NADPH oxidase


NADPH oxidase is a membrane-bound enzyme complex that faces the extracellular space. It can be found in the plasma membrane as well as in the membranes of phagosomes used by neutrophil white blood cells to engulf microorganisms. Human isoforms of the catalytic component of the complex include NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1, and DUOX2.

Reaction

NADPH oxidase catalyzes the production of a superoxide free radical by transferring one electron to oxygen from NADPH. During this process O2 is transported from the extracellular space to the cell interior and the H+ is exported.

Biological function

The NADPH oxidase complex is dormant under normal circumstances, but is activated to assemble in the membranes during respiratory burst. The activated NADPH oxidase generates superoxide which has roles in animal immune response and plant signalling.
Superoxide can be produced in phagosomes which have ingested bacteria and fungi, or it can be produced outside of the cell. Superoxide kills bacteria and fungi by mechanisms that are not yet fully understood. It is presumed that superoxide kills bacteria directly, as the virulence of many pathogens is dramatically attenuated when their superoxide dismutase genes are deleted. However, superoxide can also spontaneously form hydrogen peroxide that undergoes further reactions to generate other reactive oxygen species like hypochlorous acid. It may also inactivate critical metabolic enzymes, initiate lipid peroxidation, damage iron-sulphur clusters, and liberate redox-active iron, which allows the generation of indiscriminate oxidants such as the hydroxyl radical.

Regulation

Careful regulation of NADPH oxidase activity is crucial to maintain a healthy level of ROS in the body. The enzyme is dormant in resting cells but becomes rapidly activated by several stimuli, including bacterial products and cytokines. Vascular NADPH oxidases are regulated by a variety of hormones and factors known to be important players in vascular remodeling and disease. These include thrombin, platelet-derived growth factor, tumor necrosis factor, lactosylceramide, interleukin-1, and oxidized LDL. It is also stimulated by agonists and arachidonic acid. Conversely, assembly of the complex can be inhibited by apocynin and diphenylene iodonium. Apocynin decreases influenza-induced lung inflammation in mice in vivo and so may have clinical benefits in the treatment of influenza.

Types

In animals, NADPH oxidase is found in two types: one in white blood cells and the other in vascular cells, differing in biochemical structure and functions. Neutrophilic NADPH oxidase produces superoxide almost instantaneously, where as the vascular enzyme produces superoxide in minutes to hours. Moreover, in white blood cells superoxide have been found to transfer electrons across the membrane to extracellular oxygen, while in vascular cells the radical anion appears to be released mainly intracellularly.

Pathology

Superoxides are crucial in killing foreign bacteria in the human body. Consequently, under-activity can lead to an increased susceptibility to organisms such as catalase-positive microbes, and over-activity can lead to oxidative stress and cell damage.
Excessive production of ROS in vascular cells causes many forms of cardiovascular disease including hypertension, atherosclerosis, myocardial infarction, and ischemic stroke. Atherosclerosis is caused by the accumulation of macrophages containing cholesterol in artery walls. ROS produced by NADPH oxidase activate an enzyme that makes the macrophages adhere to the artery wall. This process is counterbalanced by NADPH oxidase inhibitors, and by antioxidants. An imbalance in favor of ROS produces atherosclerosis. In vitro studies have found that the NADPH oxidase inhibitors apocynin and diphenyleneiodonium, along with the antioxidants N-acetyl-cysteine and resveratrol, depolymerized the actin, broke the adhesions, and allowed foam cells to migrate out of the intima.
One study suggests a role for NADPH oxidase in ketamine-induced loss of neuronal parvalbumin and GAD67 expression. Similar loss is observed in schizophrenia, and the results may point at the NADPH oxidase as a possible player in the pathophysiology of the disease. Nitro blue tetrazolium is used in a diagnostic test, in particular, for chronic granulomatous disease, a disease in which there is a defect in NADPH oxidase; therefore, the phagocyte is unable to make the reactive oxygen species or radicals required for bacterial killing, resulting in bacteria thriving within the phagocyte. The higher the blue score the better the cell is at producing reactive oxygen species.
It has also been shown that NADPH oxidase plays a role in the mechanism that induces the formation of sFlt-1, a protein that deactivates certain proangiogenic factors that play a role in the development of the placenta, by facilitating the formation of reactive oxygen species, which are suspected intermediaries in sFlt-1 formation. These effects are in part responsible for inducing pre-eclampsia in pregnant women

Mutations

Mutations in the NADPH oxidase subunit genes cause several Chronic Granulomatous Diseases, characterized by extreme susceptibility to infection. These include:
In these diseases, cells have a low capacity for phagocytosis, and persistent bacterial infections occur. Areas of infected cells are common, granulomas. A similar disorder called neutrophil immunodeficiency syndrome is linked to a mutation in the RAC2, also a part of the complex.

Inhibition

NADPH oxidase can be inhibited by apocynin, nitric oxide, and diphenylene iodonium. Apocynin acts by preventing the assembly of the NADPH oxidase subunits. Apocynin decreases influenza-induced lung inflammation in mice in vivo and so may have clinical benefits in the treatment of influenza.
Inhibition of NADPH oxidase by NO blocks the source of oxidative stress in the vasculature. NO donor drugs have therefore been used for more than a century to treat coronary artery disease, hypertension, and heart failure by preventing excess superoxide from deteriorating healthy vascular cells.
More advanced NADPH oxidase inhibitors include GKT-831, a dual Inhibitor of isoforms NOX4 and NOX1 which was patented in 2007. The compound was initially developed for Idiopathic pulmonary fibrosis and obtained orphan drug designation by the FDA and EMA at end of 2010.

Structure

The membrane-bound vascular enzyme is composed of five parts: two cytosolic subunits, a cytochrome b558 which consists of gp91phox, p22phox and a small G protein Rac. Generation of the superoxide in vascular NADPH occurs by a one-electron reduction of oxygen via the gp91phox subunit, using reduced NADPH as the electron donor. The small G protein carries an essential role in the activation of the oxidase by switching between a GDP-bound and GTP-linked forms.

Neutrophilic type

The isoform found in neutrophils is made up of six subunits. These subunits are:
There are several vascular isoforms of the complex which use paralogs the NOX2 subunit:
There are two further paralogs of NOX2 subunit in the thyroid: