistan. This mutation leads to the deletion of the C-terminal 120 amino acids of the myosin VI cargo-binding domain, which includes the WWY-binding motif for the adaptor proteins LMTK2, Tom1 as well as Dab2. Interestingly, compromising myosin VI vesicle-binding ability by expressing myosin VI with the R1166X mutation or with single point mutations in the adaptor-binding sites leads to increased F-actin binding of this myosin in vitro and in vivo. As our results highlight the importance of cargo attachment for regulating actin binding to the motor domain, we perform a detailed characterisation of adaptor protein binding and identify single amino acids within myosin VI required for binding to cargo adaptors. We not only show that the adaptor proteins can directly interact with the cargo-binding tail of myosin VI, but our in vitro studies also suggest that multiple adaptor proteins can bind simultaneously to non-overlapping sites in the myosin VI tail. In conclusion, our characterisation of the human myosin VI deafness mutant (R1166X) suggests that defects in cargo binding may leave myosin VI in a primed/activated state with an increased actin-binding ability.

scholarly journals Metalloproteinase-like, disintegrin-like, cysteine-rich proteins MDC2 and MDC3: novel human cellular disintegrins highly expressed in the brain

1998 ◽  
Vol 334 (1) ◽  
pp. 93-98 ◽  
Author(s):  
Koji SAGANE ◽  
Yukio OHYA ◽  
Yoshikazu HASEGAWA ◽  
Isao TANAKA
Keyword(s):  
Snake Venom ◽  
Sequence Similarity ◽  
Amino Acid Sequences ◽  
Cytogenet Cell ◽  
Binding Motif ◽  
High Sequence Similarity ◽  
Cell Matrix ◽  
A Disintegrin And Metalloproteinase ◽  
Cell Matrix Interactions ◽  
The Brain

Cellular disintegrins are a family of membrane-anchored proteins structurally related to snake venom disintegrins, and are potential regulators of cell–cell and cell–matrix interactions. The members of this protein family are also called ADAMs (a disintegrin and metalloproteinase) or MDC proteins (metalloproteinase-like disintegrin-like cysteine-rich), because they all contain disintegrin-like and metalloproteinase-like domains. In this paper, we report the cloning and sequence analysis of two novel additional members of this family, which we have termed MDC2 and MDC3. The deduced amino acid sequences reveal that the two proteins possess typical cellular disintegrin structures [that is, pro-, metalloproteinase-like, disintegrin-like, cysteine-rich, epidermal growth factor-like, transmembrane, and cytoplasmic domains] and exhibit high sequence similarity with human MDC/ADAM11 protein [Katagiri, Harada, Emi and Nakamura (1995) Cytogenet. Cell Genet. 68, 39–44]. A zinc-binding motif, which is critical for proteinase activity, is disrupted in the metalloproteinase-like domain of MDC2 and MDC3, as well as MDC/ADAM11. In the disintegrin-like domain of snake venom short disintegrins, the RDG-containing loops are critical for integrin binding. These three MDCs do not contain the RDG sequences, but the corresponding loops in these proteins are similar to each other. Northern blot analysis revealed that the mRNAs of MDC2, MDC3 and MDC/ADAM11 are highly expressed in the brain. These findings suggest that these proteins may function as integrin ligands in the brain.

Nucleotide sequences of the 2-oxoacid ferredoxin oxidoreductase and ferredoxin genes fromFrankiastrain EuIK1, a symbiont ofElaeagnus umbellataroot nodules

1999 ◽  
Vol 77 (9) ◽  
pp. 1279-1286
Author(s):  
Won Young Yoo ◽  
Si Bum Sung ◽  
Chung Sun An
Keyword(s):  
Amino Acids ◽  
Nucleotide Sequence ◽  
Genomic Library ◽  
Sequence Similarity ◽  
Root Nodules ◽  
Amino Acid Sequences ◽  
Elaeagnus Umbellata ◽  
Ferredoxin Oxidoreductase ◽  
B Subunit ◽  
A Subunit

A genomic clone, pEuNIFII, was isolated by screening a genomic library of Frankia strain EuIK1, a symbiont of Elaeagnus umbellata Thunb. root nodules. A 1.5-kb fragment of pEuNIF4.0, which contained ORF2 and N-terminal part of nifS, was used as a probe. A 7.2-kb BamHI fragment of pEuNIFII, which was proven to be adjacent to the probe, was subjected to sequence determination. The sequence analysis suggested one partial ORF followed by three open reading frames (ORFs). Two ORFs next to nifS encodes an a subunit (672 amino acids) and b subunit (347 amino acids) of a 2-oxoacid ferredoxin oxidoreducatase (OR), respectively. The third ORF encodes 114 amino acids of a 7Fe-type ferredoxin (Fdx). All ORFs are transcribed in the same direction as other nif genes. Alignment of the deduced amino acid sequences from frankiae OR revealed the motifs of gamma and alpha domains seen in other ORs in the a subunit, and the beta domain in the b subunit. Frankia or shows about 44% nucleotide sequence similarity with nifJ from Klebsiella pneumoniae, while frankiae fdx shows about 56% similarity with fdxI from Azotobacter vinelandii. These genes are reported for the first time in Frankia, and putative roles of their products in symbiosis is discussed in relation to nitrogen fixation and carbohydrate metabolism.Key words: 2-oxoacid ferredoxin oxidoreductase, ferredoxin, nucleotide sequence, Frankia EuIK1.

scholarly journals Three determinants in ezrin are responsible for cell extension activity.

