Kei Miyano

The NADPH Oxidase Nox3 Constitutively Produces Superoxide in a p22phox-dependent Manner ITS REGULATION BY OXIDASE ORGANIZERS AND ACTIVATORS

Nox3, a member of the superoxide-producing NADPH oxidase (Nox) family, participates in otoconia formation in mouse inner ears, which is required for perception of balance and gravity. The activity of other Nox enzymes such as gp91phox/Nox2 and Nox1 is known to absolutely require both an organizer protein (p47phox or Noxo1) andanactivatorprotein (p67phox or Noxa1); for the p47phox-dependent activation of these oxidases, treatment of cells with stimulants such as phorbol 12-myristate 13-acetate is also indispensable. Here we show that ectopic expression of Nox3 in various types of cells leads to phorbol 12-myristate 13-acetate-independent constitutive production of a substantial amount of superoxide under the conditions where gp91phox and Nox1 fail to generate superoxide, i.e. in the absence of the oxidase organizers and activators. Nox3 likely forms a functional complex with p22phox; Nox3 physically interacts with and stabilizes p22phox, and the Nox3-dependent superoxide production is totally dependent on p22phox. The organizers p47phox and Noxo1 are capable of enhancing the superoxide production by Nox3 in the absence of the activators, and the enhancement requires the interaction of the organizers with p22phox, further indicating a link between Nox3 and p22phox. The p47phox-enhanced Nox3 activity is further facilitated by p67phox or Noxa1, whereas the activators cancel the Noxo1-induced enhancement. On the other hand, the small GTPase Rac, essential for the gp91phox activity, is likely dispensable to the Nox3 system. Thus Nox3 functions together with p22phox as an enzyme constitutively producing superoxide, which can be distinctly regulated by combinatorial use of the organizers and activators.

Direct Involvement of the Small GTPase Rac in Activation of the Superoxide-producing NADPH Oxidase Nox1

Activation of the non-phagocytic superoxide-producing NADPH oxidase Nox1, complexed with p22phox at the membrane, requires its regulatory soluble proteins Noxo1 and Noxa1. However, the role of the small GTPase Rac remained to be clarified. Here we show that Rac directly participates in Nox1 activation via interacting with Noxa1. Electropermeabilized HeLa cells, ectopically expressing Nox1, Noxo1, and Noxa1, produce superoxide in a GTP-dependent manner, which is abrogated by expression of a mutant Noxa1(R103E), defective in Rac binding. Superoxide production in Nox1-expressing HeLa and Caco-2 cells is decreased by depletion or sequestration of Rac; on the other hand, it is enhanced by expression of the constitutively active Rac1(Q61L), but not by that of a mutant Rac1 with the A27K substitution, deficient in binding to Noxa1. We also demonstrate that Nox1 activation requires membrane recruitment of Noxa1, which is normally mediated via Noxa1 binding to Noxo1, a protein tethered to the Nox1 partner p22phox: the Noxa1-Noxo1 and Noxo1-p22phox interactions are both essential for Nox1 activity. Rac likely facilitates the membrane localization of Noxa1: although Noxa1(W436R), defective in Noxo1 binding, neither associates with the membrane nor activates Nox1, the effects of the W436R substitution are restored by expression of Rac1(Q61L). The Rac-Noxa1 interaction also serves at a step different from the Noxa1 localization, because the binding-defective Noxa1(R103E), albeit targeted to the membrane, does not support superoxide production by Nox1. Furthermore, a mutant Noxa1 carrying the substitution of Ala for Val-205 in the activation domain, which is expected to undergo a conformational change upon Rac binding, fully localizes to the membrane but fails to activate Nox1.

