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Berlin Brandenburg

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  • 1
    Language: English
    In: The Journal of biological chemistry, 01 July 2016, Vol.291(27), pp.14095-108
    Description: The thyroid stimulating hormone receptor (TSHR) is a G protein-coupled receptor (GPCR) with a characteristic large extracellular domain (ECD). TSHR activation is initiated by binding of the hormone ligand TSH to the ECD. How the extracellular binding event triggers the conformational changes in the transmembrane domain (TMD) necessary for intracellular G protein activation is poorly understood. To gain insight in this process, the knowledge on the relative positioning of ECD and TMD and the conformation of the linker region at the interface of ECD and TMD are of particular importance. To generate a structural model for the TSHR we applied an integrated structural biology approach combining computational techniques with experimental data. Chemical cross-linking followed by mass spectrometry yielded 17 unique distance restraints within the ECD of the TSHR, its ligand TSH, and the hormone-receptor complex. These structural restraints generally confirm the expected binding mode of TSH to the ECD as well as the general fold of the domains and were used to guide homology modeling of the ECD. Functional characterization of TSHR mutants confirms the previously suggested close proximity of Ser-281 and Ile-486 within the TSHR. Rigidifying this contact permanently with a disulfide bridge disrupts ligand-induced receptor activation and indicates that rearrangement of the ECD/extracellular loop 1 (ECL1) interface is a critical step in receptor activation. The experimentally verified contact of Ser-281 (ECD) and Ile-486 (TMD) was subsequently utilized in docking homology models of the ECD and the TMD to create a full-length model of a glycoprotein hormone receptor.
    Keywords: G Protein-Coupled Receptor (Gpcr) ; Cell Signaling ; Computer Modeling ; Mass Spectrometry (MS) ; Protein Cross-Linking ; Receptor Structure-Function ; Site-Directed Mutagenesis ; Structural Biology ; Surface Plasmon Resonance (SPR) ; Receptors, Thyrotropin -- Metabolism
    E-ISSN: 1083-351X
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  • 2
    Language: English
    In: Biomaterials, December 2012, Vol.33(35), pp.8975-8985
    Description: , bone formation is a complex, tightly regulated process, influenced by multiple biochemical and physical factors. To develop a vital bone tissue engineering construct, all of these individual components have to be considered and integrated to gain an -like stimulation of target cells. The purpose of the present studies was to investigate the synergistic role of defined biochemical and physical microenvironments with respect to osteogenic differentiation of human mesenchymal stem cells (MSCs). Biochemical microenvironments have been designed using artificial extracellular matrices (aECMs), containing collagen I (coll) and glycosaminoglycans (GAGs) like chondroitin sulfate (CS), or a high-sulfated hyaluronan derivative (sHya), formulated as coatings on three-dimensional poly(caprolactone- -lactide) (PCL) scaffolds. As part of the physical microenvironment, cells were exposed to pulsed electric fields via transformer-like coupling (TC). Results showed that aECM containing sHya enhanced osteogenic differentiation represented by increases in ALP activity and gene-expression (RT-qPCR) of several bone-related proteins (RUNX-2, ALP, OPN). Electric field stimulation alone did not influence cell proliferation, but osteogenic differentiation was enhanced if osteogenic supplements were provided, showing synergistic effects by the combination of sHya and electric fields. These results will improve the understanding of bone regeneration processes and support the development of effective tissue engineered bone constructs.
