scholarly journals Topological assignment of the N-terminal extension of plasma gelsolin to the gelsolin surface

2005 ◽  
Vol 385 (3) ◽  
pp. 659-665 ◽  
Author(s):  
Ulrike FOCK ◽  
Brigitte M. JOCKUSCH ◽  
Wolf-Dieter SCHUBERT ◽  
Horst HINSSEN
Keyword(s):  
Tertiary Structure ◽  
Limited Proteolysis ◽  
Cell Types ◽  
Actin Binding ◽  
Plasma Gelsolin ◽  
Equine Plasma ◽  
Binding Epitope ◽  
N Terminus ◽  
Immunological Approach ◽  
Cytoplasmic Gelsolin

The actin-binding protein gelsolin is highly conserved in vertebrates and exists in two isoforms, a cytoplasmic and an extracellular variant, generated by alternative splicing. In mammals, these isoforms differ only by an N-terminal extension in plasma gelsolin, a short sequence of up to 25 amino acids. Cells and tissues may contain both variants, as plasma gelsolin is secreted by many cell types. The tertiary structure of equine plasma gelsolin has been elucidated, but without any information on the N-terminal extension. In this paper, we present topographical data on the N-terminal extension, derived using a biochemical and immunological approach. For this purpose, a monoclonal antibody was generated that exclusively recognizes cytoplasmic gelsolin but not the extracellular variant and thus allows isoform-specific immunodetection and quantification of cytoplasmic gelsolin in the presence of plasma gelsolin. Using limited proteolysis and pepscan analysis, we mapped the binding epitope and localized it within two regions in segment 1 of the cytoplasmic gelsolin sequence: Tyr34–Ile45 and Leu64–Ile78. In the tertiary structure of the cytoplasmic variant, these sequences are mutually adjacent and located in the proximity of the N-terminus. We therefore conclude that the binding site of the antibody is covered by the N-terminal extension in plasma gelsolin and thus sterically hinders antibody binding. Our results allow for a topological model of the N-terminal extension on the surface of the gelsolin molecule, which was unknown previously.

scholarly journals Distinct autoinhibitory mechanisms regulate vinculin binding by αT-catenin and αE-catenin

2020 ◽  
Author(s):  
Jonathon A. Heier ◽  
Sabine Pokutta ◽  
Ian W. Dale ◽  
Sun Kyung Kim ◽  
Andrew P. Hinck ◽  
...  
Keyword(s):  
Cardiac Function ◽  
Binding Site ◽  
Limited Proteolysis ◽  
Biochemical Properties ◽  
Intramolecular Interactions ◽  
Actin Binding ◽  
Titration Calorimetry ◽  
Open Conformation ◽  
Actin Binding Domain ◽  
N Terminus

ABSTRACTα-catenin binds directly to β-catenin and connects the cadherin-catenin complex to the actin cytoskeleton. Tension regulates α-catenin conformation: actomyosin-generated force stretches the middle(M)-region to relieve autoinhibition and reveal a binding site for the actin-binding protein vinculin. Here we describe the biochemical properties of αT(testes)-catenin, an α-catenin isoform critical for cardiac function, and how intramolecular interactions regulate vinculin binding autoinhibition. Isothermal titration calorimetry (ITC) showed that αT-catenin binds the β-catenin/N-cadherin complex with a similar low nanomolar affinity to that of αE-catenin. Limited proteolysis revealed that the αT-catenin M-region adopts a more open conformation than αE-catenin. The αT-catenin M-region binds the vinculin N-terminus with low nanomolar affinity, indicating that the isolated αT-catenin M-region is not autoinhibited and thereby distinct from αE-catenin. However, the αT-catenin head (N- and M-regions) binds vinculin 1000-fold more weakly (low micromolar affinity), indicating that the N-terminus regulates M-region binding to vinculin. In cells, αT-catenin recruitment of vinculin to cell-cell contacts requires the actin-binding domain and actomyosin-generated tension, indicating that force regulates vinculin binding. Together, our results indicate that the αT-catenin N-terminus is required to maintain M-region autoinhibition and modulate vinculin binding. We postulate that the unique molecular properties of αT-catenin allow it to function as a scaffold for building specific adhesion complexes.

scholarly journals Expression of human plasma gelsolin in Escherichia coli and dissection of actin binding sites by segmental deletion mutagenesis.

