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Basement membranes (BMs), a specialized form of extracellular matrix, underlie nearly all cell layers and provide structural support for tissues and interact with cell surface receptors to determine cell behavior. Both macromolecular composition and stiffness of the BM influence cell-BM interactions. Collagen IV is a major constituent of the BM that forms an extensively cross-linked oligomeric network. Its deficiency leads to BM mechanical instability, as observed with glomerular BM in Alport syndrome. These findings have led to the hypothesis that collagen IV and its cross-links determine BM stiffness. A sulfilimine bond (S = N) between a methionine sulfur and a lysine nitrogen cross-links collagen IV and is formed by the matrix enzyme peroxidasin. In peroxidasin knockout mice with reduced collagen IV sulfilimine cross-links, we find a reduction in renal tubular BM stiffness. Thus this work provides the first direct experimental evidence that collagen IV sulfilimine cross-links contribute to BM mechanical properties and provides a foundation for future work on the relationship of BM mechanics to cell function in renal disease.
Copyright © 2017 the American Physiological Society.

The collagen IV sulfilimine cross-link and its catalyzing enzyme, peroxidasin, represent a dyad critical for tissue development, which is conserved throughout the animal kingdom. Peroxidasin forms novel sulfilimine bonds between opposing methionine and hydroxylysine residues to structurally reinforce the collagen IV scaffold, a function critical for basement membrane and tissue integrity. However, the molecular mechanism underlying cross-link formation remains unclear. In this work, we demonstrate that the catalytic domain of peroxidasin and its immunoglobulin (Ig) domains are required for efficient sulfilimine bond formation. Thus, these molecular features underlie the evolutionarily conserved function of peroxidasin in tissue development and integrity and distinguish peroxidasin from other peroxidases, such as myeloperoxidase (MPO) and eosinophil peroxidase (EPO).
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Bromine is ubiquitously present in animals as ionic bromide (Br(-)) yet has no known essential function. Herein, we demonstrate that Br(-) is a required cofactor for peroxidasin-catalyzed formation of sulfilimine crosslinks, a posttranslational modification essential for tissue development and architecture found within the collagen IV scaffold of basement membranes (BMs). Bromide, converted to hypobromous acid, forms a bromosulfonium-ion intermediate that energetically selects for sulfilimine formation. Dietary Br deficiency is lethal in Drosophila, whereas Br replenishment restores viability, demonstrating its physiologic requirement. Importantly, Br-deficient flies phenocopy the developmental and BM defects observed in peroxidasin mutants and indicate a functional connection between Br(-), collagen IV, and peroxidasin. We establish that Br(-) is required for sulfilimine formation within collagen IV, an event critical for BM assembly and tissue development. Thus, bromine is an essential trace element for all animals, and its deficiency may be relevant to BM alterations observed in nutritional and smoking-related disease. PAPERFLICK:
Copyright © 2014 Elsevier Inc. All rights reserved.

Basement membrane, a specialized ECM that underlies polarized epithelium of eumetazoans, provides signaling cues that regulate cell behavior and function in tissue genesis and homeostasis. A collagen IV scaffold, a major component, is essential for tissues and dysfunctional in several diseases. Studies of bovine and Drosophila tissues reveal that the scaffold is stabilized by sulfilimine chemical bonds (S = N) that covalently cross-link methionine and hydroxylysine residues at the interface of adjoining triple helical protomers. Peroxidasin, a heme peroxidase embedded in the basement membrane, produces hypohalous acid intermediates that oxidize methionine, forming the sulfilimine cross-link. We explored whether the sulfilimine cross-link is a fundamental requirement in the genesis and evolution of epithelial tissues by determining its occurrence and evolutionary origin in Eumetazoa and its essentiality in zebrafish development; 31 species, spanning 11 major phyla, were investigated for the occurrence of the sulfilimine cross-link by electrophoresis, MS, and multiple sequence alignment of de novo transcriptome and available genomic data for collagen IV and peroxidasin. The results show that the cross-link is conserved throughout Eumetazoa and arose at the divergence of Porifera and Cnidaria over 500 Mya. Also, peroxidasin, the enzyme that forms the bond, is evolutionarily conserved throughout Metazoa. Morpholino knockdown of peroxidasin in zebrafish revealed that the cross-link is essential for organogenesis. Collectively, our findings establish that the triad-a collagen IV scaffold with sulfilimine cross-links, peroxidasin, and hypohalous acids-is a primordial innovation of the ECM essential for organogenesis and tissue evolution.

