Comparing automated cell imaging with conventional methods of measuring cell proliferation and viability.

The ability to assess cell proliferation and viability is essential for assessing new drug treatments, particularly in cancer drug discovery. This study describes a new method that uses a plate reader digital microscopy cell imaging and analysis system to assess cell proliferation and viability. This imaging system utilizes high throughput fluorescence microscopy with two fluorescent probes: cell membrane-impermeable SYTOX green and nuclear binding Hoechst-33342. Here we compare this technology to other known viability assays, namely: propidium iodide (PI)-based flow cytometry, and sulforhodamine B (SRB) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) based plate reader assays. These methods were assessed based on their effectiveness in detecting the cell numbers of two adherent cell lines and one suspension cell line. Automated cell imaging was most accurate at measuring cell number in both adherent and suspension cell lines. The PI-based flow cytometry method was more difficult to use with adherent cells, while the SRB and MTT assays had difficulties when monitoring cells in suspension. Despite these challenges, it was possible to obtain similar results when quantifying the effect of cytotoxic compounds. This study demonstrates that the digital microscopy automated cell imaging system is an effective method for assessing cell proliferation and the cytotoxic effect of compounds on both adherent and suspension cell lines.

Peroxidasin Inhibition by Phloroglucinol and Other Peroxidase Inhibitors.

Human peroxidasin (PXDN) is a ubiquitous peroxidase enzyme expressed in most tissues in the body. PXDN represents an interesting therapeutic target for inhibition, as it plays a role in numerous pathologies, including cardiovascular disease, cancer and fibrosis. Like other peroxidases, PXDN generates hypohalous acids and free radical species, thereby facilitating oxidative modifications of numerous biomolecules. We have studied the inhibition of PXDN halogenation and peroxidase activity by phloroglucinol and 14 other peroxidase inhibitors. Although a number of compounds on their own potently inhibited PXDN halogenation activity, only five were effective in the presence of a peroxidase substrate with IC50 values in the low µM range. Using sequential stopped-flow spectrophotometry, we examined the mechanisms of inhibition for several compounds. Phloroglucinol was the most potent inhibitor with a nanomolar IC50 for purified PXDN and IC50 values of 0.95 µM and 1.6 µM for the inhibition of hypobromous acid (HOBr)-mediated collagen IV cross-linking in a decellularized extracellular matrix and a cell culture model. Other compounds were less effective in these models. Most interestingly, phloroglucinol was identified to irreversibly inhibit PXDN, either by mechanism-based inhibition or tight binding. Our work has highlighted phloroglucinol as a promising lead compound for the design of highly specific PXDN inhibitors and the assays used in this study provide a suitable approach for high-throughput screening of PXDN inhibitors.

Peroxidasin protein expression and enzymatic activity in metastatic melanoma cell lines are associated with invasive potential.

Peroxidasin, a heme peroxidase, has been shown to play a role in cancer progression. mRNA expression has been reported to be upregulated in metastatic melanoma cell lines and connected to the invasive phenotype, but little is known about how peroxidasin acts in cancer cells. We have analyzed peroxidasin protein expression and activity in eight metastatic melanoma cell lines using an ELISA developed with an in-house peroxidasin binding protein. RNAseq data analysis confirmed high peroxidasin mRNA expression in the five cell lines classified as invasive and low expression in the three non-invasive cell lines. Protein levels of peroxidasin were higher in the cell lines with an invasive phenotype. Active peroxidasin was secreted to the cell culture medium, where it accumulated over time, and peroxidasin protein levels in the medium were also much higher in invasive than non-invasive cell lines. The only well-established physiological role of peroxidasin is in the formation of a sulfilimine bond, which cross-links collagen IV in basement membranes via catalyzed oxidation of bromide to hypobromous acid. We found that peroxidasin secreted from melanoma cells formed sulfilimine bonds in uncross-linked collagen IV, confirming peroxidasin activity and hypobromous acid formation. Moreover, 3-bromotyrosine, a stable product of hypobromous acid reacting with tyrosine residues, was detected in invasive melanoma cells, substantiating that their expression of peroxidasin generates hypobromous acid, and showing that it does not exclusively react with collagen IV, but also with other biomolecules.

Peroxidasin mediates bromination of tyrosine residues in the extracellular matrix.

Peroxidasin is a heme peroxidase that oxidizes bromide to hypobromous acid (HOBr), a powerful oxidant that promotes the formation of the sulfilimine crosslink in collagen IV in basement membranes. We investigated whether HOBr released by peroxidasin leads to other oxidative modifications of proteins, particularly bromination of tyrosine residues, in peroxidasin-expressing PFHR9 cells. Using stable isotope dilution LC-MS/MS, we detected the formation of 3-bromotyrosine, a specific biomarker of HOBr-mediated protein modification. The level of 3-bromotyrosine in extracellular matrix proteins from normally cultured cells was 1.1 mmol/mol tyrosine and decreased significantly in the presence of the peroxidasin inhibitor, phloroglucinol. A negligible amount of 3-bromotyrosine was detected in peroxidasin-knockout cells. 3-Bromotyrosine formed both during cell growth in culture and in the isolated decellularized extracellular matrix when embedded peroxidasin was supplied with hydrogen peroxide and bromide. The level of 3-bromotyrosine was significantly higher in extracellular matrix than intracellular proteins, although a low amount was detected intracellularly. 3-Bromotyrosine levels increased with higher bromide concentrations and decreased in the presence of physiological concentrations of thiocyanate and urate. However, these peroxidase substrates showed moderate to minimal inhibition of collagen IV crosslinking. Our findings provide evidence that peroxidasin promotes the formation of 3-bromotyrosine in proteins. They show that HOBr produced by peroxidasin is selective for, but not limited to, the crosslinking of collagen IV. Based on our findings, the use of 3-bromotyrosine as a specific biomarker of oxidative damage by HOBr warrants further investigation in clinical conditions linked to high peroxidasin expression.

The leucine-rich repeat domain of human peroxidasin 1 promotes binding to laminin in basement membranes.

Human peroxidasin 1 (PXDN) is a homotrimeric multidomain heme peroxidase and essential for tissue development and architecture. It has a biosynthetic function and catalyses the hypobromous acid-mediated formation of specific covalent sulfilimine (SN) bonds, which cross-link type IV collagen chains in basement membranes. Currently, it is unknown whether and which domain(s) [i.e. leucine-rich repeat domain (LRR), immunoglobulin domains, peroxidase domain, von Willebrand factor type C domain] of PXDN interact with the polymeric networks of the extracellular matrix (ECM), and how these interactions integrate and regulate the enzyme's cross-linking activity, without imparting oxidative damage to the ECM. In this study, we probed the interactions of four PXDN constructs with different domain compositions with components of a basement membrane extract by immunoprecipitation. Strong binding of the LRR-containing construct was detected with the major ECM protein laminin. Analysis of these interactions by surface plasmon resonance spectroscopy revealed similar kinetics and affinities of binding of the LRR-containing construct to human and murine laminin-111, with calculated dissociation constants of 1.0 and 1.5 µM, respectively. The findings are discussed with respect to the recently published in-solution structures of the PXDN constructs and the proposed biological role of this peroxidase.

Reaction of human peroxidasin 1 compound I and compound II with one-electron donors.

Human peroxidasin 1 (hsPxd01) is a homotrimeric multidomain heme peroxidase embedded in the extracellular matrix. It catalyses the two-electron oxidation of bromide by hydrogen peroxide to hypobromous acid which mediates the formation of essential sulfilimine cross-links between methionine and hydroxylysine residues in collagen IV. This confers critical structural reinforcement to the extracellular matrix. This study presents for the first time transient kinetic measurements of the reactivity of hsPxd01 compound I and compound II with the endogenous one-electron donors nitrite, ascorbate, urate, tyrosine and serotonin using the sequential stopped-flow technique. At pH 7.4 and 25 °C compound I of hsPxd01 is reduced to compound II with apparent second-order rate constants ranging from (1.9 ± 0.1) × 104 M-1 s-1 (urate) to (4.8 ± 0.1) × 105 M-1 s-1 (serotonin). Reduction of compound II to the ferric state occurs with apparent second-order rate constants ranging from (4.3 ± 0.2) × 102 M-1 s-1 (tyrosine) to (7.7 ± 0.1) × 103 M-1 s-1 (serotonin). The relatively fast rates of compound I reduction suggest that these reactions may take place in vivo and modulate bromide oxidation due to formation of compound II. Urate is shown to inhibit the bromination activity of hsPxd01, whereas nitrite stimulates the formation of hypobromous acid. The results are discussed with respect to known kinetic data of homologous mammalian peroxidases and to the physiological role of human peroxidasin 1.

Monomeric and homotrimeric solution structures of truncated human peroxidasin 1 variants.

Human peroxidasin 1 is a multidomain peroxidase situated in the basement membrane. The iron enzyme with covalently bound heme oxidizes bromide to hypobromous acid which facilitates the formation of distinct sulfilimine cross-links in the collagen IV network and therefore contributes to its mechanical stability. Additional to the catalytically active peroxidase domain peroxidasin comprises a leucine rich repeat domain, four Ig domains and a C-terminal von Willebrand factor type C module (VWC). Peroxidasin has been shown to form homotrimers involving two redox-sensitive cysteine residues and to undergo posttranslational C-terminal proteolytic cleavage. The present study on several recombinantly produced truncated peroxidasin variants showed that the VWC is not required for trimer formation whereas the alpha-helical linker region located between the peroxidase domain and the VWC is crucial for trimerization. Our data furthermore implies that peroxidasin oligomerization occurs intracellularly before C-terminal cleavage. For the first time we present overall solution structures of monomeric and trimeric truncated peroxidasin variants which were determined by rotary shadowing combined with transmission electron microscopy and by small-angle X-ray scattering (SAXS). A triangular arrangement of the peroxidase domains to each other within the homotrimer was revealed and this structure was confirmed by a model of trimeric peroxidase domains. Our SAXS data showed that the Ig domains are highly flexible and interact with the peroxidase domain and that within the homotrimer each alpha-helical linker region interacts with the respective adjacent peroxidase domain. The implications of our findings on the structure-function relationship of peroxidasin are discussed.

Human peroxidasin 1 promotes angiogenesis through ERK1/2, Akt, and FAK pathways.

Aims: The term angiogenesis refers to sprouting of new blood vessels from pre-existing ones. The angiogenic process involves cell migration and tubulogenesis requiring interaction between endothelial cells and the extracellular matrix. Human peroxidasin 1 (hsPxd01) is a multidomain heme peroxidase found embedded in the basement membranes. As it promotes the stabilization of extracellular matrix, we investigated its possible role in angiogenesis both in vitro and in vivo. Methods and results: We analysed the effects of peroxidasin 1 gene silencing and supplementation by recombinant hsPxd01 in TeloHAEC endothelial cells on cell migration, tubulogenesis in matrigel, and intracellular signal transduction as assessed by kinase phosphorylation and expression of pro-angiogenic genes as measured by qRT-PCR. We further evaluated the angiogenic potential of recombinant peroxidasin in a chicken chorioallantoic membrane model. RNA silencing of endogenous hsPxd01 significantly reduced tube formation and cell migration, whereas supplementation by the recombinant peroxidase promoted tube formation in vitro and stimulated vascularization in vivo through its catalytic activity. Moreover, recombinant hsPxd01 promoted phosphorylation of Extracellular signal-Regulated Kinases (ERK1/2), Protein kinase B (Akt), and Focal Adhesion Kinase (FAK), and induced the expression of pro-angiogenic downstream genes: Platelet Derived Growth Factor Subunit B (PDGFB), endothelial-derived Heparin Binding EGF-like growth factor (HB-EGF), CXCL-1, Hairy-Related Transcription Factor 1 (HEY-1), DNA-binding protein inhibitor (ID-2), Snail Family Zinc Finger 1 (SNAI-1), as well as endogenous hsPxd01. However, peroxidasin silencing significantly reduced Akt and FAK phosphorylation but induced ERK1/2 activation after supplementation by recombinant hsPxd01. While hsPxd01 silencing significantly reduced expression of HEY-1, ID-2, and PDGFB, it did not affect expression of SNAI-1, HB-EGF, and CXCL-1 after supplementation by recombinant hsPxd01. Conclusion: Our findings suggest a role of enzymatically active peroxidasin 1 as a pro-angiogenic peroxidase and a modulator of ERK1/2, Akt and FAK signalling.

Characterisation of peroxidasin activity in isolated extracellular matrix and direct detection of hypobromous acid formation.