1989 ◽  
Vol 109 (2) ◽  
pp. 593-605 ◽  
Author(s):  
M Way ◽  
J Gooch ◽  
B Pope ◽  
A G Weeds
Keyword(s):  
Escherichia Coli ◽  
Human Plasma ◽  
Binding Sites ◽  
Current Knowledge ◽  
Limited Proteolysis ◽  
Actin Binding ◽  
High Yield ◽  
Soluble Form ◽  
Proteolytic Fragment ◽  
Plasma Gelsolin

Human plasma gelsolin has been expressed in high yield and soluble form in Escherichia coli. The protein has nucleating and severing activities identical to those of plasma gelsolin and is fully calcium sensitive in its interactions with monomeric actin. A number of deletion mutants have been expressed to explore the function of the three actin binding sites. Their design is based on the sixfold segmental repeat in the protein sequence. (These sites are located in segment 1, segments 2-3, and segments 4-6). Two mutants, S1-3 and S4-6, are equivalent to the NH2- and COOH-terminal halves of the molecule obtained by limited proteolysis. S1-3 binds two actin monomers in the presence or absence of calcium, it severs and caps filaments but does not nucleate polymerization. S4-6 binds a single actin monomer but only in calcium. These observations confirm and extend current knowledge on the properties of the two halves of gelsolin. Two novel constructs have also been studied that provide a different pairwise juxtaposition of the three sites. S2-6, which lacks the high affinity site of segment 1 (equivalent to the 14,000-Mr proteolytic fragment) and S1,4-6, which lacks segments 2-3 (the actin filament binding domain previously identified using the 28,000-Mr proteolytic fragment). S2-6 binds two actin monomers in calcium and nucleates polymerization; it associates laterally with filaments in the presence or absence of calcium and has a weak calcium-dependent fragmenting activity. S1,4-6 also binds two actin monomers in calcium and one in EGTA, has weak severing activity but does not nucleate polymerization. A model is presented for the involvement of the three binding sites in the various activities of gelsolin.

scholarly journals Sequence and domain organization of scruin, an actin-cross-linking protein in the acrosomal process of Limulus sperm.

1995 ◽  
Vol 128 (1) ◽  
pp. 51-60 ◽  
Author(s):  
M Way ◽  
M Sanders ◽  
C Garcia ◽  
J Sakai ◽  
P Matsudaira
Keyword(s):  
Amino Acid ◽  
Actin Filaments ◽  
Limited Proteolysis ◽  
Open Reading Frames ◽  
Actin Binding ◽  
Molar Ratio ◽  
Domain Organization ◽  
Ring Canals ◽  
Drosophila Protein ◽  
Reading Frames

The acrosomal process of Limulus sperm is an 80-microns long finger of membrane supported by a crystalline bundle of actin filaments. The filaments in this bundle are crosslinked by a 102-kD protein, scruin present in a 1:1 molar ratio with actin. Recent image reconstruction of scruin decorated actin filaments at 13-A resolution shows that scruin is organized into two equally sized domains bound to separate actin subunits in the same filament. We have cloned and sequenced the gene for scruin from a Limulus testes cDNA library. The deduced amino acid sequence of scruin reflects the domain organization of scruin: it consists of a tandem pair of homologous domains joined by a linker region. The domain organization of scruin is confirmed by limited proteolysis of the purified acrosomal process. Three different proteases cleave the native protein in a 5-kD Protease-sensitive region in the middle of the molecule to generate an NH2-terminal 47-kD and a COOH-terminal 56-kD protease-resistant domains. Although the protein sequence of scruin has no homology to any known actin-binding protein, it has similarities to several proteins, including four open reading frames of unknown function in poxviruses, as well as kelch, a Drosophila protein localized to actin-rich ring canals. All proteins that show homologies to scruin are characterized by the presence of an approximately 50-amino acid residue motif that is repeated between two and seven times. Crystallographic studies reveal this motif represents a four beta-stranded fold that is characteristic of the "superbarrel" structural fold found in the sialidase family of proteins. These results suggest that the two domains of scruin seen in EM reconstructions are superbarrel folds, and they present the possibility that other members of this family may also bind actin.

scholarly journals Synthesis of p36 and p35 is increased when U-937 cells differentiate in culture but expression is not inducible by glucocorticoids.

