Interaction of ascorbate peroxidase with substrates: a mechanistic and structural analysis

Previous work [Sharp, K. H., et al. (2003) Nat. Struct. Biol. 10, 303-307] has revealed the location of the ascorbate binding site in ascorbate peroxidase and has identified hydrogen-bonding interactions to Arg172, Lys30, and the heme 6-propionate as important in formation of the enzyme-substrate complex. In this work, the individual and collective contributions of these hydrogen bond interactions have been dissected using site-directed mutagenesis, steady-state and pre-steady-state kinetics, X-ray crystallography, and modified substrate analogues. Steady-state and pre-steady-state kinetic data reveal that the hydrogen bonds to Arg172 and the heme 6-propionate play a major part in stabilization of the bound ascorbate but that the interaction with Lys30 plays only a minor role. Binding of aromatic substrates is not affected by substitutions at Arg172/Lys30. Neutralization or removal of electrostatic charge at (Lys30) or adjacent to (Lys31) the ascorbate site does not substantially disrupt the binding interaction. Substrate oxidation and reduction of Compounds I and II is still possible in the absence of Arg172, but at a much reduced level. Crystallographic data (to 1.8 A) for the R172A variant indicate that the molecular structure of the proposed [Sharp, K. H., et al. (2004) Biochemistry 43, 8644-8651] proton transfer pathway from the ascorbate to the heme is conserved, which accounts for the residual activity. The results are discussed in terms of our wider understanding of the structural features that control substrate binding specificity in other peroxidase enzymes.     

Tubulin polymerization promoting proteins (TPPPs): members of a new family with distinct structures and functions

TPPP/p25 is a brain-specific protein, which induces tubulin polymerization and microtubule (MT) bundling and is enriched in Lewy bodies characteristic of Parkinson's disease [Tirián et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 13976-13981]. We identified two human gene sequences, CG1-38 and p25beta, which encoded homologous proteins, that we termed p20 and p18, respectively. These homologous proteins display 60% identity with tubulin polymerization promoting protein/p25 (TPPP/p25); however, the N-terminal segment of TPPP/p25 is missing. They could be clustered into three subfamilies present in mammals and other vertebrates. We cloned, isolated, and characterized the structural and functional properties of the recombinant human proteins at molecular, ultrastructural, and cellular levels using a number of tools. These data revealed that, while p20 behaved as a disorganized protein similarly to TPPP/p25, which was described as a flexible and inherently dynamic protein with a long unstructured N-terminal tail, p18 was featured in more ordered fashion. TPPP/p25 and p20 specifically attached to MTs causing MT bundling both in vitro and in vivo; p18 protein did not cross-link MTs, and it distributed homogeneously within the cytosol of the transfected HeLa cells. These data indicate that the two shorter homologues display distinct structural features that determine their associations to MTs. The properties of p20 resemble TPPP/p25. The bundling activity of these two proteins results in the stabilization of the microtubular network, which is likely related to their physiological functions.     

A proton delivery pathway in the soluble fumarate reductase from Shewanella frigidimarina

The mechanism for fumarate reduction by the soluble fumarate reductase from Shewanella frigidimarina involves hydride transfer from FAD and proton transfer from the active-site acid, Arg-402. It has been proposed that Arg-402 forms part of a proton transfer pathway that also involves Glu-378 and Arg-381 but, unusually, does not involve any bound water molecules. To gain further insight into the importance of this proton pathway we have perturbed it by substituting Arg-381 by lysine and methionine and Glu-378 by aspartate. Although all the mutant enzymes retain measurable activities, there are orders-of-magnitude decreases in their k(cat) values compared with the wild-type enzyme. Solvent kinetic isotope effects show that proton transfer is rate-limiting in the wild-type and mutant enzymes. Proton inventories indicate that the proton pathway involves multiple exchangeable groups. Fast scan protein-film voltammetric studies on wild-type and R381K enzymes show that the proton transfer pathway delivers one proton per catalytic cycle and is not required for transporting the other proton, which transfers as a hydride from the reduced, protonated FAD. The crystal structures of E378D and R381M mutant enzymes have been determined to 1.7 and 2.1 A resolution, respectively. They allow an examination of the structural changes that disturb proton transport. Taken together, the results indicate that Arg-381, Glu-378, and Arg-402 form a proton pathway that is completely conserved throughout the fumarate reductase/succinate dehydrogenase family of enzymes.     

