Oxidative stress plays an important role in the development of cardiovascular diseases such as hypertension and atherosclerosis. The main source of the superoxide free radical in vascular cells is the enzyme NADPH oxidase 1 (Nox1). One of the activators of Nox1 is the protein disulfide isomerase (PDI), a ubiquitous protein that performs an array of cellular functions, and is involved in redox signaling and homeostasis. Its most well known function is the folding of nascent proteins in the endoplasmic reticulum.

Now scientists from the Center for Research of Redox Processes in Biomedicine (RIDC Redoxoma) led by Professor Lucia Rossetti Lopes at the Instituto de Ciências Biomédicas at USP, have revealed the molecular mechanism by which PDI activates Nox1 in vascular smooth muscle cells (VSMC), demonstrating that PDI is an important novel Nox1 redox regulator in atherosclerosis.

“Our data show the redox interaction between PDI and the regulatory subunit p47^phox cysteines, which facilitates p47^phox phosphorylation and Nox1 activation. We performed a redox proteomics study and identified the cysteine residues required for this interaction,” said Lopes.

The study was conducted in collaboration with researchers Francis J. Miller Jr. of Duke University and an international collaborator of Redoxoma; Luis ES Netto, of the Instituto de Biociências at USP and member of the Redoxoma; José César Rosa, from the Escola de Medicina de Ribeirão Preto, USP; and Ralf P. Brandes of the University of Frankfurt. The results were published in the journal Arteriosclerosis, Thrombosis, and Vascular Biology.

Mechanism

NADPH oxidase (Nox) comprises a family of enzymes linked to the cell membrane that catalyzes the reduction of molecular oxygen generating the superoxide radical anion, which, in turn, participates in the generation of other oxidants such as hydroxyl radical and hydrogen peroxide. The enzymatic complex is composed of several isoforms, with different functions. The Nox2 isoform, for example, is found in neutrophils and plays a key role in the control of infections. Nox1, present in the cardiovascular system, is involved in cardiovascular diseases. When its expression is increased, as in atherosclerosis, increased production of superoxide causes proliferation and migration of vascular smooth muscle cell (VSMC) that contribute vascular remodeling.

Noxes are regulated by protein-protein interactions and signaling molecules and are activated only after appropriate physiological stimuli. The Nox system contains seven isoforms and the catalytic subunits (Nox1 and Nox2) are associated with regulatory subunits, p47^phox and p67^phox in the presence of a stimulus. Thus, in the absence of this stimulus the catalytic and regulatory subunits are located in different compartments: the first on the membrane and the others on the cytosol.

The p47^phox is a cytosolic subunit present in Noxs1 and 2. It is an organizer of Nox and it is with it that the PDI interacts, forming disulfide bonds between the redox cysteines, which facilitates its phosphorylation and consequently activates Nox. With these findings, the researchers identified a new regulatory mechanism by which PDI regulates Nox activity in the vascular system.

Phosphorylation of p47phox leads to coupling of the cytosolic subunits required for assembly and activation of Nox1 and Nox2. The p47^phox is a carrier, it carries other subunits to the membrane. In general, this subunit organizes Nox. P67^phox is the activating subunit that binds to p47^phox to reach the membrane and transfer electrons from NADPH to molecular oxygen. “We are showing that PDI can be a new organizer of Nox,” explains Lopes.

According to the researcher, for the interaction between PDI and p47^phox to occur, the former needs to be reduced and the latter, oxidized. The process is dynamic. “These redox states are important. We observed the formation of different disulfide bridges, and one of them is intramolecular, occurs between cysteines 196 and 378 of the p47^phox subunit. PDI binds to p47^phox, breaks the intramolecular bridge, causing a conformational modification of the subunit and exposing serine 379, which is then phosphorylated.” The intramolecular disulfide bridge is only observed when PDI interacts with p47^phox.

In the study, the researchers showed that PDI levels are increased in atherosclerotic aortas of non-human primates. They analyzed samples from the aorta artery of monkeys submitted to an atherosclerotic diet, that is, rich in lipids, in which the increase of Nox1 had already been verified, and they also verified that there was an increase of the PDI.

Then, using recombinant proteins, they identified a redox interaction between the PDI and the p47^phox cytosolic subunit in vitro. Mutations in the four redox cysteines of PDI prevented interaction with p47^phox, showing that dimer formation depends on these cysteines. The formation of the redox-dependent disulfide bonds was confirmed by mass spectrometry. The researchers also noted the critical role of cysteine 400 located at the PDI active site a’ (redox) for PDI binding to cysteine 196 of p47^phox.

Using the Proximity ligation assay technique, they confirmed in vivo the interaction between PDI and p47^phox in carotid arteries of mice after wire injury, which is a model of vascular remodeling which, similar to what occurs after the placement of a stent in the coronary arteries of a patient. Finally, the researchers did a translational study, in which they performed an analysis of gene expression in arteries with and without atheroma plaques, from a database obtained from human arteries. Comparing arteries with and without lesion, they observed that the expression of p47^phox on the plaques correlates with the expression of the PDI having the redox domain a’ This result indicates the positive correlation between PDI and p47^phox in human atherosclerotic arteries.

The characterization of the interaction between the PDI and the p47^phox subunit of NADPH oxidase 1 suggests the existence of a cytosolic pool of PDI. “To show that the PDI is not only in the endoplasmic reticulum is an important contribution of our work. In that sense, an earlier work by Francisco Laurindo [investigator of InCor and RIDC Redoxoma], showing the pool of PDI in the cytoskeleton, which he called pecPDI [peri / epicellular disulfide protein isomerase], was very important” Lopes said.

The article Redox Activation of Nox1 (NADPH Oxidase 1) Involves an Intermolecular Disulfide Bond Between Protein Disulfide Isomerase and p47^phox in Vascular Smooth Muscle Cells, by Marcela Gimenez, Sidney Verissimo-Filho, Ilka Wittig, Brandon M. Schickling, Fabian Hahner, Christoph Schürmann , Luis ES Netto, José César Rosa, Ralf P. Brandes, Simone Sartoretto, Lívia De Lucca Camargo, Fernando Abdulkader, Francis J. Miller and Lucia Rossetti Lopes, can be read by subscribers here