Characterization of Follistatin-Type Domains and Their Contribution to Myostatin and Activin A Antagonism

Abstract Follistatin (FST)-type proteins are important antagonists of some members of the large TGF-β family of cytokines. These include myostatin, an important negative regulator of muscle growth, and the closely related activin A, which is involved in many physiological functions, including maintenance of a normal reproductive axis. FST-type proteins, including FST and FST-like 3 (FSTL3), differentially inhibit various TGF-β family ligands by binding each ligand with two FST-type molecules. In this study, we sought to examine features that are important for ligand antagonism by FST-type proteins. Previous work has shown that a modified construct consisting of the FST N-terminal domain (ND) followed by two repeating follistatin domains (FSD), herein called FST ND-FSD1-FSD1, exhibits strong specificity for myostatin over activin A. Using cell-based assays, we show that FST ND-FSD1-FSD1 is unique in its specificity for myostatin as compared with similar constructs containing domains from FSTL3 and that the ND is critical to its activity. Furthermore, we demonstrate that FSD3 of FST provides affinity to ligand inhibition and confers resistance to perturbations in the ND and FSD2, likely through the interaction of FSD3 of one FST molecule with the ND of the other FST molecule. Additionally, our data suggest that this contact provides cooperativity to ligand antagonism. Cross-linking studies show that this interaction also potentiates formation of 1:2 ligand-FST complexes, whereas lack of FSD3 allows formation of 1:1 complexes. Altogether, these studies support that domain differences generate FST-type molecules that are each uniquely suited ligand antagonists.

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Characterization of two myostatin genes in pufferfish Takifugu bimaculatus: sequence, genomic structure, and expression

PeerJ ◽

10.7717/peerj.9655 ◽

2020 ◽

Vol 8 ◽

pp. e9655

Author(s):

Yinzhen Sheng ◽

Yulong Sun ◽

Xin Zhang ◽

Haifu Wan ◽

Chengjie Yao ◽

...

Keyword(s):

Skeletal Muscle ◽

Genomic Structure ◽

Muscle Growth ◽

Negative Regulator ◽

Pigment Formation ◽

Formation Stage ◽

Proliferation And Differentiation ◽

The Brain

Myostatin (MSTN) is a negative regulator of muscle growth, which restrains the proliferation and differentiation of myoblasts. To understand the role of two mstn genes of Takifugu bimaculatus, the full-length cDNAs of 1131 bp Tbmstn1 and 1,080 bp Tbmstn2 were obtained from the T. bimaculatus’ genomic database, which encodes 376 and 359 amino acids, respectively. The results of qRT-PCR showed that Tbmstn1 was expressed in the eye, kidney, spleen, skeletal muscle, gill, and brain, and the expression level in the skeletal muscle was extremely significantly higher than in other examined tissues. Tbmstn2 was expressed in the skin, skeletal muscle, gill, and brain, and had the highest expression in the skeletal muscle, followed by expression in the brain. Meanwhile, in different stages of embryonic development, the expression of Tbmstn1 started from the gastrula stage. Its expression in the eye-pigment formation stage and hatching stage was significantly higher than that in other stages. The Tbmstn2 was expressed in all examined embryonic stages with different levels, and the highest expression was detected in the eye-pigment formation stage. These results suggested that Tbmstn1 and Tbmstn2 may involve in the development of skeletal muscle, and Tbmstn2 may be related to the formation of nervous system.

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Characterization of cis-regulatory elements of the c-myc promoter responding to human GM-CSF or mouse interleukin 3 in mouse proB cell line BA/F3 cells expressing the human GM-CSF receptor.

