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89 Publications visible to you, out of a total of 89
Collagen breaks at weak sacrificial bonds taming its mechanoradicals

Abstract (Expand)

Collagen is a force-bearing, hierarchical structural protein important to all connective tissue. In tendon collagen, high load even below macroscopic failure level creates mechanoradicals by homolytic … bond scission, similar to polymers. The location and type of initial rupture sites critically decide on both the mechanical and chemical impact of these micro-ruptures on the tissue, but are yet to be explored. We here use scale-bridging simulations supported by gel electrophoresis and mass spectrometry to determine breakage points in collagen. We find collagen crosslinks, as opposed to the backbone, to harbor the weakest bonds, with one particular bond in trivalent crosslinks as the most dominant rupture site. We identify this bond as sacrificial, rupturing prior to other bonds while maintaining the material’s integrity. Also, collagen’s weak bonds funnel ruptures such that the potentially harmful mechanoradicals are readily stabilized. Our results suggest this unique failure mode of collagen to be tailored towards combatting an early onset of macroscopic failure and material ageing.

Authors: Benedikt Rennekamp, Christoph Karfusehr, Markus Kurth, Aysecan Ünal, Kai Riedmiller, Ganna Gryn’ova, David M. Hudson, Frauke Gräter

Date Published: 12th Apr 2023

Publication Type: Journal

Disulfide Bond Reduction and Exchange in von-Willebrand-Factor’s C4-Domain Undermines Platelet Binding

Abstract

Not specified

Authors: Fabian Kutzki, Diego Butera, Angelina J. Lay, Denis Maag, Joyce Chiu, Heng-Giap Woon, Tomáš Kubař, Marcus Elstner, Camilo Aponte-Santamaría, Philip J. Hogg, Frauke Gräter

Date Published: 12th Apr 2023

Publication Type: Journal

Mechano-Redox Control of Macrophage-1 Antigen De-Adhesion From ICAM-1 (Intercellular Adhesion Molecule 1) by Protein Disulfide Isomerase Promotes Directional Movement Under Flow

Abstract (Expand)

BACKGROUND: Neutrophil migration is critical to the initiation and resolution of inflammation. Macrophage-1 antigen (Mac-1; CD11b/CD18, αMβ2) is a leukocyte integrin essential for firm adhesion to …firm adhesion to endothelial ICAM-1 (intercellular adhesion molecule 1) and migration of neutrophils in the shear forces of the circulation. PDI (protein disulfide isomerase) has been reported to influence neutrophil adhesion and migration. We aimed to elucidate the molecular mechanism of PDI control of Mac-1 affinity for ICAM-1 during neutrophil migration under fluid shear. METHODS: Neutrophils isolated from whole blood were perfused over microfluidic chips coated with ICAM-1. Colocalization of Mac-1 and PDI on neutrophils was visualized by fluorescently labeled antibodies and confocal microscopy. The redox state of Mac-1 disulfide bonds was mapped by differential cysteine alkylation and mass spectrometry. Wild-type or disulfide mutant Mac-1 was expressed recombinantly in Baby Hamster Kidney cells to measure ligand affinity. Mac-1 conformations were measured by conformation-specific antibodies and molecular dynamics simulations. Neutrophils crawling on immobilized ICAM-1 were measured in presence of oxidized or reduced PDI, and the effect of PDI inhibition using isoquercetin on neutrophil crawling on inflamed endothelial cells was examined. Migration indices in the X- and Y-direction were determined and the crawling speed was calculated. RESULTS: PDI colocalized with high-affinity Mac-1 at the trailing edge of stimulated neutrophils when crawling on ICAM-1 under fluid shear. PDI cleaved 2 allosteric disulfide bonds, C169-C176 and C224-C264, in the βI domain of the β2 subunit, and cleavage of the C224-C264 disulfide bond selectively controls Mac-1 disengagement from ICAM-1 under fluid shear. Molecular dynamics simulations and conformation-specific antibodies reveal that cleavage of the C224-C264 bond induces conformational change and mechanical stress in the βI domain. This allosterically alters the exposure of an αI domain epitope associated with a shift of Mac-1 to a lower-affinity state. These molecular events promote neutrophil motility in the direction of flow at high shear stress. Inhibition of PDI by isoquercetin reduces neutrophil migration in the direction of flow on endothelial cells during inflammation. CONCLUSIONS: Shear-dependent PDI cleavage of the neutrophil Mac-1 C224-C264 disulfide bond triggers Mac-1 de-adherence from ICAM-1 at the trailing edge of the cell and enables directional movement of neutrophils during inflammation.

