Qualitative LC-MS/MS identification, formation, and stability of adducts and cross-links arising from the reactions of glutathione with the model enal systems

2021 ◽  
Vol 25 ◽  
Author(s):  
Kinga Salus ◽  
Donata Pluskota-Karwatka
Keyword(s):  
Lipid Peroxidation ◽  
Michael Addition ◽  
Carbonyl Compounds ◽  
Cross Linking ◽  
Amino Group ◽  
Cross Links ◽  
Enzymatic Detoxification

: Glutathione (GSH), due to the ability to capture the reactive electrophiles of exo- and endogenous origin, is expected to prevent cross-linking induced by these compounds. However, it may instead become cross-linked itself. We subjected glutathione to reactions with model α,β-unsaturated carbonyl systems resulting from the interactions of adenosine with bifunctional aldehyde products of lipid peroxidation, and identified a range of adducts and cross-linked products. We found that the S-conjugated adducts, initially formed in the typical for GSH Michael addition to α,β-unsaturated carbonyl system, unexpectedly undergo gradual degradation giving rise to the final N-conjugated products, in which formation of the peptide amino group was involved instead of the sulfhydryl functionality. This finding shows that the role of the GSH amino group in the non-enzymatic detoxification is underestimated and that reactions between cellular α,β-unsaturated carbonyl compounds, and GSH may be more complex than are presently perceived.

scholarly journals Overhydroxylation of Lysine of Collagen Increases Uterine Fibroids Proliferation: Roles of Lysyl Hydroxylases, Lysyl Oxidases, and Matrix Metalloproteinases

2017 ◽  
Vol 2017 ◽  
pp. 1-13 ◽  
Author(s):  
Marwa Kamel ◽  
Mohamed Wagih ◽  
Gokhan S. Kilic ◽  
Concepcion R. Diaz-Arrastia ◽  
Mohamed A. Baraka ◽  
...  
Keyword(s):  
Matrix Metalloproteinases ◽  
Treatment Options ◽  
Uterine Fibroids ◽  
Cross Linking ◽  
Ki 67 ◽  
Subsequent Formation ◽  
Lysyl Oxidases ◽  
Cross Links ◽  
Collagen Cross Linking

The role of the extracellular matrix (ECM) in uterine fibroids (UF) has recently been appreciated. Overhydroxylation of lysine residues and the subsequent formation of hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) cross-links underlie the ECM stiffness and profoundly affect tumor progression. The aim of the current study was to investigate the relationship between ECM of UF, collagen and collagen cross-linking enzymes [lysyl hydroxylases (LH) and lysyl oxidases (LOX)], and the development and progression of UF. Our results indicated that hydroxyl lysine (Hyl) and HP cross-links are significantly higher in UF compared to the normal myometrial tissues accompanied by increased expression of LH (LH2b) and LOX. Also, increased resistance to matrix metalloproteinases (MMP) proteolytic degradation activity was observed. Furthermore, the extent of collagen cross-links was positively correlated with the expression of myofibroblast marker (α-SMA), growth-promoting markers (PCNA; pERK1/2;FAKpY397; Ki-67; and Cyclin D1), and the size of UF. In conclusion, our study defines the role of overhydroxylation of collagen and collagen cross-linking enzymes in modulating UF cell proliferation, differentiation, and resistance to MMP. These effects can establish microenvironment conducive for UF progression and thus represent potential target treatment options of UF.

scholarly journals Thyrotropin cross-links to the thyrotropin receptor through both the α and β subunits

1986 ◽  
Vol 235 (3) ◽  
pp. 879-882 ◽  
Author(s):  
P R Buckland ◽  
C R Rickards ◽  
R D Howells ◽  
B R Smith
Keyword(s):  
Tsh Receptor ◽  
Alpha Subunit ◽  
Receptor Interaction ◽  
Cross Linking ◽  
Thyrotropin Receptor ◽  
Cross Link ◽  
Receptor Binding Site ◽  
Form Part ◽  
Cross Links

We have recently shown that the beta subunit of thyrotropin (TSH) can be cross-linked to the TSH receptor [Buckland, Strickland, Pierce & Rees Smith (1985) Endocrinology (Baltimore) 116, 2122-2124; Buckland, Strickland & Rees Smith (1985) Biochem. Soc. Trans. 13, 942-943]. We failed, however, to cross-link the alpha subunit to the receptor, leaving the role of this subunit in the TSH-TSH-receptor interaction uncertain. We now report the successful cross-linking of the TSH alpha subunit to the receptor by the use of two different cross-linking reagents. Our studies suggest therefore that both subunits of TSH form part of the hormone's receptor-binding site.

