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Highlights: [•] Human Nox1 undergoes glycosylation at Asn-162 and Asn-236. [•] Nox1 can be transported to the cell surface as no or high mannose N-glycan form. [•] Nox1 N-glycosylation is dispensable for its catalytic function. [•] p22 phox allows maturation of Nox1 N-glycans into more complex oligosaccharides. [•] p22 phox directly contributes to Nox1 activation independently of Nox1 glycosylation. [ABSTRACT FROM AUTHOR]

NADPH oxidases of the Nox family exist in various supergroups of eukaryotes but not in prokaryotes, and play crucial roles in a variety of biological processes, such as host defense, signal transduction, and hormone synthesis. In conjunction with NADPH oxidation, Nox enzymes reduce molecular oxygen to superoxide as a primary product, and this is further converted to various reactive oxygen species. The electron-transferring system in Nox is composed of the C-terminal cytoplasmic region homologous to the prokaryotic (and organelle) enzyme ferredoxin reductase and the N-terminal six transmembrane segments containing two hemes, a structure similar to that of cytochrome b of the mitochondrial bc1 complex. During the course of eukaryote evolution, Nox enzymes have developed regulatory mechanisms, depending on their functions, by inserting a regulatory domain (or motif) into their own sequences or by obtaining a tightly associated protein as a regulatory subunit. For example, one to four Ca2+-binding EF-hand motifs are present at the N-termini in several subfamilies, such as the respiratory burst oxidase homolog (Rboh) subfamily in land plants (the supergroup Plantae), the NoxC subfamily in social amoebae (the Amoebozoa), and the Nox5 and dual oxidase (Duox) subfamilies in animals (the Opisthokonta), whereas an SH3 domain is inserted into the ferredoxin–NADP+ reductase region of two Nox enzymes in Naegleria gruberi, a unicellular organism that belongs to the supergroup Excavata. Members of the Nox1–4 subfamily in animals form a stable heterodimer with the membrane protein p22 phox, which functions as a docking site for the SH3 domain-containing regulatory proteins p47 phox, p67 phox, and p40 phox; the small GTPase Rac binds to p67 phox (or its homologous protein), which serves as a switch for Nox activation. Similarly, Rac activates the fungal NoxA via binding to the p67 phox-like protein Nox regulator (NoxR). In plants, on the other hand, this GTPase directly interacts with the N-terminus of Rboh, leading to superoxide production. Here I describe the regulation of Nox-family oxidases on the basis of three-dimensional structures and evolutionary conservation. [ABSTRACT FROM AUTHOR]

Abstract: The superoxide-producing phagocyte NADPH oxidase gp91 phox /Nox2 and the non-phagocytic oxidases Nox1 and Nox3 each form a complex in the membrane with p22 phox , which provides both stabilization and a docking site for organizer proteins. The p22 phox -complexed Nox2 and Nox1 are dormant on their own, and their activation requires soluble supportive proteins such as a Nox organizer (p47 phox or Noxo1) and a Nox activator (p67 phox or Noxa1). The small GTPase Rac directly binds to the activators, and thus plays an essential role in the Nox2-based oxidase containing p47 phox and p67 phox or a positive role in Nox1 activity supported by Noxo1 and Noxa1. Although Nox3 complexed with p22 phox constitutively produce superoxide, the production can be enhanced by supportive proteins. Here we compare the roles of Rac in these p22 phox -dependent oxidases using the organizer and activator in different combinations. Expression of constitutively active Rac1(Q61L) is essential for activation of the Nox2- or Nox1-based oxidase containing the organizer p47 phox and either p67 phox or Noxa1. When these oxidases use Noxo1 as an organizer instead of p47 phox , they produce a small but significant amount of superoxide without expression of Rac1(Q61L), although the production is enhanced by Rac1(Q61L). Thus p47 phox is likely related to strict dependence on Rac. The Nox3-based oxidase has a similar tendency in the change of the dependence: Rac plays a positive role in Nox3 activation in the presence of p47 phox and either p67 phox or Noxa1, whereas Rac fails to upregulate Nox3 activity when p47 phox is replaced with Noxo1. We also demonstrate that, in the Nox3-based oxidase containing solely p67 phox as supportive protein, expression of Rac1(Q61L) enhances not only superoxide production but also membrane translocation of p67 phox . Since the enhancements are not observed with a mutant p67 phox defective in binding to Rac, this GTPase appear to directly recruit p67 phox to the membrane. [Copyright &y& Elsevier]

Abstract: Reactive oxygen species (ROS) are conventionally regarded as inevitable deleterious by-products in aerobic metabolism with a few exceptions such as their significant role in host defense. The phagocyte NADPH oxidase, dormant in resting cells, becomes activated during phagocytosis to deliberately produce superoxide, a precursor of other microbicidal ROS, thereby playing a crucial role in killing pathogens. The catalytic center of this oxidase is the membrane-integrated protein gp91 phox , tightly complexed with p22 phox , and its activation requires the association with p47 phox , p67 phox , and the small GTPase Rac, which normally reside in the cytoplasm. Since recent discovery of non-phagocytic gp91 phox -related enzymes of the NAD(P)H oxidase (Nox) family—seven homologues identified in humans—deliberate ROS production has been increasingly recognized as important components of various cellular events. Here, we describe a current view on the molecular composition and post-translational regulation of Nox-family oxidases in animals. [Copyright &y& Elsevier]

Cytochrome P-450 CYP4F3A catalyzes the inactivation of leukotriene B4 by ω-hydroxylation, an activity of which is specifically expressed in human neutrophils. Here, we examined expression of the LTB4 ω-hydroxylating activity during the differentiation of HL60 cells, an acute promyelocytic leukemia cell line, in the presence of various inducers. Among the inducers used, all-trans-retinoic acid (ATRA) most strongly induces the LTB4 ω-hydroxylating activity in a dose-dependent manner. The time course of the induction of the ω-hydroxylating activity correlates well with that of the superoxide-generating activity, indicative of cell differentiation. ATRA-treated cell microsomes convert LTB4 to its 20-hydroxyl derivative under aerobic conditions in the present of NADPH. The reaction is inhibited by carbon monoxide, an inhibitor of cytochrome P-450, and by antibodies raised against NADPH-P-450 reductase. CYP4F3A appears to be responsible for the LTB4 ω-hydroxylase activity, based on the following observations: expression of the mRNA for CYP4F3A is observed together with the induction of LTB4 ω-hydroxylating activity in ATRA-treated HL60 cells; and the apparent Km values for the ω-hydroxylation of LTB4 and lipoxin B4 by ATRA-treated cell microsomes are essentially the same as those of CYP4F3A in human neutrophil microsomes. [Copyright &y& Elsevier]

The membrane-integrated protein gp91phox, existing as a heterodimer with p22phox functions as the catalytic core of the phagocyte NADPH oxidase, which plays a crucial role in host defence. The oxidase, dormant in resting cells, becomes activated to produce superoxide, a precursor of microbicidal oxidants. by interacting with the adaptor proteins p47phox and p67phox as well as the small GTPase Rac. In the past few years, several proteins homologous to gp91phox were discovered as superoxide-producing NAD(P)H oxidases (Nox's) in non-phagocytic cells; however, regulatory mechanisms for the novel oxidases have been largely unknown. Current identification of proteins highly related to p47phox and p67phox, designated Noxol (Nox organizer 1) and Noxa1 (Nox activator 1), respectively, has shed lights on common and distinct mechanisms underlying activations of Nox family oxidases. [ABSTRACT FROM AUTHOR]