Visualization of Phagosomal Hydrogen Peroxide Production by a Novel Fluorescent Probe That Is Localized via SNAP-tag Labeling

Hydrogen peroxide (H2O2), a member of reactive oxygen species (ROS), plays diverse physiological roles including host defense and cellular signal transduction. During ingestion of invading microorganisms, professional phagocytes such as macrophages release H2O2 specifically into the phagosome to direct toxic ROS toward engulfed microbes. Although H2O2 is considered to exert discrete effects in living systems depending on location of its production, accumulation, and consumption, there have been limitations of techniques for probing this oxygen metabolite with high molecular specificity at the subcellular resolution. Here we describe the development of an O(6)-benzylguanine derivative of 5-(4-nitrobenzoyl)carbonylfluorescein (NBzF-BG), a novel H2O2-specific fluorescent probe; NBzF-BG is covalently and selectively conjugated with the SNAP-tag protein, leading to formation of the fluorophore-protein conjugate (SNAP-NBzF). SNAP-NBzF rapidly reacts with H2O2 and thereby shows a 9-fold enhancement in fluorescence. When SNAP-tag is expressed in HEK293T cells and RAW264.7 macrophages as a protein C-terminally fused to the transmembrane domain of platelet-derived growth factor receptor (PDGFR), the tag is presented on the outside of the plasma membrane; conjugation of NBzF-BG with the cell surface SNAP-tag enables detection of H2O2 added exogenously. We also demonstrate molecular imaging of H2O2 that is endogenously produced in phagosomes of macrophages ingesting IgG-coated latex beads. Thus, NBzF-BG, combined with the SNAP-tag technology, should be useful as a tool to measure local production of H2O2 in living cells.

A region N-terminal to the tandem SH3 domain of p47phox plays a crucial role in the activation of the phagocyte NADPH oxidase.

The superoxide-producing NADPH oxidase in phagocytes is crucial for host defence; its catalytic core is the membrane-integrated protein gp91phox [also known as Nox2 (NADPH oxidase 2)], which forms a stable heterodimer with p22phox. Activation of the oxidase requires membrane translocation of the three cytosolic proteins p47phox, p67phox and the small GTPase Rac. At the membrane, these proteins assemble with the gp91phox-p22phox heterodimer and induce a conformational change of gp91phox, leading to superoxide production. p47phox translocates to membranes using its two tandemly arranged SH3 domains, which directly interact with p22phox, whereas p67phox is recruited in a p47phox-dependent manner. In the present study, we show that a short region N-terminal to the bis-SH3 domain is required for activation of the phagocyte NADPH oxidase. Alanine substitution for Ile152 in this region, a residue that is completely conserved during evolution, results in a loss of the ability to activate the oxidase; and the replacement of Thr153 also prevents oxidase activation, but to a lesser extent. In addition, the corresponding isoleucine residue (Ile155) of the p47phox homologue Noxo1 (Nox organizer 1) participates in the activation of non-phagocytic oxidases, such as Nox1 and Nox3. The I152A substitution in p47phox, however, does not affect its interaction with p22phox or with p67phox. Consistent with this, a mutant p47phox (I152A), as well as the wild-type protein, is targeted upon cell stimulation to membranes, and membrane recruitment of p67phox and Rac normally occurs in p47phox (I152A)-expressing cells. Thus the Ile152-containing region of p47phox plays a crucial role in oxidase activation, probably by functioning at a process after oxidase assembly.

The insert region of the Rac GTPases is dispensable for activation of superoxide-producing NADPH oxidases.

