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Spotlight
Priming and activation of NADPH oxidases in plants and animals Johnathan Canton1 and Sergio Grinstein1,2* 1
Program in Cell Biology, Hospital for Sick Children, 686 Bay Street, Toronto, Ontario, M5G 0A4 Keenan Research Centre of the Li Ka Shing Knowledge Institute, St. Michael’s Hospital, 290 Victoria Street, Toronto, Ontario, M5C 1N8 Canada
2
In mammals, engagement of Toll-like receptors by microbe-associated molecular patterns enhances the responsiveness of NADPH oxidases. Two recent papers report a similar ‘priming’ mechanism for the plant oxidase RbohD. Despite lacking structural homology, the functional parallels between plants and animals reveal that a common regulatory logic arose by convergent evolution. The nicotinamide adenine dinucleotide phosphate (NADPH) oxidases transfer electrons from a cytosolic electron donor, NADPH, to an electron acceptor, O2. The catalytic subunit of the oxidases spans the membrane and transfers electrons across the bilayer, generating a transmembrane voltage. In mammals, the NADPH oxidases have been classified into three subfamilies: the ancestral-type NOX enzymes (NOX1-4), the NOX5-like isoforms (NOX5) and the DUOX isoforms (DUOX1-2) [1]. They carry out a range of important functions, notably host defense, which is accomplished by generating potent microbicidal reactive oxygen species (ROS) such as superoxide and hydrogen peroxide. Much of what we know regarding ROS production in host defense has been gleaned from studies of the phagocyte oxidase, a multimeric complex consisting of several regulatory subunits (p22phox, p47phox, p40phox, p67phox, and the small GTPase Rac) that control the activity of NOX2, the transmembrane component that effects electron transfer (see Figure 1). The immuno-compromised status of chronic granulomatous disease patients, who have genetic defects in the phagocyte oxidase complex and suffer from recurrent bacterial and fungal infections, is testament to the central role of NOX2 in pathogen clearance. The immune function of NOX proteins is not limited to myeloid (phagocytic) cells. Several other members of the NOX family are expressed in epithelial cells. The apical surfaces of epithelial cells, such as those lining the colon, serve as a barrier and are in constant contact with potential pathogens; the epithelial NOX isoforms – which are
Corresponding author: Grinstein, S. ([email protected]). Keywords: NADPH oxidase; NOX; RbohD; reactive oxygen species; ROS; PAMP; TLR; pattern recognition receptor; priming. * Program in Cell Biology, The Hospital for Sick Children, 555 University Ave, Toronto, ON, M5G 1X8 Canada. Tel.: +(416)813-5727; Fax: +(416)813-5028 1471-4906/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.it.2014.07.007
expressed on the apical membrane – seemingly also play a role in immunity [2]. In mammalian systems, the expression level of NOX2, as well as the expression and phosphorylation status of its various regulatory subunits, can be positively regulated by immune signals. One such example is the ligation of the leucine-rich repeat receptor (LRR) TLR4 by lipopolysaccharide, a microbe-associated molecular pattern (MAMP). In phagocytes, TLR4 engagement causes increased expression of surface NOX2, promotes phosphorylation of p47phox, and facilitates translocation of p67phox, p47phox and Rac2 to the plasma membrane [3]. This combination of slow (transcriptional) and acute (signaling) responses, referred to collectively as ‘priming’, has the overall effect of lowering the threshold of activation by other stimuli and increasing the overall production of ROS by the oxidase. Importantly, the priming stimulus itself does not result in a substantial production of ROS; significant activation of the oxidase requires another, coincident signal provided by a second stimulus, such as formyl-methionyl-leucyl-phenylalanine (FMLP) in the case of neutrophils. As such, priming allows for a powerful burst of ROS in the presence of pathogenderived material, while limiting potentially harmful ROS release in the absence of such a threat. Although less is known for other members of the NOX family, similar priming has been documented to occur for NOX1, NOX4, DUOX1 