Nucleic acid-dependent cross-linking of the nucleocapsid protein of Sindbis virus.
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The assembly of the alphavirus nucleocapsid core is a multistep event requiring the association of the nucleocapsid protein with nucleic acid and the subsequent oligomerization of capsid proteins into an assembled core particle. Although the mechanism of assembly has been investigated extensively both in vivo and in vitro, no intermediates in the core assembly pathway have been identified. Through the use of both truncated and mutant Sindbis virus nucleocapsid proteins and a variety of cross-linking reagents, a possible nucleic acid-protein assembly intermediate has been detected. The cross-linked species, a covalent dimer, has been detected only in the presence of nucleic acid and with capsid proteins capable of binding nucleic acid. Optimum nucleic acid-dependent cross-linking was seen at a protein-to-nucleic-acid ratio identical to that required for maximum binding of the capsid protein to nucleic acid. Identical results were observed when cross-linking in vitro assembled core particles of both Sindbis and Ross River viruses. Purified cross-linked dimers of truncated proteins and of mutant proteins that failed to assemble were found to incorporate into assembled core particles when present as minor components in assembly reactions, suggesting that the cross-linking traps an authentic intermediate in nucleocapsid core assembly. Endoproteinase Lys-C mapping of the position of the cross-link indicated that lysine 250 of one capsid protein was cross-linked to lysine 250 of an adjacent capsid protein. Examination of the position of the cross-link in relation to the existing model of the nucleocapsid core suggests that the cross-linked species is a cross-capsomere contact between a pentamer and hexamer at the quasi-threefold axis or is a cross-capsomere contact between hexamers at the threefold axis of the icosahedral core particle and suggests several possible assembly models involving a nucleic acid-bound dimer of capsid protein as an early step in the assembly pathway. (+info)
The Tctex1/Tctex2 class of dynein light chains. Dimerization, differential expression, and interaction with the LC8 protein family.
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The Tctex1/Tctex2 family of dynein light chains associates with the intermediate chains at the base of the soluble dynein particle. These components are essential for dynein assembly and participate in specific motor-cargo interactions. To further address the role of these light chains in dynein activity, the structural and biochemical properties of several members of this polypeptide class were examined. Gel filtration chromatography and native gel electrophoresis indicate that recombinant Chlamydomonas flagellar Tctex1 exists as a dimer in solution. Furthermore, yeast two-hybrid analysis suggests that this association also occurs in vivo. In contrast, both murine and Chlamydomonas Tctex2 are monomeric. To investigate protein-protein interactions involving these light chains, outer arm dynein from Chlamydomonas flagella was cross-linked using dimethylpimelimidate. Immunoblot analysis of the resulting products revealed the interaction of LC2 (Tctex2) with LC6, which is closely related to the highly conserved LC8 protein found in many enzyme systems, including dynein. Northern dot blot analysis demonstrated that Tctex1/Tctex2 family light chains are differentially expressed both in a tissue-specific and developmentally regulated manner in humans. These data provide further support for the existence of functionally distinct populations of cytoplasmic dynein with differing light chain content. (+info)
Spatial arrangement of proteins within the small subunit of rat liver ribosomes studied by cross-linking.
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Cross-linking of proteins within the small subunit of rat liver ribosomes by the bifunctional reagent dimethyl 4,7-dioxo-5,6-dihydroxy-3,8-diazadecanbisimidate produced numerous covalently linked protein dimers which could be separated by a combination of ion-exchange chromatography on carboxymethyl cellulose and polyacrylamide gel electrophoresis. The protein components of the dimers were identified electrophoretically after periodate cleavage of the cross-link(s). The analysis revealed 42 cross-linked dimers involving 25 different proteins. Among these, proteins S3, S4 and S20 occurred in combinations with six, eight and seven different proteins, respectively. For proteins S13, S14 and S17 five protein neighbours could be identified, while 13 of the remaining proteins were linked to three or four different protein partners. The involvement of the majority of proteins in the formation of multiple cross-linked dimers implies that a large number of protein-protein interaction sites exist within the ribosomal subunit. A preliminary model illustrating the arrangement of 16 proteins in the small ribosomal subunit is presented and discussed with respect to possible functions, especially in the event of translation initiation. (+info)
The ribosome modulation factor (RMF) binding site on the 100S ribosome of Escherichia coli.
