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Biomedical subjects

Hideki Sumimoto

Publications and source records attributed to Hideki Sumimoto.

At least 55 records · Page 3Linked to original sources
PubMed2004

Solution structure of atypical protein kinase C PB1 domain and its mode of interaction with ZIP/p62 and MEK5.

Atypical protein kinase C (aPKC) has been implicated in several signaling pathways such as cell polarity, cell survival, and cell differentiation. In contrast to other PKCs, aPKC is unique in having the PB1 (Phox and Bem 1) domain in the N terminus. The aPKC PB1 domain binds with ZIP/p62, Par6, or MEK5 through a PB1-PB1 domain interaction that controls the localization of aPKC. Here, we determined the three-dimensional structure of the PB1 domain of PKCiota by NMR and found that the PB1 domain adopts a ubiquitin fold. The OPCA (OPR, PC, and AID) motif inserted into the ubiquitin fold was presented as a betabetaalpha fold in which the side chains of conserved Asp residues were oriented to the same direction to form an acidic surface. This structural feature suggested that the acidic surface of the PKCiota PB1 domain interacted with the basic surface of the target PB1 domains, and this was confirmed in the case of the PKCiota-ZIP/p62 complex by mutational analysis. Interestingly, in the PKCiota PB1 domain a conserved lysine residue was located on the side opposite to the OPCA motif-presenting surface, suggesting dual roles for the PKCiota PB1 domain in that it could interact with either the conserved lysine residue or the acidic residues on the OPCA motif of the target PB1 domains.

Amino Acid Sequence↗
PubMed2004

Solution structure of the tandem Src homology 3 domains of p47phox in an autoinhibited form.

The phagocyte NADPH oxidase is a multisubunit enzyme responsible for the generation of superoxide anions (O(2).) that kill invading microorganisms. p47(phox) is a cytosolic subunit of the phagocyte NADPH oxidase, which plays a crucial role in the assembly of the activated NADPH oxidase complex. The molecular shapes of the p47(phox) tandem SH3 domains either with or without a polybasic/autoinhibitory region (PBR/AIR) at the C terminus were studied using small angle x-ray scattering. The tandem SH3 domains with PBR/AIR formed a compact globular structure, whereas the tandem SH3 domains lacking the PBR/AIR formed an elongated structure. Alignment anisotropy analysis by NMR based on the residual dipolar couplings revealed that the tandem SH3 domains with PBR/AIR were in good agreement with a globular module corresponding to the split half of the intertwisted dimer in crystalline state. The structure of the globular module was elucidated to represent a solution structure of the tandem SH3 domain in the autoinhibited form, where the PBR/AIR bundled the tandem SH3 domains and the linker forming a closed structure. Once PBR/AIR is released by phosphorylation, rearrangements of the SH3 domains may occur, forming an open structure that binds to the cytoplasmic proline-rich region of membrane-bound p22(phox).

Dimerization↗
PubMed2004

Binding of FAD to cytochrome b558 is facilitated during activation of the phagocyte NADPH oxidase, leading to superoxide production.

The superoxide-producing phagocyte NADPH oxidase can be reconstituted in a cell-free system. The activity of NADPH oxidase is dependent on FAD, but the physiological status of FAD in the oxidase is not fully elucidated. To clarify the role of FAD in NADPH oxidase, FAD-free full-length recombinant p47(phox), p67(phox), p40(phox), and Rac were prepared, and the activity was reconstituted with these proteins and purified cytochrome b(558) (cyt b(558)) with different amounts of FAD. A remarkably high activity, over 100 micromol/s/micromol heme, was obtained in the oxidase with purified cyt b(558), ternary complex (p47-p67-p40(phox)), and Rac. From titration with FAD of the activity of NADPH oxidase reconstituted with purified FAD-devoid cyt b, the dissociation constant K(d) of FAD in cyt b(558) of reconstituted oxidase was estimated as nearly 1 nm. We also examined addition of FAD on the assembly process in reconstituted oxidase. The activity was remarkably enhanced when FAD was present during assembly process, and the efficacy of incorporating FAD into the vacant FAD site in purified cyt b(558) increased, compared when FAD was added after assembly processes. The absorption spectra of reconstituted oxidase under anaerobiosis showed that incorporation of FAD into cyt b(558) recovered electron flow from NADPH to heme. From both K(d) values of FAD and the amount of incorporated FAD in cyt b(558) of reconstituted oxidase, in combination with spectra, we propose the model in which the K(d) values of FAD in cyt b(558) is changeable after activation and FAD binding works as a switch to regulate electron transfer in NADPH oxidase.

