Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The non-A beta component (NAC) of Alzheimer's disease amyloid is a newly discovered 35 amino acid peptide found to be closely linked to the beta-amyloid fibrils in senile plaques. Apolipoprotein E (apoE) is another prominent constituent of senile plaques. In vitro studies have shown that apoE binds beta-amyloid (A beta) with high avidity, but it is unknown to what extent apoE interacts with NAC. We examined the interactions between apoE and NAC and found that apoE bound synthetic NAC, forming a complex that resisted reducing agents and separation on SDS-PAGE. The complex could be formed using apoE from either purified human very low density lipoprotein (VLDL) particles, unfractionated human cerebrospinal fluid (CSF), or recombinant protein. The binding was established within 15 min upon mixing, and the interaction between NAC and apoE was dose-dependent and specific as revealed by competition experiments. The NAC-apoE complex was affected by non-physiological pH, but not by reducing agents such as DTT or beta-mercaptoethanol. ApoE exists in different isoforms of which the apoE3 genotype is the most frequent. Notably, the apoE4 genotype has been linked to late-onset Alzheimer's disease. This study presents evidence that apoE3 as well as apoE4 bind NAC, but the binding to apoE4 is about twice as strong as to apoE3. The isoform-specific binding of NAC to apoE may thus play an important role in amyloidogenesis and in the sequestering of apoE in senile plaques during the progress of Alzheimer's disease.
Brain Res Mol Brain Res 1997 Feb
PMID:Isoform-specific binding of human apolipoprotein E to the non-amyloid beta component of Alzheimer's disease amyloid. 903 Jul 4

The successful completion of the fertilization process requires the properly choreographed unsheathing of the tightly packaged sperm once it has been fully incorporated into the egg's cytoplasm. The nuclear and accessory structures of mammalian sperm become stabilized by disulfide bonds (S-S) during epididymal maturation. This stabilization is reversed during fertilization by the reduction of S-S cross-linking, but little is known about the effect of S-S reduction on individual disulfide-hardened structures such as the sperm's connecting piece, fibrous sheath, and mitochondria. Here, we demonstrate the action of the S-S-reducing environment on the mitochondrial sheath of mammalian sperm, visualized by the vital fluorescent probe MitoTracker and by electron microscopy. In both human and bull sperm, mitochondria form a compact helix (mitochondrial sheath) wrapped around the midpiece and connecting piece that can be fluorescently labelled by a short incubation with 100 nM MitoTracker. Exposure of bull sperm to 0.1-10 mM dithiothreitol (DTT; a disulfide bond-reducing agent) induced a time and dose-dependent sliding of the mitochondrial sheath down the axoneme, accompanied by the excision of the sperm tail and decondensation of the sperm nucleus. Increasing the concentration of DTT to 100 mM accelerated mitochondrial movement, causing a completed stripping of sperm mitochondria and partial disassembly of the connecting piece. Likewise, human sperm responded to DTT treatment by the sliding or removal of the mitochondrial sheath and decondensation of the sperm chromatin. These events were not observed in the sperm of lower vertebrates and invertebrates (Xenopus laevis and Lytechinus pictus, respectively) exposed to an excess of DTT. Thus the sensitivity of sperm mitochondria to the S-S reducing environment seems to be an exclusive feature of mammalian sperm. The movement of sperm mitochondria induced by S-S reduction may be an initial critical step in the disassembly of the mammalian sperm tail during fertilization.
Mol Reprod Dev 1997 May
PMID:Mitochondrial sheath movement and detachment in mammalian, but not nonmammalian, sperm induced by disulfide bond reduction. 911 Mar 18

