Soluble Regulatory Proteins for Activation of NOX Family NADPH Oxidases.

NOX family NADPH oxidases deliberately produce reactive oxygen species and thus contribute to a variety of biological functions. Of seven members in the human family, the three oxidases NOX2, NOX1, and NOX3 form a heterodimer with p22phox and are regulated by soluble regulatory proteins: p47phox, its related organizer NOXO1; p67phox, its related activator NOXA1; p40phox; and the small GTPase Rac. Activation of the phagocyte oxidase NOX2 requires p47phox, p67phox, and GTP-bound Rac. In addition to these regulators, p40phox plays a crucial role when NOX2 is activated during phagocytosis. On the other hand, NOX1 activation prefers NOXO1 and NOXA1, although Rac is also involved. NOX3 constitutively produces superoxide, which is enhanced by regulatory proteins such as p47phox, NOXO1, and p67phox. Here we describe mechanisms for NOX activation with special attention to the soluble regulatory proteins.

Visualization of phagosomal hydrogen peroxide production by a novel fluorescent probe that is localized via SNAP-tag labeling.

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

Membrane phospholipid metabolism during phagocytosis in human neutrophils.

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)P(2)), 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)P(2), 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)P(2), PtdIns(3,4,5)P(3) 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.

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

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

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

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

Targeted disruption of the cardiac troponin T gene causes sarcomere disassembly and defects in heartbeat within the early mouse embryo.

Cardiac troponin T (cTnT) is a component of the troponin (Tn) complex in cardiac myocytes, and plays a regulatory role in cardiac muscle contraction by anchoring two other Tn components, troponin I (TnI) and troponin C, to tropomyosin (Tm) on the thin filaments. In order to determine the in vivo function of cTnT, we created a null cTnT allele in the mouse TNNT2 locus. In cTnT-deficient (cTnT(-/-)) cardiac myocytes, the thick and thin filaments and alpha-actinin-positive Z-disk-like structures were not assembled into sarcomere, causing early embryonic lethality due to a lack of heartbeats. TnI was dissociated from Tm in the thin filaments without cTnT. In spite of loss of Tn on the thin filaments, the cTnT(-/-) cardiac myocytes showed regular Ca(2+)-transients. These findings indicate that cTnT plays a critical role in sarcomere assembly during myofibrillogenesis in the embryonic heart, and also indicate that the membrane excitation and intracellular Ca(2+) handling systems develop independently of the contractile system. In contrast, heterozygous cTnT(+/-) mice had a normal life span with no structural and functional abnormalities in their hearts, suggesting that haploinsufficiency could not be a potential cause of cardiomyopathies, known to be associated with a variety of mutations in the TNNT2 locus.

Knock-in mouse model of dilated cardiomyopathy caused by troponin mutation.

We created knock-in mice in which a deletion of 3 base pairs coding for K210 in cardiac troponin (cTn)T found in familial dilated cardiomyopathy patients was introduced into endogenous genes. Membrane-permeabilized cardiac muscle fibers from mutant mice showed significantly lower Ca(2+) sensitivity in force generation than those from wild-type mice. Peak amplitude of Ca(2+) transient in cardiomyocytes was increased in mutant mice, and maximum isometric force produced by intact cardiac muscle fibers of mutant mice was not significantly different from that of wild-type mice, suggesting that Ca(2+) transient was augmented to compensate for decreased myofilament Ca(2+) sensitivity. Nevertheless, mutant mice developed marked cardiac enlargement, heart failure, and frequent sudden death recapitulating the phenotypes of dilated cardiomyopathy patients, indicating that global functional defect of the heart attributable to decreased myofilament Ca(2+) sensitivity could not be fully compensated by only increasing the intracellular Ca(2+) transient. We found that a positive inotropic agent, pimobendan, which directly increases myofilament Ca(2+) sensitivity, had profound effects of preventing cardiac enlargement, heart failure, and sudden death. These results verify the hypothesis that Ca(2+) desensitization of cardiac myofilament is the absolute cause of the pathogenesis of dilated cardiomyopathy associated with this mutation and strongly suggest that Ca(2+) sensitizers are beneficial for the treatment of dilated cardiomyopathy patients affected by sarcomeric regulatory protein mutations.