1989 ◽  
Vol 9 (1) ◽  
pp. 232-240 ◽  
Author(s):  
C M Isacke ◽  
R A Lindberg ◽  
T Hunter
Keyword(s):  
Tyrosine Kinases ◽  
Myeloid Cell ◽  
Cell Types ◽  
Culture Conditions ◽  
Actin Binding ◽  
Myeloid Cell Line ◽  
Related Proteins ◽  
Cortical Cytoskeleton ◽  
Inflammatory Action ◽  
Human Specific

p36 and p35 are distinct but related proteins that share many structural and biochemical features which were first identified as major substrates for protein-tyrosine kinases. Subsequently, both proteins have been shown to be Ca2+-, phospholipid-, and F-actin-binding proteins that underlie the plasma membrane and are associated with the cortical cytoskeleton. Recent reports have claimed that these proteins function as lipocortins, i.e., phospholipase A2 inhibitors that mediate the anti-inflammatory action of glucocorticoids. To investigate this possibility and to learn more about the functions of p36 and p35, we used human-specific anti-p36 and anti-p35 monoclonal antibodies to determine whether the expression or secretion of either protein was inducible by dexamethasone in the human U-937 myeloid cell line and in other human cell types. Additionally, we examined the levels of mRNA for both proteins. No effect of dexamethasone was observed on p36 or p35 expression at either the mRNA or protein level, nor were these proteins secreted under any of the culture conditions investigated. However, it was observed that in these cells the rate of synthesis and accumulation of both proteins was increased when the U-937 cells were induced to differentiate in culture to adherent macrophagelike cells. This offers a model system with which to study the control of p36 and p35 expression.

What Can Epitope Specific Antibodies Tell us About the Organization of Caveolin in Cells?

2001 ◽  
Vol 7 (S2) ◽  
pp. 1030-1031
Author(s):  
J.M. Robinson
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Biochemical Characterization ◽  
Cell Types ◽  
Membrane Microdomains ◽  
Truncated Form ◽  
Coated Pits ◽  
N Terminus ◽  
Plasma Membrane Microdomains

There are three members of the caveolin (CAV) gene family that give rise to four polypeptides. These polypeptides are CAV-1α, CAV-1β, CAV-2, and CAV-3. The CAV-1β isoform is a truncated form of CAV-1α that lacks 31 amino acids at the N-terminus of the molecule. The CAV- 1β molecule arises through an alternative splicing mechanism.Caveolae are specialized plasma membrane microdomains that are expressed at high levels in some cell types (e.g., endothelium, adipocytes, fibroblasts). These specialized regions of the plasma membrane have a characteristic omega-shaped appearance with diameters ranging from 40-90 run. They are distinct from clathrin-coated pits since they lack the characteristic coated appearance in electron microscopy. Caveolae were among the first structures to be discovered by biological electron microscopy. However, biochemical characterization of these structures did not begin in earnest until a marker protein was identified. The initial marker was the 22-kDa protein known as caveolin.

scholarly journals Structure of the N Terminus of a Nonmuscle α-Tropomyosin in Complex with the C Terminus: Implications for Actin Binding†‡

Biochemistry ◽  
2009 ◽  
Vol 48 (6) ◽  
pp. 1272-1283 ◽  
Author(s):  
Norma J. Greenfield ◽  
Lucy Kotlyanskaya ◽  
Sarah E. Hitchcock-DeGregori
Keyword(s):  
Actin Binding ◽  
C Terminus ◽  
N Terminus

scholarly journals Gelsolin variant (Asn-187) in familial amyloidosis, Finnish type

1990 ◽  
Vol 272 (3) ◽  
pp. 827-830 ◽  
Author(s):  
J Ghiso ◽  
M Haltia ◽  
F Prelli ◽  
J Novello ◽  
B Frangione
Keyword(s):  
Amyloid Fibrils ◽  
Corneal Dystrophy ◽  
Binding Activity ◽  
Actin Binding ◽  
Cranial Neuropathy ◽  
Protein Subunit ◽  
Plasma Gelsolin ◽  
Sequence Identity ◽  
Finnish Type ◽  
Repetitive Motif

Familial amyloidosis, Finnish type (FAF), is an inherited form of systemic amyloidosis clinically characterized by cranial neuropathy and lattice corneal dystrophy. We have demonstrated that the protein subunit isolated from amyloid fibrils shows considerable sequence identity with gelsolin, an actin-binding protein. We have purified the amyloid subunit from a second case and further analysed different fractions from the previous one. Sequence analysis shows that, in both cases, the amyloid subunit starts at position 173 of the mature molecule; it has a heterogeneous N-terminus and contains one amino acid substitution, namely asparagine for aspartic acid, at position 15 (gelsolin residue 187), that is due to a guanine-to-adenine transversion corresponding to nucleotide-654 of human plasma gelsolin cDNA. The substitution maps in a fragment with actin-binding activity and is located in a repetitive motif highly conserved among species. Thus FAF is the first human disease known to be caused by an internal abnormal degradation of a gelsolin variant. We designate this variant of gelsolin-associated amyloidosis ‘Agel Asn-187’.