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During the stationary growth phase, Escherichia coli 70S ribosomes are converted to 100S ribosomes, and translational activity is lost. This conversion is caused by the binding of the ribosome modulation factor (RMF) to 70S ribosomes. In order to elucidate the mechanisms by which 100S ribosomes form and translational inactivation occurs, the shape of the 100S ribosome and the RMF ribosomal binding site were investigated by electron microscopy and protein-protein cross-linking, respectively. We show that (i) the 100S ribosome is formed by the dimerization of two 70S ribosomes mediated by face-to-face contacts between their constituent 30S subunits, and (ii) RMF binds near the ribosomal proteins S13, L13, and L2. The positions of these proteins indicate that the RMF binding site is near the peptidyl transferase center or the P site (peptidyl-tRNA binding site). These observations are consistent with the translational inactivation of the ribosome by RMF binding. After the "Recycling" stage, ribosomes can readily proceed to the "Initiation" stage during exponential growth, but during stationary phase, the majority of 70S ribosomes are stored as 100S ribosomes and are translationally inactive. We suggest that this conversion of 70S to 100S ribosomes represents a newly identified stage of the ribosomal cycle in stationary phase cells, and we have termed it the "Hibernation" stage. (+info)
Induction of cytochrome P450, generation of oxidative stress and in vitro cell-transforming and DNA-damaging activities by glucoraphanin, the bioprecursor of the chemopreventive agent sulforaphane found in broccoli.
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The reduced cancer risk that appears to be linked to a diet rich in fruits and vegetables has fueled the belief that regular intake of isolated phytochemicals could potentially prevent cancer. In recent years, the glucosinolate metabolites derived from cruciferous vegetables, such as the isothiocyanate sulforaphane in broccoli, have gained much attention as potential cancer chemopreventive agents. The protective effect of sulforaphane, which is liberated from its glucosinolate precursor glucoraphanin (GRP) by myrosinase hydrolysis, is conventionally thought to involve the induction of Phase-II metabolizing enzymes. These Phase-II enzymes are implicated in the detoxication of many carcinogens and reactive oxygen species (ROS), thereby protecting cells against DNA damage and subsequent malignant transformation. While the induction of Phase-II enzymes is usually considered beneficial, in some cases these enzymes also bioactivate several hazardous chemicals. Furthermore, despite its projected benefits, the unknown effect of sulforaphane on Phase-I enzyme systems, which are involved in the bioactivation of a variety of carcinogens, should not be overlooked. Here we show that, in rat lungs, while GRP, the bioprecursor of the chemopreventive agent sulforaphane, slightly induced Phase-II detoxifying enzymes, it powerfully induced Phase-I carcinogen-activating enzymes, including activators of carcinogenic polycyclic aromatic hydrocarbons (PAHs). Concomitant with this Phase-I induction, GRP also over-generated ROS. Additionally, in a cell-transforming assay, GRP facilitated the metabolic activation of the PAH benzo[a]pyrene to reactive carcinogenic forms and in a yeast genotoxicity test it damaged DNA. This suggests that regular administration of GRP could actually increase rather than decrease cancer risk, especially in individuals exposed to environmental mutagens and carcinogens such as those found in tobacco smoke and in certain industrial settings. (+info)
Dietary approach to attenuate oxidative stress, hypertension, and inflammation in the cardiovascular system.