A number of viral oncogenes target the tumour suppressor protein p53 and inactivate its function. This is an important step in tumourogenesis. The cellular oncogene hdm2 acts through a similar mechanism. It binds the N terminus of p53, thereby interfering with the ability of p53 transcriptionally to activate genes responsible for growth arrest or apoptosis after genotoxic insults. The disruption of the interaction of the two proteins therefore comprises a promising therapeutic target for treatment of the subset of human cancers in which this pathway is active. In this paper we attempt to characterize the p53-hdm2 interaction biochemically. We analyse the potential of a series of peptide inhibitors, derived from previously described mdm2 binding peptide display phage, to disrupt this interaction in ELISA assays. We conclude that F19, W23 and L26 of p53 are critical contact points for p53 binding to hdm2. Furthermore, we show the potential of the monoclonal antibody 3G5 to interfere with binding of p53 to hdm2 in ELISA assays. Consequently, we define the binding site of 3G5 on hdm2 using overlapping peptides derived from the N terminus of hdm2 and phage display libraries. The result indicates L66, Y67 and E69 on hdm2 as critical binding points for 3G5. In electrophoretic mobility shift assay we demonstrate the formation of hdm2-p53 complexes that can be disrupted in the presence of 3G5 or inhibitory peptides. Finally, we describe the effects of NEM and DTT on the interaction between the two molecules in ELISA assays. All our results are discussed in the light of the recently published crystal structure of the mdm2-p53 complex. A striking correspondence between our findings and the crystal structure is revealed.
J Mol Biol 1997 Jun 27
PMID:Molecular characterization of the hdm2-p53 interaction. 922 38

Cytochrome P-450 aromatase was purified by five chromatographic steps from adult stallion testis. It was first separated from NADPH-cytochrome P-450 reductase (reductase) on omega-aminohexyl-Sepharose 4B then purified to homogeneity on concanavalin A-Sepharose 4B, hydroxyapatite-Sepharose 4B, DEAE-Sepharose CL-6B and on a second hydroxyapatite-Sepharose 4B. On the other hand, purifications of the equine testicular and rat liver reductases, which allowed the reconstitution of aromatase activity in vitro, were achieved for each species in one chromatographic step on an adenosine 2',5'-diphosphate-agarose affinity column. Analysis on SDS/PAGE indicated single bands with apparent molecular masses of 53, 82, and 80 kDa for purified equine testicular cytochrome P-450 aromatase (eAROM), equine testicular reductase and rat liver reductase respectively. eAROM shows a time- and concentration-dependent activity that was stable for at least 2 months when stored at -78 degrees C. It is a highly hydrophobic protein composed from 505 residues and direct sequencing of its N-terminal part showed good homology when compared with human aromatase. When deglycosylated by N-glycosidase-F the apparent molecular mass of eAROM was decreased from 53 to 51 kDa as revealed by electrophoresis, its activity, however, was not impaired. eAROM exhibits much higher affinity for androgens than for 19-norandrogens, Km values were approximately 3, 16 and 170 nM for androstenedione (A), testosterone (T) and 19-nortestosterone (19-NT) respectively. However, it aromatizes 19-norandrostenedione (19-NA) slightly more efficiently than A, the estrone (E1) formed was 4.27 vs 3.54 pmol min-1 micrograms-1 respectively (P < 0.01). After incubation of eAROM with radiolabelled A and separation of steroids on HPLC, E1, 19-hydroxyandrostenedione (19-OHA) and 19-oxoandrostenedione (19-oxoA) were accumulated in the incubation medium in a time-dependent manner. The presence of 4-hydroxyandrostenedione (4-OHA), a suicide inhibitor of aromatase, cause a time-dependent inactivation of the enzyme. Whereas the activity of eAROM was unchanged in the presence of K+ (up to 250 mM), it was increased in the presence of EDTA (up to 50 mM) and decreased in the presence of DTT or Mg2+ (from 25 mM). We conclude that: (a) eAROM is a glycoprotein, however, deglycosylation by N-glycosidase-F does not appear to impair its activity, (b) eAROM aromatizes really both androgens and 19-norandrogens having a higher affinity for androgens, (c) the intermediary compounds of aromatization 19-OHA and 19-oxoA appear to be synthesized by the same active site that synthesizes E1 as the final product, (d) the inhibition of eAROM by increasing concentrations of Mg2+ and the stimulation of its activity by EDTA, taken together, indicate the importance of negatively charged residues in the polypeptide chain of equine aromatase, which play a role in enzymatic activity.
Comp Biochem Physiol B Biochem Mol Biol 1997 Sep
PMID:Purification and characterization of equine testicular cytochrome P-450 aromatase: comparison with the human enzyme. 941 12