and DUOX2 upon TLR ligation (Figure 1) [4]. Homologs of NOX family proteins also exist in plants where they are referred to as respiratory burst oxidase homologs (Rboh) and, as in mammals, play a central role in host defense. The plant oxidases all share flavin adenine dinucleotide and NADPH binding sites, six membranespanning domains and calcium-binding EF-hand motifs. This organization most closely resembles the mammalian NOX5 subfamily; no ancestral-type or DUOX isoforms have been found in plants. In contrast to many of the mammalian NOX proteins, regulation of the Rboh proteins is primarily effected through post-translational modifications, such as direct phosphorylation of the cytosolic Nterminus and conformational changes induced by Ca2+ binding to the EF-hand motifs. Similarities do exist, however, between the activation mechanisms of both plant and mammalian oxidases. Although plant cells do not express TLRs, they do have LRR-containing receptors that sense MAMPs to trigger antimicrobial defenses. One such LRRcontaining receptor is the flagellin-sensing 2 (FLS2) receptor, that upon ligation interacts with the receptor-like kinase BAK1 (brassinosteroid insensitive 1-associated Trends in Immunology xx (2014) 1–3
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LRR-containing receptor
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Trends in Immunology xxx xxxx, Vol. xxx, No. x
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TRENDS in Immunology
Figure 1. Priming of the NOX family of NADPH oxidases. The known transmembrane and cytosolic regulators, as well as the priming events downstream of LRR receptors are shown for the mammalian NADPH oxidases (NOX1-5 and DUOX1-2) and for the plant RbohD. Upon TLR4 ligation, receptor-mediated signaling events (unbroken lines) result in the phosphorylation and translocation of various cytosolic regulatory subunits to the plasma membrane, facilitating rapid activation of the respective oxidase upon further signaling. Moreover, TLR signaling affects the expression level (broken lines) of NOX and/or its regulatory subunits. For NOX1, TLR4 ligation results in: (i) the phosphatidylinositol 3-kinase (PI3K)-dependent conversion of the regulatory subunit Rac from its GDP to the active, GTP-bound form; (ii) the increased expression of the regulatory subunit NOX organizer 1 (NOXO1); (iii) Ca2+-independent phospholipase A2b (iPLA2b)-dependent increase in NOX1 expression, and (iv) the glycogen synthase kinase 3b (GSK3b)-dependent accumulation of b-catenin that also increases NOX1 expression. For NOX2, TLR4 ligation results in: (i) the Vav-dependent conversion of RacGDP to Rac-GTP that, in turn, recruits p67phox; (ii) the p38- and interleukin 1 receptor-associated kinase 4 (IRAK-4)-dependent phosphorylation of the regulatory subunit p47phox, and (iii) the increased expression of NOX2. For NOX3, TLR4-mediated signaling events result in decreased expression of NOX3. For NOX4, TLR4 regulates NOX4 activity via a direct interaction between TLR4 and NOX4 and has been shown to impact upon NOX4 expression levels. For NOX5, elevation of the intracellular Ca2+ concentration activates calcium-dependent kinases such as protein kinase Ca (PKCa) and Ca2+/calmodulin-dependent protein kinase II (CAMKII) that directly phosphorylate and therefore activate NOX5. In addition, elevated intracellular Ca2+ also binds to EF-hand motifs at the N-terminus of NOX5 and to calmodulin, which in turn binds to a calmodulin-binding domain on the C-terminus resulting in conformational changes that effect NOX5 activation. For DUOX1, TLR4 ligation has been shown to result in the PKCa/b-dependent activation, as well as increased surface expression of DUOX2. The DUOX isoforms both have EF-hand motifs that bind Ca2+ to trigger DUOX activity. Upon ligation, the FLS2-BAK1 receptor complex phosphorylates BIK1, which then directly phosphorylates RbohD. Moreover, FLS2 ligation also results in an increase in the cytosolic Ca2+ concentration, resulting in the activation of Ca2+-dependent protein kinases, such as CPK5, that can directly phosphorylate and activate RbohD. Similar to NOX5 and DUOX1/2, RbohD has EF-hand motifs that render it responsive to cytosolic Ca2+ transients.