Full-length p40phox structure suggests a basis for regulation mechanism of its membrane binding.

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.

Phagocytosis-coupled activation of the superoxide-producing phagocyte oxidase, a member of the NADPH oxidase (nox) family.

The phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a crucial role in host defense by neutrophils and macrophages. When cells ingest invading microbes, this enzyme becomes activated to reduce molecular oxygen to superoxide, a precursor of microbicidal oxidants, in the phagosome. The catalytic core of the oxidase is membrane-bound cytochrome b558, which comprises gp91phox and p22phox. gp91phox belongs to the NADPH oxidase (Nox) family, which contains the entire electron-transporting apparatus from NADPH to molecular oxygen. In resting neutrophils, cytochrome b558 is mainly present in the membrane of the specific granule, an intracellular component, and is targeted to the phagosomal membrane during phagocytosis. Activation of gp91phox involves the integrated function of cytoplasmic proteins such as p47phox, p67phox, p40phox, and the small guanosine triphosphatase Rac; these proteins translocate to the phagosomal membrane to interact with cytochrome b558, leading to superoxide production. Here we describe a current molecular model for phagocytosis-coupled activation of the NADPH oxidase.

A novel protein specifically interacting with Homer2 regulates ubiquitin-proteasome systems.

Homer family proteins are encoded by three genes, homer1, 2 and 3. Most of these proteins are expressed constitutively in nervous systems and accumulated in postsynaptic regions. However, the functional significance of these proteins, especially the significance of the distinction among the proteins encoded by homer1, 2 and 3, is still obscure. In the present study, we isolated a cDNA clone encoding a novel protein by two-hybrid system screening using the C-terminal half of Homer2b as the bait. This protein, termed 2B28, has 297 amino acid residues and contains three major domains: a UBA domain, a coiled-coil region, and a UBX domain. When expressed in HEK293T cells, 2B28 showed colocalization with uniquitin and enhanced the expression levels of IkappaB or Homer1a proteins, which are known to be degraded by proteasomes, indicating that 2B28 is involved in ubiquitin-proteasome functions. 2B28 specifically interacted and colocalized with Homer2 proteins, but not with Homer1 proteins. So far, we have identified no counterpart of 2B28 for Homer1 experimentally or in the protein databases. These results suggest that the specific interaction of 2B28 with Homer2 may play a role in regulation of protein degradation by ubiquitin-proteasome systems and that this function may be specific to Homer2 proteins among Homer family proteins.

Catalytic residues of group VIB calcium-independent phospholipase A2 (iPLA2gamma).

Although human group VIB calcium-independent phospholipase A(2) (iPLA(2)gamma) contains the lipase-consensus sequence Gly-Xaa-Ser-Xaa-Gly in the C-terminal half, its overall sequence exhibits a week similarity to those of other PLA(2)s, and thus no information on the catalytic site has been available. Here we show that the C-terminal region of human iPLA(2)gamma is responsible for the enzymatic activity. Comparison of this catalytic domain with those of the mouse homologue, human cytosolic PLA(2) (cPLA(2)), and the plant PLA(2) patatin reveals that an amino acid sequence of a short segment around Asp-627 of iPLA(2)gamma is conserved among these PLA(2)s, in addition to the Ser-483-containing lipase motif; the corresponding serine and aspartate in cPLA(2) and patatin are known to form a catalytic dyad. Since substitution of alanine for either Ser-483 or Asp-627 results in a loss of the PLA(2) activity, we propose that Ser-483 and Asp-627 of human iPLA(2)gamma constitute an active site similar to the Ser-Asp dyad in cPLA(2) and patatin.