scholarly journals Cross-linking reveals laminin coiled-coil architecture

2016 ◽  
Vol 113 (47) ◽  
pp. 13384-13389 ◽  
Author(s):  
Gad Armony ◽  
Etai Jacob ◽  
Toot Moran ◽  
Yishai Levin ◽  
Tevie Mehlman ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Self Assembly ◽  
Quaternary Structure ◽  
Coiled Coil ◽  
Surface Proteins ◽  
Cross Linking ◽  
Single Particle Reconstruction ◽  
Cross Links ◽  
Particle Reconstruction ◽  
Key Questions

Laminin, an ∼800-kDa heterotrimeric protein, is a major functional component of the extracellular matrix, contributing to tissue development and maintenance. The unique architecture of laminin is not currently amenable to determination at high resolution, as its flexible and narrow segments complicate both crystallization and single-particle reconstruction by electron microscopy. Therefore, we used cross-linking and MS, evaluated using computational methods, to address key questions regarding laminin quaternary structure. This approach was particularly well suited to the ∼750-Å coiled coil that mediates trimer assembly, and our results support revision of the subunit order typically presented in laminin schematics. Furthermore, information on the subunit register in the coiled coil and cross-links to downstream domains provide insights into the self-assembly required for interaction with other extracellular matrix and cell surface proteins.

Lysyl oxidases: from enzyme activity to extracellular matrix cross-links

2019 ◽  
Vol 63 (3) ◽  
pp. 349-364 ◽  
Author(s):  
Sylvain D. Vallet ◽  
Sylvie Ricard-Blum
Keyword(s):  
Extracellular Matrix ◽  
Molecular Mechanisms ◽  
Catalytic Domain ◽  
Lysyl Oxidase ◽  
Dimensional Structure ◽  
Cross Linking ◽  
Copper Binding ◽  
Molecular Features ◽  
Ecm Proteins ◽  
Cross Links

Abstract The lysyl oxidase family comprises five members in mammals, lysyl oxidase (LOX) and four lysyl oxidase like proteins (LOXL1-4). They are copper amine oxidases with a highly conserved catalytic domain, a lysine tyrosylquinone cofactor, and a conserved copper-binding site. They catalyze the first step of the covalent cross-linking of the extracellular matrix (ECM) proteins collagens and elastin, which contribute to ECM stiffness and mechanical properties. The role of LOX and LOXL2 in fibrosis, tumorigenesis, and metastasis, including changes in their expression level and their regulation of cell signaling pathways, have been extensively reviewed, and both enzymes have been identified as therapeutic targets. We review here the molecular features and three-dimensional structure/models of LOX and LOXLs, their role in ECM cross-linking, and the regulation of their cross-linking activity by ECM proteins, proteoglycans, and by inhibitors. We also make an overview of the major ECM cross-links, because they are the ultimate molecular readouts of LOX/LOXL activity in tissues. The recent 3D model of LOX, which recapitulates its known structural and biochemical features, will be useful to decipher the molecular mechanisms of LOX interaction with its various substrates, and to design substrate-specific inhibitors, which are potential antifibrotic and antitumor drugs.

Investigation of the quaternary structure of Neurospora pyruvate kinase by cross-linking with bifunctional reagents: the effect of substrates and allosteric ligands

1977 ◽  
Vol 55 (1) ◽  
pp. 43-49 ◽  
Author(s):  
M. Kapoor ◽  
M. D. O'Brien
Keyword(s):  
Pyruvate Kinase ◽  
Functional Groups ◽  
Conformational Changes ◽  
Quaternary Structure ◽  
Polyacrylamide Gel ◽  
Cross Linking ◽  
Complex Cross ◽  
Dimethyl Suberimidate ◽  
Cross Links

Pyruvate kinase (EC 2.7.1.40) of Neurospora, a tetramer composed of apparently identical subunits, has been shown to be a dimer of dimers by interprotomeric cross-linking experiments in which bifunctional reagents were used. An analysis of the polyacrylamide gel profiles of the enzyme after cross-linking with glutaraldehyde, dimethyl suberimidate, and dimethyl adipimidate shows that the extent of intersubunit cross-linking is influenced markedly by the ligand bound to the enzyme. Bifunctional cross-linking reagents with a shorter distance between the two functional groups form cross-links effectively in the unliganded enzyme. In the FDP – pyruvate kinase complex, cross-linking was observed over longer distances compared with the unliganded enzyme. It is demonstrated that covalent cross-linkers can be used as sensitive indicators of conformational changes induced in pyruvate kinase by substrates and allosteric ligands.

