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 Oxidative Stress Conditions Result in Trapping of PHF-Core Tau (297–391) Intermediates

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 703
Author(s):  
Mahmoud B. Maina ◽  
Youssra K. Al-Hilaly ◽  
Gunasekhar Burra ◽  
Janet E. Rickard ◽  
Charles R. Harrington ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Self Assembly ◽  
Ultra Violet ◽  
Paired Helical Filaments ◽  
The Self ◽  
Cross Linking ◽  
Post Translational Modifications ◽  
Tau Oligomers ◽  
Catalysed Oxidation ◽  
Cross Links

The self-assembly of tau into paired helical filaments (PHFs) in neurofibrillary tangles (NFTs) is a significant event in Alzheimer’s disease (AD) pathogenesis. Numerous post-translational modifications enhance or inhibit tau assembly into NFTs. Oxidative stress, which accompanies AD, induces multiple post-translational modifications in proteins, including the formation of dityrosine (DiY) cross-links. Previous studies have revealed that metal-catalysed oxidation (MCO) using Cu2+ and H2O2 leads to the formation of DiY cross-links in two misfolding proteins, Aβ and α-synuclein, associated with AD and Parkinson’s disease respectively. The effect of MCO on tau remains unknown. Here, we examined the effect of MCO and ultra-violet oxidation to study the influence of DiY cross-linking on the self-assembly of the PHF-core tau fragment. We report that DiY cross-linking facilitates tau assembly into tau oligomers that fail to bind thioflavin S, lack β-sheet structure and prevents their elongation into filaments. At a higher concentration, Cu2+ (without H2O2) also facilitates the formation of these tau oligomers. The DiY cross-linked tau oligomers do not cause cell death. Our findings suggest that DiY cross-linking of pre-assembled tau promotes the formation of soluble tau oligomers that show no acute impact on cell viability.

scholarly journals Chemistry of collagen cross-links: glucose-mediated covalent cross-linking of type-IV collagen in lens capsules

1993 ◽  
Vol 296 (2) ◽  
pp. 489-496 ◽  
Author(s):  
A J Bailey ◽  
T J Sims ◽  
N C Avery ◽  
C A Miles
Keyword(s):  
Type Iv Collagen ◽  
Cross Linking ◽  
Mechanical And Thermal Properties ◽  
Maximum Strain ◽  
Scanning Calorimetry ◽  
Melting Temperatures ◽  
Type Iv ◽  
Collagen Molecules ◽  
Cross Links

The incubation of lens capsules with glucose in vitro resulted in changes in the mechanical and thermal properties of type-IV collagen consistent with increased cross-linking. Differential scanning calorimetry (d.s.c.) of fresh lens capsules showed two major peaks at melting temperatures Tm 1 and Tm 2 at approx. 54 degrees C and 90 degrees C, which can be attributed to the denaturation of the triple helix and 7S domains respectively. Glycosylation of lens capsules in vitro for 24 weeks caused an increase in Tm 1 from 54 degrees C to 61 degrees C, while non-glycosylated, control incubated capsules increased to a Tm 1 of 57 degrees C. The higher temperature required to denature the type-IV collagen after incubation in vitro suggested increased intermolecular cross-linking. Glycosylated lens capsules were more brittle than fresh samples, breaking at a maximum strain of 36.8 +/- 1.8% compared with 75.6 +/- 6.3% for the fresh samples. The stress at maximum strain (or ‘strength’) was dramatically reduced from 12.0 to 4.7 N.mm.mg-1 after glycosylation in vitro. The increased constraints within the system leading to loss of strength and increased brittleness suggested not only the presence of more cross-links but a difference in the location of these cross-links compared with the natural lysyl-aldehyde-derived cross-links. The chemical nature of the fluorescent glucose-derived cross-link following glycosylation was determined as pentosidine, at a concentration of 1 pentosidine molecule per 600 collagen molecules after 24 weeks incubation. Pentosidine was also determined in the lens capsules obtained from uncontrolled diabetics at a level of about 1 per 100 collagen molecules. The concentration of these pentosidine cross-links is far too small to account for the observed changes in the thermal and mechanical properties following incubation in vitro, clearly indicating that another as yet undefined, but apparently more important cross-linking mechanism mediated by glucose is taking place.

scholarly journals Mapping the Contact Sites of the Escherichia coli Division-Initiating Proteins FtsZ and ZapA by BAMG Cross-Linking and Site-Directed Mutagenesis

2018 ◽  
Vol 19 (10) ◽  
pp. 2928 ◽  
Author(s):  
Winfried Roseboom ◽  
Madhvi Nazir ◽  
Nils Meiresonne ◽  
Tamimount Mohammadi ◽  
Jolanda Verheul ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Cross Linking ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Binding Interface ◽  
Tetrameric Structure ◽  
Z Ring ◽  
Cross Links ◽  
Chemical Cross Linking

