scholarly journals Direct observation of structure and dynamics during phase separation of an elastomeric protein

2017 ◽  
Vol 114 (22) ◽  
pp. E4408-E4415 ◽  
Author(s):  
Sean E. Reichheld ◽  
Lisa D. Muiznieks ◽  
Fred W. Keeley ◽  
Simon Sharpe
Keyword(s):  
Phase Separation ◽  
Matrix Protein ◽  
Molecular Mechanisms ◽  
Bulk Water ◽  
Extracellular Matrix Protein ◽  
Elastic Fibers ◽  
Cross Linking ◽  
Structure And Dynamics ◽  
Chemical Cross Linking

Despite its growing importance in biology and in biomaterials development, liquid–liquid phase separation of proteins remains poorly understood. In particular, the molecular mechanisms underlying simple coacervation of proteins, such as the extracellular matrix protein elastin, have not been reported. Coacervation of the elastin monomer, tropoelastin, in response to heat and salt is a critical step in the assembly of elastic fibers in vivo, preceding chemical cross-linking. Elastin-like polypeptides (ELPs) derived from the tropoelastin sequence have been shown to undergo a similar phase separation, allowing formation of biomaterials that closely mimic the material properties of native elastin. We have used NMR spectroscopy to obtain site-specific structure and dynamics of a self-assembling elastin-like polypeptide along its entire self-assembly pathway, from monomer through coacervation and into a cross-linked elastic material. Our data reveal that elastin-like hydrophobic domains are composed of transient β-turns in a highly dynamic and disordered chain, and that this disorder is retained both after phase separation and in elastic materials. Cross-linking domains are also highly disordered in monomeric and coacervated ELP3 and form stable helices only after chemical cross-linking. Detailed structural analysis combined with dynamic measurements from NMR relaxation and diffusion data provides direct evidence for an entropy-driven mechanism of simple coacervation of a protein in which transient and nonspecific intermolecular hydrophobic contacts are formed by disordered chains, whereas bulk water and salt are excluded.

scholarly journals Fibulin-2 Is Dispensable for Mouse Development and Elastic Fiber Formation

2007 ◽  
Vol 28 (3) ◽  
pp. 1061-1067 ◽  
Author(s):  
Francois-Xavier Sicot ◽  
Takeshi Tsuda ◽  
Dessislava Markova ◽  
John F. Klement ◽  
Machiko Arita ◽  
...  
Keyword(s):  
Matrix Protein ◽  
Elastic Fiber ◽  
Embryonic Stem ◽  
Fiber Formation ◽  
Extracellular Matrix Protein ◽  
Elastic Fibers ◽  
Mouse Development ◽  
Fibrillin 1

ABSTRACT Fibulin-2 is an extracellular matrix protein belonging to the five-member fibulin family, of which two members have been shown to play essential roles in elastic fiber formation during development. Fibulin-2 interacts with two major constituents of elastic fibers, tropoelastin and fibrillin-1, in vitro and localizes to elastic fibers in many tissues in vivo. The protein is prominently expressed during morphogenesis of the heart and aortic arch vessels and at early stages of cartilage development. To examine its role in vivo, we generated mice that do not express the fibulin-2 gene (Fbln2) through homologous recombination of embryonic stem cells. Unexpectedly, the fibulin-2-null mice were viable and fertile and did not display gross and anatomical abnormalities. Histological and ultrastructural analyses revealed that elastic fibers assembled normally in the absence of fibulin-2. No compensatory up-regulation of mRNAs for other fibulin members was detected in the aorta and skin tissue. However, in the fibulin-2 null aortae, fibulin-1 immunostaining was increased in the inner elastic lamina, where fibulin-2 preferentially localizes. The results demonstrate that fibulin-2 is not required for mouse development and elastic fiber formation and suggest possible functional redundancy between fibulin-1 and fibulin-2.

scholarly journals Antifibrotic Effect of Marine Ovothiol in an In Vivo Model of Liver Fibrosis

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Mariarita Brancaccio ◽  
Giuseppe D’Argenio ◽  
Vincenzo Lembo ◽  
Anna Palumbo ◽  
Immacolata Castellano
Keyword(s):  
Liver Fibrosis ◽  
Matrix Protein ◽  
Molecular Mechanisms ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Extracellular Matrix Protein ◽  
In Vivo Model ◽  
Protein Accumulation ◽  
Antifibrotic Effect