Molecular Biology of the Cell ◽

10.1091/mbc.6.6.627 ◽

1995 ◽

Vol 6 (6) ◽

pp. 627-636 ◽

Cited By ~ 26

Author(s):

S Watanabe ◽

S Ishida ◽

K Koike ◽

K Arai

Keyword(s):

Binding Site ◽

Cell Line ◽

Regulatory Elements ◽

Negative Regulator ◽

Beta Subunit ◽

The Other ◽

Interleukin 3 ◽

Gm Csf ◽

Myc Gene

Interleukin 3 (IL-3) or granulocyte macrophage colony-stimulating factor (GM-CSF) activates c-fos, c-jun, and c-myc genes and proliferation in both hematopoietic and nonhematopoietic cells. Using a series of deletion mutants of the beta subunit of human GM-CSF receptor (hGMR) and inhibitors of tyrosine kinase, two distinct signaling pathways, one for activation of c-fos and c-jun genes, and the other for cell proliferation and activation of c-myc gene have been elucidated. In contrast to wealth of information on the pathway leading to activation of c-fos/c-jun genes, knowledge of the latter is scanty. To clarify the mechanisms of activation of c-myc gene by cytokines, we established a transient transfection assay in mouse proB cell line BA/F3 cells expressing hGMR. Analyses of hGMR beta subunit mutants revealed two cytoplasmic regions involved in activation of the c-myc promoter, one is essential and the other is dispensable but enhances the activity. These regions are located at the membrane proximal and the distal regions covering amino acid positions 455-544 and 544-589, respectively. Characterization of cis-acting regulatory elements of the c-myc gene showed that the region containing the P2 promoter initiation site is sufficient to mediate the response to mIL-3 or hGM-CSF. Electrophoretic mobility shift assay using an oligonucleotide corresponding to the distal putative E2F binding site revealed that p107/E2F complex, the negative regulator of E2F, decreased, and free E2F increased after mIL-3 stimulation. These results support the thesis that mIL-3 or hGM-CSF regulates the c-myc promoter by altering composition of the E2F complexes at E2F binding site.

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Isolation and characterization of two novel A20-like proteins

Biochemical Journal ◽

10.1042/bj3570617 ◽

2001 ◽

Vol 357 (3) ◽

pp. 617-623 ◽

Cited By ~ 47

Author(s):

Paul C. EVANS ◽

Ellen R. TAYLOR ◽

John COADWELL ◽

Karen HEYNINCK ◽

Rudi BEYAERT ◽

...

Keyword(s):

Zinc Finger ◽

Sequence Similarity ◽

Nuclear Factor Κb ◽

Negative Regulator ◽

Tumour Necrosis Factor Receptor ◽

Isolation And Characterization ◽

Important Negative Regulator ◽

Inflammatory Processes ◽

Cellular Zinc

The transcription factor nuclear factor κB (NF-κB) plays a pivotal role in inflammatory processes through induction of adhesion molecules and chemokines. The zinc finger molecule A20 is an important negative regulator of NF-κB. The mechanism utilized by A20 is not fully understood, but A20 has been shown to bind to tumour-necrosis-factor-receptor-associated factor (TRAF) molecules, which are necessary for pro-inflammatory cytokine signalling. We report two novel genes, Cezanne (cellular zinc finger anti-NF-kB) and TRABID (TRAF-bnding domain), with sequence similarity to A20. Co-immunoprecipitation studies indicated that TRAF6 was able to interact with both Cezanne and TRABID. In contrast, reporter gene experiments revealed a specific ability of Cezanne to down-regulate NF-κB. It is likely, therefore, that Cezanne participates in the regulation of inflammatory processes.