scholarly journals Self-assembly and cross-linking of Volvox extracellular matrix glycoproteins are specifically inhibited by Ellman's reagent

2000 ◽  
Vol 267 (8) ◽  
pp. 2334-2339 ◽  
Author(s):  
Manfred Sumper ◽  
Jörg Nink ◽  
Stephan Wenzl
Keyword(s):  
Extracellular Matrix ◽  
Self Assembly ◽  
Cross Linking ◽  
Ellman's Reagent

scholarly journals Coronin Promotes the Rapid Assembly and Cross-linking of Actin Filaments and May Link the Actin and Microtubule Cytoskeletons in Yeast

1999 ◽  
Vol 144 (1) ◽  
pp. 83-98 ◽  
Author(s):  
Bruce L. Goode ◽  
Jonathan J. Wong ◽  
Anne-Christine Butty ◽  
Matthias Peter ◽  
Ashley L. McCormack ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Coiled Coil ◽  
Cross Linking ◽  
Cortical Actin ◽  
Functional Link ◽  
Microtubule Binding ◽  
Filament Assembly ◽  
Cell Extracts ◽  
Cross Links

Coronin is a highly conserved actin-associated protein that until now has had unknown biochemical activities. Using microtubule affinity chromatography, we coisolated actin and a homologue of coronin, Crn1p, from Saccharomyces cerevisiae cell extracts. Crn1p is an abundant component of the cortical actin cytoskeleton and binds to F-actin with high affinity (Kd 6 × 10−9 M). Crn1p promotes the rapid barbed-end assembly of actin filaments and cross-links filaments into bundles and more complex networks, but does not stabilize them. Genetic analyses with a crn1Δ deletion mutation also are consistent with Crn1p regulating filament assembly rather than stability. Filament cross-linking depends on the coiled coil domain of Crn1p, suggesting a requirement for Crn1p dimerization. Assembly-promoting activity is independent of cross-linking and could be due to nucleation and/or accelerated polymerization. Crn1p also binds to microtubules in vitro, and microtubule binding is enhanced by the presence of actin filaments. Microtubule binding is mediated by a region of Crn1p that contains sequences (not found in other coronins) homologous to the microtubule binding region of MAP1B. These activities, considered with microtubule defects observed in crn1Δ cells and in cells overexpressing Crn1p, suggest that Crn1p may provide a functional link between the actin and microtubule cytoskeletons in yeast.

Do Proline-rich Proteins Modulate a Transglutaminase Catalyzed Mechanism of Candidal Adhesion?

1993 ◽  
Vol 4 (3) ◽  
pp. 293-299 ◽  
Author(s):  
S.D. Bradway ◽  
M.J. Levine
Keyword(s):  
Molecular Weight ◽  
Cell Wall ◽  
High Molecular Weight ◽  
Surface Proteins ◽  
Cross Linking ◽  
Cell Wall Proteins ◽  
Epithelial Surface ◽  
High Molecular Weight Complex ◽  
Sds Page ◽  
Cross Links

Previously, we reported that a membrane-bound epithelial enzyme, transglutaminase (TGase), catalyzes the covalent cross-linking of acidic proline-rich proteins (APRPs) to surface proteins of buccal epithelial cells (BECs). The purpose of this study was twofold: (1) to provide evidence that TGase stabilizes C. albicans adhesion by covalently cross-linking C. albicans and BEC surface proteins and (2) to implicate PRPs in the modulation of this adhesive mechanism. The reactivity of candidal cell wall proteins with TGase was assessed in two separate experiments. Initially, following incubation with native BECs, the cross-linking of iodinated candidal cell wall proteins into high-molecular-weight complexes, as shown by SDS-PAGE/ autoradiography, was inhibited by the TGase inhibitor iodoacetamide. Additionally, [14C]putrescine in the presence of purified TGase, but not [14C]putrescine alone, was shown by SDS-PAGE/fluorography to be cross-linked into surface proteins of both morphogenetic forms (blastospore > hyphal forms) of C. albicans. In adherence assays, a component of both blastospore and hyphal form Candida/BEC adherence was shown to be resistant to detachment by heating adherent cells in 1% SDS at 100°C. However, pretreatment of BECs with iodoacetamide decreased SDS resistant adherence of both forms of C. albicans by =75%. When incubated with [125I]APRPs and purified TGase, both morphogenetic forms of C. albicans bound dramatically more APRP than controls without TGase. [125I]APRP binding in experimental, but not control, samples was resistant to repeated extraction (48 h) with 4% SDS/10% β-mercaptoethanol at 65°C, suggesting that [125I]APRPs were cross-linked to the Candida surface. SDS-PAGE/fluorography was used to verify that APRPs, in Lyticase digests of Candida cell walls, were cross-linked into a high-molecular-weight complex. These experiments suggest that epithelial TGase may stabilize Candida adherence by cross-linking Candida and BEC surface proteins. Additionally, because TGase cross-links APRPs to candidal and epithelial surface proteins, APRPs may interfere with TGase catalyzed mechanisms of adhesion. Supported by USPHS grants DE00185, DE07585, and OSU Seed grant.