Liver fibrosis is a complex process caused by chronic hepatic injury, which leads to an excessive increase in extracellular matrix protein accumulation and fibrogenesis. Several natural products, including sulfur-containing compounds, have been investigated for their antifibrotic effects; however, the molecular mechanisms underpinning their action are partially still obscure. In this study, we have investigated for the first time the effect of ovothiol A, π-methyl-5-thiohistidine, isolated from sea urchin eggs on an in vivo murine model of liver fibrosis. Mice were intraperitoneally injected with carbon tetrachloride (CCl4) to induce liver fibrosis and treated with ovothiol A at the dose of 50 mg/kg 3 times a week for 2 months. Treatment with ovothiol A caused a significant reduction of collagen fibers as observed by histopathological changes and serum parameters compared to mice treated with control solution. This antifibrotic effect was associated to the decrease of fibrogenic markers involved in liver fibrosis progression, such as the transforming growth factor (TGF-β), the α-smooth muscle actin (α-SMA), and the tissue metalloproteinases inhibitor (TIMP-1). Finally, we provided evidence that the attenuation of liver fibrosis by ovothiol A treatment can be regulated by the expression and activity of the membrane-bound γ-glutamyl-transpeptidase (GGT), which is a key player in maintaining intracellular redox homoeostasis. Overall, these findings indicate that ovothiol A has significant antifibrotic properties and can be considered as a new marine drug or dietary supplement in potential therapeutic strategies for the treatment of liver fibrosis.

scholarly journals Specific heparan sulfate modifications stabilize the synaptic organizer MADD-4/Punctin at C. elegans neuromuscular junctions

Genetics ◽  
2021 ◽  
Author(s):  
Mélissa Cizeron ◽  
Laure Granger ◽  
Hannes E BÜlow ◽  
Jean-Louis Bessereau
Keyword(s):  
Heparan Sulfate ◽  
Matrix Protein ◽  
Neuromuscular Junctions ◽  
Core Protein ◽  
Extracellular Matrix Protein ◽  
Inhibitory Synapses ◽  
Single Chain ◽  
C Elegans ◽  
Sugar Chains

Abstract Heparan sulfate proteoglycans contribute to the structural organization of various neurochemical synapses. Depending on the system, their role involves either the core protein or the glycosaminoglycan chains. These linear sugar chains are extensively modified by heparan sulfate modification enzymes, resulting in highly diverse molecules. Specific modifications of glycosaminoglycan chains may thus contribute to a sugar code involved in synapse specificity. Caenorhabditis elegans is particularly useful to address this question because of the low level of genomic redundancy of these enzymes, as opposed to mammals. Here, we systematically mutated the genes encoding heparan sulfate modification enzymes in C. elegans and analyzed their impact on excitatory and inhibitory neuromuscular junctions. Using single chain antibodies that recognize different heparan sulfate modification patterns, we show in vivo that these two heparan sulfate epitopes are carried by the SDN-1 core protein, the unique C. elegans syndecan orthologue, at neuromuscular junctions. Intriguingly, these antibodies differentially bind to excitatory and inhibitory synapses, implying unique heparan sulfate modification patterns at different neuromuscular junctions. Moreover, while most enzymes are individually dispensable for proper organization of neuromuscular junctions, we show that 3-O-sulfation of SDN-1 is required to maintain wild-type levels of the extracellular matrix protein MADD-4/Punctin, a central synaptic organizer that defines the identity of excitatory and inhibitory synaptic domains at the plasma membrane of muscle cells.

scholarly journals Low-Intensity Pulsed Ultrasound Promotes Autophagy-Mediated Migration of Mesenchymal Stem Cells and Cartilage Repair

2021 ◽  
Vol 30 ◽  
pp. 096368972098614
Author(s):  
Peng Xia ◽  
Xinwei Wang ◽  
Qi Wang ◽  
Xiaoju Wang ◽  
Qiang Lin ◽  
...  
Keyword(s):  
Cartilage Repair ◽  
Matrix Protein ◽  
Extracellular Matrix Protein ◽  
Histopathological Analysis ◽  
Pulsed Ultrasound ◽  
Low Intensity Pulsed Ultrasound ◽  
Low Intensity ◽  
Autophagy Inhibitor

Mesenchymal stem cell (MSC) migration is promoted by low-intensity pulsed ultrasound (LIPUS), but its mechanism is unclear. Since autophagy is known to regulate cell migration, our study aimed to investigate if LIPUS promotes the migration of MSCs via autophagy regulation. We also aimed to investigate the effects of intra-articular injection of MSCs following LIPUS stimulation on osteoarthritis (OA) cartilage. For the in vitro study, rat bone marrow-derived MSCs were treated with an autophagy inhibitor or agonist, and then they were stimulated by LIPUS. Migration of MSCs was detected by transwell migration assays, and stromal cell-derived factor-1 (SDF-1) and C-X-C chemokine receptor type 4 (CXCR4) protein levels were quantified. For the in vivo study, a rat knee OA model was generated and treated with LIPUS after an intra-articular injection of MSCs with autophagy inhibitor added. The cartilage repair was assessed by histopathological analysis and extracellular matrix protein expression. The in vitro results suggest that LIPUS increased the expression of SDF-1 and CXCR4, and it promoted MSC migration. These effects were inhibited and enhanced by autophagy inhibitor and agonist, respectively. The in vivo results demonstrate that LIPUS significantly enhanced the cartilage repair effects of MSCs on OA, but these effects were blocked by autophagy inhibitor. Our results suggest that the migration of MSCs was enhanced by LIPUS through the activation autophagy, and LIPUS improved the protective effect of MSCs on OA cartilage via autophagy regulation.