Animals↗
PubMed2004

Role of nicotinamide adenine dinucleotide phosphate oxidase 1 in oxidative burst response to Toll-like receptor 5 signaling in large intestinal epithelial cells.

The NADPH oxidase 1 (Nox1) is a gp91(phox) homologue preferentially expressed in the colon. We have established primary cultures of guinea pig large intestinal epithelial cells giving 90% purity of surface mucous cells. These cells spontaneously released superoxide anion (O(2)(-)) of 160 nmol/mg protein/h and expressed the Nox1, p22(phox), p67(phox), and Rac1 mRNAs, but not the gp91(phox), Nox4, p47(phox), p40(phox), and Rac2 mRNAs. They also expressed novel homologues of p47(phox) and p67(phox) (p41(nox) and p51(nox), respectively). Human colon cancer cell lines (T84 and Caco2 cells) expressed the Nox1, p22(phox), p51(nox), and Rac1 mRNAs, but not the other NADPH component mRNAs, and secreted only small amounts of O(2)(-) (<2 nmol/mg protein/h). Cotransfection of p41(nox) and p51(nox) cDNAs in T84 cells enhanced PMA-stimulated O(2)(-) release 5-fold. Treatment of the transfected T84 cells with recombinant flagellin (rFliC) from Salmonella enteritidis further augmented the O(2)(-) release in association with the induction of Nox1 protein. The enhanced O(2)(-) production by cotransfection of p41(nox) and p51(nox) vectors further augmented the rFliC-stimulated IL-8 release from T84 cells. T84 cells expressed the Toll-like receptor 5, and rFliC rapidly phosphorylated TGF-beta-activated kinase 1 and TGF-beta-activated kinase 1-binding protein 1. A potent inhibitor for NF-kappaB (pyrrolidine dithiocarbamate) significantly blocked the rFliC-primed increase in O(2)(-) production and induction of Nox1 protein. These results suggest that p41(nox) and p51(nox) are involved in the Nox1 activation in surface mucous cells of the colon, and besides that, epithelial cells discern pathogenicities among bacteria to appropriately operate Nox1 for the host defense.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Induction of cytochrome CYP4F3A in all-trans-retinoic acid-treated HL60 cells.

Cytochrome P-450 CYP4F3A catalyzes the inactivation of leukotriene B(4) by omega-hydroxylation, an activity of which is specifically expressed in human neutrophils. Here, we examined expression of the LTB(4) omega-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 LTB(4) omega-hydroxylating activity in a dose-dependent manner. The time course of the induction of the omega-hydroxylating activity correlates well with that of the superoxide-generating activity, indicative of cell differentiation. ATRA-treated cell microsomes convert LTB(4) 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 LTB(4) omega-hydroxylase activity, based on the following observations: expression of the mRNA for CYP4F3A is observed together with the induction of LTB(4) omega-hydroxylating activity in ATRA-treated HL60 cells; and the apparent K(m) values for the omega-hydroxylation of LTB(4) and lipoxin B(4) by ATRA-treated cell microsomes are essentially the same as those of CYP4F3A in human neutrophil microsomes.

Antineoplastic Agents↗
PubMed2004

A molecular mechanism for autoinhibition of the tandem SH3 domains of p47phox, the regulatory subunit of the phagocyte NADPH oxidase.

