NADPH oxidase

NADPH oxidase in its entirety leads to the release of reactive oxygen species; this process is called oxidative burst, where the eradication of invading microorganisms in macrophages and neutrophils ensues. NADPH oxidase is important in the maintenance of immune function, apoptosis, and cell growth. In previous times it was believed that NADPH oxidase generation of superoxides was only to happen in phagocytes, but inconsequent research studies, there are many enzymes that are responsible for reactive oxygen species production. For example, in kidneys, reactive oxygen species are produced from NOX3, and those molecules monitor renal function through sodium transport and oxygenation. Adding on, oxygen radicals increase sodium chloride absorption in the loop of Henle, which results in the regulation of sodium and hydrogen ion exchange. NADPH oxidase is known to function itself as a bacteria killer from the production of bacterial oxygen species by using oxygen and NADPH as substrates. In general, NADPH oxidase generates superoxides by moving electrons from NADPH inside of a cell and conjugating the oxygen atom to make superoxides

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Structure

The structure of NADPH oxidase is classified as a multiplex enzyme where it has more than one integral membrane proteins; glycoprotein gp9 1 Phox and adaptor protein p22(phox); the two integral membrane proteins can combine and form a heterodimeric flavocytochrome b558 that takes in the inner part of the enzyme. Furthermore, the C-terminal is a cytoplasmic domain that is similar to ferredoxin-NADP+ reductase, which holds the NADPH binding and FAD-binding sites. The N-terminal has six alpha-helical transmembrane segments, and this segment is common to all NADPH oxidase family enzymes. These transmembrane segments are also found in fungal ferric reductase, where Fre enzymes are activated to reduce Fe3+ and Cu2+ for iron and copper upregulation, however, they do not use oxygen as a substrate as NADPH oxidase would. The third and fifth helices of NADPH oxidase have two His residues that are used to give ligands for the binding of iron from two non-similar hemes. This action results in one heme facing the cytoplasmic side and the other heme facing the outer membrane side. The hemes themselves are set up perpendicular to the surface of the membrane. This allows electrons to be transferred from the cytosolic NADPH through FAD and across the cellular membrane from the hemes to reach oxygen, this is how it produces the superoxides. Again, this all happens in the N-terminal heme-containing region. The third alpha helix contains 13 amino acids, notably His101 and His115, the fifth alpha-helix on the other hand contains 12 amino acids which are His209 and His222. In any of the alpha helices, if there is any substitution in any of the four His residues (His101,His115,His209, or His222) then there is an imbalance in the attached hemes into gp91.

• Nox1 is normally found in the endothelium, fibroblasts, and smooth muscle cells. Nox1 contains an activator called Noxa1 and an organizer called p45phox, p47phox needs activation in order to regulate the enzyme activity.
• Nox2 is found in vascular cells, except in large arteries, it is also a 58 kDa protein. Nox2 is the best enzyme of the Nox family, it serves as a superoxide producer and a signaling module. Nox2 has a separate gp91 system that is found in white blood cells such as neutrophils and macrophages. Nox2 makes intracellular and extracellular superoxides. Nox2 just like Nox1 is activated with the assistance of p47phox phosphorylation. Nox4 is found in all vascular cells and is also more abundant compared to all of the other Nox subtypes. It can be specifically found in perinuclear space or the endoplasmic reticulum.
• Nox4 uses p22phox as its subunit. Nox4 is highly expresed in the kidney, but in other human tissues it is not usually found. Nox4 is commonly encoded on chromosome eleven.
• Nox5 is different than all the other Nox subtypes because it contains an added N-terminal domain that is meant for regulation. Nox5 also does not need any additional subunits, and is activated by Ca2+ binding to its N-terminal. The sensitivity to Ca2+ is increased due to calmodulin and phosphorylation. Nox5 can primarily be found in cytoskeletal components, plasma membranes, or the endoplasmic reticulum.

Function

NADPH oxidase is a major variable for the production of superoxides, there are seven types of NADPH oxidases. In general, NADPH oxidase transfers electrons from NADPH to oxygens in order to make superoxide, it does this because superoxide does not easily cross membranes. However, with the help of NADPH and location of the NADPH it can be released inside or outside of organelles. After superoxide is present, it is then turned into hydrogen peroxide (H202) to become a longer lasting and diffusible version of a superoxide. NADPH oxidase plays an assistive role in ion transport in the nephron of the kidney, proximal tubule and collecting duct. An increase in NADPH oxidase regulation can lead to a reduction in water and salt reabsorption.