Peroxidasin is a heme peroxidase that catalyses the oxidation of bromide by hydrogen peroxide to form an essential sulfilimine cross-link between methionine and hydroxylysine residues in collagen IV. We investigated cross-linking by peroxidasin embedded in extracellular matrix isolated from cultured epithelial cells and its sensitivity to alternative substrates and peroxidase inhibitors. Peroxidasin showed peroxidase activity as measured with hydrogen peroxide and Amplex red. Using a specific mass spectrometry assay that measures NADH bromohydrin, we showed definitively that the enzyme releases hypobromous acid (HOBr). Less than 1 µM of the added hydrogen peroxide was used by peroxidasin. The remainder was consumed by catalase activity that was associated with the matrix. Results from NADH bromohydrin measurements indicates that low micromolar HOBr generated by peroxidasin was sufficient for maximum sulfilimine cross-linking, whereas 100 µM reagent HOBr or taurine bromamine was less efficient. This implies selectivity for the enzymatic process. Physiological concentrations of thiocyanate and urate partially inhibited cross-link formation. 4-Aminobenzoic acid hydrazide, a commonly used myeloperoxidase inhibitor, also inhibited peroxidasin, whereas acetaminophen and a 2-thioxanthine were much less effective. In conclusion, HOBr is produced by peroxidasin in the extracellular matrix. It appears to be directed at the site of collagen IV sulfilimine formation but the released HOBr may also undergo other reactions.

Posttranslational modification of heme in peroxidases - Impact on structure and catalysis.

Four heme peroxidase superfamilies arose independently in evolution. Only in the peroxidase-cyclooxygenase superfamily the prosthetic group is posttranslationally modified (PTM). As a consequence these peroxidases can form one, two or three covalent bonds between heme substituents and the protein. This may include ester bonds between heme 1- and 5-methyl groups and glutamate and aspartate residues as well as a sulfonium ion link between the heme 2-vinyl substituent and a methionine. Here the phylogeny and physiological roles of representatives of this superfamily, their occurrence in all kingdoms of life, the relevant sequence motifs for definite identification and the available crystal structures are presented. We demonstrate the autocatalytic posttranslational maturation process and the impact of the covalent links on spectral and redox properties as well as on catalysis, including Compound I formation and reduction by one- and two-electron donors. Finally, we discuss the evolutionary advantage of these PTMs with respect to the proposed physiological functions of the metalloenzymes that range from antimicrobial defence in innate immunity to extracellular matrix formation and hormone biosynthesis.

Structure of human promyeloperoxidase (proMPO) and the role of the propeptide in processing and maturation.

Myeloperoxidase (MPO) is synthesized by neutrophil and monocyte precursor cells and contributes to host defense by mediating microbial killing. Although several steps in MPO biosynthesis and processing have been elucidated, many questions remained, such as the structure-function relationship of monomeric unprocessed proMPO versus the mature dimeric MPO and the functional role of the propeptide. Here we have presented the first and high resolution (at 1.25 Å) crystal structure of proMPO and its solution structure obtained by small-angle X-ray scattering. Promyeloperoxidase hosts five occupied glycosylation sites and six intrachain cystine bridges with Cys-158 of the very flexible N-terminal propeptide being covalently linked to Cys-319 and thereby hindering homodimerization. Furthermore, the structure revealed (i) the binding site of proMPO-processing proconvertase, (ii) the structural motif for subsequent cleavage to the heavy and light chains of mature MPO protomers, and (iii) three covalent bonds between heme and the protein. Studies of the mutants C158A, C319A, and C158A/C319A demonstrated significant differences from the wild-type protein, including diminished enzymatic activity and prevention of export to the Golgi due to prolonged association with the chaperone calnexin. These structural and functional findings provide novel insights into MPO biosynthesis and processing.

Pre-steady-state Kinetics Reveal the Substrate Specificity and Mechanism of Halide Oxidation of Truncated Human Peroxidasin 1.

Human peroxidasin 1 is a homotrimeric multidomain peroxidase that is secreted to the extracellular matrix. The heme enzyme was shown to release hypobromous acid that mediates the formation of specific covalent sulfilimine bonds to reinforce collagen IV in basement membranes. Maturation by proteolytic cleavage is known to activate the enzyme. Here, we present the first multimixing stopped-flow study on a fully functional truncated variant of human peroxidasin 1 comprising four immunoglobulin-like domains and the catalytically active peroxidase domain. The kinetic data unravel the so far unknown substrate specificity and mechanism of halide oxidation of human peroxidasin 1. The heme enzyme is shown to follow the halogenation cycle that is induced by the rapid H2O2-mediated oxidation of the ferric enzyme to the redox intermediate compound I. We demonstrate that chloride cannot act as a two-electron donor of compound I, whereas thiocyanate, iodide, and bromide efficiently restore the ferric resting state. We present all relevant apparent bimolecular rate constants, the spectral signatures of the redox intermediates, and the standard reduction potential of the Fe(III)/Fe(II) couple, and we demonstrate that the prosthetic heme group is post-translationally modified and cross-linked with the protein. These structural features provide the basis of human peroxidasin 1 to act as an effective generator of hypobromous acid, which mediates the formation of covalent cross-links in collagen IV.