Formins constitute a family of eukaryotic proteins that are considered to function as a cytoskeleton organizer to regulate morphogenesis, cell polarity and cytokinesis. Fhos is a recently identified mammalian formin, which contains the conserved domains FH (formin homology) 1 and FH2 in the middle region and the Dia-autoregulatory domain (DAD) in the C-terminus. The role of Fhos in the regulation of cytoskeleton, however, has remained unknown. Here we show that Fhos, in an active form, induces the formation of actin stress fibers and localizes to the actin-based structure. Fhos appears to normally exist in a closed inactive form via an intramolecular interaction between the N-terminal region and the C-terminal DAD. Both FH1 and FH2 domains are required for the induction of the stress fiber formation. However, the N-terminal region of Fhos is required for the targeting of this protein to stress fibers, which is probably mediated via its F-actin-binding activity. We also show that Fhos occurs as a homotypic complex in cells. The self-association of Fhos seems to be mediated via the FH2 domain: the domains bind to each other in a direct manner. Thus, the mammalian formin Fhos, which directly binds to F-actin via the N-terminal region, forms a homotypic complex via the FH2 domain to organize actin cytoskeleton. [ABSTRACT FROM AUTHOR]

The superoxide-generating NADPH oxidase, dormant in resting phagocytes, is activated during phagocytosis following assembly of the membrane-integrated protein cytochrome b558 and cytosolic factors. Among the latter are the three proteins containing Src homology 3 (SH3) domains, p67phox, p47phox and p40 phox. While the first two factors are indispensable for the activity, p40 phox is tightly associated with p67phox in resting cells and is suggested to have some modulatory role. Here we describe a systematic analysis of the interaction between p40 phox and p67phox using the yeast two-hybrid system and in vitro binding assays with recombinant proteins. Both methods unequivocally showed that the minimum requirements for stable interaction are the C-terminal region of p40 phox and the region between the two SH3 domains of p67phox. This interaction is maintained even in the presence of anionic amphiphiles used for the activation of the NADPH oxidase, raising a possibility that it mediates constitutive association of the two factors in both resting and activated cells. The C-terminal region of p40 phox responsible for the interaction contains a characteristic stretch of amino acids designated as the PC motif, that also exists in other signal-transducing proteins from yeast to human. Intensive site-directed mutagenesis to the motif in p40 phox revealed that it plays a critical role in the binding to p67 phox. Thus the PC motif appears to represent a novel module for protein-protein interaction used in a variety of signaling pathways. [ABSTRACT FROM AUTHOR]

Lipoxin B4 (LXB4) is metabolized to 20-hydroxy-LXB4 by rat liver microsomes. The ω-hydroxylation requires both molecular oxygen and NADPH, and is inhibited by carbon monoxide, indicating involvement of a cytochrome P-450 (P-450). This is supported by inhibition of the reaction by antibodies raised against NADPH—P-450 reductase. The P-450 appears to be the one responsible for leukotriene B4 ω-hydroxylation, because leukotriene B4 inhibits the formation of 20-hydroxy-LXB4 and LXB4 blocks the leukotriene B4 ω-hydroxylase activity in microsomes. Incubation of 20-hydroxy-LXB4 with both rat liver cytosol and NAD leads to formation of a more polar metabolite on high-performance liquid chromatography. The metabolite is identified as 20-carboxy-LXB4 a novel metabolite of LXB4 based on analyses by ultraviolet spectrometry and by gas chromatography/mass spectrometry. The 20-carboxy-LXB4 forming activity is localized in cytosol, with an optimal pH of 8.5. The activity is dependent on NAD+, but NADP- can not replace NAD-. The reaction is inhibited by pyrazole and 4-methylpyrazole, inhibitors of alcohol dehydrogenase, and by substrates of the enzyme such as ethanol and 20-hydroxy-leukotriene B4. Disulfiram, an inhibitor of aldehyde dehydrogenase, also blocks the 20-carboxy-LXB4 formation. These observations suggest that both alcohol dehydrogenase and aldehyde dehydrogenase participate in the oxidation of 20-hydroxy-LXB4 to 20-carboxy-LXB4. [ABSTRACT FROM AUTHOR]

Leukotriene B4 (LTB4) is converted to 20-hydroxy-LTB4 (20-OH-LTB4) which is subsequently oxidized to 20-carboxy-LTB4 (20-COOH-LTB4). The oxidation of the hydroxy LTB4 to the carboxy LTB4 by human neutrophils was associated with the reduction of NAD + and required both cytosolic and microsomal fractions. We isolated a cytosolic protein which oxidized the hydroxy LTB4 in the presence of NAD + and the microsomal fraction. It was homogeneous on SDS/PAGE, with a subunit molecular mass of 37 kDa, and may be a dimeric protein with two identical or similar subunits because its molecular mass, estimated by Sephadex G-100 column chromatography, was about 80 kDa. The protein was an alcohol dehydrogenase with high affinity for ω-hydroxy fatty acids, such as 12-hydroxylaurate and 16-hydroxypalmitate. We conclude that the cytosolic protein oxidizes 20-OH-LTB4 to 20-oxo-LTB4 and the microsomal fraction oxidizes the oxo-LTB4 to the carboxy-LTB4, based on the finding that the activity which oxidizes ω-hydroxy fatty acids is present only in the cytosol fraction, while that which oxidizes hydrophobic aldehydes is found only in the microsomal fraction and that the stoichiometry of the formation of 20-COOH-LTB4 to the reduction of NAD+ was 1: 2. The affinity of the dehydrogenase for 20-OHLTB4 may be higher than that for 12-hydroxylaurate (Km = 70 µM), because the latter inhibited the oxidation of the former by only 40%, at a concentration of 12-hydroxylaurate 10 times higher than that of 20-OH-LTB4. The enzyme activity was not affected by pyrazole and 4-methylpyrazole at millimolar concentrations. These characteristics indicate that the dehydrogenase is a unique type of alcohol dehydrogenase. [ABSTRACT FROM AUTHOR]

Leukotriene B4 (LTB4), a potent chemotactic agent, was catabolized to 20-hydroxyleukotriene B4 (20-OHLTB4) by the 150 000 × g pellet (microsomal fraction) of human neutrophil sonicate. The reaction required molecular oxygen and NADPH, and was significantly inhibited by carbon monoxide, suggesting that a cytochrome P-450 is involved. The neutrophil microsomal fraction showed a carbon monoxide difference spectrum with a peak at 450 nm in the presence of NADPH or dithionite, indicating the presence of a cytochrome P-450. The addition of LTB4 to the microsomal fraction gave a type-1 spectral change with a peak at around 390 nm and a trough at 422 nm, indicating a direct interaction of LTB4 with the cytochrome P-450. The dissociation constant of LTB4, determined from the difference spectra, is 0.40 µM, in agreement with the kinetically determined apparent Km value for LTB4 (0.30 µM). Such a spectral change was not observed with prostaglandins A1, E1 and F2, or laurie acid, none of which inhibited the LTB4 ω-hydroxylation. The inhibition of the LTB4 ω-hydroxylation by carbon monoxide was effectively reversed by irradiation with monochromatic light of 450 nm wavelength. The photochemical action spectrum of the light reversal of the inhibition corresponded remarkably welt with the carbon monoxide difference spectrum. These observations provide direct evidence that the oxygen-activating component of the LTB4 ω-hydroxylase system is a cytochrome P-450. Ferricytochrome c inhibited the hydroxylation of LTB4 and the inhibition was fortified by cytochrome oxidase. An antibody raised against rat liver NADPH-cytochrome-P-450 reductase inhibited both LTB4 ω-hydroxylase activity and the NADPH-cytochrome-c reductase activity of human neutrophil microsomal fraction. These observations indicate that NADPH-cytochrome-P-450 reductase acts as an electron carrier in LTB4 ω-hydroxylase. On the other hand, an antibody raised against rat liver microsomal cytochrome b5 inhibited the NADH-cytochrome-c reductase activity but not the LTB4 ω-hydroxylase activity of human neutrophil microsomal fraction, suggesting that cytochrome b5 does not participate in the LTB4-hydroxylating system. These characteristics indicate that the isoenzyme of cytochrome P-450 in human neutrophils, LTB4 ω-hydroxylase, is different from the ones reported to be involved in ω-hydroxylation reactions of prostaglandins and fatty acids. [ABSTRACT FROM AUTHOR]

