Ivan C. Baines

Mammalian Peroxiredoxin Isoforms Can Reduce Hydrogen Peroxide Generated in Response to Growth Factors and Tumor Necrosis Factor-α

Mammalian tissues express three immunologically distinct peroxiredoxin (Prx) proteins (Prx I, II, and III), which are the products of distinct genes. With the use of recombinant proteins Prx I, II, and III, all have now been shown to possess peroxidase activity and to rely on Trx as a source of reducing equivalents for the reduction of H2O2. Prx I and II are cytosolic proteins, whereas Prx III is localized in mitochondria. Transient overexpression of Prx I or II in cultured cells showed that they were able to eliminate the intracellular H2O2 generated in response to growth factors. Moreover, the activation of nuclear factor κB (NFκB) induced by extracellularly added H2O2 or tumor necrosis factor-α was blocked by overproduction of Prx II. These results suggest that, together with glutathione peroxidase and catalase, Prx enzymes likely play an important role in eliminating peroxides generated during metabolism. In addition, Prx I and II might participate in the signaling cascades of growth factors and tumor necrosis factor-α by regulating the intracellular concentration of H2O2.

Characterization of a Mammalian Peroxiredoxin That Contains One Conserved Cysteine

A new type of peroxidase enzyme, named thioredoxin peroxidase (TPx), that reduces H2O2 with the use of electrons from thioredoxin and contains two essential cysteines was recently identified. TPx homologs, termed peroxiredoxin (Prx), have also been identified and include several proteins, designated 1-Cys Prx, that contain only one conserved cysteine. Recombinant human 1-Cys Prx expressed in and purified from Escherichia coli has now been shown to reduce H2O2 with electrons provided by dithiothreitol. Furthermore, human 1-Cys Prx transiently expressed in NIH 3T3 cells was able to remove intracellular H2O2 generated in response either to the addition of exogenous H2O2 or to treatment with platelet-derived growth factor. The conserved Cys47–SH group was shown to be the site of oxidation by H2O2. Thus, mutation of Cys47 to serine abolished peroxidase activity. Moreover, the oxidized intermediate appears to be Cys–SOH. In contrast to TPx, in which one of the two conserved cysteines is oxidized to Cys–SOH and then immediately reacts with the second conserved cysteine of the second subunit of the enzyme homodimer to form an intermolecular disulfide, the Cys–SOH of 1-Cys Prx does not form a disulfide. Neither thioredoxin, which reduces the disulfide of TPx, nor glutathione, which reduces the Cys–SeOH of oxidized glutathione peroxidase, was able to reduce the Cys–SOH of 1-Cys Prx and consequently could not support peroxidase activity. Human 1-Cys Prx was previously shown to exhibit a low level of phospholipase A2 activity at an acidic pH; the enzyme was thus proposed to be lysosomal, and Ser32 was proposed to be critical for lipase function. However, the mutation of Ser32 or Cys47 has now been shown to have no effect on the lipase activity of 1-Cys Prx, which was also shown to be a cytosolic protein. Thus, the primary cellular function of 1-Cys Prx appears to be to reduce peroxides with the use of electrons provided by an as yet unidentified source; the enzyme therefore represents a new type of peroxidase.

Identification of a New Type of Mammalian Peroxiredoxin That Forms an Intramolecular Disulfide as a Reaction Intermediate

Peroxidases of the peroxiredoxin (Prx) family contain a Cys residue that is preceded by a conserved sequence in the NH2-terminal region. A new type of mammalian Prx, designated PrxV, has now been identified as the result of a data base search with this conserved Cys-containing sequence. The 162-amino acid PrxV shares only ∼10% sequence identity with previously identified mammalian Prx enzymes and contains Cys residues at positions 73 and 152 in addition to that (Cys48) corresponding to the conserved Cys. Analysis of mutant human PrxV proteins in which each of these three Cys residues was individually replaced with serine suggested that the sulfhydryl group of Cys48 is the site of oxidation by peroxides and that oxidized Cys48 reacts with the sulfhydryl group of Cys152 to form an intramolecular disulfide linkage. The oxidized intermediate of PrxV is thus distinct from those of other Prx enzymes, which form either an intermolecular disulfide or a sulfenic acid intermediate. The disulfide formed by PrxV is reduced by thioredoxin but not by glutaredoxin or glutathione. Thus, PrxV mutants lacking Cys48 or Cys152 showed no detectable thioredoxin-dependent peroxidase activity, whereas mutation of Cys73 had no effect on activity. Immunoblot analysis revealed that PrxV is widely expressed in rat tissues and cultured mammalian cells and is localized intracellularly to cytosol, mitochondria, and peroxisomes. The peroxidase function of PrxV in vivo was demonstrated by the observations that transient expression of the wild-type protein, but not that of the Cys48 mutant, in NIH 3T3 cells inhibited H2O2 accumulation and activation of c-Jun NH2-terminal kinase induced by tumor necrosis factor-α.