    Keywords: Bone Tissue Engineering ; Human Mesenchymal Stem Cells (Mscs) ; Extracellular Matrices (Ecms) ; Glycosaminoglycans (Gags) ; Electrical Stimulation ; Transformer-Like Coupling (TC) ; Medicine ; Engineering
    ISSN: 0142-9612
    E-ISSN: 1878-5905
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  • 3
    Language: English
    In: Biomaterials, 2011, Vol.32(34), pp.8938-8946
    Description: The application of native extracellular matrix (ECM) components is a promising approach for biomaterial design. Here, we investigated artificial ECM (aECM) consisting of collagen I (coll) and the glycosaminoglycans (GAGs) hyaluronan (HA) or chondroitin sulfate (CS). Additionally, GAGs were chemically modified by the introduction of sulfate groups to obtain low-sulfated and high-sulfated GAG derivatives. Sulfate groups are expected to bind and concentrate growth factors and improve their bioactivity. In this study we analyzed the effect of aECM on initial adhesion, proliferation, ECM synthesis and differentiation of human dermal fibroblasts (dFb) within 8–48 h. We show that initial adhesion and cell proliferation of dFb progressively increased in a sulfate dependent manner. In contrast, synthesis of ECM components coll and HA was decreased on high-sulfated aECM coll/HA3.0 and coll/CS3.1. Furthermore, the matrix metallo-proteinase-1 (MMP-1) was down-regulated on coll/HA3.0 and coll/CS3.1 on mRNA and protein level. The fibroblast differentiation marker α-smooth muscle actin (αSMA) is not affected by aECM on mRNA level. Artificial ECM consisting of coll and high-sulfated GAGs proves to be a suitable biomaterial for dFb adhesion and proliferation that induces a “proliferative phenotype” of dFb found in the early stages of cutaneous wound healing.
    Keywords: Wound Healing ; Collagen I ; Sulfated Glycosaminoglycan ; Hyaluronan ; Chondroitin Sulfate ; Human Dermal Fibroblasts ; Medicine ; Engineering
    ISSN: 0142-9612
    E-ISSN: 1878-5905
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  • 4
    Language: English
    In: Biomacromolecules, 11 August 2014, Vol.15(8), pp.3083-92
    Description: Sulfated glycosaminoglycans (GAGs) can direct cellular processes by interacting with proteins of the extracellular matrix (ECM). In this study we characterize the interaction profiles of chemically sulfated hyaluronan (HA) and chondroitin sulfate (CS) derivatives with bone morphogenetic protein-2 (BMP-2) and investigate their relevance for complex formation with the receptor BMPR-IA. These goals were addressed by surface plasmon resonance (SPR) and ELISA in combination with molecular modeling and dynamics simulation. We found not only the interaction of BMP-2 with GAGs to be dependent on the type and sulfation of GAGs but also BMP-2/GAG/BMPR-IA complex formation. The conformational plasticity of the BMP-2 N-termini plays a key role in the structural and thermodynamic characteristics of the BMP-2/GAG/BMPR-IA system. Hence we propose a model that provides direct insights into the importance of the structural and dynamical properties of the BMP-2/BMPR-IA system for its regulation by sulfated GAGs, in which structural asymmetry plays a key role.
    Keywords: Bone Morphogenetic Protein 2 -- Chemistry ; Bone Morphogenetic Protein Receptors, Type I -- Chemistry ; Glycosaminoglycans -- Chemistry
    ISSN: 15257797
    E-ISSN: 1526-4602
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  • 5
    Language: English
    In: Biomaterials, November 2012, Vol.33(33), pp.8418-8429
    Description: To meet the growing need for bone replacement of our aging population, development of new adaptive biomaterials is essential. Collagen and glycosaminoglycans (GAGs) such as hyaluronan (HA) and chondroitin sulfate (CS) are major components of the extracellular matrix (ECM) in bone. We manufactured native and sulfate-modified GAG matrices, evaluated how these components modulate different functions of osteoclasts, the cells that resorb bone, and analyzed the underlying mechanisms. GAGs were tested for their effects on osteoclast adhesion, viability, differentiation, morphology, and resorption as well as proteome alterations using murine RAW264.7 cells and primary human osteoclasts. Native and sulfated GAGs were stable and largely non-cytotoxic. Sulfation of GAGs led to a significant inhibition of osteoclast differentiation and resorption, which was largely dependent on the degree of sulfation of GAGs rather than the monosaccharide composition. Sulfation significantly reduced resorptive function by 14% (CS) and 43% (HA). Highly sulfated GAGs dose-dependently suppressed osteoclast differentiation, osteoclast-specific expression of TRAP, cathepsin K, SWAP-70, and OSCAR by 63–95%, and inhibited proteins involved in cytoskeletal rearrangement. In conclusion, highly sulfated GAGs significantly inhibit various functions of bone-resorbing osteoclasts. Whether these properties locally contribute to improved fracture or bone defect healing needs to be validated .