kinase 1) and the cytosolic kinase Botrytis-induced kinase 1 (BIK1), to bring about a variety of microbicidal responses including the activation of the oxidase RbohD. Until recently, FLS2-BAK1-BIK1 was thought to activate RbohD solely by eliciting an influx of extracellular Ca2+. The resulting elevation of cytosolic [Ca2+] was envisaged to have a dual effect: (i) it alters the conformation of the oxidase by directly binding to its EF-hand motifs and (ii) it activates Ca2+-dependent protein kinases that in turn phosphorylate the N-terminus of RbohD [5]. Two new papers have now demonstrated the existence of additional, Ca2+-independent means of activation of RbohD by pattern-recognition receptors. Specifically, Li et al. [6] and Kadota et al. [7] recently reported that, when activated by FLS2, RbohD can be directly phosphorylated by BIK1. Upon FLS2 ligation, RbohD – which is in a complex with FLS2 prior to sensing flagellin – is phosphorylated by BIK1 at various sites, including serine residues 39 and 343. Of note, phosphorylation at these sites is Ca2+independent and their mutation to non-phosphorylatable residues abolished flagellin-induced ROS production. Interestingly, the converse (phosphomimetic) mutations were not sufficient to trigger ROS production in the absence of FLS2 ligation. These observations imply that the Ca2+-independent BIK1-mediated phosphorylation of RbohD is required, but not sufficient to trigger its activity. Kadota et al. proposed a two-step mechanism in which the 2
rapid phosphorylation of RbohD by BIK1 serves to prime the oxidase by increasing its sensitivity to the coincident Ca2+-dependent stimulation. This could result from conformational changes that increase the affinity of the EFhand motifs for Ca2+ and/or by increasing the ability of Ca2+-dependent protein kinases to phosphorylate RbohD [7]. Thus, a common logic emerges for the acute response to MAMPs for both plants and mammals in which (i) a MAMP is sensed by an LRR-containing receptor, (ii) a series of phosphorylation and/or subcellular translocation events result in a primed NADPH oxidase and (iii) a coincident triggering stimulus, such as a cytosolic Ca2+ transient, triggers a burst of ROS production that effects pathogen clearance. This is more likely to be a case of convergent evolution than of a signaling cassette inherited from a common ancestor, as the various signaling and regulatory components of plants and animals have no structural homology [8]. These intriguing similarities raise the possibility that knowledge gained from studies of the plant oxidases may be extended to mammalian systems and vice versa. Because it was identified most recently, remarkably little is known about the function and regulation of NOX5, the closest homolog of the Rboh proteins. Investigators studying this mammalian isoform stand to benefit the most from the lessons learned in plants. Indeed, the presence of EF-hand motifs and Ca2+-dependent phosphorylation sites
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Spotlight in NOX5 suggests that additional similarities with Rboh may exist [9]. An aspect that appears to have been neglected in describing the plant oxidases is whether, like their animal counterparts, they are electrogenic, i.e. separate negative and positive charges in the process of transferring electrons from NADPH to O2. This has been abundantly documented for mammalian oxidases, specifically NOX2. The electrical potential can limit the catalytic activity of the oxidase and must be dissipated for continued, optimal activity. In the prototypical case of phagocytes, this is accomplished by the parallel activation of voltage-gated H+ channels, known as HV1 (reviewed in [10]). No comparable voltage-gated H+ channels have been identified in plants. It is noteworthy, however, that in the absence of charge compensation, the large depolarization generated by an electrogenic oxidase would depress the influx of Ca2+, which seems to be essential for the activation of Rboh. Whether such depolarization in fact occurs and serves as a regulatory (negative) feedback mechanism remains to be established. Alternatively, unidentified charge compensatory mechanisms may exist, which could support the continued entry of Ca2+. Future studies should resolve these uncertainties. In closing, it is worth emphasizing that, despite their structural divergence, the plant and animal oxidases show surprising functional commonalities that should be