Regulation of NADPH Oxidase

NADPH oxidase is regulated in many ways when it comes to superoxide formation. In one case, many families of NADPH oxidase are regulated by Ca2+. For instance, thyroid oxidase and sea urchin NADPH oxidase are both regulated reversibly by Ca2+. Proteolysis with alpha chymotrypsin regulates thyroid NADPH oxidase and irreversibly activates independently of Ca2+. Another example is with Nox5, Nox5 itself is regulated by Ca2+ extensively, superoxide formation from Nox5 has membrane fractions that require Ca2+ to be present. Cells that are activating Nox5 are producing superoxides because of Ca2+ ionophore ionomycin. In previous terms, the binding of Ca2+ formed a conformational change that ultimately leads to interactions of the N-terminal Ca2+ binding domain with the C-terminal NADPH domain. In skeletal muscle cells, Nox1, Nox2, and Nox4 are all found in skeletal muscle cells. Nox2 superoxide formation is regulated in the skeletal muscle cell by phosphorylation of p47phox and p67phox as well as the activation of Rac1. Although Nox4 is regularly active, it is regulated as well. Increased concentration of Nox4 cman result in diseases such as cancer and sarcopenia. The regulation of Nox4 comes from Nrf2,SMAD3, and histone deacetylases. These agents can decrease or regulate the mRNA transcription of Nox4.

Insulin-Signaling Pathway

Reactive oxygen species can regulate insulin signaling and glucose transport in skeletal muscles, more importantly Nox reactive oxygen species are important in insulin signaling. More specifically, Nox2 produced superoxides serve as regulators of skeletal muscle glucose uptake. It entirely depends on the concentration of the superoxides/reactive oxygen species. High concentration levels of superoxides damage insulin signaling and cause insulin resistance. Specifically, Nox2 superoxides regulate insulin resistance from angiotensin II. Another circumstance is seen in increased fat diet, Nox2 has an important role in insulin resistance from increased fat diets. Damage can be done to insulin signaling from an increased fat diet that is not regulated by Nox2.

NADPH Oxidase and Renal Function

In the kidney, any reactive oxygen species that is made by NADPH oxidase is meant to monitor renal glucose formation. The kidney plays a vital role in glucose metabolism by making glucose from gluconeogenesis. The amount of glucose that is released is equal to the amount of glucose made by the liver. For patients with diabetes (Type II), there is a disruption in the rate of glucose transfer. To combat this issue, apocynin which is an NADPH oxidase inhibitor and reactive oxygen species forager is used to control NADPH oxidase since the role of apocynin is to target NADPH oxidase and reactive oxygen species in order to re-balance glucose metabolism in the kidney.

Diseases of NADPH Oxidase

NADPH oxidase is associated with diseases, an example involves a mutation in the gene that encodes the NADPH oxidase complex, this mutation can cause chronic granulomatous disease. Chronic granulomatous disease is an uncommon immunodeficiency disease that is inherited from through X-linked recessive or autosomal recessive pathways. Through the mutations, there is a reduction in Nox2 expression that leads to deficiencies in phagocyte function. Another disease that is impacted by NADPH oxidase is stroke, stroke in general is caused by the exhaustion of oxygen and the input of oxygen. Higher levels of reactive oxygen species has been shown to have a significant impact on stroke. Nox2 and Nox4 are the common forms of NADPH oxidase that are associated with superoxide formation and pathology in stroke. NADPH oxidase is important for neutrotoxic components after microglia activation. During stroke it is shown that there is an increase in Nox2 levels and superoxide formation. Multiple sclerosis also has NADPH oxidase involvement. Multiple Sclerosis is another autoimmune disease that results in demyelination and neurodegeneration in humans. The production of superoxides is vital in multiple sclerosis pathogenesis. Research also presented that Nox2 is upregulated in microglia, this represents that activated microglia can aid in the formation of reactive oxygen species.

3D structures of NADPH oxidase

NADPH oxidase 3D structures