The extracellular matrix is an intricate network composed of a set of macromolecules that unites and fixes cells to form tissues, performing vital functions for cellular support, intercellular communications, and regulation of tissue development. In a study published in the journal Redox Biology, researchers identified extracellular matrix proteins modified by the action of hypobromous acid in normal and fibrotic lung tissues, suggesting a possible physiological role for protein bromination.

“This work showed that bromination is found physiologically and that peroxidasin, the enzyme that catalyzes the formation of hypobromous acid, has other targets than collagen, which was one of the biggest questions in the field,” said researcher Litiele Cezar Cruz, first author of the article. She carried out the research during her postdoctoral studies, under the supervision of Professor Flavia Meotti, from the Instituto de Química at Universidade de São Paulo (USP) and a member of the RIDC Redoxoma. Part of the study was carried out with a Research Internship Abroad Grant (BEPE) from FAPESP, in the laboratory of Professor Albert van der Vliet, at the University of Vermont, in the United States.

Halogenation is a chemical reaction in which a halogen atom – mainly fluorine, chlorine, bromine, or iodine – is incorporated into a molecule. Halogen modifications of biological molecules are increasingly recognized in several areas of biology, but their relevance in the mammalian organism is still poorly studied. The role of bromide ions in the oxidative cross-linking of collagen IV within the extracellular matrix was the first evidence that bromine plays an essential role in living organisms.

“We know better about chlorination, a modification that depends on myeloperoxidase, an inflammatory enzyme. In general, this type of halogenation associated with disease processes is more studied, because inflammation is at the genesis of the most diseases. In inflammatory diseases with myeloperoxidase activity, we tend to find an increase in proteins with the addition of chlorine. This modification by bromine is very similar, as chlorine and bromine are very similar chemical elements, and the modification occurs at the same amino acids, including tyrosine. Despite the fact that plasma concentration of bromine is a thousand times lower than that of chlorine, the latter is not a good substrate for peroxidasin, therefore hypobromous acid, rather than hypochlorous acid, is produced in the extracellular matrix” Meotti explained.

Peroxidasin

Peroxidasin (PXDN) is a heme peroxidase enzyme expressed in the extracellular matrix in various tissues. It catalyzes the formation of hypobromous acid (HOBr) from hydrogen peroxide and bromide. Hypobromous acid oxidizes amino acid residues to form covalent cross-links between collagen IV molecules, generating a collagen network that structures the basement membrane of the extracellular matrix.

According to the researchers, recent studies have suggested that peroxidasin could modify other extracellular matrix proteins in addition to collagen IV, with the incorporation of bromide into tyrosine residues, through hypobromous acid. Since hypobromous acid is a strong oxidant constantly formed in the matrix, they decided to investigate other protein targets for this oxidant. To this end, they used the mass spectrometry proteomic analysis technique to identify proteins containing brominated tyrosines in different types of samples.

Initially, they worked with PHFR9 cells, which overproduce the extracellular matrix. “We isolated the matrix, treated it with hypobromous acid, and carried out a proteomic search. Then came a giant list, with many brominated proteins, and the one that stood out the most, with the greatest number of modified peptides, was laminin”, says Cruz. Laminins are the main non-collagenous proteins of the basement membrane and bind to other proteins, including peroxidasin. They also have a support function and are involved in cell proliferation signaling. The basement membrane is the part of the matrix closest to the epithelial, endothelial, and muscle cells, and is composed mainly of collagen IV and laminins.

Then, in collaboration with Vilet’s group, the researcher analyzed lung tissues from healthy mice and mice model of pulmonary fibrosis. One of the main characteristics of pulmonary fibrosis is the increased deposition of extracellular matrix. In the lungs of mice with fibrosis, she observed that peroxidasin, laminins, collagens, and several other extracellular membrane proteins were upregulated. Among the proteins with brominated tyrosines, laminins stood out again. Brominated proteins were also found in the lungs of healthy mice.

To expand the findings, Cruz investigated the presence of bromotyrosine-modified proteins in lung tissues obtained from healthy humans and patients with idiopathic pulmonary fibrosis. Additionally, she reanalyzed previously published proteomic data from lung tissue. In both tissue samples and proteomic data, the researcher found modifications in several extracellular membrane proteins, even in healthy lungs.

This is the first study to identify brominated proteins in pulmonary fibrosis and non-fibrotic lung tissue.

“We have detected in a parallel study that, to produce hypobromous acid, peroxidasin needs very low concentrations of hydrogen peroxide, which our cells produce constantly. So, there is no need for an inflammatory event to produce hypobromous acid. Hence the importance of finding this bromination in normal tissues, without fibrosis,” Meotti explained.

According to Cruz, “An interesting aspect recently shown is that the presence of bromine in the basement membrane is dependent on the presence of peroxidasin. When peroxidasin is inhibited, bromine and brominated peptides disappear.“

Although the biological relevance of these protein bromination is still unclear, this is a post-translational modification that can affect the structure of the extracellular membrane and the interactions with adjacent cells.

The article Identification of tyrosine brominated extracellular matrix proteins in normal and fibrotic lung tissues, de Litiele Cezar Cruz, Aida Habibovic, Bianca Dempsey, Mariana P. Massafera, Yvonne Janssen-Heininger, Miao-Chong Joy Lin, Evan T. Hoffman, Daniel J . Weiss, Steven K.Huang, Albert van der Vliet, Flavia C. Meotti can be accessed here.