Kimie Murayama

Inactivation of Human Manganese-superoxide Dismutase by Peroxynitrite Is Caused by Exclusive Nitration of Tyrosine 34 to 3-Nitrotyrosine

Peroxynitrite has recently been implicated in the inactivation of many enzymes. However, little has been reported on the structural basis of the inactivation reaction. This study proposes that nitration of a specific tyrosine residue is responsible for inactivation of recombinant human mitochondrial manganese-superoxide dismutase (Mn-SOD) by peroxynitrite. Mass spectroscopic analysis of the peroxynitrite-inactivated Mn-SOD showed an increased molecular mass because of a single nitro group substituted onto a tyrosine residue. Single peptides that had different elution positions between samples from the native and peroxynitrite-inactivated Mn-SOD on reverse-phase high performance liquid chromatography were isolated after successive digestion of the samples by staphylococcal serine protease and lysylendopeptidase and subjected to amino acid sequence and molecular mass analyses. We found that tyrosine 34 of the enzyme was exclusively nitrated to 3-nitrotyrosine by peroxynitrite. This residue is located near manganese and in a substrate O⨪2 gateway in Mn-SOD.

Altered quinone biosynthesis in the long-lived clk-1 mutants of Caenorhabditis elegans.

Mutations in theclk-1 gene of Caenorhabditis elegans result in an extended life span and an average slowing down of developmental and behavioral rates. However, it has not been possible to identify biochemical changes that might underlie the extension of life span observed in clk-1 mutants, and therefore the function of CLK-1 in C. elegans remains unknown. In this report, we analyzed the effect of clk-1 mutation on ubiquinone (UQ9) biosynthesis and show that clk-1 mutants mitochondria do not contain detectable levels of UQ9. Instead, the UQ9 biosynthesis intermediate, demethoxyubiquinone (DMQ9), is present at high levels. This result demonstrates that CLK-1 is absolutely required for the biosynthesis of UQ9 in C. elegans. Interestingly, the activity levels of NADH-cytochrome creductase and succinate-cytochrome c reductase in mutant mitochondria are very similar to those in the wild-type, suggesting that DMQ9 can function as an electron carrier in the respiratory chain. To test this possibility, the short side chain derivative DMQ2 was chemically synthesized. We find that DMQ2 can act as an electron acceptor for both complex I and complex II in clk-1 mutant mitochondria, while another ubiquinone biosynthesis precursor, 3-hydroxy-UQ2, cannot. The accumulation of DMQ9 and its use in mutant mitochondria indicate, for the first time in any organism, a link between the alteration in the quinone species used in respiration and life span.

Differential involvement of μ-opioid receptor subtypes in endomorphin-1- and -2-induced antinociception

We investigated the role of mu-opioid receptor subtypes in both endomorphin-1 and endomorphin-2 induced antinociception in mice using supraspinally mediated behavior. With tail pressure as a mechanical noxious stimulus, both intracerebroventricularly (i.c.v.) and intrathecally (i.t.) injected-endomorphins produced potent and significant antinociceptive activity. Antinociception induced by i.t. and i.c.v. injection of endomorphin-1 was not reversed by pretreatment with a selective mu1-opioid receptor antagonist, naloxonazine (35 mg/kg, s.c.). By contrast, antinociception induced by i.t. and i.c.v. endomorphin-2 was significantly decreased by mu1-opioid receptor antagonist. Antinociception of both i.t. and i.c.v. endomorphin-1 and -2 was completely reversed by pretreatment with beta-funaltrexamine (40 mg/kg, s.c.). The results indicate that endomorphins may produce antinociception through the distinct mu1 and mu2 subtypes of mu-opioid receptor.

Mite serine protease activates protease-activated receptor-2 and induces cytokine release in human keratinocytes

Background:  House dust mites produce serine and cysteine proteases. Mite‐derived proteases have been suggested to be involved in the pathogenesis of allergies; however, whether mite‐derived serine protease activity can stimulate keratinocytes remains unknown.

TANDEM REPEAT STRUCTURE OF RHAMNOSE-BINDING LECTIN FROM CATFISH (SILURUS ASOTUS) EGGS

The primary structure of catfish (Silurus asotus) egg lectin (SAL) was determined. SAL cDNA contained 1448-bp nucleotides and 308 amino acid residues, deduced from open reading frame. The SAL mature protein composed of 285-amino acid residues was followed by a predicted signal sequence having 23 residues. The mRNA of SAL was found to be expressed in eggs, but not in liver. SAL is composed of three tandem repeat domain structures divided into exactly 95 amino acid residues each, and all cysteine positions of each domain were completely conserved. Sequence homologies between the three domains, termed D1 (1-95), D2 (96-190) and D3 (191-285), were as follows; D1-D2, 28%; D2-D3, 33%; D1-D3, 43%. Two conserved peptide motifs, -(AN)YGR(TD)S(T)XCS(TGR)P- and -DPCX(G)T(Y)KY(L)-, appear to exist at the N- and C-terminal regions of each domain, respectively. The kinetic parameters of SAL obtained by measuring surface plasmon resonance were as follows: K(a) (M(-1)) for neohesperidosyl-BSA, 7. 1 x 10(6); for melibiosyl-BSA, 4.9 x 10(6); and for lactosyl-BSA, 5. 2 x 10(5). These results show that RBLs including SAL comprise a family of alpha-galactosyl binding lectins having characteristic tandem repeat domain structures.