Age-related hearing loss (ARHL) is a common sensory impairment with complex underlying mechanisms. In our previous study, we performed a meta-analysis of genome-wide association studies (GWAS) in mice and identified a novel locus on chromosome 18 associated with ARHL specifically linked to a 32 kHz tone burst stimulus. Consequently, we investigated the role of Formin Homology 2 Domain Containing 3 (Fhod3), a newly discovered candidate gene for ARHL based on the GWAS results. We observed Fhod3 expression in auditory hair cells (HCs) primarily localized at the cuticular plate (CP). To understand the functional implications of Fhod3 in the cochlea, we generated Fhod3 overexpression mice (Pax2-Cre+/-; Fhod3Tg/+) (TG) and HC-specific conditional knockout mice (Atoh1-Cre+/-; Fhod3fl/fl) (KO). Audiological assessments in TG mice demonstrated progressive high-frequency hearing loss, characterized by predominant loss of outer hair cells, and a decreased phalloidin intensities of CP. Ultrastructural analysis revealed loss of the shortest row of stereocilia in the basal turn of the cochlea, and alterations in the cuticular plate surrounding stereocilia rootlets. Importantly, the hearing and HC phenotype in TG mice phenocopied that of the KO mice. These findings suggest that balanced expression of Fhod3 is critical for proper CP and stereocilia structure and function. Further investigation of Fhod3 related hearing impairment mechanisms may lend new insight towards the myriad mechanisms underlying ARHL, which in turn could facilitate the development of therapeutic strategies for ARHL. [ABSTRACT FROM AUTHOR]

The hydrogen peroxide (H2O2)‐producing NADPH oxidase Nox4, forming a heterodimer with p22phox, is expressed in a variety of cells including those in the heart to mediate adaptive responses to cellular stresses such as hypoxia. Since Nox4 is constitutively active, H2O2 production is controlled by its protein abundance. Hypoxia‐induced Nox4 expression is observed in various types of cells and generally thought to be regulated at the transcriptional level. Here we show that hypoxia upregulates the Nox4 protein level and Nox4‐catalyzed H2O2 production without increasing the Nox4 mRNA in rat H9c2 cardiomyocytes. In these cells, the Nox4 protein is stabilized under hypoxic conditions in a manner dependent on the presence of p22phox. Cell treatment with the proteasome inhibitor MG132 results in a marked decrease of the Nox4 protein under both normoxic and hypoxic conditions, indicating that the proteasome pathway does not play a major role in Nox4 degradation. The decrease is partially restored by the autophagy inhibitor 3‐methyladenine. Furthermore, the Nox4 protein level is upregulated by the lysosome inhibitors bafilomycin A1 and chloroquine. Thus, in cardiomyocytes, Nox4 appears to be degraded via an autophagy‐related pathway, and its suppression by hypoxia likely stabilizes Nox4, leading to upregulation of Nox4‐catalyzed H2O2 production. [ABSTRACT FROM AUTHOR]

The adaptor protein LGN interacts via the N-terminal domain comprising eight tetratricopeptide-repeat (TPR) motifs with its partner proteins mInsc, NuMA, Frmpd1 and Frmpd4 in a mutually exclusive manner. Here, the crystal structure of the LGN TPR domain in complex with human Frmpd4 is described at 1.5 Å resolution. In the complex, the LGN-binding region of Frmpd4 (amino-acid residues 990-1011) adopts an extended structure that runs antiparallel to LGN along the concave surface of the superhelix formed by the TPR motifs. Comparison with the previously determined structures of the LGN-Frmpd1, LGN-mInsc and LGN-NuMA complexes reveals that these partner proteins interact with LGN TPR1-6 via a common core binding region with consensus sequence (E/Q) XE X4-5(E/D/Q) X1-2(K/R) X0-1(V/I). In contrast to Frmpd1, Frmpd4 makes additional contacts with LGN via regions N- and C-terminal to the core sequence. The N-terminal extension is replaced by a specific α-helix in mInsc, which drastically increases the direct contacts with LGN TPR7/8, consistent with the higher affinity of mInsc for LGN. A crystal structure of Frmpd4-bound LGN in an oxidized form is also reported, although oxidation does not appear to strongly affect the interaction with Frmpd4. [ABSTRACT FROM AUTHOR]

The nuclear protein I κ B ζ activates transcription of a subset of NF- κ B-dependent innate immune genes such as Lcn2 encoding the antibacterial protein lipocalin-2. I κ B ζ functions as a coactivator via its interaction with NF- κ B p50, which contains a DNA-binding Rel-homology domain but lacks a transcriptional activation domain. However cis-regulatory elements involved in I κ B ζ function have remained unknown. Here, we show that, although I κ B ζ by itself is unable to associate with the Lcn2 promoter, I κ B ζ interacts with the promoter via p50 binding to the NF- κ B-binding site ( κ B site) and the interaction also requires the pyrimidine-rich site ( CCCCTC) that localizes seven bases downstream of the κ B site. The pyrimidine-rich site is also essential for I κ B ζ-mediated activation of the Lcn2 gene. Introduction of both sites into an I κ B ζ-independent gene culminates in I κ B ζ-p50- DNA complex formation and transcriptional activation. Furthermore, spacing between the two sites is crucial for both I κ B ζ- DNA interaction and I κ B ζ-mediated gene activation. Thus, the pyrimidine-rich I κ B ζ-responsive site plays an essential role in productive interaction of I κ B ζ with the p50- DNA complex. [ABSTRACT FROM AUTHOR]

Phagocytosis by macrophages is essential for host defense, i.e. preventing invasion of pathogens and foreign materials. Macrophages engulf immunoglobulin G (IgG)-opsonized particles through the action of the receptors for the Fc of IgG (FcγRs). Leukotriene B4 (LTB4) is a classical lipid chemoattractant derived from arachidonic acid. Leukotriene B4 receptor 1 (BLT1), a high affinity LTB4 receptor, is expressed in a variety of immune cells such as neutrophils, macrophages, and dendritic cells. Although LTB4 has been shown to enhance macrophage phagocytosis, few studies have investigated the intracellular mechanisms involved in this in detail. Furthermore, there have been no reports of the direct cross-talk between LTB4-BLT1 and IgG-FcγRs signaling. Here, we show that FcγRs-dependent phagocytosis was attenuated in BLT1-deficient macrophages as compared with wild-type (WT) cells. Moreover, cross-talk between LTB4-BLT1 and IgG-FcγRs signaling was identified at the level of phosphatidylinositol 3-OH kinase (PI3K) and Rac, downstream of Syk. In addition, the trimeric Gi protein (Gi) was found to be essential for BLT1-dependent phagocytosis. Surprisingly, we found that LTB4-BLT1 signaling restores phagocytosis in the absence of FcγRs signaling. These data indicate that LTB4-BLT1 signaling plays a pivotal role in macrophage phagocytosis and innate immunity. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