scholarly journals Elastin as a self–organizing biomaterial: use of recombinantly expressed human elastin polypeptides as a model for investigations of structure and self–assembly of elastin

2002 ◽  
Vol 357 (1418) ◽  
pp. 185-189 ◽  
Author(s):  
F. W. Keeley ◽  
C. M. Bellingham ◽  
K. A. Woodhouse
Keyword(s):  
Self Assembly ◽  
Matrix Protein ◽  
Extracellular Matrix Protein ◽  
Cross Linking ◽  
Sufficient Information ◽  
Elastic Recoil ◽  
Membrane Structures ◽  
Self Assembling ◽  
Cross Links ◽  
Organizational Ability

Elastin is the major extracellular matrix protein of large arteries such as the aorta, imparting characteristics of extensibility and elastic recoil. Once laid down in tissues, polymeric elastin is not subject to turnover, but is able to sustain its mechanical resilience through thousands of millions of cycles of extension and recoil. Elastin consists of ca . 36 domains with alternating hydrophobic and cross–linking characteristics. It has been suggested that these hydrophobic domains, predominantly containing glycine, proline, leucine and valine, often occurring in tandemly repeated sequences, are responsible for the ability of elastin to align monomeric chains for covalent cross–linking. We have shown that small, recombinantly expressed polypeptides based on sequences of human elastin contain sufficient information to self–organize into fibrillar structures and promote the formation of lysine–derived cross–links. These cross–linked polypeptides can also be fabricated into membrane structures that have solubility and mechanical properties reminiscent of native insoluble elastin. Understanding the basis of the self–organizational ability of elastin–based polypeptides may provide important clues for the general design of self–assembling biomaterials.

scholarly journals Dityrosine cross-link trapping of amyloid-β intermediates reveals that self-assembly is required for Aβ-induced cytotoxicity

2020 ◽  
Author(s):  
Mahmoud B. Maina ◽  
Kurtis Mengham ◽  
Gunasekhar K. Burra ◽  
Youssra A. Al-Hilaly ◽  
Louise C. Serpell
Keyword(s):  
Self Assembly ◽  
Neuroblastoma Cells ◽  
Amyloid Β ◽  
Cross Linking ◽  
Wild Type ◽  
Catalysed Oxidation ◽  
Mechanism Of Toxicity ◽  
Cross Links ◽  
Multiple Chemical

AbstractMultiple chemical reactions, such as the production of reactive oxygen species (ROS) can lead to dityrosine (DiY) formation via the cross-linking of closely spaced tyrosine residues and this can serve as a marker for aging. Amyloid-β (Aβ) has been found to be DiY cross-linked in the brains of AD patients. In vitro, Aβ forms DiY cross-links via metal-catalysed oxidation (Cu2+ and H202) (MCO) leading to the formation of fibrils that are resistant to formic acid denaturation. However, copper is well known to influence and enhance self-assembly. Here, to investigate the interplay between self-assembly and DiY cross-linking we have utilised a non-assembly competent variant of Aβ (vAβ). MCO and UV oxidation experiments using vAβ and wild-type Aβ, revealed that DiY cross-linking stabilises, but does not induce or promote Aβ assembly. Cu2+ alone, without H202, facilitates the formation and DiY cross-linking of wild-type Aβ into long-lived oligomers. Our work reveals DiY formation halts further Aβ self-assembly. DiY cross-linked Aβ is non-toxic to neuroblastoma cells at all stages of self-assembly in contrast to oligomeric non-cross-linked Aβ. These findings point to a mechanism of toxicity that necessitates continuing self-assembly of the Aβ peptide, whereby trapped DiY Aβ assemblies and assembly incompetent variant Aβ are unable to result in cell death.