Trends in Immunology xxx xxxx, Vol. xxx, No. x
exploited in an effort to better understand the role and regulation of these fascinating enzymes. References 1 Bedard, K. et al. (2007) NOX family NADPH oxidases: not just in mammals. Biochimie 89, 1107–1112 2 Leto, T.L. and Geiszt, M. (2006) Role of Nox family NADPH oxidases in host defense. Antioxid. Redox Signal. 8, 1549–1561 3 DeLeo, F.R. et al. (1998) Neutrophils exposed to bacterial lipopolysaccharide upregulate NADPH oxidase assembly. J. Clin. Invest. 101, 455–463 4 Ogier-Denis, E. et al. (2008) NOX enzymes and Toll-like receptor signaling. Semin. Immunopathol. 30, 291–300 5 Kobayashi, M. et al. (2007) Calcium-dependent protein kinases regulate the production of reactive oxygen species by potato NADPH oxidase. Plant Cell 19, 1065–1080 6 Li, L. et al. (2014) The FLS2-associated kinase BIK1 directly phosphorylates the NADPH oxidase RbohD to control plant immunity. Cell Host Microbe 15, 329–338 7 Kadota, Y. et al. (2014) Direct regulation of the NADPH oxidase RBOHD by the PRR-associated kinase BIK1 during plant immunity. Mol. Cell 54, 43–55 8 Ausubel, F.M. (2005) Are innate immune signaling pathways in plants and animals conserved? Nat. Immunol. 6, 973–979 9 Jagnandan, D. et al. (2007) Novel mechanism of activation of NADPH oxidase 5. calcium sensitization via phosphorylation. J. Biol. Chem. 282, 6494–6507 10 DeCoursey, T.E. (2013) Voltage-gated proton channels: molecular biology, physiology, and pathophysiology of the H(V) family. Physiol. Rev. 93, 599–652
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Spotlight
Priming and activation of NADPH oxidases in plants and animals Johnathan Canton1 and Sergio Grinstein1,2* 1
Program in Cell Biology, Hospital for Sick Children, 686 Bay Street, Toronto, Ontario, M5G 0A4 Keenan Research Centre of the Li Ka Shing Knowledge Institute, St. Michael’s Hospital, 290 Victoria Street, Toronto, Ontario, M5C 1N8 Canada
2
In mammals, engagement of Toll-like receptors by microbe-associated molecular patterns enhances the responsiveness of NADPH oxidases. Two recent papers report a similar ‘priming’ mechanism for the plant oxidase RbohD. Despite lacking structural homology, the functional parallels between plants and animals reveal that a common regulatory logic arose by convergent evolution. The nicotinamide adenine dinucleotide phosphate (NADPH) oxidases transfer electrons from a cytosolic electron donor, NADPH, to an electron acceptor, O2. The catalytic subunit of the oxidases spans the membrane and transfers electrons across the bilayer, generating a transmembrane voltage. In mammals, the NADPH oxidases have been classified into three subfamilies: the ancestral-type NOX enzymes (NOX1-4), the NOX5-like isoforms (NOX5) and the DUOX isoforms (DUOX1-2) [1]. They carry out a range of important functions, notably host defense, which is accomplished by generating potent microbicidal reactive oxygen species (ROS) such as superoxide and hydrogen peroxide. Much of what we know regarding ROS production in host defense has been gleaned from studies of the phagocyte oxidase, a multimeric complex consisting of several regulatory subunits (p22phox, p47phox, p40phox, p67phox, and the small GTPase Rac) that control the activity of NOX2, the transmembrane component that effects electron transfer (see Figure 1). The immuno-compromised status of chronic granulomatous disease patients, who have genetic defects in the phagocyte oxidase complex and suffer from recurrent bacterial and fungal infections, is testament to the central role of NOX2 in pathogen clearance. The immune function of NOX proteins is not limited to myeloid (phagocytic) cells. Several other members of the NOX family are expressed in epithelial cells. The apical surfaces of epithelial cells, such as those lining the colon, serve as a barrier and are in constant contact with potential pathogens; the epithelial NOX isoforms – which are