Rac1 and Rac2, which belong to the Rho subfamily of Ras-related GTPases, play an essential role in activation of gp91phox/Nox2 (cytochrome b-245, beta polypeptide; also known as Cybb), the catalytic core of the superoxide-producing NADPH oxidase in phagocytes. Rac1 also contributes to activation of the non-phagocytic oxidases Nox1 (NADPH oxidase 1) and Nox3 (NADPH oxidase 3), each related closely to gp91phox/Nox2. It has remained controversial whether the insert region of Rac (amino acids 123-135), unique to the Rho subfamily proteins, is involved in gp91phox/Nox2 activation. In the present study we show that removal of the insert region from Rac1 neither affects activation of gp91phox/Nox2, which is reconstituted under cell-free and whole-cell conditions, nor blocks its localization to phagosomes during ingestion of IgG-coated beads by macrophage-like RAW264.7 cells. The insert region of Rac2 is also dispensable for gp91phox/Nox2 activation at the cellular level. Although Rac2, as well as Rac1, is capable of enhancing superoxide production by Nox1 and Nox3, the enhancements by the two GTPases are both independent of the insert region. We also demonstrate that Rac3, a third member of the Rac family in mammals, has an ability to activate the three oxidases and that the activation does not require the insert region. Thus the insert region of the Rac GTPases does not participate in regulation of the Nox family NADPH oxidases.

TRPC3 positively regulates reactive oxygen species driving maladaptive cardiac remodeling

Reactive oxygen species (ROS) produced by NADPH oxidase 2 (Nox2) function as key mediators of mechanotransduction during both physiological adaptation to mechanical load and maladaptive remodeling of the heart. This is despite low levels of cardiac Nox2 expression. The mechanism underlying the transition from adaptation to maladaptation remains obscure, however. We demonstrate that transient receptor potential canonical 3 (TRPC3), a Ca2+-permeable channel, acts as a positive regulator of ROS (PRROS) in cardiomyocytes, and specifically regulates pressure overload-induced maladaptive cardiac remodeling in mice. TRPC3 physically interacts with Nox2 at specific C-terminal sites, thereby protecting Nox2 from proteasome-dependent degradation and amplifying Ca2+-dependent Nox2 activation through TRPC3-mediated background Ca2+ entry. Nox2 also stabilizes TRPC3 proteins to enhance TRPC3 channel activity. Expression of TRPC3 C-terminal polypeptide abolished TRPC3-regulated ROS production by disrupting TRPC3-Nox2 interaction, without affecting TRPC3-mediated Ca2+ influx. The novel TRPC3 function as a PRROS provides a mechanistic explanation for how diastolic Ca2+ influx specifically encodes signals to induce ROS-mediated maladaptive remodeling and offers new therapeutic possibilities.

DOCK2 and DOCK5 Act Additively in Neutrophils To Regulate Chemotaxis, Superoxide Production, and Extracellular Trap Formation

Neutrophils are highly motile leukocytes that play important roles in the innate immune response to invading pathogens. Neutrophils rapidly migrate to the site of infections and kill pathogens by producing reactive oxygen species (ROS). Neutrophil chemotaxis and ROS production require activation of Rac small GTPase. DOCK2, an atypical guanine nucleotide exchange factor (GEF), is one of the major regulators of Rac in neutrophils. However, because DOCK2 deficiency does not completely abolish fMLF-induced Rac activation, other Rac GEFs may also participate in this process. In this study, we show that DOCK5 acts with DOCK2 in neutrophils to regulate multiple cellular functions. We found that fMLF- and PMA-induced Rac activation were almost completely lost in mouse neutrophils lacking both DOCK2 and DOCK5. Although β2 integrin–mediated adhesion occurred normally even in the absence of DOCK2 and DOCK5, mouse neutrophils lacking DOCK2 and DOCK5 exhibited a severe defect in chemotaxis and ROS production. Similar results were obtained when human neutrophils were treated with CPYPP, a small-molecule inhibitor of these DOCK GEFs. Additionally, we found that DOCK2 and DOCK5 regulate formation of neutrophil extracellular traps (NETs). Because NETs are involved in vascular inflammation and autoimmune responses, DOCK2 and DOCK5 would be a therapeutic target for controlling NET-mediated inflammatory disorders.

Assessment of the role for Rho family GTPases in NADPH oxidase activation.

