BBA - General Subjects (v.1722, #3)
Editorial Board (ii).
Nine new human Rhodopsin family G-protein coupled receptors: identification, sequence characterisation and evolutionary relationship by David E.I. Gloriam; Helgi B. Schiöth; Robert Fredriksson (235-246).
We report nine new members of the Rhodopsin family of human G protein-coupled receptors (GPCRs) found by searches in the genome databases. BLAST searches and phylogenetic analyses showed that only four of the receptors are closely related to previously characterised GPCRs, GPR150 and GPR154 to oxytocin/vasopressin receptors, GPR152 to CRTH2/FPRs and GPR165 to GPR72/NPYR. Four of the receptors, GPR139, GPR146, GPR153 and GPR162, have one other orphan GPCRs as close relative while GPR148 lacks close relatives. We have identified in total 37 orthologues for the new receptors, primarily from rat, mouse, chicken, fugu and zebrafish. GPR162 and GPR139 are remarkably well conserved while GPR148 seems to be evolving rapidly. Analyses using expressed sequence tags (ESTs) indicate that all the new receptors except GPR153 have the CNS as a major site of expression.
Keywords: Rhodopsin; G-protein coupled receptor; Glutamate, Rhodopsin, adhesion, frizzled/taste2 and secretin; Monoamine; Orphan; Phylogeny;
Apolipoprotein E3 (apoE3) safeguards pig proximal tubular LLC-PK1 cells against reduction in SGLT1 activity induced by gentamicin C by Kozo Takamoto; Manabu Kawada; Daishiro Ikeda; Motonobu Yoshida (247-253).
Megalin, a family of endocytic receptors related to the low-density lipoprotein (LDL) receptor, is a major pathway for proximal tubular aminoglycoside accumulation. We previously reported that aminoglycoside antibiotics reduce SGLT1-dependent glucose transport in pig proximal tubular epithelial LLC-PK1 cells in parallel with the order of their nephrotoxicity. In this study, using a model of gentamicin C (GMC)-induced reduction in SGLT1 activity, we examined whether ligands for megalin protect LLC-PK1 cells from the GMC-induced reduction in SGLT1 activity. We employed apolipoprotein E3 (apoE3) and lactoferrin as ligands for megalin. Then the cells were treated with various concentrations of apoE3, lactoferrin and bovine serum albumin with or without 100 μg/ml of GMC, and the SGLT1-dependent methyl α-d-glucopyranoside (AMG) uptake and levels of SGLT1 expression were determined. As a result, we demonstrated that the apoE3 significantly protects these cells from GMC-induced reduction in AMG uptake, but neither lactoferrin nor albumin does. In accord with a rise in AMG uptake activity, the mRNA and protein levels of SGLT1 were apparently up-regulated in the presence of apoE3. Furthermore, we found that the uptake of [3H] gentamicin is decreased by apoE3, and that apoE3 showed obvious protection against the GMC-dependent N-acetyl-β-d-glucosamidase (NAG) release from LLC-PK1 cells. Thus, these results indicate that apoE3 could be a valuable tool for the prevention of aminoglycoside nephrotoxicity.
Keywords: Apolipoprotein E3; SGLT1; Nephrotoxicity; LLC-PK1 cell; N-acetyl-β-d-glucosamidase;
Expression and purification of the catalytic domain of human vascular endothelial growth factor receptor 2 for inhibitor screening by Li Zhong; Xiao-Ning Guo; Xiu-Hua Zhang; Zhi-Xing Wu; Xiao-min Luo; Hua-Liang Jiang; Li-Ping Lin; Xiong-Wen Zhang; Jian Ding (254-261).
Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, can act in tumor-induced angiogenesis by binding to specific receptors on the surface of endothelial cells. One such receptor, VEGFR-2/KDR, plays a key role in VEGF-induced angiogenesis. Here, we expressed the catalytic domain of VEGFR-2 as a soluble active kinase using Bac-to-Bac expression system, and investigated correlations between VEGFR-2 activity and enzyme concentration, ATP concentration, substrate concentration and divalent cation type. We used these data to establish a convenient, effective and non-radioactive ELISA screening technique for the identification and evaluation of potential inhibitors for VEGFR-2 kinase. We screened 200 RTK target-based compounds and identified one (TKI-31) that potently inhibited VEGFR-2 kinase activity (IC50=0.596 μM). Treatment of NIH3T3/KDR cells with TKI-31 blocked VEGF-induced phosphorylation of KDR in a dose-dependent manner. Moreover, TKI-31 dose-dependently suppressed HUVEC tube formation. Thus, we herein report a novel, efficient method for identifying VEGFR-2 kinase inhibitors and introduce one, TKI-31, that may prove to be a useful new angiogenesis inhibitor.