Corresponding author: Grinstein, S. ([email protected]). Keywords: NADPH oxidase; NOX; RbohD; reactive oxygen species; ROS; PAMP; TLR; pattern recognition receptor; priming. * Program in Cell Biology, The Hospital for Sick Children, 555 University Ave, Toronto, ON, M5G 1X8 Canada. Tel.: +(416)813-5727; Fax: +(416)813-5028 1471-4906/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.it.2014.07.007
expressed on the apical membrane – seemingly also play a role in immunity [2]. In mammalian systems, the expression level of NOX2, as well as the expression and phosphorylation status of its various regulatory subunits, can be positively regulated by immune signals. One such example is the ligation of the leucine-rich repeat receptor (LRR) TLR4 by lipopolysaccharide, a microbe-associated molecular pattern (MAMP). In phagocytes, TLR4 engagement causes increased expression of surface NOX2, promotes phosphorylation of p47phox, and facilitates translocation of p67phox, p47phox and Rac2 to the plasma membrane [3]. This combination of slow (transcriptional) and acute (signaling) responses, referred to collectively as ‘priming’, has the overall effect of lowering the threshold of activation by other stimuli and increasing the overall production of ROS by the oxidase. Importantly, the priming stimulus itself does not result in a substantial production of ROS; significant activation of the oxidase requires another, coincident signal provided by a second stimulus, such as formyl-methionyl-leucyl-phenylalanine (FMLP) in the case of neutrophils. As such, priming allows for a powerful burst of ROS in the presence of pathogenderived material, while limiting potentially harmful ROS release in the absence of such a threat. Although less is known for other members of the NOX family, similar priming has been documented to occur for NOX1, NOX4, DUOX1 and DUOX2 upon TLR ligation (Figure 1) [4]. Homologs of NOX family proteins also exist in plants where they are referred to as respiratory burst oxidase homologs (Rboh) and, as in mammals, play a central role in host defense. The plant oxidases all share flavin adenine dinucleotide and NADPH binding sites, six membranespanning domains and calcium-binding EF-hand motifs. This organization most closely resembles the mammalian NOX5 subfamily; no ancestral-type or DUOX isoforms have been found in plants. In contrast to many of the mammalian NOX proteins, regulation of the Rboh proteins is primarily effected through post-translational modifications, such as direct phosphorylation of the cytosolic Nterminus and conformational changes induced by Ca2+ binding to the EF-hand motifs. Similarities do exist, however, between the activation mechanisms of both plant and mammalian oxidases. Although plant cells do not express TLRs, they do have LRR-containing receptors that sense MAMPs to trigger antimicrobial defenses. One such LRRcontaining receptor is the flagellin-sensing 2 (FLS2) receptor, that upon ligation interacts with the receptor-like kinase BAK1 (brassinosteroid insensitive 1-associated Trends in Immunology xx (2014) 1–3
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LRR-containing receptor
NOX proteins Receptor-mediated Cytosolic and transmembrane signaling regulatory subunits regulatory subunits
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Trends in Immunology xxx xxxx, Vol. xxx, No. x
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TRENDS in Immunology