1997 ◽  
Vol 8 (8) ◽  
pp. 1543-1557 ◽  
Author(s):  
M Martin ◽  
C Roy ◽  
P Montcourrier ◽  
A Sahuquet ◽  
P Mangeat
Keyword(s):  
Amino Acids ◽  
Amino Acid Sequences ◽  
Full Length ◽  
Actin Binding ◽  
Cell Extension ◽  
Erm Proteins ◽  
Terminal Domain ◽  
Extension Activity ◽  
Dependent Cell

The ERM proteins--ezrin, radixin, and moesin--are key players in membrane-cytoskeleton interactions. In insect cells infected with recombinant baculoviruses, amino acids 1-115 of ezrin were shown to inhibit an actin- and tubulin-dependent cell-extension activity located in ezrin C-terminal domain (ezrin310-586), whereas full-length ezrin1-586 did not induce any morphological change. To refine the mapping of functional domains of ezrin, 30 additional constructs were overexpressed in Sf9 cells, and the resulting effect of each was qualitatively and semiquantitatively compared. The removal of amino acids 13-30 was sufficient to release a cell-extension phenotype. This effect was abrogated if the 21 distal-most C-terminal amino acids were subsequently deleted (ezrin31-565), confirming the existence of a head-to-tail regulation in the whole molecule. Surprisingly, the deletion in full-length ezrin of the same 21 amino acids provided strong cell-extension competence to ezrin1-565, and this property was recovered in N-terminal constructs as short as ezrin1-310. Within ezrin1-310, amino acid sequences 13-30 and 281-310 were important determinants and acted in cooperation to induce cytoskeleton mobilization. In addition, these same residues are part of a new actin-binding site characterized in vitro in ezrin N-terminal domain.

scholarly journals Cloning of the Schizosaccharomyces pombe gene encoding diadenosine 5′,5′′′-P1,P4-tetraphosphate (Ap4A) asymmetrical hydrolase: sequence similarity with the histidine triad (HIT) protein family

1995 ◽  
Vol 312 (3) ◽  
pp. 925-932 ◽  
Author(s):  
Y Huang ◽  
P N Garrison ◽  
L D Barnes
Keyword(s):  
Amino Acids ◽  
Schizosaccharomyces Pombe ◽  
Sequence Similarity ◽  
Enzymic Activity ◽  
Protein Family ◽  
Partial Purification ◽  
Binding Motif ◽  
Local Similarity ◽  
Multicopy Plasmid

Diadenosine 5′,5‴-P1,P4-tetraphosphate (Ap4A) asymmetric hydrolase (EC 3.6.1.17) is a specific catabolic enzyme of Ap4A found in Schizosaccharomyces pombe. We have previously described the partial purification of Ap4A hydrolase from S. pombe [Robinson, de la Peña and Barnes (1993) Biochim. Biophys. Acta 1161, 139-148]. We determined the sequence of the N-terminal 20 amino acids of Ap4A hydrolase and designed two degenerate PCR primers based on the sequence. The 60 bp DNA fragment obtained by PCR, which is specific to Ap4A hydrolase, was used to isolate the Ap4A hydrolase gene, aph1, from S. pombe by screening a genomic DNA library in a multicopy plasmid. Ap4A hydrolase activity from the crude supernatant of a positive S. pombe transformant was about 25-fold higher than the control. There was no detectable stimulation of enzymic activity by phosphate. The aph1 gene from S. pombe contains three introns. The intron boundaries were confirmed by sequencing the cDNA of the aph1 gene from a S. pombe cDNA library. The deduced open reading frame of the aph1 gene codes for 182 amino acids. Two regions of significant local similarity were identified between the Ap4A hydrolase and the histidine triad (HIT) protein family [Séraphin (1992) DNA Sequence 3, 177-179]. HIT proteins are present in prokaryotes, yeast, plants and mammals. Their functions are unknown, except that the bovine protein inhibits protein kinase C in vitro. All four histidine residues which are conserved among the HIT proteins, including the HxHxH putative Zn(2+)-binding motif, are conserved in the Ap4A hydrolase. In addition, there are two regions of similarity between the Ap4A phosphorylases I and II from Saccharomyces cerevisiae and Ap4A hydrolase from S. pombe. These regions overlap with the HIT protein similarity regions. The aph1 gene from S. pombe is the first asymmetrical Ap4A hydrolase gene to be cloned and sequenced.

scholarly journals Hereditary elliptocytosis due to both qualitative and quantitative defects in membrane skeletal protein 4.1