Differential antinociceptive effects induced by intrathecally administered endomorphin-1 and endomorphin-2 in the mouse

Two highly selective mu-opioid receptor agonists, endomorphin-1 and endomorphin-2, have been identified and postulated to be endogenous ligands for mu-opioid receptors. Intrathecal (i.t.) administration of endomorphin-1 and endomorphin-2 at doses from 0.039 to 5 nmol dose-dependently produced antinociception with the paw-withdrawal test. The paw-withdrawal inhibition rapidly reached its peak at 1 min, rapidly declined and returned to the pre-injection levels in 20 min. The inhibition of the paw-withdrawal responses to endomorphin-1 and endomorphin-2 at a dose of 5 nmol observed at 1 and 5 min after injection was blocked by pretreatment with a non-selective opioid receptor antagonist naloxone (1 mg/kg, s.c.). The antinociceptive effect of endomorphin-2 was more sensitive to the mu (1)-opioid receptor antagonist, naloxonazine than that of endomorphin-1. The endomorphin-2-induced paw-withdrawal inhibition at both 1 and 5 min after injection was blocked by pretreatment with kappa-opioid receptor antagonist nor-binaltorphimine (10 mg/kg, s.c.) or the delta(2)-opioid receptor antagonist naltriben (0.6 mg/kg, s.c.) but not the delta(1)-opioid receptor antagonist 7-benzylidine naltrexone (BNTX) (0.6 mg/kg s.c.). In contrast, the paw-withdrawal inhibition induced by endomorphin-1 observed at both 1 and 5 min after injection was not blocked by naloxonazine (35 mg/kg, s.c.), nor-binaltorphimine (10 mg/kg, s.c.), naltriben (0.6 mg/kg, s.c.) or BNTX (0.6 mg/kg s.c.). The endomorphin-2-induced paw-withdrawal inhibition was blocked by the pretreatment with an antiserum against dynorphin A-(1-17) or [Met(5)]enkephalin, but not by antiserum against dynorphin B-(1-13). Pretreatment with these antisera did not affect the endomorphin-1-induced paw-withdrawal inhibition. Our results indicate that endomorphin-2 given i.t. produces its antinociceptive effects via the stimulation of mu (1)-opioid receptors (naloxonazine-sensitive site) in the spinal cord. The antinociception induced by endomophin-2 contains additional components, which are mediated by the release of dynorphin A-(1-17) and [Met(5)]enkephalin which subsequently act on kappa-opioid receptors and delta(2)-opioid receptors to produce antinociception.

Differential antagonism of endomorphin-1 and endomorphin-2 spinal antinociception by naloxonazine and 3-methoxynaltrexone

To determine the role of spinal mu-opioid receptor subtypes in antinociception induced by intrathecal (i.t.) injection of endomorphin-1 and -2, we assessed the effects of beta-funaltrexamine (a selective mu-opioid receptor antagonist) naloxonazine (a selective antagonist at the mu(1)-opioid receptor) and a novel receptor antagonist (3-methoxynaltrexone) using the paw-withdrawal test. Antinociception of i.t. endomorphins and [D-Ala(2), MePhe(4), Gly(ol)(5)]enkephalin (DAMGO) was completely reversed by pretreatment with beta-funaltrexamine (40 mg/kg s.c.). Pretreatment with a variety of doses of i.t. or s.c. naloxonazine 24 h before testing antagonized the antinociception of endomorphin-1, -2 and DAMGO. Judging from the ID(50) values of naloxonazine, the antinociceptive effect of endomorphin-2 was more sensitive to naloxonazine than that of endomorphin-1 or DAMGO. The selective morphine-6beta-glucuronide antagonist, 3-methoxynaltrexone, which blocked endomorphin-2-induced antinociception at each dose (0.25 mg/kg s.c. or 2.5 ng i.t.) that was inactive against DAMGO, did not affect endomorphin-1-induced antinociception but shifted the dose-response curve of endomorphin-2 3-fold to the right. These findings may be interpreted as indicative of the existence of a novel mu-opioid receptor subtype in spinal sites, where antinociception of morphine-6beta-glucuronide and endomorphin-2 are antagonized by 3-methoxynaltrexone. The present results suggest that endomorphin-1 and endomorphin-2 may produce antinociception through different subtypes of mu-opioid receptor.