Cell division in bacteria is initiated by the polymerization of FtsZ at midcell in a ring-like structure called the Z-ring. ZapA and other proteins assist Z-ring formation and ZapA binds ZapB, which senses the presence of the nucleoids. The FtsZ–ZapA binding interface was analyzed by chemical cross-linking mass spectrometry (CXMS) under in vitro FtsZ-polymerizing conditions in the presence of GTP. Amino acids residue K42 from ZapA was cross-linked to amino acid residues K51 and K66 from FtsZ, close to the interphase between FtsZ molecules in protofilaments. Five different cross-links confirmed the tetrameric structure of ZapA. A number of FtsZ cross-links suggests that its C-terminal domain of 55 residues, thought to be largely disordered, has a limited freedom to move in space. Site-directed mutagenesis of ZapA reveals an interaction site in the globular head of the protein close to K42. Using the information on the cross-links and the mutants that lost the ability to interact with FtsZ, a model of the FtsZ protofilament–ZapA tetramer complex was obtained by information-driven docking with the HADDOCK2.2 webserver.

scholarly journals Self-Assembled Metal–Organic Biohybrids (MOBs) Using Copper and Silver for Cell Studies

Nanomaterials ◽  
2019 ◽  
Vol 9 (9) ◽  
pp. 1282 ◽  
Author(s):  
Neha Karekar ◽  
Anik Karan ◽  
Elnaz Khezerlou ◽  
Neela Prajapati ◽  
Chelsea D. Pernici ◽  
...  
Keyword(s):  
Self Assembly ◽  
Neuroblastoma Cells ◽  
The Novel ◽  
Culture Methods ◽  
Synthesis Conditions ◽  
Tissue Constructs ◽  
Silver Sulfate ◽  
Significant Toxicity ◽  
Metal Organic

The novel synthesis of metal-containing biohybrids using self-assembly methods at physiological temperatures (37 °C) was compared for copper and silver using the amino acid dimer cystine. Once assembled, the copper containing biohybrid is a stable, high-aspect ratio structure, which we call CuHARS. Using the same synthesis conditions, but replacing copper with silver, we have synthesized cystine-capped silver nanoparticles (AgCysNPs), which are shown here to form stable colloid solutions in contrast to the CuHARS, which settle out from a 1 mg/mL solution in 90 min. Both the copper and silver biohybrids, as synthesized, demonstrate very low agglomeration which we have applied for the purpose of applications with cell culture methods, namely, for testing as anti-cancer compounds. AgCysNPs (1000 ng/mL) demonstrated significant toxicity (only 6.8% viability) to glioma and neuroblastoma cells in vitro, with concentrations as low as 20 ng/mL causing some toxicity. In contrast, CuHARS required at least 5 μg/mL. For comparative purposes, silver sulfate at 100 ng/mL decreased viability by 52% and copper sulfate at 100 ng/mL only by 19.5% on glioma cells. Using these methods, the novel materials were tested here as metal–organic biohybrids (MOBs), and it is anticipated that the functionalization and dynamics of MOBs may result in building a foundation of new materials for cellular applications, including cell engineering of both normal and diseased cells and tissue constructs.

scholarly journals Characterization of Anchorless Human PrP With Q227X Stop Mutation Linked to Gerstmann-Sträussler-Scheinker Syndrome In Vivo and In Vitro

2020 ◽  
Vol 58 (1) ◽  
pp. 21-33
Author(s):  
Pingping Shen ◽  
Johnny Dang ◽  
Zerui Wang ◽  
Weiguanliu Zhang ◽  
Jue Yuan ◽  
...  
Keyword(s):  
Protein Misfolding ◽  
Neuroblastoma Cells ◽  
Cellular Prion Protein ◽  
Wild Type ◽  
Gpi Anchor ◽  
Two Dimensional Gel Electrophoresis ◽  
Molecular Weights ◽  
Prion Propagation

AbstractAlteration in cellular prion protein (PrPC) localization on the cell surface through mediation of the glycosylphosphatidylinositol (GPI) anchor has been reported to dramatically affect the formation and infectivity of its pathological isoform (PrPSc). A patient with Gerstmann-Sträussler-Scheinker (GSS) syndrome was previously found to have a nonsense heterozygous PrP-Q227X mutation resulting in an anchorless PrP. However, the allelic origin of this anchorless PrPSc and cellular trafficking of PrPQ227X remain to be determined. Here, we show that PrPSc in the brain of this GSS patient is mainly composed of the mutant but not wild-type PrP (PrPWt), suggesting pathological PrPQ227X is incapable of recruiting PrPWt in vivo. This mutant anchorless protein, however, is able to recruit PrPWt from humanized transgenic mouse brain but not from autopsied human brain homogenates to produce a protease-resistant PrPSc-like form in vitro by protein misfolding cyclic amplification (PMCA). To further investigate the characteristics of this mutation, constructs expressing human PrPQ227X or PrPWt were transfected into neuroblastoma cells (M17). Fractionation of the M17 cells demonstrated that most PrPWt is recovered in the cell lysate fraction, while most of the mutant PrPQ227X is recovered in the medium fraction, consistent with the results obtained by immunofluorescence microscopy. Two-dimensional gel-electrophoresis and Western blotting showed that cellular PrPQ227X spots clustered at molecular weights of 22–25 kDa with an isoelectric point (pI) of 3.5–5.5, whereas protein spots from the medium are at 18–26 kDa with a pI of 7–10. Our findings suggest that the role of GPI anchor in prion propagation between the anchorless mutant PrP and wild-type PrP relies on the cellular distribution of the protein.