TISSUE FIXATION WITH DIIMIDOESTERS AS AN ALTERNATIVE TO ALDEHYDES I. COMPARISON OF CROSS-LINKING AND ULTRASTRUCTURE OBTAINED WITH DIMETHYLSUBERIMIDATE AND GLUTARALDEHYDE

1974 ◽  
Vol 22 (4) ◽  
pp. 223-239 ◽  
Author(s):  
JOHN HASSELL ◽  
ARTHUR R. HAND
Keyword(s):  
Smooth Endoplasmic Reticulum ◽  
Light And Electron Microscopy ◽  
Assay Method ◽  
Cross Linking ◽  
Amino Group ◽  
Tissue Fixation ◽  
Optimal Conditions ◽  
Insoluble Protein ◽  
Cross Links ◽  
Per Se

Diimidoesters are bifunctional reagents which react with the ε-amino group of lysine, forming intermolecular cross-links with minimal alteration of the native properties of proteins. To determine the optimal conditions for tissue fixation with diimidoesters, a cross-linking assay was developed based on the assumption that protein aggregates formed by intermolecular cross-links would be water-insoluble. With this assay method, the proportion of insoluble protein in fresh liver was 16%, while optimal conditions for cross-linking with the diimidoester, dimethylsuberimidate (DMS) (16-20 mg/ml, 0.02 M Ca++ and 0.15 M Tris-HCl, pH 9.5), rendered 92.1% insoluble and permitted only 3.3% of the protein to diffuse into the fixative solution. The smaller diimidoesters, dimethyladipimate and dimethylmalonimidate, rendered 74 and 16% of the protein insoluble, respectively. Fixation with various aldehydes produced insoluble fractions varying from 74% to over 90%. Ultrastructurally, hepatocytes of DMS-fixed liver and glutaraldehyde-fixed liver were similar except that with the former fixative the Golgi saccules and smooth endoplasmic reticulum were dilated and mitochondrial matrices exhibited an increased electron density. Furthermore, the appearance of glutaraldehyde- and DMS-fixed liver was more readily correlated with the degree of cross-linking than with the pH of the fixative solution per se. The results of this study indicate that DMS is a potentially useful fixative for light and electron microscopy.

scholarly journals Role of endopeptidases in peptidoglycan synthesis mediated by alternative cross-linking enzymes in Escherichia coli

2021 ◽  
Author(s):  
Henri Voedts ◽  
Delphine Dorchêne ◽  
Adam Lodge ◽  
Waldemar Vollmer ◽  
Michel Arthur ◽  
...  
Keyword(s):  
Turgor Pressure ◽  
Cross Linking ◽  
Penicillin Binding Proteins ◽  
Peptidoglycan Synthesis ◽  
Dual Specificity ◽  
Common Motif ◽  
Cross Links ◽  
Hydrolysis Of ◽  
Controlled Hydrolysis

ABSTRACTBacteria resist to the turgor pressure of the cytoplasm through a net-like macromolecule, the peptidoglycan, made of glycan strands connected via peptides cross-linked by penicillin-binding proteins (PBPs). We recently reported the emergence of β-lactam resistance resulting from a bypass of PBPs by the YcbB L,D-transpeptidase (LdtD), which form chemically distinct 3→3 cross-links compared to 4→3 formed by PBPs. Here we show that peptidoglycan expansion requires controlled hydrolysis of cross-links and identify amongst eight endopeptidase paralogues the minimum enzyme complements essential for bacterial growth with 4→3 (MepM) and 3→3 (MepM and MepK) cross-links. Purified Mep endopeptidases unexpectedly displayed a 4→3 and 3→3 dual specificity implying recognition of a common motif in the two cross-link types. Uncoupling of the polymerization of glycan chains from the 4→3 cross-linking reaction was found to facilitate the bypass of PBPs by YcbB. These results illustrate the plasticity of the peptidoglycan polymerization machinery in response to the selective pressure of β-lactams.

scholarly journals Function of the N-Terminal Cap of the PAS Domain in Signaling by the Aerotaxis Receptor Aer

2006 ◽  
Vol 188 (6) ◽  
pp. 2154-2162 ◽  
Author(s):  
Kylie J. Watts ◽  
Kirsten Sommer ◽  
Sheena L. Fry ◽  
Mark S. Johnson ◽  
Barry L. Taylor
Keyword(s):  
Terminal Segment ◽  
Cross Linking ◽  
Pas Domain ◽  
Mutant Proteins ◽  
Dimer Interface ◽  
E Coli ◽  
Cross Links ◽  
Input Domain

ABSTRACT Aer, the Escherichia coli receptor for behavioral responses to oxygen (aerotaxis), energy, and redox potential, contains a PAS sensory-input domain. Within the PAS superfamily, the N-terminal segment (N-cap) is poorly conserved and its role is not well understood. We investigated the role of the N-cap (residues 1 to 19) in the Aer PAS domain by missense and truncation mutagenesis. Aer-PAS N-cap truncations and an Aer-M21P substitution resulted in low cellular levels of the mutant proteins, suggesting that the N-terminal region was important for stabilizing the structure of the PAS domain. The junction of the N-cap and PAS core was critical for signaling in Aer. Mutations and truncations in the sequence encoding residues 15 to 21 introduced a range of phenotypes, including defects in FAD binding, constant tumbling motility, and an inverse response in which E. coli cells migrated away from oxygen concentrations to which they are normally attracted. The proximity of two N-cap regions in an Aer dimer was assessed in vivo by oxidatively cross-linking serial cysteine substitutions. Cross-linking of several cysteine replacements at 23°C was attenuated at 10°C, indicating contact was not at a stable dimer interface but required lateral mobility. We observed large multimers of Aer when we combined cross-linking of N-cap residues with a cysteine replacement that cross-links exclusively at the Aer dimer interface. This suggests that the PAS N-cap faces outwards in a dimer and that PAS-PAS contacts can occur between adjacent dimers.

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.

Sign in / Sign up

Export Citation Format

Share Document