Collagen IV comprises the predominant protein network of basement membranes, a specialized extracellular matrix, which underlie epithelia and endothelia. These networks assemble through oligomerization and covalent crosslinking to endow mechanical strength and shape cell behavior through interactions with cell-surface receptors. A recently discovered sulfilimine (S=N) bond between a methionine sulfur and hydroxylysine nitrogen reinforces the collagen IV network. We demonstrate that peroxidasin, an enzyme found in basement membranes, catalyzes formation of the sulfilimine bond. Drosophila peroxidasin mutants have disorganized collagen IV networks and torn visceral muscle basement membranes, pointing to a critical role for the enzyme in tissue biogenesis. Peroxidasin generates hypohalous acids as reaction intermediates, suggesting a paradoxically anabolic role for these usually destructive oxidants. This work highlights sulfilimine bond formation as what is to our knowledge the first known physiologic function for peroxidasin, a role for hypohalous oxidants in tissue biogenesis, and a possible role for peroxidasin in inflammatory diseases.

The γ-hydroxy-1,N(2)-propano-2'-deoxyguanosine adduct (γ-OH-PdG) was introduced into 5'-d(GCTAGCXAGTCC)-3'·5'-d(GGACTCGCTAGC)-3' (X = γ-OH-PdG). In the presence of excess peptide KWKK, (13)C isotope-edited NMR revealed the formation of two spectroscopically distinct DNA-KWKK conjugates. These involved the reaction of the KWKK N-terminal amino group with the N(2)-dG propylaldehyde tautomer of the γ-OH-PdG lesion. The guanine N1 base imino resonance at the site of conjugation was observed in isotope-edited (15)N NMR experiments, suggesting that the conjugated guanine was inserted into the duplex and that the guanine imino proton was protected from exchange with water. The conjugates could be reduced in the presence of NaCNBH(3), suggesting that they existed, in part, as imine (Schiff base) linkages. However, (13)C isotope-edited NMR failed to detect the imine linkages, suggesting that these KWKK conjugates existed predominantly as diastereomeric carbinolamines, in equilibrium with trace amounts of the imines. The structures of the diastereomeric DNA-KWKK conjugates were predicted from potential energy minimization of model structures derived from the refined structure of the fully reduced cross-link [ Huang, H., Kozekov, I. D., Kozekova, A., Rizzo, C. J., McCullough, A., Lloyd, R. S., and Stone, M. P. ( 2010 ) Biochemistry , 49 , 6155 -6164 ]. Molecular dynamics calculations carried out in explicit solvent suggested that the conjugate bearing the S-carbinolamine linkage was the major species due to its potential for intramolecular hydrogen bonding. These carbinolamine DNA-KWKK conjugates thermally stabilized duplex DNA. However, the DNA-KWKK conjugates were chemically reversible and dissociated when the DNA was denatured. In this 5'-CpX-3' sequence, the DNA-KWKK conjugates slowly converted to interstrand N(2)-dG:N(2)-dG DNA cross-links and ring-opened γ-OH-PdG derivatives over a period of weeks.
© 2011 American Chemical Society