The phagocyte NADPH oxidase is a multisubunit enzyme responsible for the production of reactive oxygen species. p47(phox) is a cytosolic component of the NADPH oxidase and plays an important role in the assembly of the activated complex. The structural determination of the tandem SH3 domains of p47(phox) is crucial for elucidation of the molecular mechanism of the activation of p47(phox). We determined the X-ray crystal structure of the tandem SH3 domains with the polybasic/autoinhibitory region (PBR/AIR) of p47(phox). The GAPPR sequence involved in PBR/AIR forms a left-handed polyproline type-II helix (PPII) and interacts with the conserved SH3 binding surfaces of the SH3 domains simultaneously. These SH3 domains are related by a 2-fold pseudosymmetry axis at the centre of the binding groove and interact with the single PPII helix formed by the GAPPR sequence with opposite orientation. In addition, a number of intra-molecular interactions among the SH3 domains, PBR/AIR and the linker tightly hold the architecture of the tandem SH3 domains into the compact structure and stabilize the autoinhibited form synergistically. Phosphorylation of the serine residues in PBR/AIR could destabilize and successively release the intra-molecular interactions. Thus, the overall structure could be rearranged from the autoinhibitory conformation to the active conformation and the PPII ligand binding surfaces on the SH3 domains are now unmasked, which enables their interaction with the target sequence in p22(phox).

Amino Acid Sequence↗
PubMed2004

Molecular mechanism underlying activation of superoxide-producing NADPH oxidases: roles for their regulatory proteins.

The phagocyte NADPH oxidase is dormant in resting cells but becomes activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants, thereby playing a crucial role in host defence. The catalytic core of this enzyme comprises the two membranous subunits gp91phox/Nox2 and p22phox. The oxidase activation requires the small GTPase Rac and the SH3 domain-containing proteins p47phox and p67phox; they normally exist in the cytoplasm and translocate upon cell stimulation to the membrane. The translocation depends on a stimulus-induced conformational change of p47phox, which leads to the SH3 domain-mediated interaction with p22phox, a binding required for the gp91phox/Nox2-dependent superoxide production. Activation of Nox1, an oxidase that is likely involved in host defence at the colon, requires novel proteins homologous to p47phox and p67phox, designated Noxo1 and Noxa1, respectively. Noxo1 and Noxa1, both expressed abundantly in the colon, are capable of constitutively activating Nox1. The constitutive activation may be due to the property of Noxo1: in contrast with p47phox, Noxo1 seems to normally associate with p22phox, which is required for the Nox1 activation. We will also describe the mechanism underlying regulation of the third oxidase Nox3, which exits in fetal kidney and inner ears.

Animals↗
PubMed2003

Molecular mechanism for activation of superoxide-producing NADPH oxidases.

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 Noxal (Nox activator 1), respectively, has shed lights on common and distinct mechanisms underlying activations of Nox family oxidases.

Animals↗
PubMed2003

Fhos, a mammalian formin, directly binds to F-actin via a region N-terminal to the FH1 domain and forms a homotypic complex via the FH2 domain to promote actin fiber formation.

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.

Actins↗
PubMed2003

The PB1 domain and the PC motif-containing region are structurally similar protein binding modules.

The PC motif is evolutionarily conserved together with the PB1 domain, a binding partner of the PC motif-containing protein. For interaction with the PB1 domain, the PC motif-containing region (PCCR) comprising the PC motif and its flanking regions is required. Because the PB1 domain and the PCCR are novel binding modules found in a variety of signaling proteins, their structural and functional characterization is crucial. Bem1p and Cdc24p interact through the PB1-PCCR interaction and regulate cell polarization in budding yeast. Here, we determined a tertiary structure of the PCCR of Cdc24p by NMR. The tertiary structure of the PCCR is similar to that of the PB1 domain of Bem1p, which is classified into a ubiquitin fold. The PC motif portion takes a compact betabetaalpha-fold, presented on the ubiquitin scaffold. Mutational studies indicate that the PB1-PCCR interaction is mainly electrostatic. Based on the structural information, we group the PB1 domains and the PCCRs into a novel family, named the PB1 family. Thus, the PB1 family proteins form a specific dimer with each other.

Amino Acid Motifs↗
PubMed2003

Molecular recognition in dimerization between PB1 domains.