    Keywords: Extracellular Matrix (Ecm) ; Chondroitin Sulfate ; Hyaluronan ; Osteoclast ; Sulfated Glycosaminoglycans ; Quantitative Proteomics ; Medicine ; Engineering
    ISSN: 0142-9612
    E-ISSN: 1878-5905
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  • 6
    Language: English
    In: Biomaterials, October 2013, Vol.34(31), pp.7653-7661
    Description: In order to improve bone regeneration, in particular in aged and multimorbid patients, the development of new adaptive biomaterials and their characterization in terms of their impact on bone biology is warranted. Glycosaminoglycans (GAGs) such as hyaluronan (HA) are major extracellular matrix (ECM) components in bone and may display osteogenic properties that are potentially useful for biomaterial coatings. Using native and synthetically derived sulfate-modified HA, we evaluated how GAG sulfation modulates the activity of two main regulators of osteoclast function: receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG). GAGs were tested for their capability to bind to OPG and RANKL using surface plasmon resonance (SPR), ELISA and molecular modeling techniques. Results were validated in an model of osteoclastogenesis. Sulfated GAGs bound OPG but not RANKL in a sulfate-dependent manner. Furthermore, OPG pre-incubated with different GAGs displayed a sulfate- and dose-dependent loss in bioactivity, possibly due to competition of GAGs for the RANKL/OPG binding site revealing a potential GAG interaction site at the RANKL/OPG interface. In conclusion, high-sulfated GAGs might significantly control osteoclastogenesis via interference with the physiological RANKL/OPG complex formation. Whether these properties can be utilized to improve bone regeneration and fracture healing needs to be validated .
    Keywords: Osteoprotegerin (Opg) ; Receptor Activator of Nf-Κb Ligand (Rankl) ; Hyaluronic Acid/Hyaluronan (Ha) Sulfate ; Surface Plasmon Resonance (SPR) ; Osteoclast (OC) ; Medicine ; Engineering
    ISSN: 0142-9612
    E-ISSN: 1878-5905
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  • 7
    Language: English
    In: Biomaterials, October 2015, Vol.67, pp.335-345
    Description: In order to improve bone defect regeneration, the development of new adaptive biomaterials and their functional and biological validation is warranted. Glycosaminoglycans (GAGs) are important extracellular matrix (ECM) components in bone and may display osteogenic properties that are potentially useful for biomaterial coatings. Using hyaluronan (HA), chondroitin sulfate (CS) and chemically modified highly sulfated HA and CS derivatives (sHA3 and sCS3; degree of sulfation ∼3), we evaluated how GAG sulfation modulates Wnt signaling, a major regulator of osteoblast, osteoclast and osteocyte biology. GAGs were tested for their capability to bind to sclerostin, an inhibitor of Wnt signaling, using surface plasmon resonance and molecular modeling to characterize their interactions. GAGs bound sclerostin in a concentration- and sulfate-dependent manner at a common binding region. These findings were confirmed in an LRP5/sclerostin interaction study and an model of Wnt activation. Here, pre-incubation of sclerostin with different GAGs led to a sulfate- and dose-dependent loss of its bioactivity. Using GAG-biotin derivatives in a competitive ELISA approach sclerostin was shown to be the preferred binding partner over Wnt3a. In conclusion, highly sulfated GAGs might control bone homeostasis via interference with sclerostin/LRP5/6 complex formation. Whether these properties can be utilized to improve bone regeneration needs to be validated .