The effect of regucalcin, a Ca2+-binding protein, on Ca2+ transport system in rat renal cortex microsomes was investigated. The presence of regucalcin (10[-8] to 10[-6] M) in the reaction mixture caused a significant increase in Ca2+-ATPase activity and ATP-dependent 45Ca2+ uptake in the microsomes. Regucalcin (10[-7] M) increased Ca2+-ATPase activity independently of increasing concentrations of CaCl2. The microsomal Ca2+-ATPase activity and 45Ca2+ uptake were markedly decreased by the presence of vanadate (0.1 mM) or N-ethylmaleimide (NEM; 5 mM) in the absence or presence of regucalcin. Dithiothreitol (DTT; 5 mM) markedly elevated Ca2+-ATPase activity and 45Ca2+ uptake in the microsomes. The DTT effects were not further enhanced by regucalcin (10[-7] M). Meanwhile, the microsomal Ca2+-ATPase activity and 45Ca2+ uptake were significantly decreased by the presence of dibutyryl cyclic AMP (DcAMP; 10[-5] and 10[-3] M) or inositol 1,4, 5-trisphosphate (IP3; 10[-7] and 10[-5] M). The effect of regucalcin (10[-7] M) on Ca2+ATPase activity and 45Ca2+ uptake was weakened in the presence of DcAMP or IP3. The present results demonstrate that regucalcin has a stimulatory effect on ATP-dependent Ca2+ uptake in the microsomes of rat renal cortex due to acting on the thiol groups of Ca2+-ATPase.
Mol Cell Biochem 1997 Dec
PMID:Regucalcin increases Ca2+-ATPase activity and ATP-dependent calcium uptake in the microsomes of rat kidney cortex. 945 Jun 63

This study aims (i) to ascertain whether oxidative-stress-induced disturbances in chromosomal distribution in the metaphase-II spindle of mouse oocytes can be counteracted by supplementing culture medium with antioxidants; and (ii) to determine whether supplemental intake of antioxidants neutralizes the disturbing effects of maternal ageing on segregation of chromosomes during the first meiotic division and distribution of chromosomes in the metaphase-II spindle. (i): Germinal vesicle oocytes from unstimulated 10-12 week old mice were matured in vitro in the presence or absence of diamide and/or dithiothreitol. Metaphase-II oocytes were fixed and stained with 4',6-diamidino-2-phenylindole (DAPI) to detect abnormalities in chromosomal distribution. The percentage of oocytes arrested in metaphase I (12.9% vs 28.4%; P < or = 0.05) or with a telophase-I chromosome configuration (0.0% vs 8.2%; P < or = 0.0005) was decreased in diamide-DTT-treated oocytes when compared to diamide-treated oocytes. (ii): Mice were fed, from the first day of weaning until their death, a diet supplemented or not with an antioxidant mixture of vitamin C and vitamin E. Ovulated oocytes were fixed and stained with DAPI or C-banded for chromosome analysis. The percentage of abnormal (chromosome scattering and nulloploidy) or asynchronous (anaphase I or telophase I) oocytes was 2.7-fold higher in controls than in females fed an antioxidant diet (24.4% vs 8.9%, P < or = 0.05). Furthermore, the percentage of aneuploidy (2.2% vs 0.0%; P < or = 0.01) and diploidy (5.8% vs 1.7%; P < or = 0.05) was significantly higher in controls than in females fed an antioxidant diet. These findings support Tarin's oxidative stress hypothesis of aneuploidy and have clinical implications for preventing both laboratory-induced and maternal-age-associated aneuploidy in human beings.
Mol Hum Reprod 1998 Mar
PMID:Antioxidant therapy counteracts the disturbing effects of diamide and maternal ageing on meiotic division and chromosomal segregation in mouse oocytes. 957 Feb 74

A soman-hydrolyzing enzyme (somanase) was purified from human liver. The human somanase is capable of hydrolyzing pinacolyl methylphosphonofluoridate (soman), diisopropylphosphorofluoridate (DFP), and ethyl-N-dimethyl phosphoramidocyanidate (Tabun) with P-F or P-CN bonding, but not ethyl (S-2-diisopropylaminoethyl) methylphosphonothiolate (VX) and diethyl-p-nitro-phosphenylphosphate (paraoxon) with P-S or P-O bonding. The somanase has been purified 1570-fold with a specific activity of 41.4 mumol/min/mg protein. Its molecular weight is around 58 kDa determined by SDS-PAGE. The somanase could be stimulated by the divalent cations Mn+2, Mg+2, and Co+2, where CO+2 activation is the highest. The requirement of disulfide bonds for the enzyme activity was demonstrated by the inhibition effect of DTT.
J Biochem Mol Toxicol 1998
PMID:Purification and properties of soman-hydrolyzing enzyme from human liver. 958 Aug 73