Authors: Alexander Dupuy, Camilo Aponte Santamaría, Adva Yeheskel, Elinor Hortle, Stefan H. Oehlers, Frauke Gräter, Philip J. Hogg, Freda H. Passam, Joyce Chiu

Date Published: 6th Apr 2023

Publication Type: Journal

DOPA Residues Endow Collagen with Radical Scavenging Capacity

Abstract (Expand)

Here we uncover collagen, the main structural protein of all connective tissues, as a redox-active material. We identify dihydroxyphenylalanine (DOPA) residues, post-translational oxidation products … of tyrosine residues, to be common in collagen derived from different connective tissues. We observe that these DOPA residues endow collagen with substantial radical scavenging capacity. When reducing radicals, DOPA residues work as redox relay: they convert to the quinone and generate hydrogen peroxide. In this dual function, DOPA outcompetes its amino acid precursors and ascorbic acid. Our results establish DOPA residues as redox-active side chains of collagens, probably protecting connective tissues against radicals formed under mechanical stress and/or inflammation.

Authors: Markus Kurth, Uladzimir Barayeu, Hassan Gharibi, Andrei Kuzhelev, Kai Riedmiller, Jennifer Zilke, Kasimir Noack, Vasyl Denysenkov, Reinhard Kappl, Thomas F. Prisner, Roman A. Zubarev, Tobias P. Dick, Frauke Gräter

Date Published: 3rd Apr 2023

Publication Type: Journal

Energetics and kinetics of membrane permeation of photoresists for bioprinting

Abstract

Not specified

Authors: Lucas Diedrich, Matthias Brosz, Tobias Abele, Salome Steinke, Frauke Gräter, Kerstin Göpfrich, Camilo Aponte-Santamaría

Date Published: 28th Mar 2023

Publication Type: Journal

FERM domains recruit ample PI(4,5)P2s to form extensive protein-membrane attachments

Abstract (Expand)

The four-point-one ezrin-radixin-moesin homology (FERM) protein domain is a multifunctional protein-lipid binding site, constituting an integral part of numerous membrane-associated proteins. Its … interaction with the lipid phosphatidylinositol-4,5-bisphosphate (PIP2), located at the inner leaflet of eukaryotic plasma membranes, is important for localization, anchorage, and activation of FERM-containing proteins. FERM-PIP2 complexes structurally determined so far exclusively feature a 1:1 binding stoichiometry of protein and lipid, with a few basic FERM residues neutralizing the −4 charge of the bound PIP2. Whether this picture from static crystal structures also applies to the dynamic interaction of FERM domains on PIP2 membranes is unknown. We here quantified the stoichiometry of FERM-PIP2 binding in a lipid bilayer using atomistic molecular dynamics simulations and experiments on solid supported membranes for the FERM domains of focal adhesion kinase and ezrin. In contrast to the structural data, we find much higher average stoichiometries of FERM-PIP2 binding, amounting to 1:3 or 1:4 ratios, respectively. In simulations, the full set of basic residues at the membrane interface, 7 and 15 residues for focal adhesion kinase and ezrin, respectively, engages in PIP2 interactions. In addition, Na ions enter the FERM-membrane binding interface, compensating negative PIP2 charges in case of high charge surpluses from bound PIP2. We propose the multivalent binding of FERM domains to PIP2 in lipid bilayers to significantly enhance the stability of FERM-membrane binding and to render the FERM-membrane linkage highly adjustable.

Authors: Thomas Ehret, Tim Heißenberg, Svenja de Buhr, Camilo Aponte-Santamaría, Claudia Steinem, Frauke Gräter

Date Published: 21st Feb 2023

Publication Type: Journal

Intrinsically disordered region of talin’s FERM domain functions as an initial PIP2 recognition site

Abstract (Expand)

Focal adhesions (FAs) mediate the interaction of the cytoskeleton with the extracellular matrix in a highly dynamic fashion. Talin is a central regulator, adaptor protein, and mechano-sensor of FA … complexes. For recruitment and firm attachment at FAs, talin’s N-terminal FERM domain binds to phosphatidylinositol 4,5-bisphosphate (PIP2)-enriched membranes. A newly published autoinhibitory structure of talin-1, where the known PIP2 interaction sites are covered up, lead us to hypothesize that a hitherto less examined loop insertion of the FERM domain acts as an additional and initial site of contact. We evaluated direct interactions of talin-1 with a PIP2 membrane by means of atomistic molecular dynamics simulations. We show that this unstructured, 33-residue-long loop strongly interacts with PIP2 and can facilitate further membrane contacts, including the canonical PIP2 interactions, by serving as a flexible membrane anchor. Under force as present at FAs, the extensible FERM loop ensures talin maintains membrane contacts when pulled away from the membrane by up to 7 nm. We identify key basic residues of the anchor mediating the highly dynamic talin-membrane interaction. Our results put forward an intrinsically disordered loop as a key and highly adaptable PIP2 recognition site of talin and potentially other PIP2-binding mechano-proteins.

Authors: Jannik Buhr, Florian Franz, Frauke Gräter

Date Published: 21st Feb 2023

Publication Type: Journal

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