Glucose-induced β-cell action potential (AP) repolarization is regulated by potassium efflux through voltage gated (Kv) and calcium activated (K(Ca)) potassium channels. Thus, ablation of the primary Kv channel of the β-cell, Kv2.1, causes increased AP duration. However, Kv2.1(-/-) islet electrical activity still remains sensitive to the potassium channel inhibitor tetraethylammonium. Therefore, we utilized Kv2.1(-/-) islets to characterize Kv and K(Ca) channels and their respective roles in modulating the β-cell AP. The remaining Kv current present in Kv2.1(-/-) β-cells is inhibited with 5 μM CP 339818. Inhibition of the remaining Kv current in Kv2.1(-/-) mouse β-cells increased AP firing frequency by 39.6% but did not significantly enhance glucose stimulated insulin secretion (GSIS). The modest regulation of islet AP frequency by CP 339818 implicates other K(+) channels, possibly K(Ca) channels, in regulating AP repolarization. Blockade of the K(Ca) channel BK with slotoxin increased β-cell AP amplitude by 28.2%, whereas activation of BK channels with isopimaric acid decreased β-cell AP amplitude by 30.6%. Interestingly, the K(Ca) channel SK significantly contributes to Kv2.1(-/-) mouse islet AP repolarization. Inhibition of SK channels decreased AP firing frequency by 66% and increased AP duration by 67% only when Kv2.1 is ablated or inhibited and enhanced GSIS by 2.7-fold. Human islets also express SK3 channels and their β-cell AP frequency is significantly accelerated by 4.8-fold with apamin. These results uncover important repolarizing roles for both Kv and K(Ca) channels and identify distinct roles for SK channel activity in regulating calcium- versus sodium-dependent AP firing.

Although acetaminophen is the most widely used analgesic in the world, it is also a leading cause of toxic drug overdoses. Beyond normal therapeutic doses, the drug is hepatotoxic and genotoxic. All of the harmful effects of acetaminophen have been attributed to the production of its toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI). Since many of the cytotoxic/genotoxic events triggered by NAPQI are consistent with the actions of topoisomerase II-targeted drugs, the effects of this metabolite on human topoisomerase IIalpha were examined. NAPQI was a strong topoisomerase II poison and increased levels of enzyme-mediated DNA cleavage >5-fold at 100 microM. The compound induced scission at a number of DNA sites that were similar to those observed in the presence of the topoisomerase II-targeted anticancer drug etoposide; however, the relative site utilization differed. NAPQI strongly impaired the ability of topoisomerase IIalpha to reseal cleaved DNA molecules, suggesting that inhibition of DNA religation is the primary mechanism underlying cleavage enhancement. In addition to its effects in purified systems, NAPQI appeared to increase levels of DNA scission mediated by human topoisomerase IIalpha in cultured CEM leukemia cells. In contrast, acetaminophen did not significantly affect the DNA cleavage activity of the human enzyme in vitro or in cultured CEM cells. Furthermore, the analgesic did not interfere with the actions of etoposide against the type II enzyme. These results suggest that at least some of the cytotoxic/genotoxic effects caused by acetaminophen overdose may be mediated by the actions of NAPQI as a topoisomerase II poison.

The labile protons of two 32-base-pair, four-arm models of immobile Holliday junctions have been studied by two-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy. Overlap of resonances in the imino-imino region of two-dimensional nuclear Overhauser enhancement (NOE) spectra necessitates the use of a multi-pathway approach for obtaining sequence-specific assignments wherein all possible NOE connectivities to the labile protons are utilized, including those from the 2H of adenine, 5CH3 of thymine, and 5H of cytosine. Resonance assignments are obtained for all slowly exchanging imino and cytosine amino protons. Base-pairing up to and including the junction point is found in all four arms of both Holliday junctions. Several cross-arm NOE connectivities are identified and can be used to infer the geometry of the helical stacking domains. The two Holliday junctions studied, which differ only by the exchange of two base pairs at the branch point, appear to have opposite arm stacking geometries. These assignments form an important part of the critical background for detailed NMR analysis of Holliday junction structure and dynamics.