    Keywords: Sclerostin ; Low-Density Lipoprotein Receptor-Related Protein 5/6 (Lrp5/6) ; Hyaluronic Acid/Hyaluronan (Ha) Sulfate ; Chondroitin Sulfate (CS) ; Surface Plasmon Resonance (SPR) ; Molecular Docking ; Medicine ; Engineering
    ISSN: 0142-9612
    E-ISSN: 1878-5905
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  • 8
    Language: English
    In: Acta Biomaterialia, 2012, Vol.8(2), pp.659-666
    Description: Sulfated glycosaminoglycans (GAG) are multifunctional components of the extracellular matrix and are involved in the regulation of adhesion, proliferation and differentiation of cells. The effects of GAG are mediated in general by their interactions with cations and water, and in particular by their binding to growth factors. The aim of this study was to generate artificial extracellular matrices (aECM) containing collagen I and hyaluronan sulfate (HyaS), which are capable of adsorbing and releasing transforming growth factor β1 (TGF-β1), and to promote collagen synthesis of cultured human mesenchymal stromal cells (hMSC). For the preparation of aECM, monosulfated Hya (HyaS1) or trisulfated Hya (HyaS3) were used; the natural chondroitin-4-sulfate was used as a control. As applied for the in vitro experiments, the resulting matrices were composed of 93–98% collagen I and 2–7% GAG derivative. Adsorption of TGF-β1 to the aECM and release from the aECM was dependent on the degree of sulfation of hyaluronan. Collagen synthesis of hMSC was promoted only by aECM with adsorbed TGF-β1; the bare aECM had a slightly inhibitory effect on collagen synthesis. The promoting effect did not correlate either to the amount of adsorbed TGF-β1 nor to the release of TGF-β1, indicating that the correct presentation of TGF-β1 to the cells might be critical. The results indicate that sulfated hyaluronan-containing aECM have the potential to control both the adsorption and release of TGF-β1, and thereby promote collagen synthesis of hMSC. Thus, these aECM might be a useful tool for different tissue-engineering applications to enhance bone formation when used for biomaterial coating.
    Keywords: Artificial Extracellular Matrix ; Collagen Synthesis ; Glycosaminoglycans ; Mesenchymal Stromal Cells ; Sulfated Hyaluronan ; Engineering
    ISSN: 1742-7061
    E-ISSN: 1878-7568
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  • 9
    Language: English
    In: Biomacromolecules, 10 October 2016, Vol.17(10), pp.3252-3261
    Description: Sulfated glycosaminoglycans (sGAGs) modulate cellular processes via their interaction with extracellular matrix (ECM) proteins. We revealed a direct binding of tissue inhibitor of metalloproteinase-3 (TIMP-3) to the endocytic receptor low-density lipoprotein receptor-related protein (LRP-1) clusters II and IV using surface plasmon resonance. Sulfated hyaluronan (sHA) and chondroitin sulfate (sCS) derivatives interfered with TIMP-3/LRP-1 complex formation in a sulfation-dependent manner stronger than heparin. Electrostatic potential calculations suggested a competition between negatively charged GAGs and highly negatively charged complement-like domains of LRP-1 for the binding to a positively charged area of TIMP-3 as an underlying mechanism. In vitro studies revealed increased amounts of pericellular TIMP-3 in the presence of sHA as a consequence of the blocked protein uptake. GAG derivatives as part of biomaterials might post-translationally modulate TIMP-3 levels stronger than native GAGs, thus exhibiting catabolic effects on the ECM, which could prevent extensive pathological matrix degradation and promote wound healing.
    Keywords: Glycosaminoglycans -- Administration & Dosage ; Hyaluronic Acid -- Administration & Dosage ; Low Density Lipoprotein Receptor-Related Protein-1 -- Biosynthesis ; Tissue Inhibitor of Metalloproteinase-3 -- Biosynthesis
    ISSN: 15257797
    E-ISSN: 1526-4602
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  • 10
    Language: English
    In: Biomatter, 01 July 2012, Vol.2(3), pp.132-141
    Description: Construction of biomaterials with the ability to guide cell function is a topic of high interest in biomaterial development. One approach is using components native to the ECM of the target tissue to generate in vitro a microenvironment that can also elicit specific responses in cells and tissues-an...
    Keywords: Artificial Extracellular Matrix ; Bioactive Coatings ; Collagen ; Immobilization ; Implants ; Surface Functionalization ; Engineering
    E-ISSN: 2159-2535
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