The basic function of the Src homology 3 (SH3) domain is considered to be binding to proline-rich sequences containing a PxxP motif. Recently, many SH3 domains, including those from Grb2 and Pex13p, were reported to bind sequences lacking a PxxP motif. We report here that the 22 residue peptide lacking a PxxP motif, derived from p47phox, binds to the C-terminal SH3 domain from p67phox. We applied the NMR cross-saturation method to locate the interaction sites for the non-PxxP peptides on their cognate SH3 domains from p67phox, Grb2 and Pex13p. The binding site of the Grb2 SH3 partially overlapped the conventional PxxP-binding site, whereas those of p67Pphox and Pex13p SH3s are located in different surface regions. The non-PxxP peptide from p47phox binds to the p67Pphox SH3 more tightly when it extends to the N-terminus to include a typical PxxP motif, which enabled the structure determination of the complex, to reveal that the non-PxxP peptide segment interacted with the p67phox SH3 in a compact helix-turn-helix structure (PDB entry 1K4U). [ABSTRACT FROM AUTHOR]

Superoxide production by NADPH oxidase is essential for bactericidal properties of neutrophils. However, molecular mechanisms underlying the activation of this enzyme remain largely unknown. Here, using bovine neutrophils we examined the role of p38 mitogen-activated protein kinase (p38 MAPK) in the signaling pathways of the NADPH oxidase activation. Superoxide production was induced by stimulation with serum-opsonized zymosan (OZ) and attenuated by p38 MAPK inhibitor, SB203580. OZ stimulation induced the translocation of p47phox and Rac to the plasma membrane and SB203580 completely blocked the translocation of Rac, but only partially blocked that of p47phox. Furthermore, SB203580 abolished the OZ-elicited activation of Rac, which was assessed by detecting the GTP-bound form of this protein. Phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin and LY294002, blocked not only p38 MAPK activation but also Rac activation. However, SB203580 showed no effect on the PI3K activity. These results suggested that PI3K/p38 MAPK/Rac pathway was present in the activation of NADPH oxidase in bovine neutrophils. [Copyright &y& Elsevier]

The NADPH-dependent superoxide-generating oxidase of pig neutrophils is activated by sodium dodecyl sulfate in a cell-free system, The activation requires both membrane and cytosolic components. The membrane component was effectively extracted with 0.75% octyl glucoside and the extract was fractionated by wheat-germ-agglutinin—agarose column chromatography. The chromatography resulted in loss of the O2--generating activity in the cell-free system. The activity, however, was restored by the reconstitution with the fraction which passed through the column (fraction A) and the one eluted with N-acetylglucosamine (fraction B) using an octyl glucose dilution procedure: both fractions were pre-mixed in the presence of 0.75% octyl glucoside and diluted by putting the mixture into the detergent-free assay mixture. The latter fraction was copurified with cytochrome b558, the content of which is 2.12 ± 0.53 nmol/mg protein (mean ± SD, n = 5). The potency of fraction B in the reconstitution of the O2--generating activity was lost by heat treatment and decreased by protease treatment, whereas that of fraction A was not affected. Fraction A in the reconstitution of the O2--generating activity was replaced by lipid extracted from fraction A, furthermore, by exogenous phospholipid, azolectin. The O2--generating activity reconstituted with azolectin and the partially purified component in fraction B was dependent on SDS, cytosol and the concentrations of azolectin and FAD. The activity was sensitive to p-chloromercuribenzoate but not to azide. The maximal activity was obtained at pH 7.0–7.5, The Km values for NADPH and NADH were 0.024 mM and 0.57 mM, respectively. These properties were consistent with those of the NADPH oxidase responsible for the respiratory burst. The activity in the reconstitution system was 20.5 ± 3.5 μmol O2- · min-1 mg-1 membrane-derived protein (mean ± SD, n = 5) which shows that the membrane component was purified about 100-fold. These findings indicate that cytochrome b558 is probably a membrane component of the O2--generating NADPH oxidase and its activation in the cell-free system requires the reconstitution with phospholipids. [ABSTRACT FROM AUTHOR]

Transmembrane glycoproteins, synthesized at the endoplasmic reticulum (ER), generally reach the Golgi apparatus in COPII‐coated vesicles en route to the cell surface. Here, we show that the bona fide nonglycoprotein Nox5, a transmembrane superoxide‐producing NADPH oxidase, is transported to the cell surface in a manner resistant to co‐expression of Sar1 (H79G), a GTP‐fixed mutant of the small GTPase Sar1, which blocks COPII vesicle fission from the ER. In contrast, Sar1 (H79G) effectively inhibits ER‐to‐Golgi transport of glycoproteins including the Nox5‐related oxidase Nox2. The trafficking of Nox2, but not that of Nox5, is highly sensitive to over‐expression of syntaxin 5 (Stx5), a t‐SNARE required for COPII ER‐to‐Golgi transport. Thus, Nox2 and Nox5 mainly traffic via the Sar1/Stx5‐dependent and ‐independent pathways, respectively. Both participate in Nox1 trafficking, as Nox1 advances to the cell surface in two differentially N‐glycosylated forms, one complex and one high mannose, in a Sar1/Stx5‐dependent and ‐independent manner, respectively. Nox2 and Nox5 also can use both pathways: a glycosylation‐defective mutant Nox2 is weakly recruited to the plasma membrane in a less Sar1‐dependent manner; N‐glycosylated Nox5 mutants reach the cell surface in part as the complex form Sar1‐dependently, albeit mainly as the high‐mannose form in a Sar1‐independent manner. [ABSTRACT FROM AUTHOR]

Correct cyst morphogenesis of epithelial cells requires apical-basal polarization, which is partly regulated by mitotic spindle orientation, a process dependent on the heterotrimeric G protein subunit Gαi and its binding protein LGN. Here, we show that in three-dimensional culture of mammalian epithelial Madin-Darby canine kidney (MDCK) cells, the Gαi-activating protein Ric-8A is crucial for orientation of the mitotic spindle and formation of normal cysts that comprise a single layer of polarized cells with their apical surfaces lining an inner lumen. Consistent with the involvement of LGN, cystogenesis can be well organized by ADP-ribosylated Gαi, retaining the ability to interact with LGN, but not by the interaction-defective mutant protein Gαi2 (N150I). In monolayer culture of MDCK cells, functional tight junction (TJ) assembly, a process associated with epithelial cell polarization, is significantly delayed in Ric-8A-depleted cells as well as in Gαi-depleted cells in a mitosis-independent manner. Ric-8A knockdown results in a delayed cortical delivery of Gαi and the apical membrane protein gp135, and an increased formation of intercellular lumens surrounded by membranes rich in Gαi3 and gp135. TJ development also involves LGN and its related protein AGS3. Thus, Ric-8A regulates mammalian epithelial cell polarity for TJ assembly and cystogenesis probably in concert with Gαi and LGN/AGS3. [ABSTRACT FROM AUTHOR]

The heterotrimeric G protein subunit Gαi can be activated by G protein-coupled receptors and the cytosolic protein Ric-8A, the latter of which is also known to prevent ubiquitin-dependent degradation of Gαi. Here we show that the amounts of the three Gαi-related proteins Gαi1, Gαi2, and Gαi3, but not that of Gαq, are rapidly decreased by cell treatment with pertussis toxin (PTX). The decrease appears to be due to ADP-ribosylation of Gαi, because PTX treatment does not affect the amount of a mutant Gαi2 carrying alanine substitution for Cys352, the residue that is ADP-ribosylated by the toxin. The presence of endogenous and exogenous Ric-8A increases Gαi stability as shown in cells treated with the protein synthesis inhibitor cycloheximide; however, Ric-8A fails to efficiently stabilize ADP-ribosylated Gαi. The failure agrees with the inability of Ric-8A to bind to ADP-ribosylated Gαi both in vitro and in vivo . Thus PTX appears to exert its pathological effects at least in part by converting Gαi to an unstable ADP-ribosylated form, in addition to the well-known inability of ADP-ribosylated Gαi to transduce signals triggered by G protein-coupled receptors. [ABSTRACT FROM AUTHOR]