The amino acid sequence of the light chain of Acanthamoeba myosin IC

The amino acid sequence of the light chain of Acanthamoebamyosin IC deduced from the cDNA sequence comprises 149 aminoacids with a calculated molecular weight of 16739. All but the 3N-terminal residues were also determined by amino acid sequencingof the purified protein, which also showed the N-terminus to beblocked. Phylogenetic analysis shows Acanthamoeba myosin IC lightchain to be more similar to the calmodulin subfamily of EF-handcalcium-modulated proteins than to the myosin II essential lightchain or regulatory light chain subfamilies. In pairwisecomparisons, the myosin IC light chain sequence is most similarto sequences of calmodulins (∼50% identical) and a squidcalcium-binding protein (∼43% identical); the sequence is∼37% identical to the calcium-binding essential light chainof Physarum myosin II and ∼30% identical to the essentiallight chain of Acanthamoeba myosin II, and the essential lightchain and regulatory light chain of Dictyostelium myosin II. Thesequence predicts four helix-loop-helix domains with possiblecalcium-binding sites in domains I and III, suggesting thatcalcium may affect the activity of this unconventional myosin.This is the first report of the sequence of an unconventionalmyosin light chain other than calmodulin

Localization of actobindin, profilin I, profilin II, and phosphatidylinositol‐4,5‐bisphosphate (PIP2) in Acanthamoeba castellanii

Specific polyclonal antisera were raised against purified Acanthamoeba actobindin and synthetic peptides corresponding to regions of maximum charge differences in Acanthamoeba profilin I and profilin II. Immunofluorescence studies with these antibodies showed profilin I to be distributed throughout the Acanthamoeba cytoplasm, except for lamellipodia, with the highest fluorescence intensity in cortical regions in which monomeric actin also was present, as shown by labeling with fluorescent DNase. In contrast, profilin II appeared to be uniformly associated with the plasma membrane except at sites of pseudopod extension, where the concentration was frequently decreased, in addition to cortical regions. Immunofluorescence studies using a monoclonal antibody specific for phosphatidylinositol-4,5-bisphosphate (PIP2) suggested that its distribution is mostly limited to the plasma membrane. In contrast to the distribution of profilin II, PIP2 immunofluorescence was prominent at the leading edge of cells, including the plasma membrane of lamellipodia. Quantitative immunoelectron microscopy showed that profilin II was approximately 36 times more likely to localize to the plasma membrane than profilin I. Immunofluorescence and confocal microscopy localized actobindin to the base of lamellipodia. The differential localization of the three actin monomer-binding proteins suggests that they have different biologic functions in Acanthamoeba and is consistent with the hypotheses that (1) profilin I functions predominantly as an actin monomer-binding protein; (2) profilin II regulates, or is regulated by, PIP2; and (3) actobindin inhibits nucleation of new filaments and facilitates elongation of existing polarized filaments in actively motile regions.

[2] Purification of myosin I and myosin I heavy chain kinase from Acanthamoeba castellanii

Publisher Summary The myosins I are structurally distinct from conventional myosins (myosins II) in that they are globular, single-headed proteins whose native molecular masses range from 140,000 to 160,000 Da. The activity of the myosin I heavy chain kinase is enhanced by its autophosphorylation which in turn is stimulated by phosphatidylserine. Myosin I and its heavy chain kinase are isolated from Acanthamoeba castellanii by conventional chromatographic methods. This chapter presents detailed procedures for isolating both. A cell extract is adsorbed to DE-52 and the kinase and myosin I are step eluted in a single pool. This material is applied to a phosphocellulose column, which is then eluted with a salt gradient in order to resolve the kinase and all three myosin I isozymes. The myosins I are further purified (individually) on ADP- or ATP-agarose and Mono Q columns, both eluted with salt gradients. Myosin I is assayed during its purification by its (K + ,EDTA)-ATPase activity. A radioisotopic form of this assay utilizing [γ- 32 P]ATP is necessary, at least in the early stages, because of the presence of phosphate liberated from pyrophosphate. It is difficult to estimate recovery and the extent of purification, but this procedure should yield 500 μg or more of myosin I heavy chain kinase.