Contrasting migratory response of astrocytoma cells to tenascin mediated by different integrins

1996 ◽  
Vol 109 (8) ◽  
pp. 2161-2168 ◽  
Author(s):  
A. Giese ◽  
M.A. Loo ◽  
S.A. Norman ◽  
S. Treasurywala ◽  
M.E. Berens
Keyword(s):  
Cell Adhesion ◽  
Matrix Protein ◽  
Glioma Cells ◽  
Extracellular Matrix Protein ◽  
Integrin Receptors ◽  
Migration Assay ◽  
Dose Dependent Fashion ◽  
Beta 1 Integrin

Tenascin, an extracellular matrix protein, is expressed in human gliomas in vitro and in vivo. The distribution of tenascin at the invasive edge of these tumors, even surrounding solitary invading cells, suggests a role for this protein as a regulator of glioma cell migration. We tested whether purified tenascin, passively deposited on surfaces, influenced the adhesion or migration of a human gliomaderived cell line, SF-767. Adhesion of glioma cells to tenascin increased in a dose-dependent fashion up to a coating concentration of 10 micrograms/ml. Higher coating concentrations resulted in progressively fewer cells attaching. Cell adhesion could be blocked to basal levels using anti-beta 1 integrin antibodies. In contrast, when anti-alpha v antibodies were added to the medium of cells on tenascin, cell adhesion was enhanced slightly. Using a microliter scale migration assay, we found that cell motility on tenascin was dose dependently stimulated at coating concentrations of 1 and 3 micrograms/ml, but migration was inhibited below levels of non-specific motility when tested at coating concentrations of 30 and 100 micrograms/ml. Migration on permissive concentrations of tenascin could be reversibly inhibited with anti-beta 1, while treatment with anti-alpha v antibodies increased migration rates. We conclude that SF-767 glioma cells express two separate integrin receptors that mediate contrasting adhesive and migratory responses to tenascin.

Initial characterization of anosmin-1, a putative extracellular matrix protein synthesized by definite neuronal cell populations in the central nervous system

1996 ◽  
Vol 109 (7) ◽  
pp. 1749-1757 ◽  
Author(s):  
N. Soussi-Yanicostas ◽  
J.P. Hardelin ◽  
M.M. Arroyo-Jimenez ◽  
O. Ardouin ◽  
R. Legouis ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Extracellular Matrix ◽  
Nervous System ◽  
Cell Membrane ◽  
Heparan Sulfate ◽  
Matrix Protein ◽  
Neuronal Cell ◽  
Extracellular Matrix Protein ◽  
Cell Populations

The KAL gene is responsible for the X-chromosome linked form of Kallmann's syndrome in humans. Upon transfection of CHO cells with a human KAL cDNA, the corresponding encoded protein, KALc, was produced. This protein is N-glycosylated, secreted in the cell culture medium, and is localized at the cell surface. Several lines of evidence indicate that heparan-sulfate chains of proteoglycan(s) are involved in the binding of KALc to the cell membrane. Polyclonal and monoclonal antibodies to the purified KALc were generated. They allowed us to detect and characterize the protein encoded by the KAL gene in the chicken central nervous system at late stages of embryonic development. This protein is synthesized by definite neuronal cell populations including Purkinje cells in the cerebellum, mitral cells in the olfactory bulbs and several subpopulations in the optic tectum and the striatum. The protein, with an approximate molecular mass of 100 kDa, was named anosmin-1 in reference to the deficiency of the sense of smell which characterizes the human disease. Anosmin-1 is likely to be an extracellular matrix component. Since heparin treatment of cell membrane fractions from cerebellum and tectum resulted in the release of the protein, we suggest that one or several heparan-sulfate proteoglycans are involved in the binding of anosmin-1 to the membranes in vivo.

scholarly journals Stromal protein βig-h3 reprogrammes tumour microenvironment in pancreatic cancer

Gut ◽  
2018 ◽  
Vol 68 (4) ◽  
pp. 693-707 ◽  
Author(s):  
Delphine Goehrig ◽  
Jérémy Nigri ◽  
Rémi Samain ◽  
Zhichong Wu ◽  
Paola Cappello ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Tumour Growth ◽  
Matrix Protein ◽  
Immune Escape ◽  
Interaction Mechanism ◽  
Tumour Microenvironment ◽  
Extracellular Matrix Protein ◽  
Ductal Adenocarcinoma ◽  
The Impact