Fhod3 is a cardiac member of the formin family proteins that play pivotal roles in actin filament assembly in various cellular contexts. The targeted deletion of mouse Fhod3 gene leads to defects in cardiogenesis, particularly during myofibrillogenesis, followed by lethality at embryonic day (E) 11.5. However, it remains largely unknown how Fhod3 functions during myofibrillogenesis. In this study, to assess the mechanism whereby Fhod3 regulates myofibrillogenesis during embryonic cardiogenesis, we generated transgenic mice expressing Fhod3 selectively in embryonic cardiomyocytes under the control of the β-myosin heavy chain (MHC) promoter. Mice expressing wild-type Fhod3 in embryonic cardiomyocytes survive to adulthood and are fertile, whereas those expressing Fhod3 (I1127A) defective in binding to actin die by E11.5 with cardiac defects. This cardiac phenotype of the Fhod3 mutant embryos is almost identical to that observed in Fhod3 null embryos, suggesting that the actin-binding activity of Fhod3 is crucial for embryonic cardiogenesis. On the other hand, the β-MHC promoter-driven expression of wild-type Fhod3 sufficiently rescues cardiac defects of Fhod3-null embryos, indicating that the Fhod3 protein expressed in a transgenic manner can function properly to achieve myofibril maturation in embryonic cardiomyocytes. Using the transgenic mice, we further examined detailed localization of Fhod3 during myofibrillogenesis in situ and found that Fhod3 localizes to the specific central region of nascent sarcomeres prior to massive rearrangement of actin filaments and remains there throughout myofibrillogenesis. Taken together, the present findings suggest that, during embryonic cardiogenesis, Fhod3 functions as the essential reorganizer of actin filaments at the central region of maturating sarcomeres via the actin-binding activity of the FH2 domain. [ABSTRACT FROM AUTHOR]

Superoxide production by Nox1, a member of the Nox family NAPDH oxidases, requires expression of its regulatory soluble proteins Noxo1 (Nox organizer 1) and Noxa1 (Nox activator 1) and is markedly enhanced upon cell stimulation with phorbol 12-myristate 13-acetate ( PMA), a potent activator of protein kinase C ( PKC). The mechanism underlying PMA-induced enhancement of Nox1 activity, however, remains to be elucidated. Here we show that, in response to PMA, Noxo1 undergoes phosphorylation at multiple sites, which is inhibited by the PKC inhibitor GF109203X. Among them, Thr341 in Noxo1 is directly phosphorylated by PKC in vitro, and alanine substitution for this residue reduces not only PMA-induced Noxo1 phosphorylation but also PMA-dependent enhancement of Nox1-catalyzed superoxide production. Phosphorylation of Thr341 allows Noxo1 to sufficiently interact with Noxa1, an interaction that participates in Nox1 activation. Thus phosphorylation of Noxo1 at Thr341 appears to play a crucial role in PMA-elicited activation of Nox1, providing a molecular link between PKC-mediated signal transduction and Nox1-catalyzed superoxide production. Furthermore, Ser154 in Noxo1 is phosphorylated in both resting and PMA-stimulated cells, and the phosphorylation probably participates in a PMA-independent constitutive activity of Nox1. Ser154 may also be involved in protein kinase A ( PKA) mediated regulation of Nox1; this serine is the major residue that is phosphorylated by PKA in vitro. Thus phosphorylation of Noxo1 at Thr341 and at Ser154 appears to regulate Nox1 activity in different manners. Structured digital abstract Noxo1 binds to p22phox by pull down (1, 2, 3), Noxo1 binds to Noxo1 by pull down (View interaction), Noxa1 binds to Noxo1 by pull down (1, 2, 3, 4, 5) [ABSTRACT FROM AUTHOR]

Summary: Successful chemotaxis requires not only increased motility but also sustained directionality. Here, we show that, during neutrophil chemotaxis via receptors coupled with the Gi family of heterotrimeric G proteins, directional movement is regulated by mInsc, a mammalian protein distantly related to the Drosophila polarity-organizer Inscuteable. The GDP-bound, Gβγ-free Gαi subunit accumulates at the front of chemotaxing neutrophils to recruit mInsc—complexed with the Par3-aPKC evolutionarily conserved polarity complex—via LGN/AGS3 that simultaneously binds to Gαi-GDP and mInsc. Both mInsc-deficient and aPKC-blocked neutrophils exhibit a normal motile activity but migrate in an undirected manner. mInsc deficiency prevents neutrophils from efficiently stabilizing pseudopods at the leading edge; the stability is restored by wild-type mInsc, but not by a mutant protein defective in binding to LGN/AGS3. Thus, mInsc controls directional migration via noncanonical G protein signaling, in which Gβγ-free Gαi-GDP, a product from Gαi-GTP released after receptor activation, plays a central role. [ABSTRACT FROM AUTHOR]

Highlights: [•] Ric-8A interacts with and stabilizes Gαi2, Gαq, Gα12, but not Gαs in cells. [•] Gαq as well as Gαi2 undergoes polyubiquitination. [•] The ubiquitination of Gαi2 and Gαq is suppressed by expression of Ric-8A. [•] The suppression appears to require Ric-8A interaction with Gαi2 and Gαq. [ABSTRACT FROM AUTHOR]

Bipolar spindle assembly in mitotic cells is a prerequisite to ensure correct alignment of chromosomes for their segregation to each daughter cell; spindle microtubules are tethered at plus ends to chromosomes and focused at minus ends to either of the two spindle poles. NuMA (nuclear mitotic apparatus protein) is present solely in the nucleus in interphase cells, but relocalizes during mitosis to the spindle poles to play a crucial role in spindle assembly via focusing spindle microtubules to each pole. In the present study we show that the kinesin-5 family motor Eg5 is a protein that directly interacts with NuMA, using a proteomics approach and various binding assays both in vivo and in vitro. During mitosis Eg5 appears to interact with NuMA in the vicinity of the spindle poles, whereas the interaction does not occur in interphase cells, where Eg5 is distributed throughout the cytoplasm but NuMA exclusively localizes to the nucleus. Slight, but significant, depletion of Eg5 in HeLa cells by RNA interference results in formation of less-focused spindle poles with misaligned chromosomes in metaphase; these phenotypes are similar to those induced by depletion of NuMA. Since NuMA is less accumulated at the spindle poles in Eg5- depleted cells, Eg5 probably contributes to spindle assembly via regulating NuMA localization. Furthermore, depletion of cytoplasmic dynein induces mislocalization of NuMA and phenotypes similar to those observed in NuMA-depleted cells, without affecting Eg5 localization to the spindles. Thus dynein appears to control NuMA function in conjunction with Eg5. [ABSTRACT FROM AUTHOR]

Formation of apico-basal polarity in epithelial cells is crucial for both morphogenesis (e.g., cyst formation) and function (e.g., tight junction development). Atypical protein kinase C (aPKC), complexed with Par6, is considered to translocate to the apical membrane and function in epithelial cell polarization. However, the mechanism for translocation of the Par6-aPKC complex has remained largely unknown. Here, we show that the WD40 protein Morgl (mitogen-activated protein kinase organizer 1) directly binds to Par6 and thus facilitates apical targeting of Par6-aPKC in Madin-Darby canine kidney epithelial cells. Morgl also interacts with the apical transmembrane protein Crumbs3 to promote Par6- aPKC binding to Crumbs3, which is reinforced with the apically localized small GTPase Cdc42. Depletion of Morgl disrupted both tight junction development in monalayer culture and cyst formation in three-dimensional culture; apico-basal polarity was notably restored by forced targeting of aPKC to the apical surface. Thus, Par6-aPKC recruitment to the premature apical membrane appears to be required for definition of apical identity of epithelial cells. [ABSTRACT FROM AUTHOR]