We describe a conserved yeast gene, ERO1, that is induced by the unfolded protein response and encodes a novel glycoprotein required for oxidative protein folding in the ER. In a temperature-sensitive ero1-1 mutant, newly synthesized carboxypeptidase Y is retained in the ER and lacks disulfide bonds, as shown by thiol modification with AMS. ERO1 apparently determines cellular oxidizing capacity since mutation of ERO1 causes hypersensitivity to the reductant DTT, whereas overexpression of ERO1 confers resistance to DTT. Moreover, the oxidant diamide can restore growth and secretion in ero1 mutants. Genetic tests distinguish the essential function of ERO1 from that of PDI1. We show that glutathione is not required for CPY folding and conclude that Ero1p functions in a novel mechanism that sustains the ER oxidizing potential, supporting net formation of protein disulfide bonds.
Mol Cell 1998 Jan
PMID:The ERO1 gene of yeast is required for oxidation of protein dithiols in the endoplasmic reticulum. 965 13

The structure of many proteins entering the secretory pathway is dependent on stabilization by disulfide bonds. To support disulfide-linked folding, the endoplasmic reticulum (ER) must maintain a strongly oxidizing environment compared to the highly reduced environment of the cytosol. We report here the identification and characterization of Ero1p, a novel and essential ER-resident protein. Mutations in Ero1p cause extreme sensitivity to the reducing agent DTT, whereas overexpression confers DTT resistance. Strikingly, compromised Ero1p function results in ER retention of disulfide-stabilized proteins in a reduced, nonnative form, while not affecting structural maturation of a disulfide-free protein. We conclude that there exists a specific cellular redox machinery required for disulfide-linked protein folding in the ER and that Ero1p is an essential component of this machinery.
Mol Cell 1998 Jan
PMID:Ero1p: a novel and ubiquitous protein with an essential role in oxidative protein folding in the endoplasmic reticulum. 965 14

Full-length (67 kDa) immunoreactive estrogen receptor (ER) extracted from a third of untreated ER-positive primary breast tumors appears unable to bind to its cognate estrogen response element (ERE). We have observed partial reversibility of this ER DNA-binding defect upon treatment of these tumor extracts with excess thiol reducing agent (DTT), suggesting that ER DNA-binding is subject to redox modulation as is reported for other zinc-finger proteins and transcriptional activators. Treatment of recombinant ER DNA-binding domain (ER-DBD) or ER-enriched extracts from CHO(ER) and MCF-7 cells with thiol-reacting oxidants (diamide, iodosobenzoate, H2O2) or alkylator (iodoacetamide) produces a dose-dependent loss in ER DNA-binding capacity. Thiol-specific oxidative loss in ER DNA-binding is fully reversible by DTT reduction, unlike the defect caused by thiol-specific alkylation. Circular dichroism spectrometry shows that both forms of treatment substantially modify ER secondary structure, inducing loss of alpha-helical content within the ER-DBD that is reversible after thiol oxidation but not after thiol alkylation. Oxidant (H2O2, menadione) exposure of cultured CHO(ER) or MCF-7 cells impairs the ability of endogenous ER to bind DNA and transactivate an ER-responsive reporter gene (ERE-tk-CAT), demonstrating that extracellular redox stress can modulate intracellular ER function. Since these thiol-specific oxidant and alkylator treatments have no significant effect on either recombinant ER ligand-binding or intracellular immunoreactive ER content, our findings suggest that DNA-binding and transactivation are the most sensitive intracellular ER functions impaired by oxidant stress in some ER-positive human breast tumors.
Mol Cell Endocrinol 1998 Nov 25
PMID:Oxidant stress impaired DNA-binding of estrogen receptor from human breast cancer. 1002 73


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