The PB1 (Phox and Bem 1) domain is a recently identified module that mediates formation of a heterodimeric complex with other PB1 domain, e.g. the complexes between the phagocyte oxidase activators p67phox and p40phox and between the yeast polarity proteins Bem1p and Cdc24p. These PB1 domains harbor either a conserved lysine residue on one side or an acidic OPCA (OPR/PC/AID) motif around the other side; the lysine of p67phox or Bem1p likely binds to the OPCA of p40phox or Cdc24p, respectively, via electrostatic interactions. To further understand molecular recognition by PB1 domains, here we investigate the interactions mediated by proteins presenting both the lysine and OPCA on a single PB1 domain, namely Par6, atypical protein kinase C (aPKC), and ZIP. Par6 and aPKC form a complex via the interaction of the Par6 lysine with aPKC-OPCA but not via that between the aPKC lysine and Par6-OPCA, thereby localizing to the tight junction of epithelial cells. aPKC also uses its OPCA to interact with ZIP, another protein that has a PB1 domain presenting both the lysine and OPCA, whereas aPKC binds via the conserved lysine to MEK5 in the same manner as ZIP interacts with MEK5. In addition, ZIP can form a homotypic complex via the conserved electrostatic interactions. Thus the PB1 domain appears to be a protein module that fully exploits its two mutually interacting elements in molecular recognition to expand its repertoire of protein-protein interactions.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Crystallization and preliminary crystallographic analysis of the autoinhibited form of the tandem SH3 domain of p47(phox).

p47(phox) is a cytosolic component of the phagocyte NADPH oxidase, which is responsible for the production of the superoxide which kills invasive microorgamisms. A recombinant form of a histidine-tagged tandem SH3 domain of the p47(phox)-containing polybasic autoinhibited region was expressed in Escherichia coli and purified and crystallized by the sitting-drop vapour-diffusion method at 293 K using polyethylene glycol 6000 as a precipitant. Diffraction data were collected to 2.15 A resolution at 100 K using synchrotron radiation. The crystal belongs to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 100.02, c = 44.94 A. The presence of one molecule per asymmetric unit gives a crystal volume per protein mass (V(M)) of 2.6 A(3) Da(-1) and a solvent content of 52% by volume.

Crystallography, X-Ray↗
PubMed2003

Novel human homologues of p47phox and p67phox participate in activation of superoxide-producing NADPH oxidases.

The catalytic core of a superoxide-producing NADPH oxidase (Nox) in phagocytes is gp91phox/Nox2, a membrane-integrated protein that forms a heterodimer with p22phox to constitute flavocytochrome b558. The cytochrome becomes activated by interacting with the adaptor proteins p47phox and p67phox as well as the small GTPase Rac. Here we describe the cloning of human cDNAs for novel proteins homologous to p47phox and p67phox, designated p41nox and p51nox, respectively; the former is encoded by NOXO1 (Nox organizer 1), and the latter is encoded by NOXA1 (Nox activator 1). The novel homologue p41nox interacts with p22phox via the two tandem SH3 domains, as does p47phox. The protein p51nox as well as p67phox can form a complex with p47phox and with p41nox via the C-terminal SH3 domain and binds to GTP-bound Rac via the N-terminal domain containing four tetratricopeptide repeat motifs. These bindings seem to play important roles, since p47phox and p67phox activate the phagocyte oxidase via the same interactions. Indeed, p41nox and p51nox are capable of replacing the corresponding classical homologue in activation of gp91phox. Nox1, a homologue of gp91phox, also can be activated in cells, when it is coexpressed with p41nox and p51nox, with p41nox and p67phox, or with p47phox and p51nox; in the former two cases, Nox1 is partially activated without any stimulants added, suggesting that p41nox is normally in an active state. Thus, the novel homologues p41nox and p51nox likely function together or in combination with a classical one, thereby activating the two Nox family oxidases.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Phosphorylation of p47phox directs phox homology domain from SH3 domain toward phosphoinositides, leading to phagocyte NADPH oxidase activation.

Protein-phosphoinositide interaction participates in targeting proteins to membranes where they function correctly and is often modulated by phosphorylation of lipids. Here we show that protein phosphorylation of p47(phox), a cytoplasmic activator of the microbicidal phagocyte oxidase (phox), elicits interaction of p47(phox) with phosphoinositides. Although the isolated phox homology (PX) domain of p47(phox) can interact directly with phosphoinositides, the lipid-binding activity of this protein is normally suppressed by intramolecular interaction of the PX domain with the C-terminal Src homology 3 (SH3) domain, and hence the wild-type full-length p47(phox) is incapable of binding to the lipids. The W263R substitution in this SH3 domain, abrogating the interaction with the PX domain, leads to a binding of p47(phox) to phosphoinositides. The findings indicate that disruption of the intramolecular interaction renders the PX domain accessible to the lipids. This conformational change is likely induced by phosphorylation of p47(phox), because protein kinase C treatment of the wild-type p47(phox) but not of a mutant protein with the S303304328A substitution culminates in an interaction with phosphoinositides. Furthermore, although the wild-type p47(phox) translocates upon cell stimulation to membranes to activate the oxidase, neither the kinase-insensitive p47(phox) nor lipid-binding-defective proteins, one lacking the PX domain and the other carrying the R90K substitution in this domain, migrates. Thus the protein phosphorylation-driven conformational change of p47(phox) enables its PX domain to bind to phosphoinositides, the interaction of which plays a crucial role in recruitment of p47(phox) from the cytoplasm to membranes and subsequent activation of the phagocyte oxidase.