The isolated right half ( RH) or left half ( LH) of Xenopus embryos can undergo regulation so as to form well-proportioned larvae. To assess how the combined actions of maternal determinants and cell-cell interactions contribute to form the well-proportioned larvae, we quantitatively compared four-cell stage blastomere fate between normal larvae and regulated larvae from RH embryos. In normal larvae, the clones of the right dorsal blastomere ( RD) and right ventral blastomere ( RV) were located unilaterally. In contrast, in regulated larvae: (i) the RD clone exclusively occupied the anterior endomesoderm ( AE) derivatives, coinciding no RV progeny in those derivatives of normal larvae. The clone bilaterally populated tissues along the dorsal midline, which characteristically included the medial regions of both somites adjoining the notochord, with higher percentages on the right and anterior sides. (ii) The RV clone extensively compensated for the missing left side at the expense of its right side contribution, and bilaterally occupied the ventroposterior and also dorsal regions excluding the AE derivatives. This clone considerably populated, with altered orientations, the derivatives of the left half gastrocoel roof plate ( GRP), the left half GRP being essential for laterality determination. These results show that the high cell-autonomy in the AE constitutes a mechanism common to both normal and regulative development. In regulated larvae, cell-cell interactions shifted the midlines on the dorsal side slightly and the ventral side to a greater extent. The cell lineage difference in the left half GRP could result in a different utilization of maternal determinants in that area. [ABSTRACT FROM AUTHOR]

The Phox homology (PX) domain is a functional module that targets membranes through specific interactions with phosphoinositides. The p47phox PX domain preferably binds phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2) and plays a pivotal role in the assembly of phagocyte NADPH oxidase. We describe the PI(3,4)P2 binding mode of the p47phox PX domain as identified by a transferred cross-saturation experiment. The identified PI(3,4)P2-binding site, which includes the residues of helices a1 and a1' and the following loop up to the distorted left-handed PPII helix, is located at a unique position, as compared with the phosphoinositide-binding sites of all other PX domains characterized thus far. Mutational analyses corroborated the results of the transferred cross-saturation experiments. Moreover, experiments with intact cells demonstrated the importance of this unique binding site for the function of the NADPH oxidase. The low affinity and selectivity of the atypical phosphoinositide-binding site on the p47phox PX domain suggest that different types of phosphoinositides sequentially bind to the p47phox PX domain, allowing the regulation of the multiple events that characterize the assembly and activation of phagocyte NADPH oxidase. [ABSTRACT FROM AUTHOR]

Summary: Tissue infiltration of activated lymphocytes is a hallmark of transplant rejection and organ-specific autoimmune diseases. Migration and activation of lymphocytes depend on DOCK2, an atypical Rac activator predominantly expressed in hematopoietic cells. Although DOCK2 does not contain Dbl homology domain typically found in guanine nucleotide exchange factors, DOCK2 mediates the GTP-GDP exchange reaction for Rac through its DHR-2 domain. Here, we have identified 4-[3′-(2″-chlorophenyl)-2′-propen-1′-ylidene]-1-phenyl-3,5-pyrazolidinedione (CPYPP) as a small-molecule inhibitor of DOCK2. CPYPP bound to DOCK2 DHR-2 domain in a reversible manner and inhibited its catalytic activity in vitro. When lymphocytes were treated with CPYPP, both chemokine receptor- and antigen receptor-mediated Rac activation were blocked, resulting in marked reduction of chemotactic response and T cell activation. These results provide a rational of and a chemical scaffold for development of the DOCK2-targeting immunosuppressant. [Copyright &y& Elsevier]

The formin family proteins play pivotal roles in actin filament assembly via the FH2 domain. The mammalian formin Fhod3 is highly expressed in the heart, and its mRNA in the adult heart contains exons 11, 12, and 25, which are absent from nonmuscle Fhod3 isoforms. In cultured neonatal cardiomyocytes, Fhod3 localizes to the middle of the sarcomere and appears to function in its organization, although it is suggested that Fhod3 localizes differently in the adult heart. Here we show, using immunohistochemical analysis with three different antibodies, each recognizing distinct regions of Fhod3, that Fhod3 localizes as two closely spaced bands in middle of the sarcomere in both embryonic and adult hearts. The bands are adjacent to the M-line that crosslinks thick myosin filaments at the center of a sarcomere but distant from the Z-line that forms the boundary of the sarcomere, which localization is the same as that observed in cultured cardiomyocytes. Detailed immunohistochemical and immuno-electron microscopic analyses reveal that Fhod3 localizes not at the pointed ends of thin actin filaments but to a more peripheral zone, where thin filaments overlap with thick myosin filaments. We also demonstrate that the embryonic heart of mice specifically expresses the Fhod3 mRNA isoform harboring the three alternative exons, and that the characteristic localization of Fhod3 in the sarcomere does not require a region encoded by exon 25, in contrast to an essential role of exons 11 and 12. Furthermore, the exon 25-encoded region appears to be dispensable for actin-organizing activities both in vivo and in vitro, albeit it is inserted in the catalytic FH2 domain. [ABSTRACT FROM AUTHOR]

Abstract: Noxa1 was discovered as an activating factor for Nox1, an -generating enzyme. Subsequent studies have shown that Noxa1 is colocalized with Nox2 in several cell types, including vascular cells. Nox2 activation by Noxa1 has been examined in reconstituted model cells. However, little is known about the kinetic properties of Noxa1 in Nox2 activation. In the present study, we used purified cyt.b 558 (Nox2 plus p22phox), Rac(Q61L), and Noxo1 to examine the ability of Noxa1 to activate Nox2. In the pure reconstitution system, Noxa1 activated Nox2 with lower efficiency than p67phox, a canonical activator of Nox2. The EC 50 value of Noxa1 was considerably higher than that of p67phox. The V max value with Noxa1 and Noxo1 was one-third of that with p67phox and p47phox. The EC 50 value of Noxo1 or Rac(Q61L) was also higher when Noxa1 was used. The affinity of FAD for the oxidase and the stability of the active complex were remarkably low when Noxa1 and Noxo1 were used compared with p67phox and p47phox. The stability was not improved by fusion of Noxa1 with Rac(Q61L). These findings show that Noxa1 has quite different kinetic properties from p67phox and suggest that Noxa1 may function as a moderate activator of Nox2. [Copyright &y& Elsevier]

