A Nedd8 conjugation pathway is essential for proteolytic targeting of p27Kip1 by ubiquitination.

Temporal control of p27(Kip1) (p27) degradation imposes periodicity in its activity during cell cycle progression and its accumulation during cell cycle exit. Degradation of p27 is initiated by phosphorylation of p27 at Thr-187, which marks the protein for ubiquitination by SCF(Skp2) and subsequent proteolysis by the 26S proteasome. Here we show that the p27 ubiquitination activity in cell extracts depends on the presence of the ubiquitin-like protein Nedd8 and enzymes that catalyze Nedd8 conjugation to proteins. Moreover, we show that reconstitution of the p27 ubiquitination activity of recombinant SCF(Skp2) also requires Nedd8 conjugation pathway components. Inactivation of the Nedd8 conjugation pathway by a dominant negative mutant of the Nedd8-conjugating enzyme Nce1/Ubc12 blocks the ubiquitination and degradation of p27 in cell extracts. Consistent with a role in cell-cycle progression, Nedd8 is expressed in proliferating cells and is itself down-regulated upon cellular differentiation. These results suggest that the Nedd8 conjugation pathway may regulate the turnover of p27(Kip1), independently of p27 phosphorylation, and further establishes the identity of protein components involved in p27 ubiquitination. Finally, these findings provide a direct demonstration of a function for Nedd8 in a biological process.

Nedd8 modification of cul-1 activates SCF(beta(TrCP))-dependent ubiquitination of IkappaBalpha.

Regulation of NF-kappaB occurs through phosphorylation-dependent ubiquitination of IkappaBalpha, which is degraded by the 26S proteasome. Recent studies have shown that ubiquitination of IkappaBalpha is carried out by a ubiquitin-ligase enzyme complex called SCF(beta(TrCP)). Here we show that Nedd8 modification of the Cul-1 component of SCF(beta(TrCP)) is important for function of SCF(beta(TrCP)) in ubiquitination of IkappaBalpha. In cells, Nedd8-conjugated Cul-1 was complexed with two substrates of SCF(beta(TrCP)), phosphorylated IkappaBalpha and beta-catenin, indicating that Nedd8-Cul-1 conjugates are part of SCF(beta(TrCP)) in vivo. Although only a minute fraction of total cellular Cul-1 is modified by Nedd8, the Cul-1 associated with ectopically expressed betaTrCP was highly enriched for the Nedd8-conjugated form. Moreover, optimal ubiquitination of IkappaBalpha required Nedd8 and the Nedd8-conjugating enzyme, Ubc12. The site of Nedd8 ligation to Cul-1 is essential, as SCF(beta(TrCP)) containing a K720R mutant of Cul-1 only weakly supported IkappaBalpha ubiquitination compared to SCF(beta(TrCP)) containing WT Cul-1, suggesting that the Nedd8 ligation of Cul-1 affects the ubiquitination activity of SCF(beta(TrCP)). These observations provide a functional link between the highly related ubiquitin and Nedd8 pathways of protein modification and show how they operate together to selectively target the signal-dependent degradation of IkappaBalpha.

Identification of an essential cysteinyl residue in the ArsC arsenate reductase of plasmid R773.

The ArsC protein encoded by the arsenical resistance operon of plasmid R773 catalyzes the reduction of arsenate to arsenite in Escherichia coli. The reductase has been shown to require glutathione and glutaredoxin, suggesting that thiol chemistry might be involved in the reaction mechanism. The ArsC arsenate reductase has two cysteinyl residues, Cys12 and Cys106. By a combination of random and site-specific mutagenesis, Cys12 was altered to four other amino acid residues. Cells expressing any of those arsC genes were sensitive to arsenate. The ArsCC12S protein was purified and found to be catalytically inactive. Cys106 was altered separately to seryl, glycyl, and valyl residues. Cells expressing arsCC106S, arsCC106G, and arsCC106V genes retained arsenate resistance, and the purified C106S and C106G proteins had reductase activity. Both wild-type ArsC and C106S proteins were inactivated by iodoacetate. In the native enzyme only Cys12 was alkylate by iodoacetate; Cys106 was alkylated only if the enzyme was first denatured. In the presence of the substrate, arsenate, or competitive inhibitors, phosphate or sulfate, the rate of alkylation was reduced. Reductase activity was inhibited by N-ethylmaleimide and could be protected by arsenate. These results suggest Cys12 is an active-site residue essential for catalysis by the arsenate reductase.

Crystallization and preliminary X-ray diffraction analysis of the ArsC protein from the Escherichia coli arsenical resistance plasmid, R773.

Diffraction data to 3.0 A resolution were collected on crystals of ArsC protein from the conjugative resistance factor R773 which mediates arsenical resistance in the Gram negative bacterium Escherichia coli. The crystal system is tetragonal, a = 116.2 A, c = 145.0 A and the space group is either P4(1)2(1)2 or P4(3)2(1)2. The most probable range for the contents of the asymmetric unit is four to eight ArsC molecules (M(r) = 15,811).

Properties of the arsenate reductase of plasmid R773.

