Nox enzymes from fungus to fly to fish and what they tell us about Nox function in mammals

Abstract

The production of reactive oxygen species (ROS) in a highly regulated fashion is a hallmark of members of the NADPH oxidase (Nox) family of enzymes. Nox enzymes are present in most eukaryotic groups such as the amebozoid, fungi, algae and plants, and animals, in which they are involved in seemingly diverse biological processes. However, a comprehensive survey of Nox functions throughout biology reveals common functional themes. Noxes are often activated in response to stressful conditions such as nutrient starvation, physical damage, or pathogen attack. Although the end result varies depending on the organism and tissue, Nox-produced ROS mediate the response to the adverse stimuli, such as innate immunity responses in plants and animals or cell differentiation in Dictyostelium, fungi, and plants. These responses involve ROS-mediated signaling mechanisms occurring at intracellular or cell-to-cell levels and sometimes involve cell wall or extracellular matrix cross-linking. Indeed, Noxes are involved in local and systemic signaling from plants to fish and in cross-linking of the plant hair-cell wall, synthesis of the nematode cuticle, and formation of the sea urchin fertilization envelope. The extensive use of Nox enzymes in biology to regulate cell-to-cell signaling and morphogenesis suggests that additional functions in mammalian signaling and development remain to be discovered.

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.