Interaction between the mammalian cell polarity proteins mInsc (mammalian homologue of Inscuteable) and Leu-Gly-Asn repeat-enriched protein (LGN), as well as that between their respective Drosophila homologues Inscuteable and Partner of Inscuteable (Pins), plays crucial roles in mitotic spindle orientation, a process contributing to asymmetric cell division. Here, we report a crystal structure of the LGN-binding domain (LBD) of human mInsc complexed with the N-terminal tetratricopeptide repeat (TPR) motifs of human LGN at 2.6-Å resolution. In the complex, mInsc-LBD adopts an elongated structure with three binding modules—an α-helix, an extended region, and a β-sheet connected with a loop—that runs antiparallel to LGN along the concave surface of the superhelix formed by the TPRs. Structural analysis and structure-based mutagenesis define residues that are critical for mInsc-LGN association, and reveal that the activator of G-protein signaling 3 (AGS3)-binding protein Frmpd1 [4.1/ezrin/radixin/moesin (FERM) and PSD-95/Dlg/ZO-1 (PDZ) domain-containing protein 1] and its relative Frmpd4 interact with LGN via a region homologous to a part of mInsc-LBD, whereas nuclear mitotic apparatus protein (NuMA) and the C terminus of LGN recognize the TPR domain in a manner different from that by mInsc. mInsc binds to LGN with the highest affinity (KD ≈ 2.4 nM) and effectively replaces the Frmpd proteins, NuMA, and the LGN C terminus, suggesting the priority of mInsc in binding to LGN. We also demonstrate, using mutant proteins, that mInsc-LGN interaction is vital for stabilization of LGN and for intracellular localization of mInsc. [ABSTRACT FROM AUTHOR]

Regulated synthesis of reactive oxygen species (ROS) by membrane-bound fungal NADPH oxidases (Nox) plays a key role in fungal morphogenesis, growth, and development. Generation of reactive oxygen species (ROS) by the plant symbiotic fungus, Epichloë festucae, requires functional assembly of a multisubunit complex composed of NoxA, a regulatory component, NoxR, and the small GTPase RacA. However, the mechanism for assembly and activation of this complex at the plasma membrane is unknown. We found by yeast two-hybrid and coimmunoprecipitation assays that E. festucae NoxR interacts with homologs of the yeast polarity proteins, Bem1 and Cdc24, and that the Phox and Bem1 (PB1) protein domains found in these proteins are essential for these interactions. GFP fusions of BemA, Cdc24, and NoxR preferentially localized to actively growing hyphal tips and to septa. These proteins interact with each other in vivo at these same cellular sites as shown by bimolecular fluorescent complementation assays. The PB1 domain of NoxR is essential for localization to the hyphal tip. An E. festucae ΔbemA mutant was defective in hyphal morphogenesis and growth in culture and in planta. The changes in fungal growth in planta resulted in a defective symbiotic interaction phenotype. Our inability to isolate a Δcdc24 mutant suggests this gene is essential. These results demonstrate that BemA and Cdc24 play a critical role in localizing NoxR protein to sites of fungal hyphal morphogenesis and growth. Our findings identify a potential shared ancestral link between the protein machinery required for fungal polarity establishment and the Nox complex controlling cellular differentiation. [ABSTRACT FROM AUTHOR]

Pertussis toxin (PTX) is recognized as a specific tool that uncouples receptors from Gi and Go through ADP-ribosylation. During the study analyzing the effects of PTX on Ang II type 1 receptor (AT1R) function in cardiac fibroblasts, we found that PTX increases the number of AT1Rs and enhances AT1R-mediated response. Microarray analysis revealed that PTX increases the induction of interleukin (IL)-1β among cytokines. Inhibition of IL-1β suppressed the enhancement of AT1R-mediated response by PTX. PTX increased the expression of IL-1β and AT1R through NF-κB, and a small GTP-binding protein, Rac, mediated PTX-induced NF-κB activation through NADPH oxidase-dependent production of reactive oxygen species. PTX induced biphasic increases in Rac activity, and the Rac activation in a late but not an early phase was suppressed by IL-1β siRNA, suggesting that IL-1β-induced Rac activation contributes to the amplification of Rac-dependent signaling induced by PTX. Furthermore, inhibition of TLR4 (Toll-like receptor 4) abolished PTX-induced Rac activation and enhancement of AT1R function. However, ADP-ribosylation of Gi/Go by PTX was not affected by inhibition of TLR4. Thus, PTX binds to two receptors; one is TLR4, which activates Rac, and another is the binding site that is required for ADP-ribosylation of Gi/Go. [ABSTRACT FROM AUTHOR]

Neutrophils play an essential role via phagocytosis in host defense against microbial infections. However, little is known about molecular mechanisms underlying phagocytosis in neutrophils, because of the difficulty in genetically manipulating these cells. Here, we provide the first comprehensive description of phospholipid metabolism during phagocytosis in human neutrophils, which we have efficiently transfected with cDNAs encoding lipid-probing protein modules. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), an F-actin organizer abundant in the plasma membrane, diminishes progressively from phagosomes during phagosome formation and vanishes after phagosome closure with F-actin disappearance. Diacylglycerol, a metabolite of PtdIns(4,5)P2, appears at phagocytic cups and remains associated with nascent (closed) phagosomes; it may function with phosphatidylserine, present in both plasma and phagosomal membranes, to recruit phagocytosis-associated proteins. From PtdIns(4,5)P2, PtdIns(3,4,5)P3 is also produced at phagocytic cups but becomes undetectable shortly after phagosome sealing, consistent with its proposed roles in pseudopod extension and phagosome closure. PtdIns(3)P, a putative participant in phagosome maturation, emerges at closed phagosomes as does the class III PtdIns 3-kinase Vps34. Although the small GTPases Rab5 and Rab7 are thought to contribute to phagosome maturation in macrophages, Rab5 but not Rab7 fails to accumulate at phagosomes in neutrophils, suggesting a difference in phagocytic mechanism between the two phagocytes. [ABSTRACT FROM AUTHOR]

Background: Most in vivo studies that have addressed the role of actin dynamics in NADPH oxidase function in phagocytes have used toxins to modulate the polymerization state of actin and mostly effects on actin has been evaluated by end point measurements of filamentous actin, which says little about actin dynamics, and without consideration for the subcellular distribution of the perturbed actin cytoskeleton. Results: Here, we in addition to toxins use conditional expression of the major actin regulatory protein LIM kinase- 1 (LIMK1), and shRNA knock-down of cofilin to modulate the cellular F/G-actin ratio in the Ra2 microglia cell line, and we use Fluorescence Recovery after Photobleaching (FRAP) in β-actin-YFP-transduced cells to obtain a dynamic measure of actin recovery rates (actin turn-over rates) in different F/G-actin states of the actin cytoskeleton. Our data demonstrate that stimulated NADPH oxidase function was severely impaired only at extreme actin recovery rates and F/G-actin ratios, and surprisingly, that any moderate changes of these parameters of the actin cytoskeleton invariably resulted in an increased NADPH oxidase activity. Conclusion: moderate actin polymerization and depolymerization both increase the FMLP and PMA-stimulated NADPH oxidase activity of microglia, which is directly correlated with neither actin recovery rate nor F/G- actin ratio. Our results indicate that NADPH oxidase functions in an enhanced state of activity in stimulated phagocytes despite widely different states of the actin cytoskeleton. [ABSTRACT FROM AUTHOR]

Actin filament assembly in nonmuscle cells is regulated by the actin polymerization machinery, including the Arp2/3 complex and formins. However, little is known about the regulation of actin assembly in muscle cells, where straight actin filaments are organized into the contractile unit sarcomere. Here, we show that Fhod3, a myocardial formin that localizes to thin actin filaments in a striated pattern, regulates sarcomere organization in cardiomyocytes. RNA interference-mediated depletion of Fhod3 results in a marked reduction in filamentous actin and disruption of the sarcomeric structure. These defects are rescued by expression of wild-type Fhod3 but not by that of mutant proteins carrying amino acid substitution for conserved residues for actin assembly. These findings suggest that actin dynamics regulated by Fhod3 are critical for sarcomere organization in striated muscle cells. [ABSTRACT FROM AUTHOR]

