Review ArticleNox enzymes from fungus to fly to fish and what they tell us about Nox function in mammals
Introduction
Nox and Duox enzymes are membrane flavocytochromes that catalyze the NADPH-dependent reduction of molecular oxygen to generate superoxide and/or hydrogen peroxide. For the first 25 years of the field, however, the only known mammalian NADPH oxidase was the phagocyte NADPH oxidase. Also referred to as the respiratory burst oxidase, the enzyme uses Nox2 (a.k.a. gp91phox) as its catalytic subunit. The enzyme is activated in neutrophils and other inflammatory cells upon exposure to microbes or inflammatory products, resulting in the generation of high levels of superoxide, with secondary production of hydrogen peroxide, hydroxyl radical, and, in the presence of myeloperoxidase, HOCl. Together, this witch's brew of radicals and oxidants functions (along with other nonoxidative mechanisms) to kill or damage invading microbes. The importance of the Nox2 system in innate immunity is illustrated by the genetic condition chronic granulomatous disease, wherein affected patients suffer frequent and severe infections, often resulting from normally innocuous microbes such as the common mold Aspergillus niger.
Around the turn of the 21st century, it was discovered that Nox enzymes in mammals represent a family of homologous enzymes consisting in humans of seven gene products (six in rodents) plus additional splice variants, indicated in Fig. 1. These can be organized into two broad classes: the p22phox-requiring Nox enzymes (Nox1, Nox2, Nox3, and Nox4) and the Ca2+-regulated Nox enzymes (Nox5, Duox1, and Duox2). In addition, they can be grouped into three subfamilies based on their domain structure. Nox1, Nox2, Nox3, and Nox4 consist solely of the catalytic subunit, which is made up of an N-terminal transmembrane domain that binds two heme groups plus a C-terminal dehydrogenase domain that binds FAD and NADPH. All four bind to the small membrane-associated subunit p22phox, which both stabilizes the flavocytochrome and provides a binding site for regulatory subunits in the cases of Nox1, Nox2, and Nox3 (but not Nox4). Nox5 is the sole representative of the second group: this enzyme contains an EF-hand-containing Ca2+-binding domain N-terminal to the catalytic domain and is regulated by Ca2+ and protein kinase C. Duox1 and Duox2 build on the Nox5 structure in that they have an additional domain that is homologous to heme-containing peroxidases at their extreme N-termini; this is then linked via an additional transmembrane α-helix to the Nox5-like structure. Like Nox5, Duox enzymes are activated by Ca2+.
The realization that the Nox enzymes in mammals represent a family, the members of which are widely expressed in many tissues, has raised important questions about their normal biological roles. Although it has been tempting to try to assign immunity-based functions based upon analogy to the phagocyte NADPH oxidase, their typically considerably lower expression levels and lower output of ROS raise questions about whether they function in an analogous manner. A growing literature focusing on the signaling roles of ROS has supported a role for Nox-derived ROS in various signaling processes. Although gene-deleted or mutant mice have in some cases been informative (for example, in demonstrating that Nox3 plays a key role in otolith formation in the inner ear), some knockout mice fail to demonstrate an obvious phenotype, perhaps because of isoform redundancy or adaptation. In addition, in most of the published animals, the knockout has been expressed in all tissues, making it impossible to delineate tissue- or cell-specific phenotypes.
With this in mind, this review explores Nox enzymes throughout biology, reviewing biological functions in simpler systems, and attempts to determine whether common functional themes exist. Recently, we reviewed the occurrence of Nox enzymes in biology [1], comparing 105 Nox sequences. These occur in plants and algae, fungi, amoeba, nematode worms, echinoderms, urochordates, insects, fish, reptiles, birds, and mammals and can be organized into seven distinct subfamilies based on sequence similarities of the catalytic domains. The enzymes have not been reported in prokaryotes, but rather evolved more or less at the same time as single-cell eukaryotes, predating multicellularity by some 1.4 billion years. Some of these nonmammalian systems—for example, Caenorhabditis elegans and Drosophila—have experimental advantages in comparison with mammalian systems, in that they express a small number of Nox enzymes thereby reducing the likelihood of redundant functions. In addition, the use of these model organisms is extremely powerful with regard to the availability of elegant genetic tools, allowing, for example, exploration of the biological function of a given gene at a particular stage in development and in a specific tissue. The following sections are organized phylogenetically, more or less according to the evolutionary distance from mammals, starting with plants and ending with fish. The final sections attempt to discern common themes or patterns that are present in many species.