ObjectivePancreatic cancer is associated with an abundant stromal reaction leading to immune escape and tumour growth. This massive stroma drives the immune escape in the tumour. We aimed to study the impact of βig-h3 stromal protein in the modulation of the antitumoural immune response in pancreatic cancer.DesignWe performed studies with p48-Cre;KrasG12D, pdx1-Cre;KrasG12D;Ink4a/Arffl/fl, pdx1-Cre;KrasG12D; p53R172H mice and tumour tissues from patients with pancreatic ductal adenocarcinoma (PDA). Some transgenic mice were given injections of anti-βig-h3, anti-CD8, anti-PD1 depleting antibodies. Tumour growth as well as modifications in the activation of local immune cells were analysed by flow cytometry, immunohistochemistry and immunofluorescence. Tissue stiffness was measured by atomic force microscopy.ResultsWe identified βig-h3 stromal-derived protein as a key actor of the immune paracrine interaction mechanism that drives pancreatic cancer. We found that βig-h3 is highly produced by cancer-associated fibroblasts in the stroma of human and mouse. This protein acts directly on tumour-specific CD8+ T cells and F4/80 macrophages. Depleting βig-h3 in vivo reduced tumour growth by enhancing the number of activated CD8+ T cell within the tumour and subsequent apoptotic tumour cells. Furthermore, we found that targeting βig-h3 in established lesions released the tissue tension and functionally reprogrammed F4/80 macrophages in the tumour microenvironment.ConclusionsOur data indicate that targeting stromal extracellular matrix protein βig-h3 improves the antitumoural response and consequently reduces tumour weight. Our findings present βig-h3 as a novel immunological target in pancreatic cancer.

Glucocorticoids upregulate tropoelastin expression during late stages of fetal lung development

1995 ◽  
Vol 268 (3) ◽  
pp. L491-L500 ◽  
Author(s):  
R. A. Pierce ◽  
W. I. Mariencheck ◽  
S. Sandefur ◽  
E. C. Crouch ◽  
W. C. Parks
Keyword(s):  
Lung Development ◽  
Matrix Protein ◽  
Fetal Lung ◽  
Extracellular Matrix Protein ◽  
Transferase Activity ◽  
Mrna Levels ◽  
Pregnant Rats ◽  
Fetal Lung Development ◽  
Chloramphenicol Acetyl Transferase Activity

The production of elastin, an essential extracellular matrix protein of terminal airway interstitium, occurs mostly during early development. Because glucocorticoids influence airway maturation, we studied the effect of dexamethasone (Dex) on tropoelastin expression during fetal lung development. Timed-pregnant rats were treated with Dex (1 mg/kg daily), and fetal lungs were collected 3 days later at 17, 19, and 21 days of gestation. Dex treatment resulted in about a threefold increase in tropoelastin mRNA levels at 19 days concomitant with accelerated airway development. By in situ hybridization, Dex treatment increased the number of tropoelastin-expressing cells and the level of tropoelastin mRNA per cell. In organ culture, Dex increased lung tropoelastin expression and augmented cortisol stimulation of tropoelastin expression. In fetal pulmonary artery smooth muscle cells, 10(-8) M Dex upregulated tropoelastin mRNA expression and increased tropoelastin promoter-chloramphenicol acetyl transferase activity in transient transfections. These data indicate that pharmacologically administered glucocorticoids transcriptionally upregulate fetal lung tropoelastin expression and suggest that steroid hormones may be important regulators of elastin production in vivo.

scholarly journals An Elastin-Derived Self-Assembling Polypeptide

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Antonietta Pepe ◽  
Brigida Bochicchio
Keyword(s):  
Elastic Properties ◽  
Matrix Protein ◽  
Extracellular Matrix Protein ◽  
Elastic Fibers ◽  
Hydrophobic Region ◽  
Beta Turn ◽  
Self Assembling ◽  
Polypeptide Sequence ◽  
Hydrophilic Region ◽  
Self Aggregation

Elastin is an extracellular matrix protein responsible for the elastic properties of organs and tissues, the elastic properties being conferred to the protein by the presence of elastic fibers. In the perspective of producing tailor-made biomaterials of potential interest in nanotechnology and biotechnology fields, we report a study on an elastin-derived polypeptide. The choice of the polypeptide sequence encoded by exon 6 of Human Tropoelastin Gene is dictated by the peculiar sequence of the polypeptide. As a matter of fact, analogously to elastin, it is constituted of a hydrophobic region (GLGAFPAVTFPGALVPGG) and of a more hydrophilic region rich of lysine and alanine residues (VADAAAAYKAAKA). The role played by the two different regions in triggering the adoption of beta-turn and beta-sheet conformations is herein discussed and demonstrated to be crucial for the self-aggregation properties of the polypeptide.

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