Multidomain human peroxidasin 1 is a highly glycosylated and stable homotrimeric high spin ferric peroxidase.

Human peroxidasin 1 (hsPxd01) is a multidomain heme peroxidase that uses bromide as a cofactor for the formation of sulfilimine cross-links. The latter confers critical structural reinforcement to collagen IV scaffolds. Here, hsPxd01 and various truncated variants lacking nonenzymatic domains were recombinantly expressed in HEK cell lines. The N-glycosylation site occupancy and disulfide pattern, the oligomeric structure, and unfolding pathway are reported. The homotrimeric iron protein contains a covalently bound ferric high spin heme per subunit with a standard reduction potential of the Fe(III)/Fe(II) couple of -233 ± 5 mV at pH 7.0. Despite sequence homology at the active site and biophysical properties similar to human peroxidases, the catalytic efficiency of bromide oxidation (kcat/KM(app)) of full-length hsPxd01 is rather low but increased upon truncation. This is discussed with respect to its structure and proposed biosynthetic function in collagen IV cross-linking.

Inactivation of human myeloperoxidase by hydrogen peroxide.

Human myeloperoxidase (MPO) uses hydrogen peroxide generated by the oxidative burst of neutrophils to produce an array of antimicrobial oxidants. During this process MPO is irreversibly inactivated. This study focused on the unknown role of hydrogen peroxide in this process. When treated with low concentrations of H2O2 in the absence of reducing substrates, there was a rapid loss of up to 35% of its peroxidase activity. Inactivation is proposed to occur via oxidation reactions of Compound I with the prosthetic group or amino acid residues. At higher concentrations hydrogen peroxide acts as a suicide substrate with a rate constant of inactivation of 3.9 × 10(-3) s(-1). Treatment of MPO with high H2O2 concentrations resulted in complete inactivation, Compound III formation, destruction of the heme groups, release of their iron, and detachment of the small polypeptide chain of MPO. Ten of the protein's methionine residues were oxidized and the thermal stability of the protein decreased. Inactivation by high concentrations of H2O2 is proposed to occur via the generation of reactive oxidants when H2O2 reacts with Compound III. These mechanisms of inactivation may occur inside neutrophil phagosomes when reducing substrates for MPO become limiting and could be exploited when designing pharmacological inhibitors.

Ceruloplasmin is an endogenous inhibitor of myeloperoxidase.

Myeloperoxidase is a neutrophil enzyme that promotes oxidative stress in numerous inflammatory pathologies. It uses hydrogen peroxide to catalyze the production of strong oxidants including chlorine bleach and free radicals. A physiological defense against the inappropriate action of this enzyme has yet to be identified. We found that myeloperoxidase oxidized 75% of the ascorbate in plasma from ceruloplasmin knock-out mice, but there was no significant loss in plasma from wild type animals. When myeloperoxidase was added to human plasma it became bound to other proteins and was reversibly inhibited. Ceruloplasmin was the predominant protein associated with myeloperoxidase. When the purified proteins were mixed, they became strongly but reversibly associated. Ceruloplasmin was a potent inhibitor of purified myeloperoxidase, inhibiting production of hypochlorous acid by 50% at 25 nm. Ceruloplasmin rapidly reduced Compound I, the Fe(V) redox intermediate of myeloperoxidase, to Compound II, which has Fe(IV) in its heme prosthetic groups. It also prevented the fast reduction of Compound II by tyrosine. In the presence of chloride and hydrogen peroxide, ceruloplasmin converted myeloperoxidase to Compound II and slowed its conversion back to the ferric enzyme. Collectively, our results indicate that ceruloplasmin inhibits myeloperoxidase by reducing Compound I and then trapping the enzyme as inactive Compound II. We propose that ceruloplasmin should provide a protective shield against inadvertent oxidant production by myeloperoxidase during inflammation.