Biology subjects

Winterbourn, C. C.

Publications and source records attributed to Winterbourn, C. C..

3 recordsLinked to original sources

bioRxiv2024

Peroxidasin is associated with a mesenchymal-like transcriptional phenotype and promotes invasion in metastatic melanoma

Cutaneous melanoma is a highly invasive, heterogeneous and treatment resistant cancer. Its ability to dynamically shift between transcriptional states or phenotypes results in an adaptive cell plasticity that may drive cancer cell invasion or the development of therapy resistance. The expression of peroxidasin (PXDN), an extracellular matrix peroxidase, has been proposed to be associated with the invasive metastatic melanoma phenotype. We have confirmed this association by analysing the transcriptomes of 70 metastatic melanoma cell lines with variable levels of PXDN expression. This analysis highlighted a strong association between high PXDN expression and the undifferentiated invasive melanoma phenotype. To assess the functional role of PXDN in melanoma invasion, we performed a knockout of PXDN in a highly invasive cell line (NZM40). PXDN knockout decreased the invasive potential by [~]50% and decreased the expression of epithelial-mesenchymal transition and invasive marker genes as determined by RNAseq and substantiated by proteomics analysis. Bioinformatics analysis of differentially expressed genes following PXDN knockout highlighted decreases in genes linked to extracellular matrix formation, organisation and degradation as well as signalling pathways such as the WNT pathway. This study provides compelling evidence that PXDN plays a functional role in melanoma invasion by promoting an invasive, mesenchymal-like transcriptional phenotype. Research HighlightsPXDN expression is strongly associated with the invasive melanoma phenotype. Knockout of PXDN decreased invasion and expression of EMT marker genes concomitant with vast transcriptional changes relevant to many aspects of melanoma biology.

cancer biology↗

bioRxiv2021

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.

cancer biology↗

bioRxiv2020

Intra-dimer cooperativity between the active site cysteines during the oxidation of peroxiredoxin 2

Peroxiredoxin 2 (Prdx2) and other typical 2-Cys Prdxs function as homodimers in which hydrogen peroxide oxidizes each active site cysteine to a sulfenic acid which then condenses with the resolving cysteine on the alternate chain. Previous kinetic studies have considered both sites as equally reactive. Here we have studied Prdx2 using a combination of non-reducing SDS-PAGE to separate reduced monomers and dimers with one and two disulfide bonds, and stopped flow analysis of tryptophan fluorescence, to investigate whether there is cooperativity between the sites. We have observed positive cooperativity when H2O2 is added as a bolus and oxidation of the second site occurs while the first site is present as a sulfenic acid. Modelling of this reaction showed that the second site reacts 2.2 {+/-} 0.1 times faster. In contrast, when H2O2 was generated slowly and the first active site condensed to a disulfide before the second site reacted, no cooperativity was evident. Conversion of the sulfenic acid to the disulfide showed negative cooperativity, with modelling of the exponential rise in tryptophan fluorescence yielding a rate constant of 0.75 {+/-} 0.08 s-1 when the alternate active site was present as a sulfenic acid and 2.29 {+/-} 0.08-fold lower when it was a disulfide. No difference in the rate of hyperoxidation at the two sites was detected. Our findings imply that oxidation of one active site affects the conformation of the second site and influences which intermediate forms of the protein are favored under different cellular conditions.

biochemistry↗