Section snippets
Plants
The first plant Nox gene was identified in rice as a homologue of mammalian gp91phox (Nox2) and was named rbohA for respiratory burst oxidase homologue [2]. A single plant species contains multiple rboh genes, as for example the 10 Atrboh genes seen in Arabidopsis thaliana, suggesting multiple functions and/or localizations in the plant. Characterization of rbohA in A. thaliana showed that RbohA was localized at the plasma membrane and predicted two EF-hand Ca2+-binding domains within the RbohA
Algae
Algae represent a diverse group of photosynthetic unicellular and multicellular eukaryotic organisms that range from phytoplankton to seaweed such as kelp, but in modern classifications exclude cyanobacteria, which are considered to be prokaryotic. In the red alga Chondrous crispus, the Nox homologue Ccrboh was originally identified as a gene encoding a Nox2-like respiratory burst oxidase homologue [29]. In contrast to other Nox homologues, the predicted sequence of Ccrboh contains an insertion
Amebozoa
Regulated production of superoxide is seen during early multicellular development in the slime mold Dictyostelium discoideum [32]. The presence of three Nox genes (noxA, noxB, and noxC) as well as homologues of p22phox and p67phox in D. discoideum [32], [33], [34] suggested the involvement of Noxes in this process [34]. Although NoxA, NoxB, p22phox and p67phox homologues show similarities to the mammalian Nox2 system, no homologues of p47phox and p40phox were detected in the D. discoideum
Fungi
The phylogenetic analysis of nox genes in fungi showed that unicellular and some dimorphic fungi lack nox genes, whereas other fungi contain one, two, or three genes (noxA–noxC), depending on the species [33], [35]. According to their structure, Noxes encoded by these genes have been grouped into the NoxA/NoxB and the NoxC/NoxD subfamilies of NADPH oxidases [1]. Members of the NoxA/NoxB subfamily show structures related to the subunit-regulated Nox enzymes such as mammalian Nox2, but NoxB
Insects
Insects encode several types of Nox enzymes. The genome of the fruit fly Drosophila melanogaster encodes two calcium-regulated Nox enzymes: a Nox5 homologue termed d-Nox (d for Drosophila) and a Duox known as d-Duox. In addition to Nox enzymes containing calcium-binding domains, the malaria mosquito Anopheles gambiae, as well as Aedes aegypti, the principal vector of yellow and dengue fevers, also encodes a unique Nox gene, “Nox-mosquito” or NoxM [1], which has only the Nox domain and no
Echinodermata
Sea urchins express at least two Nox enzymes: Nox-U1 [66] is an ancestor of Nox1, Nox2, and Nox3, whereas Udx-1 (short for urchin Duox1) is a member of the Duox subfamily. In addition, ancestors of both p47phox and p67phox are present, whereas an ancestor of p22phox has not yet been identified [42]. Whereas there seems to be no functional information available for Nox-U1, Udx-1 plays a dual role in both egg fertilization and development of early stage embryos.
During fertilization, Udx-1
Urochordates
The genome of the sea squirt Ciona intestinalis—among the most primitive of chordates—encodes a Nox2-like common ancestor of Nox1, Nox2, and Nox3, along with the first appearance in evolution of Nox4. In addition to the Nox2-like enzyme, Ciona also possesses homologues of p22phox, p67phox, and p47phox, but not p40phox [42]. Based on the expression of Nox2 and its presumed regulatory subunits in blood cells, the system was proposed to have a host defense function analogous to that in mammalian
Fish
The genome of zebrafish encodes Nox1, Nox2, Nox4, Nox5, and a single Duox. In addition, zebrafish have the gene for p22phox, those for the regulators of Nox1 (NOXO1 and NOXA1), and those for Nox2 (p47phox, p67phox, and p40phox). Although the functions of most of these have not been investigated in fish, Duox has recently been found to play a surprising role in wound healing and chemotaxis of neutrophils. When the epithelial layer is disrupted by physical trauma in fish or human,
Stress response
Consideration of the above reveals that one of the most frequent and general processes in which Nox enzymes participate can be considered to be a “stress response,” which can include a response to noxious stimuli such as toxins or harsh physical conditions, physical damage to tissue or cells, and invasion by foreign organisms. The end response of the cells or tissues varies depending on the organism and tissue. In plants, AtrbohD and AtrbohF mediate stomatal closure, helping tissues retain
Concluding remarks
Noxes have been retained throughout eukaryotic evolution. Nox regulation and Nox multiplicity allows for the choreographed production of ROS in time and space, allowing their use for a variety of coordinated functions. In diverse biological systems and even within the same organism, Nox enzymes play seemingly diverse roles, but several common themes can be discerned. These include killing pathogens, triggering apoptotic processes, protein cross-linking of the extracellular matrix, induction and
Acknowledgments
This work was supported by NIH Grants CA084138 and CA105116 and EHS Grant ES011163 to J.D.L. and by PAPIIT-UNAM IN201709 and CONACYT 49667Q to J.A.
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