Rac, a member of the Rho family small GTPases, plays a crucial role in activation of Nox family NADPH oxidases in animals, enzymes dedicated to production of reactive oxygen species such as superoxide. The phagocyte oxidase Nox2, crucial for microbicidal activity during phagocytosis, is activated in a manner completely dependent on Rac. Rac in the GTP-bound form directly binds to the oxidase activator p67( phox ), which in turn interacts with Nox2, leading to superoxide production. Rac also participates in activation of the nonphagocytic oxidase Nox1; in this case, GTP-bound Rac functions by interacting with Noxa1, a p67( phox )-related protein that is required for Nox1 activation. On the other hand, in the presence of either p67( phox ) or Noxa1, Rac facilitates superoxide production by Nox3, which is responsible in the inner ear for formation of otoconia, tiny mineralized structures that are required for sensing balance and gravity. All the three mammalian homologs of Rac (Rac1, Rac2, and Rac3), but not Cdc42 or RhoA, are capable of serving as an activator of Nox1-3. Here, we describe methods for the assay of Rac binding to p67( phox ) and Noxa1 and for the reconstitution of Rac-dependent Nox activity in cell-free and whole-cell systems.

A Conserved Region between the TPR and Activation Domains of p67phox Participates in Activation of the Phagocyte NADPH Oxidase

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The membrane-integrated protein gp91phox serves as the catalytic core, because it contains a complete electron-transporting apparatus from NADPH to molecular oxygen for superoxide production. Activation of gp91phox requires the cytosolic proteins p67phox, p47phox, and Rac (a small GTPase). p67phox, comprising 526 amino acids, moves upon cell stimulation to the membrane together with p47phox and there interacts with Rac; these processes are prerequisite for gp91phox activation. Here we show that a region of p67phox (amino acids 190–200) C-terminal to the Rac-binding domain is evolutionarily well conserved and participates in oxidase activation at a later stage in conjunction with an activation domain. Alanine substitution for Tyr-198, Leu-199, or Val-204 abrogates the ability of p67phox to support superoxide production by gp91phox-based oxidase as well as its related oxidases Nox1 and Nox3; the activation also involves other invariant residues such as Leu-193, Asp-197, and Gly-200. Intriguingly, replacement of Gln-192 by alanine or that of Tyr-198 by phenylalanine or tryptophan rather enhances superoxide production by gp91phox-based oxidase, suggesting a tuning role for these residues. Furthermore, the Y198A/V204A or L199A/V204A substitution leads to not only a complete loss of the activity of the reconstituted oxidase system but also a significant decrease in p67phox interaction with the gp91phox NADPH-binding domain, although these mutations affect neither the protein integrity nor the Rac binding activity. Thus the extended activation domain of p67phox (amino acids 190–210) containing the D(Y/F)LGK motif plays an essential role in oxidase activation probably by interacting with gp91phox.

Role for the first SH3 domain of p67phox in activation of superoxide-producing NADPH oxidases

The membrane-bound NADPH oxidase in phagocytes, gp91(phox) (a.k.a. Nox2), produces superoxide, a precursor of microbicidal oxidants, thereby playing a crucial role in host defense. Activation of gp91(phox)/Nox2 requires assembly with the cytosolic proteins p67(phox) and p47(phox), each containing two SH3 domains. Although the C-terminal SH3 domain of p67(phox) is responsible for binding to p47(phox), little is known about the role for the first (N-terminal) SH3 domain [SH3(N)]. Here we show that truncation of p67(phox)-SH3(N), but not substitution of arginine for the invariant residue Trp-277 in SH3(N), results in an impaired activation of gp91(phox)/Nox2. The impairment is overcome by higher expression of an SH3(N)-defective p67(phox) in cells, suggesting that SH3(N) primarily increases the affinity of p67(phox) for the oxidase complex. On the other hand, p67(phox)-SH3(N) is not involved in activation of Nox1 and Nox3, closely-related homologues of gp91(phox)/Nox2. Thus p67(phox)-SH3(N) specifically functions in gp91(phox)/Nox2 activation probably via facilitating oxidase assembly.