Figure 1. Priming of the NOX family of NADPH oxidases. The known transmembrane and cytosolic regulators, as well as the priming events downstream of LRR receptors are shown for the mammalian NADPH oxidases (NOX1-5 and DUOX1-2) and for the plant RbohD. Upon TLR4 ligation, receptor-mediated signaling events (unbroken lines) result in the phosphorylation and translocation of various cytosolic regulatory subunits to the plasma membrane, facilitating rapid activation of the respective oxidase upon further signaling. Moreover, TLR signaling affects the expression level (broken lines) of NOX and/or its regulatory subunits. For NOX1, TLR4 ligation results in: (i) the phosphatidylinositol 3-kinase (PI3K)-dependent conversion of the regulatory subunit Rac from its GDP to the active, GTP-bound form; (ii) the increased expression of the regulatory subunit NOX organizer 1 (NOXO1); (iii) Ca2+-independent phospholipase A2b (iPLA2b)-dependent increase in NOX1 expression, and (iv) the glycogen synthase kinase 3b (GSK3b)-dependent accumulation of b-catenin that also increases NOX1 expression. For NOX2, TLR4 ligation results in: (i) the Vav-dependent conversion of RacGDP to Rac-GTP that, in turn, recruits p67phox; (ii) the p38- and interleukin 1 receptor-associated kinase 4 (IRAK-4)-dependent phosphorylation of the regulatory subunit p47phox, and (iii) the increased expression of NOX2. For NOX3, TLR4-mediated signaling events result in decreased expression of NOX3. For NOX4, TLR4 regulates NOX4 activity via a direct interaction between TLR4 and NOX4 and has been shown to impact upon NOX4 expression levels. For NOX5, elevation of the intracellular Ca2+ concentration activates calcium-dependent kinases such as protein kinase Ca (PKCa) and Ca2+/calmodulin-dependent protein kinase II (CAMKII) that directly phosphorylate and therefore activate NOX5. In addition, elevated intracellular Ca2+ also binds to EF-hand motifs at the N-terminus of NOX5 and to calmodulin, which in turn binds to a calmodulin-binding domain on the C-terminus resulting in conformational changes that effect NOX5 activation. For DUOX1, TLR4 ligation has been shown to result in the PKCa/b-dependent activation, as well as increased surface expression of DUOX2. The DUOX isoforms both have EF-hand motifs that bind Ca2+ to trigger DUOX activity. Upon ligation, the FLS2-BAK1 receptor complex phosphorylates BIK1, which then directly phosphorylates RbohD. Moreover, FLS2 ligation also results in an increase in the cytosolic Ca2+ concentration, resulting in the activation of Ca2+-dependent protein kinases, such as CPK5, that can directly phosphorylate and activate RbohD. Similar to NOX5 and DUOX1/2, RbohD has EF-hand motifs that render it responsive to cytosolic Ca2+ transients.
kinase 1) and the cytosolic kinase Botrytis-induced kinase 1 (BIK1), to bring about a variety of microbicidal responses including the activation of the oxidase RbohD. Until recently, FLS2-BAK1-BIK1 was thought to activate RbohD solely by eliciting an influx of extracellular Ca2+. The resulting elevation of cytosolic [Ca2+] was envisaged to have a dual effect: (i) it alters the conformation of the oxidase by directly binding to its EF-hand motifs and (ii) it activates Ca2+-dependent protein kinases that in turn phosphorylate the N-terminus of RbohD [5]. Two new papers have now demonstrated the existence of additional, Ca2+-independent means of activation of RbohD by pattern-recognition receptors. Specifically, Li et al. [6] and Kadota et al. [7] recently reported that, when activated by FLS2, RbohD can be directly phosphorylated by BIK1. Upon FLS2 ligation, RbohD – which is in a complex with FLS2 prior to sensing flagellin – is phosphorylated by BIK1 at various sites, including serine residues 39 and 343. Of note, phosphorylation at these sites is Ca2+independent and their mutation to non-phosphorylatable residues abolished flagellin-induced ROS production. Interestingly, the converse (phosphomimetic) mutations were not sufficient to trigger ROS production in the absence of FLS2 ligation. These observations imply that the Ca2+-independent BIK1-mediated phosphorylation of RbohD is required, but not sufficient to trigger its activity. Kadota et al. proposed a two-step mechanism in which the 2