Involvement of very long fatty acid-containing lactosylceramide in lactosylceramide-mediated superoxide generation and migration in neutrophils

The neutral glycosphingolipid lactosylceramide (LacCer) forms lipid rafts (membrane microdomains) coupled with the Src family kinase Lyn on the plasma membranes of human neutrophils; ligand binding to LacCer activates Lyn, resulting in neutrophil functions, such as superoxide generation and migration (Iwabuchi and Nagaoka, Lactosylceramide-enriched glycosphingolipid signaling domain mediates superoxide generation from human neutrophils, Blood 100, 1454–1464, 2002 and Sato et al. Induction of human neutrophil chemotaxis by Candida albicans-derived beta-1,6-long glycoside side-chain-branched beta glycan, J. Leukoc. Biol. 84, 204–211, 2006). Neutrophilic differentiated HL-60 cells (D-HL-60 cells) express almost the same amount of LacCer as neutrophils. However, D-HL-60 cells do not have Lyn-associated LacCer-enriched lipid rafts and lack LacCer-mediated superoxide-generating and migrating abilities. Here, we examined the roles of LacCer molecular species of different fatty acid compositions in these processes. Liquid chromatography-mass spectrometry analyses revealed that the very long fatty acid C24:0 and C24:1 chains were the main components of LacCer (31.6% on the total fatty acid content) in the detergent-resistant membrane fraction (DRM) from neutrophil plasma membranes. In contrast, plasma membrane DRM of D-HL-60 cells included over 70% C16:0-LacCer, but only 13.6% C24-LacCer species. D-HL-60 cells loaded with C24:0 or C24:1-LacCer acquired LacCer-mediated migrating and superoxide-generating abilities, and allowed Lyn coimmunoprecipitation by anti-LacCer antibody. Lyn knockdown by siRNA completely abolished the effect of C24:1-LacCer loading on LacCer-mediated migration of D-HL-60 cells. Immunoelectron microscopy revealed that LacCer clusters were closely associated with Lyn molecules in neutrophils and C24:1-LacCer-loaded D-HL-60 cells, but not in D-HL-60 cells or C16:0-LacCer-loaded cells. Taken together, these observations suggest that LacCer species with very long fatty acids are specifically necessary for Lyn-coupled LacCer-enriched lipid raft-mediated neutrophil superoxide generation and migration.

Sphingosine-dependent Protein Kinase-1, Directed to 14-3-3, Is Identified as the Kinase Domain of Protein Kinase Cδ

Some protein kinases are known to be activated by d-erythro-sphingosine (Sph) or N,N-dimethyl-d-erythro-sphingosine (DMS), but not by ceramide, Sph-1-P, other sphingolipids, or phospholipids. Among these, a specific protein kinase that phosphorylates Ser60, Ser59, or Ser58 of 14-3-3β, 14-3-3η, or 14-3-3ζ, respectively, was termed “sphingosine-dependent protein kinase-1” (SDK1) (Megidish, T., Cooper, J., Zhang, L., Fu, H., and Hakomori, S. (1998) J. Biol. Chem. 273, 21834–21845). We have now identified SDK1 as a protein having the C-terminal half kinase domain of protein kinase Cδ (PKCδ) based on the following observations. (i) Large-scale preparation and purification of proteins showing SDK1 activity from rat liver (by six steps of chromatography) gave a final fraction with an enhanced level of an ∼40-kDa protein band. This fraction had SDK1 activity ∼50,000-fold higher than that in the initial extract. (ii) This protein had ∼53% sequence identity to the Ser/Thr kinase domain of PKCδ based on peptide mapping using liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry data. (iii) A search for amino acid homology based on the BLAST algorithm indicated that the only protein with high homology to the ∼40-kDa band is the kinase domain of PKCδ. The kinase activity of PKCδ did not depend on Sph or DMS; rather, it was inhibited by these sphingoid bases, i.e. PKCδ did not display any SDK1 activity. However, strong SDK1 activity became detectable when PKCδ was incubated with caspase-3, which releases the ∼40-kDa kinase domain. PKCδ and SDK1 showed different lipid requirements and substrate specificity, although both kinase activities were inhibited by common PKC inhibitors. The high susceptibility of SDK1 to Sph and DMS accounts for their important modulatory role in signal transduction.

Characterization of Cln3p, the gene product responsible for juvenile neuronal ceroid lipofuscinosis, as a lysosomal integral membrane glycoprotein

Juvenile neuronal ceroid lipofuscinosis (JNCL) is an autosomal recessively inherited lysosomal storage disease involving a mutation in the CLN3 gene. The sequence of CLN3 was determined in 1995; however, the localization of the CLN3 gene product (Cln3p) was not confirmed. In this study, we investigated endogenous Cln3p using two peptide antibodies raised against two distinct epitopes of murine Cln3p. Identification of the liver 60 kDa protein as Cln3p was ascertained by amino acid sequence analysis using tandem mass spectrometry. Liver Cln3p was predominantly localized in the lysosomal membranes, not in endoplasmic reticulum (ER) or Golgi apparatus. As the tissue concentration of brain Cln3p was much lower than that of liver Cln3p, it could be detected only after purification from brain extract using anti‐Cln3p IgG Sepharose. The apparent molecular masses of liver Cln3p and brain Cln3p were determined to be about 60 kDa and 55 kDa, respectively. Both brain and liver Cln3p were deglycosylated by PNGase F treatment to form polypeptides with almost the same molecular mass (45 kDa). However, they were not affected by Endo h treatment. In addition, it was also elucidated that the amino terminal region of Cln3p faces the cytosol.