scholarly journals TGFβ-1 Induced Cross-Linking of the Extracellular Matrix of Primary Human Dermal Fibroblasts

2021 ◽  
Vol 22 (3) ◽  
pp. 984
Author(s):  
Mariya E. Semkova ◽  
J. Justin Hsuan
Keyword(s):  
Extracellular Matrix ◽  
Lysyl Oxidase ◽  
Transglutaminase 2 ◽  
Dermal Fibroblasts ◽  
Cross Linking ◽  
Human Dermal Fibroblasts ◽  
Tgfβ Signalling ◽  
A Cell ◽  
Cross Links

Excessive cross-linking is a major factor in the resistance to the remodelling of the extracellular matrix (ECM) during fibrotic progression. The role of TGFβ signalling in impairing ECM remodelling has been demonstrated in various fibrotic models. We hypothesised that increased ECM cross-linking by TGFβ contributes to skin fibrosis in Systemic Sclerosis (SSc). Proteomics was used to identify cross-linking enzymes in the ECM of primary human dermal fibroblasts, and to compare their levels following treatment with TGFβ-1. A significant upregulation and enrichment of lysyl-oxidase-like 1, 2 and 4 and transglutaminase 2 were found. Western blotting confirmed the upregulation of lysyl hydroxylase 2 in the ECM. Increased transglutaminase activity in TGFβ-1 treated ECM was revealed from a cell-based assay. We employed a mass spectrometry-based method to identify alterations in the ECM cross-linking pattern caused by TGFβ-1. Cross-linking sites were identified in collagens I and V, fibrinogen and fibronectin. One cross-linking site in fibrinogen alpha was found only in TGFβ-treated samples. In conclusion, we have mapped novel cross-links between ECM proteins and demonstrated that activation of TGFβ signalling in cultured dermal fibroblasts upregulates multiple cross-linking enzymes in the ECM.

scholarly journals Bioinspired Histidine–Zn2+ Coordination for Tuning the Mechanical Properties of Self-Healing Coiled Coil Cross-Linked Hydrogels

Biomimetics ◽  
2019 ◽  
Vol 4 (1) ◽  
pp. 25 ◽  
Author(s):  
Isabell Tunn ◽  
Matthew J. Harrington ◽  
Kerstin G. Blank
Keyword(s):  
Mechanical Properties ◽  
Self Assembly ◽  
Ethylenediaminetetraacetic Acid ◽  
Coiled Coil ◽  
Higher Order ◽  
Metal Coordination ◽  
Cross Linking ◽  
Self Healing ◽  
Poly Ethylene ◽  
Tunable Mechanical Properties

Natural biopolymeric materials often possess properties superior to their individual components. In mussel byssus, reversible histidine (His)–metal coordination is a key feature, which mediates higher-order self-assembly as well as self-healing. The byssus structure, thus, serves as an excellent natural blueprint for the development of self-healing biomimetic materials with reversibly tunable mechanical properties. Inspired by byssal threads, we bioengineered His–metal coordination sites into a heterodimeric coiled coil (CC). These CC-forming peptides serve as a noncovalent cross-link for poly(ethylene glycol)-based hydrogels and participate in the formation of higher-order assemblies via intermolecular His–metal coordination as a second cross-linking mode. Raman and circular dichroism spectroscopy revealed the presence of α-helical, Zn2+ cross-linked aggregates. Using rheology, we demonstrate that the hydrogel is self-healing and that the addition of Zn2+ reversibly switches the hydrogel properties from viscoelastic to elastic. Importantly, using different Zn2+:His ratios allows for tuning the hydrogel relaxation time over nearly three orders of magnitude. This tunability is attributed to the progressive transformation of single CC cross-links into Zn2+ cross-linked aggregates; a process that is fully reversible upon addition of the metal chelator ethylenediaminetetraacetic acid. These findings reveal that His–metal coordination can be used as a versatile cross-linking mechanism for tuning the viscoelastic properties of biomimetic hydrogels.

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