rapid phosphorylation of RbohD by BIK1 serves to prime the oxidase by increasing its sensitivity to the coincident Ca2+-dependent stimulation. This could result from conformational changes that increase the affinity of the EFhand motifs for Ca2+ and/or by increasing the ability of Ca2+-dependent protein kinases to phosphorylate RbohD [7]. Thus, a common logic emerges for the acute response to MAMPs for both plants and mammals in which (i) a MAMP is sensed by an LRR-containing receptor, (ii) a series of phosphorylation and/or subcellular translocation events result in a primed NADPH oxidase and (iii) a coincident triggering stimulus, such as a cytosolic Ca2+ transient, triggers a burst of ROS production that effects pathogen clearance. This is more likely to be a case of convergent evolution than of a signaling cassette inherited from a common ancestor, as the various signaling and regulatory components of plants and animals have no structural homology [8]. These intriguing similarities raise the possibility that knowledge gained from studies of the plant oxidases may be extended to mammalian systems and vice versa. Because it was identified most recently, remarkably little is known about the function and regulation of NOX5, the closest homolog of the Rboh proteins. Investigators studying this mammalian isoform stand to benefit the most from the lessons learned in plants. Indeed, the presence of EF-hand motifs and Ca2+-dependent phosphorylation sites
TREIMM-1114; No. of Pages 3
Spotlight in NOX5 suggests that additional similarities with Rboh may exist [9]. An aspect that appears to have been neglected in describing the plant oxidases is whether, like their animal counterparts, they are electrogenic, i.e. separate negative and positive charges in the process of transferring electrons from NADPH to O2. This has been abundantly documented for mammalian oxidases, specifically NOX2. The electrical potential can limit the catalytic activity of the oxidase and must be dissipated for continued, optimal activity. In the prototypical case of phagocytes, this is accomplished by the parallel activation of voltage-gated H+ channels, known as HV1 (reviewed in [10]). No comparable voltage-gated H+ channels have been identified in plants. It is noteworthy, however, that in the absence of charge compensation, the large depolarization generated by an electrogenic oxidase would depress the influx of Ca2+, which seems to be essential for the activation of Rboh. Whether such depolarization in fact occurs and serves as a regulatory (negative) feedback mechanism remains to be established. Alternatively, unidentified charge compensatory mechanisms may exist, which could support the continued entry of Ca2+. Future studies should resolve these uncertainties. In closing, it is worth emphasizing that, despite their structural divergence, the plant and animal oxidases show surprising functional commonalities that should be
Trends in Immunology xxx xxxx, Vol. xxx, No. x
exploited in an effort to better understand the role and regulation of these fascinating enzymes. References 1 Bedard, K. et al. (2007) NOX family NADPH oxidases: not just in mammals. Biochimie 89, 1107–1112 2 Leto, T.L. and Geiszt, M. (2006) Role of Nox family NADPH oxidases in host defense. Antioxid. Redox Signal. 8, 1549–1561 3 DeLeo, F.R. et al. (1998) Neutrophils exposed to bacterial lipopolysaccharide upregulate NADPH oxidase assembly. J. Clin. Invest. 101, 455–463 4 Ogier-Denis, E. et al. (2008) NOX enzymes and Toll-like receptor signaling. Semin. Immunopathol. 30, 291–300 5 Kobayashi, M. et al. (2007) Calcium-dependent protein kinases regulate the production of reactive oxygen species by potato NADPH oxidase. Plant Cell 19, 1065–1080 6 Li, L. et al. (2014) The FLS2-associated kinase BIK1 directly phosphorylates the NADPH oxidase RbohD to control plant immunity. Cell Host Microbe 15, 329–338 7 Kadota, Y. et al. (2014) Direct regulation of the NADPH oxidase RBOHD by the PRR-associated kinase BIK1 during plant immunity. Mol. Cell 54, 43–55 8 Ausubel, F.M. (2005) Are innate immune signaling pathways in plants and animals conserved? Nat. Immunol. 6, 973–979 9 Jagnandan, D. et al. (2007) Novel mechanism of activation of NADPH oxidase 5. calcium sensitization via phosphorylation. J. Biol. Chem. 282, 6494–6507 10 DeCoursey, T.E. (2013) Voltage-gated proton channels: molecular biology, physiology, and pathophysiology of the H(V) family. Physiol. Rev. 93, 599–652
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