Keywords: KDR; Bac-to-Bac; Angiogenesis inhibitor; TKI-31;
Loss-of-function mutations identified in the Helical domain of the G protein α-subunit, Gα2, of Dictyostelium discoideum by Robert E. Gundersen; Jianxin You; Steven Rauch; Kate Farnham; Christopher McCarty; Nicholas Willis; Alison Prince (262-270).
The guanine nucleotide binding regulatory proteins (G proteins) play essential roles in a wide variety of physiological processes, such as vision, hormone responses, olfaction, immune response, and development. The heterotrimeric G proteins consist of α-, β-, and γ-subunits and act as molecular switches to relay information from transmembrane receptors to intracellular effectors. The switch mechanism is a function of the inherent GTPase activity of the α-subunit. The α-subunit is comprised of two domains, the GTPase domain and the Helical domain. The GTPase domain performs all of the known α-subunit functions while little is know about the role of the Helical domain. To gain a better understanding of α-subunit function, we performed a screen for loss-of-function mutations, using the Gα2-subunit of Dictyostelium. Gα2 is essential for the developmental life cycle of Dictyostelium. It is known that the loss of Gα2 function results in a failure of cells to enter the developmental phase, producing a visibly abnormal phenotype. This allows the easy identification of amino acids essential to Gα2 function. A library of random point mutations in the gα2 cDNA was constructed using low fidelity polymerase chain reaction (PCR). The library was then expressed in a gα2 null cell line and screened for loss-of-function mutations. Mutations were identified in isolated clones by sequencing the gα2 insert. To date, sixteen single amino acids changes have been identified in Gα2 which result in loss-of-function. Of particular interest are seven mutations found in the Helical domain of the α-subunit. These loss-of-function mutations in the α-subunit Helical domain may provide important insight into its function.
Keywords: Signal transduction; G proteins; Dictyostelium;
Surfactant-induced refolding of lysozyme by Run-Chao Lu; Jin-Xin Xiao; Ao-Neng Cao; Lu-Hua Lai; Bu-Yao Zhu; Guo-Xi Zhao (271-281).
The surfactant–lysozyme interaction was investigated by circular dichroism, fluorescence, UV, dynamic light scattering, surface tension, turbidity measurements and lysozyme activity assay. A new way of refolding of lysozyme was found. It was shown that the lysozyme unfolded by anionic surfactants could be renatured by adding cationic surfactants. That is, lysozyme formed precipitate with anionic surfactants, the precipitates could be dissolved by adding a cationic surfactant solution, and then the lysozyme was refolded to its native state spontaneously. Different couples of anionic surfactants and cationic surfactants including C10SO3/C10NE, C12SO3/C10NE, C10SO3/C12NE, C10SO3/C12NB, C10SO4/C10NE and C12SO4/C10NE (C n SO3, C n SO4, C n NE and C n NB represent sodium alkyl sulfonate/sulfate, alkyl triethyl/butyl ammonium bromide respectively) were investigated, all of them gave similar results. The results were explained in terms of the differences between the interaction of anionic–cationic surfactants and that of surfactant–lysozyme. It was thought that the formation of mixed micelles of anionic–cationic surfactants is a more favorable process than that of lysozyme–surfactant complexes, which induces the dissociation of lysozyme–surfactant complexes when cationic surfactants were added.
Keywords: Anionic surfactant; Cationic surfactant; Unfolding; Refolding; Lysozyme; Cationic–anionic surfactant mixed micelles;
Characterization of the putative native and recombinant rat sterol transporter Niemann-Pick C1 Like 1 (NPC1L1) protein by Sai Prasad N. Iyer; Xiaorui Yao; James H. Crona; Lizbeth M. Hoos; Glen Tetzloff; Harry R. Davis; Michael P. Graziano; Scott W. Altmann (282-292).