Bem1 and Cdc24 of the budding yeast Saccharomyces cerevisiae interact with each other through PB1–PB1 heterodimer formation to regulate the establishment of cell polarity. Here we present the tertiary structure of the heterodimer of Bem1 and Cdc24 PB1 domains determined by NMR spectroscopy. To avoid ambiguity in the NMR spectral analysis, we first prepared a mutant of the Cdc24 PB1 domain that had truncated loops. The mutant provided well dispersed spectra without spectral overlapping, thus allowing unambiguous spectral assignments for structure determination. We confirmed that the loop deletion-mutant was quite similar to the wild-type in both 3D structure and binding affinity. The NMR structure of the heterodimer of the deletion-mutant of Cdc24 PB1 and Bem1 PB1 was determined using a variety of isotope labelled samples including perdeuteration. The interface between the Bem1/Cdc24 PB1 heterodimer was analysed at atomic resolution. Through a comparison with the tertiary structures of other PB1–PB1 heterodimers, we found that conserved electrostatic properties on the molecular surface were commonly used for PB1–PB1 interaction, but hydrophobic interactions were important for cognate interaction in Bem1/Cdc24 PB1 heterodimer formation. [ABSTRACT FROM PUBLISHER]

Abstract: We previously demonstrated that some fish have unique response in the form of reactive oxygen species (ROS) production. In the present study, we cloned and sequenced the full-length cDNAs for carp (Cyprinus carpio) phagocyte NADPH oxidase components: gp91phox, p22phox, p47phox, p67phox and p40phox. These amino acid sequences were compared with other teleost and mammalian homologues, to elucidate the features of ROS production of fish neutrophils. The phylogeny analysis clearly demonstrates that the radiation of phagocyte oxidase components took place in the common ancestor of teleosts and mammals. Thereafter, the overall structure and expression pattern of phagocyte oxidase have been highly conserved in two different strains. However the amino acid identity of p67phox and p47phox was relatively lower than the amino acid identities of other components. Moreover, a synteny analysis supports the hypothesis that there was strong selective pressure in the p67phox and p47phox genes. Thus, it is likely that the higher divergence of p67phox and p47phox are responsible for the difference of ROS responses between different species of teleosts. [Copyright &y& Elsevier]

Formin-family proteins, in the active state, form actin-based structures such as stress fibres. Their activation mechanisms, however, are largely unknown except that mDia and its closely related formins can be activated by direct binding of the small GTPase Rho or Cdc42. Here we show that the Rho-dependent protein kinase ROCK phosphorylates the C-terminal residues Ser1131, Ser1137, and Thr1141 of formin homology domain protein 1 (FHOD1), a major endothelial formin that is normally autoinhibited by intramolecular interaction between the N- and C-terminal regions. Phosphorylation of FHOD1 at the three residues fully disrupts the autoinhibitory interaction, which culminates in formation of stress fibres. We also demonstrate that, in vascular endothelial cells, thrombin, a vasoactive substance leading to Rho activation, elicits both FHOD1 phosphorylation and stress fibre formation in a ROCK-dependent manner, and that FHOD1 depletion by RNA interference impairs thrombin-induced stress fibre formation. Based on these findings we propose a novel mechanism for activation of formin-family proteins: ROCK, activated by G protein-coupled receptor ligands such as thrombin, directly phosphorylates FHOD1 at the C-terminal region, which renders this formin in the active form, leading to stress fibre formation. [ABSTRACT FROM AUTHOR]

The superoxide-producing phagocyte NADPH oxidase is activated during phagocytosis to destroy ingested microbes. The adaptor protein p40phox associates via the PB1 domain with the essential oxidase activator p67phox, and is considered to function by recruiting p67phox to phagosomes; in this process, the PX domain of p40phox binds to phosphatidylinositol 3-phosphate [PtdIns(3)P], a lipid abundant in the phagosomal membrane. Here we show that the PtdIns(3)P-binding activity of p40phox is normally inhibited by the PB1 domain both in vivo and in vitro. The crystal structure of the full-length p40phox reveals that the inhibition is mediated via intramolecular interaction between the PB1 and PX domains. The interface of the p40phox PB1 domain for the PX domain localizes on the opposite side of that for the p67phox PB1 domain, and thus the PB1-mediated PX regulation occurs without preventing the PB1–PB1 association with p67phox. [ABSTRACT FROM AUTHOR]

Abstract: NADPH oxidase organizer 1 (Noxo1), harboring a PX domain, two SH3 domains, and a proline-rich region (PRR), participates in activation of superoxide-producing Nox-family NADPH oxidases. Here, we show that Noxo1 supports superoxide production in a cell-free system for gp91 phox /Nox2 activation by arachidonic acid. This lipid enhances an SH3-mediated binding of Noxo1 to p22 phox , a protein complexed with Nox oxidases; the binding is known to be required for Nox activation. We also demonstrate that the bis-SH3 domain directly interacts with the Noxo1 PRR. The interaction appears to prevent the bis-SH3 domain and PRR from binding to their target proteins; disruption of the intramolecular interaction facilitates Noxo1 binding to p22 phox and also allows the PRR to associate with the Nox activator Noxa1, which association is crucial for Nox activation as well. These findings suggest that Nox activation involves a conformational change leading to disruption of the bis-SH3–PRR interaction in Noxo1. [Copyright &y& Elsevier]

Abstract: Increased oxidative stress plays a role in the pathogenesis of β-cell dysfunction and death. We studied isoforms of NADPH oxidase components in islets of Langerhans isolated from rat pancreas and tumoral rat β-cell line RINm5F cells by RT-PCR and sequencing of its products. RT-PCR revealed that isolated islets constitutively expressed mRNA of NADPH oxidase components, Nox1, Nox2, Nox4 and p22 phox as membrane-associated components and p47 phox , Noxo1 (homologue of p47 phox ), Noxa1 (homologue of p67 phox ), and p40 phox as cytosolic components. RINm5F cells showed a similar pattern of expression but Nox2 mRNA was not detected. Expression of Nox1, Nox4, Noxo1 and Noxa1 was confirmed by sequencing the PCR products. Immunohistochemistry revealed the expression of NADPH oxidase component in β-cells of rat pancreatic islets. Glucose-stimulated insulin secretion from isolated islets was suppressed by diphenyleneiodonium, a flavocytochrome inhibitor, but not by apocynin, an inhibitor of p47 phox translocation to membranes. Our results suggest that the functional significance of NADPH oxidase in insulin secretion may merit further investigation. [Copyright &y& Elsevier]

Angiotensin (Ang) II participates in the pathogenesis of heart failure through induction of cardiac hypertrophy. Ang II-induced hypertrophic growth of cardiomyocytes is mediated by nuclear factor of activated T cells (NFAT), a Ca2+-responsive transcriptional factor. It is believed that phospholipase C (PLC)-mediated production of inositol-1,4,5-trisphosphate (IP3) is responsible for Ca2+ increase that is necessary for NFAT activation. However, we demonstrate that PLC-mediated production of diacylglycerol (DAG) but not IP3 is essential for Ang II-induced NFAT activation in rat cardiac myocytes. NFAT activation and hypertrophic responses by Ang II stimulation required the enhanced frequency of Ca2+ oscillation triggered by membrane depolarization through activation of DAG-sensitive TRPC channels, which leads to activation of L-type Ca2+ channel. Patch clamp recordings from single myocytes revealed that Ang II activated DAG-sensitive TRPC-like currents. Among DAG-activating TRPC channels (TRPC3, TRPC6, and TRPC7), the activities of TRPC3 and TRPC6 channels correlated with Ang II-induced NFAT activation and hypertrophic responses. These data suggest that DAG-induced Ca2+ signaling pathway through TRPC3 and TRPC6 is essential for Ang II-induced NFAT activation and cardiac hypertrophy. [ABSTRACT FROM AUTHOR]