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.

scholarly journals Aβ(M1–40) and Wild-Type Aβ40 Self-Assemble into Oligomers with Distinct Quaternary Structures

Molecules ◽  
2019 ◽  
Vol 24 (12) ◽  
pp. 2242 ◽  
Author(s):  
Jacob L. Bouchard ◽  
Taylor C. Davey ◽  
Todd M. Doran
Keyword(s):  
Self Assembly ◽  
Quaternary Structure ◽  
Biological Activities ◽  
Amyloid Β ◽  
Long Term Potentiation ◽  
Wild Type ◽  
Molecular Weights ◽  
Term Potentiation ◽  
Quaternary Structures

Amyloid-β oligomers (AβOs) self-assemble into polymorphic species with diverse biological activities that are implicated causally to Alzheimer’s disease (AD). Synaptotoxicity of AβO species is dependent on their quaternary structure, however, low-abundance and environmental sensitivity of AβOs in vivo have impeded a thorough assessment of structure–function relationships. We developed a simple biochemical assay to quantify the relative abundance and morphology of cross-linked AβOs. We compared oligomers derived from synthetic Aβ40 (wild-type (WT) Aβ40) and a recombinant source, called Aβ(M1–40). Both peptides assemble into oligomers with common sizes and morphology, however, the predominant quaternary structures of Aβ(M1–40) oligomeric states were more diverse in terms of dispersity and morphology. We identified self-assembly conditions that stabilize high-molecular weight oligomers of Aβ(M1–40) with apparent molecular weights greater than 36 kDa. Given that mixtures of AβOs derived from both peptides have been shown to be potent neurotoxins that disrupt long-term potentiation, we anticipate that the diverse quaternary structures reported for Aβ(M1–40) oligomers using the assays reported here will facilitate research efforts aimed at isolating and identifying common toxic species that contribute to synaptic dysfunction.

scholarly journals Cross-linking modifications of HDL apoproteins by oxidized phospholipids: structural characterization, in vivo detection, and functional implications

2020 ◽  
Vol 295 (7) ◽  
pp. 1973-1984
Author(s):  
Detao Gao ◽  
Mohammad Z. Ashraf ◽  
Lifang Zhang ◽  
Niladri Kar ◽  
Tatiana V. Byzova ◽  
...  
Keyword(s):  
Density Lipoprotein ◽  
Cross Linking ◽  
Oxidized Phospholipids ◽  
Cross Link ◽  
In Vivo Detection ◽  
Lysine Residues ◽  
Cross Links ◽  
Human Atheroma

Apolipoprotein A-I (apoA-I) is cross-linked and dysfunctional in human atheroma. Although multiple mechanisms of apoA-I cross-linking have been demonstrated in vitro, the in vivo mechanisms of cross-linking are not well-established. We have recently demonstrated the highly selective and efficient modification of high-density lipoprotein (HDL) apoproteins by endogenous oxidized phospholipids (oxPLs), including γ-ketoalkenal phospholipids. In the current study, we report that γ-ketoalkenal phospholipids effectively cross-link apoproteins in HDL. We further demonstrate that cross-linking impairs the cholesterol efflux mediated by apoA-I or HDL3 in vitro and in vivo. Using LC-MS/MS analysis, we analyzed the pattern of apoprotein cross-linking in isolated human HDL either by synthetic γ-ketoalkenal phospholipids or by oxPLs generated during HDL oxidation in plasma by the physiologically relevant MPO-H2O2-NO2− system. We found that five histidine residues in helices 5–8 of apoA-I are preferably cross-linked by oxPLs, forming stable pyrrole adducts with lysine residues in the helices 3–4 of another apoA-I or in the central domain of apoA-II. We also identified cross-links of apoA-I and apoA-II with two minor HDL apoproteins, apoA-IV and apoE. We detected a similar pattern of apoprotein cross-linking in oxidized murine HDL. We further detected oxPL cross-link adducts of HDL apoproteins in plasma and aorta of hyperlipidemic LDLR−/− mice, including cross-link adducts of apoA-I His-165–apoA-I Lys-93, apoA-I His-154–apoA-I Lys-105, apoA-I His-154–apoA-IV Lys-149, and apoA-II Lys-30–apoE His-227. These findings suggest an important mechanism that contributes to the loss of HDL's atheroprotective function in vivo.

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