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Imbalance between production and scavenging of superoxide anion results in hypertension by the inactivation of nitric oxide, and the increased oxidative stress from the resultant peroxynitrite that is produced promotes inflammatory processes such as atherosclerosis. Induction of phase 2 proteins promotes oxidant scavenging. We hypothesized that intake of dietary phase 2 protein inducers would ameliorate both hypertension and atherosclerotic changes in the spontaneously hypertensive stroke-prone rat. For 5 days/week for 14 weeks, we fed rats 200 mg/day of dried broccoli sprouts that contained glucoraphanin, which is metabolized into the phase 2 protein-inducer sulforaphane (Group A), sprouts in which most of the glucoraphanin was destroyed (Group B), or no sprouts (Group C). After 14 weeks of treatment, no significant differences were seen between rats in Groups B and C. Rats in Group A had significantly decreased oxidative stress in cardiovascular and kidney tissues, as shown by increased glutathione (GSH) content and decreased oxidized GSH, decreased protein nitrosylation, as well as increased GSH reductase and GSH peroxidase activities. Decreased oxidative stress correlated with better endothelial-dependent relaxation of the aorta and significantly lower (20 mm Hg) blood pressure. Tissues from Groups B and C had considerable numbers of infiltrating activated macrophages, indicative of inflammation, whereas animals in Group A had few detectable infiltrating macrophages. There is interest in dietary phase 2 protein inducers as means of reducing cancer incidence. We conclude that a diet containing phase 2 protein inducers also reduces the risk of developing cardiovascular problems of hypertension and atherosclerosis. (+info)
The Drosophila acetylcholine receptor subunit D alpha5 is part of an alpha-bungarotoxin binding acetylcholine receptor.
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The central nervous system of Drosophila melanogaster contains an alpha-bungarotoxin-binding protein with the properties expected of a nicotinic acetylcholine receptor. This protein was purified 5800-fold from membranes prepared from Drosophila heads. The protein was solubilized with 1% Triton X-100 and 0.5 M sodium chloride and then purified using an alpha-cobratoxin column followed by a lentil lectin affinity column. The purified protein had a specific activity of 3.9 micromol of 125I-alpha-bungarotoxin binding sites/g of protein. The subunit composition of the purified receptor was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This subunit profile was identical with that revealed by in situ labeling of the membrane-bound protein using the photolyzable methyl-4-azidobenzoimidate derivative of 125I-alpha-bungarotoxin. The purified receptor reveals two different protein bands with molecular masses of 42 and 57 kDa. From sedimentation analysis of the purified protein complex in H2O and D2O and gel filtration, a mass of 270 kDa was calculated. The receptor has a s(20,w) of 9.4 and a Stoke's radius of 7.4 nm. The frictional coefficient was calculated to be 1.7 indicating a highly asymmetric protein complex compatible with a transmembrane protein forming an ion channel. The sequence of a peptide obtained after tryptic digestion of the 42-kDa protein allowed the specific identification of the Drosophila D alpha5 subunit by sequence comparison. A peptide-specific antibody raised against the D alpha5 subunit provides further evidence that this subunit is a component of an alpha-bungarotoxin binding nicotinic acetylcholine receptor from the central nervous system of Drosophila. (+info)
A formal [3,3]-sigmatropic rearrangement route to quaternary alpha-vinyl amino acids: use of allylic N-PMP trifluoroacetimidates.
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Pd(II)-mediated rearrangement of allylic N-PMP (p-methoxyphenyl) trifluoroacetimidates provides the first formal sigmatropic route to quaternary, alpha-vinylic amino acids, potential suicide substrates for PLP enzymes. The amino acid side chains enter via transition-metal-mediated C-C bond constructions, including (i) Cu(I)-mediated conjugate addition (Ala); (ii) Pd(0)/AsPh3-mediated Stille coupling (allyl-Gly, Phe, DOPA, m-Tyr); and (iii) Pd(0)/Pt-Bu3-mediated Negishi coupling (Leu). In the synthesis of the DOPA decarboxylase inactivator, alpha-vinyl-m-tyrosine, the new N-PMP trifluoroacetimidate rearranges much more efficiently than the corresponding trichloroacetimidate. (+info)