Mimicking cotranslational folding of prosubtilisin E in vitro

2020 ◽  
Vol 167 (5) ◽  
pp. 473-482 ◽  
Author(s):  
Sung-Gun Kim ◽  
Yu-Jen Chen ◽  
Liliana Falzon ◽  
Jean Baum ◽  
Masayori Inouye
Keyword(s):  
Crucial Role ◽  
Tertiary Structure ◽  
Molten Globule ◽  
Secondary Structures ◽  
Terminal Region ◽  
Folding Intermediates ◽  
C Terminus ◽  
Cotranslational Folding ◽  
N Terminus

Abstract Nascent polypeptides are synthesized on ribosomes starting at the N-terminus and simultaneously begin to fold during translation. We constructed N-terminal fragments of prosubtilisin E containing an intramolecular chaperone (IMC) at N-terminus to mimic cotranslational folding intermediates of prosubtilisin. The IMC-fragments of prosubtilisin exhibited progressive enhancement of their secondary structures and thermostabilities with increasing polypeptide length. However, even the largest IMC-fragment with 72 residues truncated from the C-terminus behaved as a molten globule, indicating the requirement of the C-terminal region to have a stable tertiary structure. Furthermore, truncation of the IMC in the IMC-fragments resulted in aggregation, suggesting that the IMC plays a crucial role to prevent misfolding and aggregation of cotranslational folding intermediates during translation of prosubtilisin polypeptide.

scholarly journals Cortactin: A Major Cellular Target of the Gastric Carcinogen Helicobacter pylori

Cancers ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 159 ◽  
Author(s):  
Irshad Sharafutdinov ◽  
Steffen Backert ◽  
Nicole Tegtmeyer
Keyword(s):  
Helicobacter Pylori ◽  
Epithelial Cells ◽  
Cell Movement ◽  
Cell Types ◽  
Eukaryotic Cell ◽  
Actin Binding ◽  
Gastric Diseases ◽  
Cell Functions ◽  
H Pylori ◽  
Cytoskeletal Rearrangements

Cortactin is an actin binding protein and actin nucleation promoting factor regulating cytoskeletal rearrangements in nearly all eukaryotic cell types. From this perspective, cortactin poses an attractive target for pathogens to manipulate a given host cell to their own benefit. One of the pathogens following this strategy is Helicobacter pylori, which can cause a variety of gastric diseases and has been shown to be the major risk factor for the onset of gastric cancer. During infection of gastric epithelial cells, H. pylori hijacks the cellular kinase signaling pathways, leading to the disruption of key cell functions. Specifically, by overruling the phosphorylation status of cortactin, H. pylori alternates the activity of molecular interaction partners of this important protein, thereby manipulating the performance of actin-cytoskeletal rearrangements and cell movement. In addition, H. pylori utilizes a unique mechanism to activate focal adhesion kinase, which subsequently prevents host epithelial cells from extensive lifting from the extracellular matrix in order to achieve chronic infection in the human stomach.

scholarly journals Actin-binding compounds, previously discovered by FRET-based high-throughput screening, differentially affect skeletal and cardiac muscle

2020 ◽  
Vol 295 (41) ◽  
pp. 14100-14110 ◽  
Author(s):  
Piyali Guhathakurta ◽  
Lien A. Phung ◽  
Ewa Prochniewicz ◽  
Sarah Lichtenberger ◽  
Anna Wilson ◽  
...  
Keyword(s):  
Cardiac Muscle ◽  
High Throughput ◽  
High Throughput Screening ◽  
Cell Types ◽  
Actin Binding ◽  
Time Resolved ◽  
Biochemical Assays ◽  
Numerous Cell ◽  
Cardiac Muscles ◽  
And Function

Actin's interactions with myosin and other actin-binding proteins are essential for cellular viability in numerous cell types, including muscle. In a previous high-throughput time-resolved FRET (TR-FRET) screen, we identified a class of compounds that bind to actin and affect actomyosin structure and function. For clinical utility, it is highly desirable to identify compounds that affect skeletal and cardiac muscle differently. Because actin is more highly conserved than myosin and most other muscle proteins, most such efforts have not targeted actin. Nevertheless, in the current study, we tested the specificity of the previously discovered actin-binding compounds for effects on skeletal and cardiac α-actins as well as on skeletal and cardiac myofibrils. We found that a majority of these compounds affected the transition of monomeric G-actin to filamentous F-actin, and that several of these effects were different for skeletal and cardiac actin isoforms. We also found that several of these compounds affected ATPase activity differently in skeletal and cardiac myofibrils. We conclude that these structural and biochemical assays can be used to identify actin-binding compounds that differentially affect skeletal and cardiac muscles. The results of this study set the stage for screening of large chemical libraries for discovery of novel compounds that act therapeutically and specifically on cardiac or skeletal muscle.

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