Amino Acid Substitution↗
PubMed2003

Protein kinase C-dependent increase in reactive oxygen species (ROS) production in vascular tissues of diabetes: role of vascular NAD(P)H oxidase.

Hyperglycemia seems to be an important causative factor in the development of micro- and macrovascular complications in patients with diabetes. Several hypotheses have been proposed to explain the adverse effects of hyperglycemia on vascular cells. Both protein kinase C (PKC) activation and oxidative stress theories have increasingly received attention in recent years. This article shows a PKC-dependent increase in oxidative stress in diabetic vascular tissues. High glucose level stimulated reactive oxygen species (ROS) production via a PKC-dependent activation of NAD(P)H oxidase in cultured aortic endothelial cells, smooth muscle cells, and renal mesangial cells. In addition, expression of NAD(P)H oxidase components were shown to be upregulated in vascular tissues and kidney from animal models of diabetes. Furthermore, several agents that were expected to block the mechanism of a PKC-dependent activation of NAD(P)H oxidase clearly inhibited the increased oxidative stress in diabetic animals, as assessed by in vivo electron spin resonance method. Taken together, these findings strongly suggest that the PKC-dependent activation of NAD(P)H oxidase may be an essential mechanism responsible for increased oxidative stress in diabetes.

Animals↗
PubMed2002

PAR3beta, a novel homologue of the cell polarity protein PAR3, localizes to tight junctions.

The cell polarity protein PAR3, conserved from the nematode to the vertebrate, forms a complex with PAR6 and atypical protein kinase C (aPKC), and the protein complex occurs at the tight junctions in mammalian epithelial cells. Here we have cloned human cDNA for a novel PAR3 homologue, designated PAR3beta, whose messages are present in a variety of tissues and most abundantly expressed in the adult and fetal kidneys. The encoded protein of 1,205 amino acids contains a region homologous to the aPKC-binding domain of PAR3alpha, another human homologue previously identified, and three PDZ domains; the first PDZ domain of PAR3alpha is considered to interact with PAR6. Unexpectedly, in contrast to other PAR3s found in various species, PAR3beta is incapable of binding to any isotypes of PAR6 or aPKC. Nevertheless PAR3beta, expressed intrinsically or extrinsically, localizes to the tight junctions, indicating that the localization does not require the ternary complex formation.

Amino Acid Sequence↗
PubMed2002

The adaptor protein p40(phox) as a positive regulator of the superoxide-producing phagocyte oxidase.

Activation of the superoxide-producing phagocyte NADPH oxidase, crucial in host defense, requires the cytosolic proteins p67(phox) and p47(phox). They translocate to the membrane upon cell stimulation and activate flavocytochrome b(558), the membrane-integrated catalytic core of this enzyme system. The activators p67(phox) and p47(phox) form a ternary complex together with p40(phox), an adaptor protein with unknown function, comprising the PX/PB2, SH3 and PC motif- containing domains: p40(phox) associates with p67(phox) via binding of the p40(phox) PC motif to the p67(phox) PB1 domain, while p47(phox) directly interacts with p67(phox) but not with p40(phox). Here we show that p40(phox) enhances membrane translocation of p67(phox) and p47(phox) in stimulated cells, which leads to facilitated production of superoxide. The enhancement cannot be elicited by a mutant p40(phox) carrying the D289A substitution in PC or a p67(phox) with the K355A substitution in PB1, each being defective in binding to its respective partner. Thus p40(phox) participates in activation of the phagocyte oxidase by regulating membrane recruitment of p67(phox) and p47(phox) via the PB1-PC interaction with p67(phox).

DNA-Binding Proteins↗