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A very rapid in situ Kikuchi technique to determine relative crystal misorientation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100106211 ◽

1988 ◽

Vol 46 ◽

pp. 830-831

Author(s):

J. I. Bennetch

Keyword(s):

Electron Beam ◽

Analytical Techniques ◽

Superplastic Forming ◽

The Other ◽

Kikuchi Pattern ◽

Kikuchi Line ◽

Line Analysis

In a recent study of the superplastic forming (SPF) behavior of certain Al-Li-X alloys, the relative misorientation between adjacent (sub)grains proved to be an important parameter. It is well established that the most accurate way to determine misorientation across boundaries is by Kikuchi line analysis. However, the SPF study required the characterization of a large number of (sub)grains in each sample to be statistically meaningful, a very time-consuming task even for comparatively rapid Kikuchi analytical techniques.In order to circumvent this problem, an alternate, even more rapid in-situ Kikuchi technique was devised, eliminating the need for the developing of negatives and any subsequent measurements on photographic plates. All that is required is a double tilt low backlash goniometer capable of tilting ± 45° in one axis and ± 30° in the other axis. The procedure is as follows. While viewing the microscope screen, one merely tilts the specimen until a standard recognizable reference Kikuchi pattern is centered, making sure, at the same time, that the focused electron beam remains on the (sub)grain in question.

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645194 ◽

1990 ◽

Vol 63 (02) ◽

pp. 193-203 ◽

Cited By ~ 9

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.

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Rapid Isolation of High Molecular Weight Urokinase from Native Human Urine

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657167 ◽

1982 ◽

Vol 47 (03) ◽

pp. 197-202 ◽

Cited By ~ 23

Author(s):

Kurt Huber ◽

Johannes Kirchheimer ◽

Bernd R Binder

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Human Urine ◽

Gel Filtration ◽

Chain Structure ◽

The Other ◽

Single Chain ◽

Apparent Homogeneity ◽

Sequential Adsorption

SummaryUrokinase (UK) could be purified to apparent homogeneity starting from crude urine by sequential adsorption and elution of the enzyme to gelatine-Sepharose and agmatine-Sepharose followed by gel filtration on Sephadex G-150. The purified product exhibited characteristics of the high molecular weight urokinase (HMW-UK) but did contain two distinct entities, one of which exhibited a two chain structure as reported for the HMW-UK while the other one exhibited an apparent single chain structure. The purification described is rapid and simple and results in an enzyme with probably no major alterations. Yields are high enough to obtain purified enzymes for characterization of UK from individual donors.

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Biopolymers with Medical Applications Optimized method for obtaining chitosan-based delivery systems

Revista de Chimie ◽

10.37358/rc.18.7.6411 ◽

2018 ◽

Vol 69 (7) ◽

pp. 1756-1759 ◽

Cited By ~ 1

Author(s):

Luminita Confederat ◽

Iuliana Motrescu ◽

Sandra Constantin ◽

Florentina Lupascu ◽

Lenuta Profire

Keyword(s):

Drug Delivery ◽

Drug Delivery Systems ◽

Average Diameter ◽

Delivery Systems ◽

Cross Linking ◽

Low Molecular Weight ◽

Polymeric Systems ◽

Chitosan Microparticles ◽

Degree Of Swelling

The aim of this study was to optimize the method used for obtaining microparticles based on chitosan � a biocompatible, biodegradable, and nontoxic polymer, and to characterize the developed systems. Chitosan microparticles, as drug delivery systems were obtained by inotropic gelation method using pentasodiumtripolyphosphate (TPP) as cross-linking agent. Chitosan with low molecular weight (CSLMW) in concentration which ranged between 0.5 and 5 %, was used while the concentration of cross-linking agent ranged between 1 and 5%. The characterization of the microparticles in terms of shape, uniformity and adhesion was performed in solution and dried state. The size of the microparticles and the degree of swelling were also determined. The structure and the morphology of the developed polymeric systems were analyzed by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM).The average diameter of the chitosan microparticles was around 522 �m. The most stable microparticles were obtained using CSLMW 1% and TPP 2% or CSLMW 0.75%and TPP 1%. The micropaticles were spherical, uniform and without flattening. Using CSLMW in concentration of 0.5 % poorly cross-linked and crushed microparticles have been obtained at all TPP concentrations. By optimization of the method, stable chitosan-based micropaticles were obtained which will be used to develop controlled release systems for drug delivery.

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