Blood ◽  
1991 ◽  
Vol 78 (9) ◽  
pp. 2438-2443
Author(s):  
JG Conboy ◽  
R Shitamoto ◽  
M Parra ◽  
R Winardi ◽  
A Kabra ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mechanical Stability ◽  
Membrane Skeleton ◽  
Actin Binding ◽  
Erythrocyte Morphology ◽  
Protein 4.1 ◽  
Hereditary Elliptocytosis ◽  
Exon Expression ◽  
Amino Acid Peptide ◽  
Skeletal Protein

Protein 4.1 is an important structural component of the membrane skeleton that helps determine erythrocyte morphology and membrane mechanical properties. In a previous study we identified a case of human hereditary elliptocytosis (HE) in which decreased membrane mechanical stability was due to deletion of 80 amino acids encompassing the entire 10-Kd spectrin-actin binding domain. A portion of this domain (21 amino acids) is encoded by an alternatively spliced exon that is expressed in late but not early erythroid cells. We now report a case of canine HE in which the abnormal phenotype is caused by failure to express this alternative peptide in the mature red blood cell (RBC) membrane skeleton, in conjunction with quantitative deficiency of protein 4.1. Western blotting of RBC membranes from a dog with HE showed a truncated protein 4.1 that did not react with antibodies directed against the alternative peptide. In addition, sequencing of cloned reticulocyte protein 4.1 cDNA showed a precise deletion of 63 nucleotides comprising this exon. Normal dog reticulocytes did express this exon. Expression of this 21 amino acid peptide during erythroid maturation is therefore essential for proper assembly of a mechanically competent membrane skeleton, because RBCs lacking this peptide have unstable membranes.

scholarly journals Molecular cloning and sequence analysis of the cDNA for ancrod, a thrombin-like enzyme from the venom of Calloselasma rhodostoma

1993 ◽  
Vol 294 (2) ◽  
pp. 387-390 ◽  
Author(s):  
L C Au ◽  
S B Lin ◽  
J S Chou ◽  
G W Teh ◽  
K J Chang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Serine Proteases ◽  
Sequence Similarity ◽  
Active Form ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Binding Reaction ◽  
Venom Glands ◽  
High Degree

The 1.54 kb cDNA for ancrod, a thrombin-like enzyme, was cloned from a lambda ZAP cDNA library derived from the venom glands of Calloselasma (Agkistrodon) rhodostoma. The cDNA sequence reveals that ancrod is synthesized as a pre-zymogen of 258 amino acids, including a putative secretory peptide of 18 amino acids and a proposed zymogen peptide of 6 amino-acid residues. The amino-acid sequence of the predicted active form of the enzyme exhibits a high degree of sequence similarity to those of mammalian serine proteases (trypsin and pancreatic kallikrein) and other thrombin-like enzymes (batroxobin and flavoxobin). Key amino-acid residues (His43, Asp88, Ser182 and Asp176) that are thought to be involved in the substrate cleavage and in the substrate-binding reaction are conserved. Ancrod contains 13 cysteine residues. Based on alignment with the amino-acid sequences of trypsin and batroxobin, six disulphide bridges can be predicted to be present in the ancrod protein. The existence of a free cysteine, which changes the common sequence surrounding the Ser182 active site from Gly-Asp-Ser-Gly-Gly-Pro to Cys-Asp-Ser-Gly-Gly-Pro, is unusual for a serine protease.

scholarly journals Equine rhinitis B virus: a new serotype

2001 ◽  
Vol 82 (11) ◽  
pp. 2641-2645 ◽  
Author(s):  
Jin-an Huang ◽  
Nino Ficorilli ◽  
Carol A. Hartley ◽  
Rebbecca S. Wilcox ◽  
Marianne Weiss ◽  
...  
Keyword(s):  
Amino Acids ◽  
Sequence Comparison ◽  
Sequence Similarity ◽  
Amino Acid Sequences ◽  
Open Reading Frame ◽  
Reading Frame ◽  
B Virus ◽  
The Family ◽  
High Degree

Equine rhinovirus serotype 3 isolate P313/75 was assigned, with an unclassified genus status, to the family Picornaviridae. The sequence from the 5′ poly(C) tract to the 3′ poly(A) tract of P313/75 was determined. The sequence is 8821 bases in length and contains a potential open reading frame for a polyprotein of 2583 amino acids. Sequence comparison and phylogenic analysis suggest that P313/75 is most closely related to the prototype equine rhinitis B virus (ERBV) strain P1436/71, formerly named equine rhinovirus type 2. A high degree of sequence similarity was found in the P2 and P3 regions of the two genomes. However, the deduced amino acid sequences of the P1 region of P313/75 and ERBV strain P1436/71 contained significant differences, which presumably account for the serological segregation of the two viruses. It is suggested that P313/75 can be classified as a new serotype of the genus Erbovirus, tentatively named ERBV2. Seroepidemiological data indicate that ERBV2 infection of horses may be common (24%) in Australia.

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