The exact mechanistic pathway of cholesterol absorption in the jejunum of the small intestines is a poorly understood process. Recently, a relatively novel gene, Niemann-Pick C1 Like 1 (NPC1L1), was identified as being critical for intestinal sterol absorption in a pathway which is sensitive to sterol absorption inhibitors such as ezetimibe. NPC1L1 is a multi-transmembrane protein, with a putative sterol sensing domain. Very little else is known about the NPC1L1 protein. In this report, we characterize the native and recombinant rat NPC1L1 protein. We show that NPC1L1 is a 145 kDa membrane protein, enriched in the brush border membrane of the intestinal enterocyte and is highly glycosylated. In addition, sequential detergent extraction of enterocytes result in highly enriched preparations of NPC1L1. An engineered Flag epitope tagged rat NPC1L1 cDNA was expressed as recombinant protein in CHO cells and demonstrated cell surface expression, similar to the native rat protein. These biochemical data indicate that NPC1L1 exists as a predominantly cell surface membrane expressed protein, consistent with its proposed role as the putative intestinal sterol transporter.
Keywords: NPC1L1; Ezetimibe; Intestinal cholesterol absorption;
Characterization of Haemonchus contortus calreticulin suggests its role in feeding and immune evasion by the parasite by Sajja Suchitra; Paritosh Joshi (293-303).
Haemonchus contortus, a gastrointestinal parasite of sheep and goat feeds on the blood of its host and causes bleeding at the biting site. In this report, we demonstrate that the Ca2+ binding protein, calreticulin (CalR), is present in excretory/secretory products of adult worms. The secreted CalR enhanced plasma coagulation time. Using recombinant fragments, this property has been mapped to C-terminal part of the molecule which has binding sites for Ca2+ as well as clotting factors. Complement protein C1q bound to immobilized CalR and C1q dependent lysis of sensitized sheep erythrocytes was inhibited by CalR, a function mapped to N-domain of the protein. Factor X and a 24 kDa polypeptide derived from prothrombin but not prothrombin bound to immobilized CalR. The binding site for 24 kDa polypeptide in the CalR molecule has been localized in the P-domain. Our results suggest at least two functions for secreted CalR: first, to prevent blood clotting by binding to Ca2+ and clotting factors thus enabling parasite to feed on the host blood and second to modulate the host immune response by binding to complement C1q thereby facilitating parasite's survival within the host.
Keywords: Haemonchus contortus; Calreticulin; C1q protein; Domain;
Metabolism of 1,8-cineole by human cytochrome P450 enzymes: identification of a new hydroxylated metabolite by Mike Duisken; Frank Sandner; Brunhilde Blömeke; Juliane Hollender (304-311).
Human metabolism of the monoterpene cyclic ether 1,8-cineole was investigated in vitro and in vivo. In vitro, the biotransformation of 1,8-cineole was investigated by human liver microsomes and by recombinant cytochrome P450 enzymes coexpressed with human CYP-reductase in Escherichia coli cells. Besides the already described metabolite 2α-hydroxy-1,8-cineole we found another metabolite produced at high rates. The structure was identified by a comparison of its mass spectrum and retention time with the reference compounds as 3α-hydroxy-1,8-cineole. There was a clear correlation between the concentration of the metabolites, incubation time and enzyme content, respectively. CYP3A4/5 antibody significantly inhibited the 2α- and 3α-hydroxylation catalyzed by pooled human liver microsomes. Further kinetic analysis revealed that the Michaelis–Menten K m and V max for oxidation of 1,8-cineole in position three were 19 μM and 64.5 nmol/min/nmol P450 for cytochrome P450 3A4, and 141 μM and 10.9 nmol/min/nmol P450 for cytochrome P450 3A5, respectively. To our knowledge, this is the first time that 3α-hydroxy-1,8-cineole is described as a human metabolite of 1,8-cineole. We confirmed these in vitro results by the investigation of human urine after the oral administration of cold medication containing 1,8-cineole. In human urine we found by GC-MS analysis the described metabolites, 2α-hydroxy-1,8-cineole and 3α-hydroxy-1,8-cineole.
Keywords: Metabolism; 1,8-Cineole; Cytochrome P450; CYP3A; 2α-Hydroxy-1,8-cineole; 3α-Hydroxy-1,8-cineole;
Stepwise binding of nickel to horseradish peroxidase and inhibition of the enzymatic activity by Jacqueline Keyhani; Ezzatollah Keyhani; Sekineh Zarchipour; Hossein Tayefi-Nasrabadi; Nahid Einollahi (312-323).