Resistance to toxic oxyanions in Escherichia coli is conferred by the ars operon carried on plasmid R773. The gene products of this operon catalyze extrusion of antimonials and arsenicals from cells of E. coli, thus providing resistance to those toxic oxyanions. In addition, resistance to arsenate is conferred by the product of the arsC gene. In this report, purified ArsC protein was shown to catalyze reduction of arsenate to arsenite. The enzymatic activity of the ArsC protein required glutaredoxin as a source of reducing equivalents. Other reductants, including glutathione and thioredoxin, were not effective electron donors. A spectrophotometric assay was devised in which arsenate reduction was coupled to NADPH oxidation. The results obtained with the coupled assay corresponded to those found by direct reduction of radioactive arsenate to arsenite. The only substrate of the reaction was arsenate (Km = 8 mM); other oxyanions including phosphate, sulfate, and antimonate were not reduced. Phosphate and sulfate were weak inhibitors, while the product, arsenite, was a stronger inhibitor (Ki = 0.1 mM). Arsenate reductase activity exhibited a pH optimum of 6.3-6.8. These results indicate that the ArsC protein is a novel reductase, and elucidation of its enzymatic mechanism should be of interest.

Arsenate reduction mediated by the plasmid-encoded ArsC protein is coupled to glutathione.

Resistance to arsenate conferred on Escherichia coli by the ars operon of plasmid R773 requires both the product of the arsC gene and reduction of arsenate to arsenite. A genetic analysis was performed to identify the source of reducing potential in vivo. In addition to the ars genes, arsenate resistance required the products of the gor gene for glutathione reductase and the gshA and gshB genes for glutathione synthesis. Mutations in the trx and grx genes for thioredoxin and glutaredoxin, respectively, had no effect on arsenate resistance. Although resistance required the arsC gene, the rate of reduction of arsenate to arsenite was nearly the same in cells lacking the ars operon. In strains deficient in glutathione biosynthesis this endogenous reduction was greatly diminished, and cells exhibited increased sensitivity to arsenate. When glutathione was supplied exogenously to such mutants, resistance was restored only to cells expressing the ars operon, and only such cells had detectable arsenate reduction after addition of glutathione. Since ArsC-catalysed reduction of arsenate provides high level resistance, physical coupling of the ArsC reaction to efflux of the resulting arsenite is hypothesised.

[The dose dependence of various loads using the rosette formation method]. / Dozovaia zavisimost' razlichnykh nagruzok v metode rozetkoobrazovaniia.

A method of the loading tests in rosette-formation was used to study the rosette-formation of lymphocytes and phagocytosis of neutrophils depending on different concentrations of T-activin (and levamysol) after preincubation of leucocytes in vitro with these medicines. It is shown that significant effect of the cells is observed up to the concentrations of the medicines 10(-10)-10(-13) g/l.

Structural rearrangements in soluble mitochondrial ATPase.

Treatment of isolated factor F1 by 1% dimethylsuberimidate in the presence of 50 mM (NH4)2SO4 leads to the formation of four different types of cross-linked dimers of the subunits, on average one dimer per molecule of the enzyme. This treatment results in 60-70% inactivation of factor F1. Factor F1 treated with dimethylsuberimidate does not show a change in the sedimentation coefficient and is not inactivated in the cold; it is not inactivated in the presence of Mg2+ either, nor is it activated by anions. Incubation of the cross-linked factor F1 with ADP does not lead to inactivation, although the ability to tightly bind ADP is retained. The total quantity of tightly bound ADP reaches 5 mol per mol of the cross-linked factor F1. Cross-linking of factor F1 also prevents the slow inactivation of the enzyme coupled with the hydrolysis of Mg-ATP and Mg-GTP. The dependence of the inactivation rate constant on the concentration of Mg-ATP and Mg-GTP at substrate concentrations of 0.05-2 mM is characterized by the same values of Km,app as those of the ATPase and GTPase activities of factor F1. The probability of the inactivation of factor F1 per turnover remains constant for all the concentrations of the substrates studied and is 2 . 10(-6) per turnover for the ATPase reaction and 2 . 10(-5) per turnover for the GTPase reaction. Moderate hydrostatic pressure (up to 150 atmospheres) greatly accelerates ATP-induced inactivation of factor F1. The activation volume (delta V*) of the inactivation process is equal to 5.1 . 10(-4) cm3/g, which is evidence of considerable changes in the extent of protein hydration during inactivation. Inactivation of the enzyme under pressure is accompanied by dissociation into subunits. Dimethyladipimidate, which does not cause intersubunit cross-linking in the molecule of factor F1, does not alter the properties of the native enzyme. It is suggested that the formation of one intersubunit cross-link in the molecule of factor F1 by dimethylsuberimidate affects the ability of the enzyme to undergo co-operative rearrangements of the quaternary structure under the influence of Mg2+, ADP, ATP, anions, and low temperature. The rate constants of ATP binding to the active site of factor F2 (k+1) = 2 . 10(8) M-1 . min-1), of ATP release from the active site (k-1 = 2 . 10(-2) min-1), and of ADP and Pi release from the active site (k2 = 5 . 10(3) min-1) have been determined. The results obtained confirm the correctness of Boyer's idea, according to which ATP is formed in the active site of mitochondrial ATPase without any external source of energy. Energy is used at the stage of the release of synthesized ATP from the active site of ATPase in the solution.