Characterization of p80, a Novel Nuclear and Cytoplasmic Protein in Dinoflagellates

The presence of myosin in dinoflagellates was tested using an anti-Acanthamoeba castellanii myosin II polyclonal antibody on the heterotrophic dinoflagellate Crypthecodinium cohnii Seligo. Western blots revealed the presence of a unique band of 80 kDa in total protein extracts and after immunoprecipitation. Expression of this 80 kDa protein appeared constant during the different phases of the cell cycle. In protein extracts from various other dinoflagellates, this 80 kDa protein was detected only in the autotrophic species Prorocentrum micans Ehr. Screening of a C. cohnii cDNA expression library with this antibody revealed a cDNA coding for an amino acid sequence without homology in the databases. However, particular regions were detected: - a polyglutamine repeat domain in the N-terminal part of the protein, - four peptide sequences associated with GTP-binding sites, - a sequence with slight homology to the rod tail of Caenorhabditis elegans myosin II, -a sequence with homology to a human kinesin motor domain. Immunocytolocalization performed on C. cohnii thin sections with a polyclonal antibody raised against the recombinant protein showed p80 to be present both within the nucleus and in the cytoplasm. Labelling was widespread in the nucleoplasm and more concentrated at the periphery of the permanently condensed chromosomes. In the cytoplasm, labelling appeared in a punctate region close to the nucleus and in the flagellum. Potential functions of this novel protein are discussed.

P75, a novel protein in Dinoflagellates

FVll is a 54 kI>a plasma serine protease zymogen glycoprotein which may be cleaved to produce FVIIa, which binds tightly to cell-exposed tissue factor (TF). The complex is responsible for the initiation of blood coagulation (Stenflo J., Suttie J. (19771 Annu. Rev. Biochern. 46, 157-172). Although FVIIa has been cocrystallised with its cofactor TF (Banner D. et al 0996) Nature 380,41-46), no structure exists for the protease. In certain clinical situations the 56kDa plasma serine protease inhibitor (Serpin) antithrombin III (ATIII) may directly inhibit FVlIa by the formation of a FVIIa/ATIII complex (Lawson J. et al. (1993) I. &of. Chem. 268,767.770).

Molecules involved in the mitosis of dinoflagellate protists

‘URA 671 CNRS, Station Zuologique. Observatoire OcCanologique. 06230 VillefiancheMer We reported earlier that phospholipase C (PLC) and phosphomositide kinase (PI kinase) involved in the turnover of polyphosphoinositides (PPI) may control mitosis in sea urchin eggs (Ciapa et al. (1994), Nature 368, 875-878). Besides PLC second messengers, Pi 3-kinase generates 3phosphorylated inositol lipids which have been proposed to affect a wide variety of cellular processes in other cellular systems (Carpenter, C. L.. Cantley, L. C. (1996), Current Opinion in Cell Biology 8, 153158). We have investigated whether PI 3-kinase could play a role in regulating the sea urchin early embryonic development. We have immunoprecipited from egg extracts a protein of YSkDa w~tb an anti-PI 3 kinase antibody. We found that proteins immunoprecipited with anti-phosphotyrosine antibodies displayed a PI kinase activity sensitive to wortmannih, a specific inhibitor of PI 3kmase. These results suggest that Pi 3-kinase is present in sea urchin eggs. We observed that treatment of eggs with wortmannin did not affect fertilization but led to arrest -of the cell cycle after chromosome condensation and nuclear envelope breakdown bave occured. Although protein and DNA synthesis were not affected, Maturation Promoting.Factor (MPF) activation was inhibited. We found that fertilization induced a transient stimulation of a mitogen-activated protein (MAP) kinase activity at the time of mitosis and during the succeeding mitdtic cycles of sea urchin eggs: We observed that wortmannin delayed MAP kinase stimulation and inhibited its inactivation normally observed when the eggsdivide. It is accepted that high levels of MAP kinase activity are responsible for metaphase arrest of oocytes of various species inch&ins Xenonus and mouse (Sag&a, N. (1996),Tr&ds in Cell B&logy-6, 22-25). Th& fits~with our observation that MAP kinase activity increased when eggs entered mitosis and was maintained at a-high level in eggs arrested by-wottmannin. How MAP kinase and MPF interact during mitosisis still unclear. Our data show that, in wortmannin treated egns, MAP kinase can be activated and remained stimulated without a full activation of MPF, suggesting that MAP kinase may be controlled during the cell cycle bv comuonents other than MPF. Bv regulating MAP kit&e activity, PI 3-k&se &ght be an important element in the control of metaphase arrest in oocytes. MAPK, cychne B et p34”“’ during the acquisition of meiotic maturation competence in goat ooqtes. Thierrv DEDIEU, Isabelle HUE, Emilie LEDAN, Laurence GALL, INRA Unite Biologie de la Fecondation 78352 Jouy-en-Josas cMex