The incubation of horseradish peroxidase C (HRPC) with millimolar concentrations of nickel, at room temperature and at pH 4.0, induced the progressive formation of a metal–enzyme complex characterized by alterations of the enzyme Soret absorption band that were time- as well as nickel concentration- dependent. For any given incubation period between 1 and 60 min, 2 values for the apparent dissociation constant (K d) were found, suggesting the presence of binding sites with different affinities for nickel. The value of each K d dropped as the incubation time increased, indicating a progressive stabilization of the metal–enzyme complex. Hill plots suggested a cooperative binding of up to four Ni2+ ions per molecule of HRPC. The inhibition of the enzymatic activity by nickel was studied by following the H2O2-mediated oxidation of o-dianisidine by HRPC under steady-state kinetic conditions. Ni2+ was found to be either a noncompetitive or a mixed inhibitor of HRPC depending both on the duration of preincubation with the enzyme and on Ni2+ concentration. The enzyme remained active only over a limited metal concentration range and data indicated that binding of one Ni2+ affected the substrate binding site, binding of a second Ni2+ affected both substrate and peroxide binding sites, and binding of more than 2 Ni2+ per HRPC molecule led to complete loss of enzymatic activity. Results pointed to the damaging effects of prolonged exposure to heavy metals and also to the existence of a critical metal concentration beyond which immediate abolishing of enzymatic activity was observed.
Keywords: Nickel; Horseradish peroxidase; Enzyme inhibition; Dissociation constant; Cooperativity; Critical metal concentration;
Correlation between the intracellular content of glutathione and the formation of germ-tubes induced by human serum in Candida albicans by Pilar González-Párraga; Francisco R. Marín; Juan-Carlos Argüelles; José A. Hernández (324-330).
The physiological role of the tripeptide glutathione (GSH) and its oxidized form (GSSG) was investigated during the initial steps of dimorphism (formation of germ-tubes), which is induced by human serum in exponential yeast-like cells (blastoconidia) of the Candida albicans strain CAI-4 (wild type) and its congenic tps1/tps1 mutant, deficient in trehalose synthesis. The content of glutathione, measured both as GSH and the ratio GSH/GSSG, underwent a moderate drop in parallel with the induction of a significant degree of germ-tube emergence. Whereas the supply of exogenous glutathione did not affect the degree of dimorphic transition, depletion of intracellular glutathione by addition of 1-chloro-2,4 dinitrobenzene (CDNB) caused a clear reduction in the percentage of hyphae formation; although this effect must be due to the severe cell mortality produced by CDNB. Simultaneous measurements of GSH-metabolizing activities revealed a moderate decrease of glutathione reductase concomitant with the activation of glutathione peroxidase. In turn, catalase activity did not show noticeable changes. The putative correlation between the redox status of glutathione and the dimorphic conversion in C. albicans is discussed.
Keywords: Glutathione; Dimorphism; Glutathione reductase; Glutathione peroxidase; Candida albicans;
Identification of a key amino acid residue of Streptomyces phospholipase D for thermostability by in vivo DNA shuffling by Tomofumi Negishi; Takafumi Mukaihara; Koichi Mori; Hiroko Nishikido; Yuko Kawasaki; Hiroyuki Aoki; Michiko Kodama; Hatsuho Uedaira; Yoshiko Uesugi; Masaki Iwabuchi; Tadashi Hatanaka (331-342).
To isolate thermostability-related amino acid residues of Streptomyces phospholipase D (PLD), we constructed a chimeral genes library between two highly homologous plds, which exhibited different thermostabilities, by an in vivo DNA shuffling method using Escherichia coli that has a mutation of a single-stranded DNA-binding protein gene. To confirm the location of the recombination site, we carried out the restriction mapping of 68 chimeral pld genes. The recombination sites were widely dispersed over the entire pld sequence. Moreover, we examined six chimeral PLDs by comparing their thermostabilities with those of parental PLDs. To identify a thermostability-related amino acid residue, we investigated the thermostability of chimera C that was the most thermolabile among the six chimeras. We identified the thermostability-related factor Gly-188, which is located in the alpha-7 helix of PLD from Streptomyces septatus TH-2 (TH-2PLD). TH-2PLD mutants, in which Gly-188 was substituted with Phe, Val or Trp, exhibited higher thermostabilities than that of the parental PLD. Gly-188 substituted with the Phe mutant, which was the most stable among the mutants, showed an enzyme activity almost the same as that of TH-2PLD as determine by kinetic analysis.
Keywords: Phospholipase D; Thermostability; Streptomyces; Biocatalyst;
Author Index (343-345).
Cumulative Contents (346-348).