Wind dispersal of soil invertebrates in the McMurdo Dry Valleys, Antarctica

Dispersal of soil organisms is crucial for their spatial distribution and adaptation to the prevailing conditions of the Antarctic Dry Valleys. This study investigated the possibility of wind dispersal of soil invertebrates within the dry valleys. Soil invertebrates were evaluated in (1) pockets of transported sediments to lake ice and glacier surfaces, (2) wind-transported dust particles in collection pans (Bundt pans) 100 cm above the soil surface, and (3) sediments transported closer to the surface (<50 cm) and collected in open top chambers (OTCs). Invertebrates were extracted and identified. Nematodes were identified to species and classified according to life stage and sex. Three species of nematodes were recovered and Scottnema lindsayae was the most dominant. There were more juveniles (∼71%) in the transported sediments than adults (29%). Tardigrades and rotifers were more abundant in sediments on lake and glacier surfaces while nematodes were more abundant in the dry sediment collections of Bundt pans and OTCs. The abundance of immobile (dead) nematodes in the Bundt pans and OTCs was three times greater than active (live) nematodes. Anhydrobiosis constitutes a survival mechanism that allows wind dispersal of nematodes in the McMurdo Dry Valleys. Our results show that soil invertebrates are dispersed by wind in the Dry Valleys and are viable in ice communities on lake surfaces and glaciers.     

The protistan origins of animals and fungi

Recent molecular studies suggest that Opisthokonta, the eukaryotic supergroup including animals and fungi, should be expanded to include a diverse collection of primitively single-celled eukaryotes previously classified as Protozoa. These taxa include corallochytreans, nucleariids, ministeriids, choanoflagellates, and ichthyosporeans. Assignment of many of these taxa to Opisthokonta remains uncorroborated as it is based solely on small subunit ribosomal RNA trees lacking resolution and significant bootstrap support for critical nodes. Therefore, important details of the phylogenetic relationships of these putative opisthokonts with each other and with animals and fungi remain unclear. We have sequenced elongation factor 1-alpha (EF-1alpha), actin, beta-tubulin, and HSP70, and/or alpha-tubulin from representatives of each of the proposed protistan opisthokont lineages, constituting the first protein-coding gene data for some of them. Our results show that members of all opisthokont protist groups encode a approximately 12-amino acid insertion in EF-1alpha, previously found exclusively in animals and fungi. Phylogenetic analyses of combined multigene data sets including a diverse set of opisthokont and nonopisthokont taxa place all of the proposed opisthokont protists unequivocally in an exclusive clade with animals and fungi. Within this clade, the nucleariid appears as the closest sister taxon to fungi, while the corallochytrean and ichthyosporean form a group which, together with the ministeriid and choanoflagellates, form two to three separate sister lineages to animals. These results further establish Opisthokonta as a bona fide taxonomic group and suggest that any further testing of the legitimacy of this taxon should, at the least, include data from opisthokont protists. Our results also underline the critical position of these "animal-fungal allies" with respect to the origin and early evolution of animals and fungi.     

Coinoculation with hepatitis B surface and core antigen promotes a Th1 immune response to a multiepitopic protein of HIV-1

It has been defined that strong and multispecific cellular immune responses correlate with a better prognosis during the course of chronic diseases. A cross-enhancing effect on the resulting immune response obtained by the coadministration of recombinant hepatitis B virus (HBV) surface and core Ag was recently observed. With the objective of studying the effect of such Ag on the immune response to coinoculated heterologous Ag and vice versa, several formulations containing the recombinant HBV Ag and a multiepitopic protein (CR3) composed by CTL and Th epitopes from HIV-1 were evaluated by s.c. and mucosal administration. Combinations of two and three Ag were evaluated for cellular and humoral immune responses. The results showed that the best Ag combination for nasal immunization was the mixture comprising the CR3 recombinant HIV protein and both HBV Ag. Similarly, it was also the best formulation for s.c. immunization in aluminium phosphate adjuvant. In conclusion, it is possible to induce a Th1 stimulation of the cellular immune response specific for a HIV-based recombinant protein by formulating this Ag with the recombinant HBV Ag.     

Perturbations in nuclear factor-kappaB or c-Jun N-terminal kinase pathways in pancreatic beta cells confer susceptibility to cytokine-induced cell death

Pro-inflammatory cytokines have been implicated in the death of pancreatic beta cells leading to type 1 diabetes. NIT-1 cells are an insulinoma cell line derived from mice expressing the SV40 large T antigen. These cells are a useful tool in analysis of beta cell death. NIT-1 cells are highly susceptible to caspase-dependent apoptosis induced by TNF-alpha alone. Primary islets are not susceptible to cell death induced by TNF-alpha alone; however, they are killed by TNF-alpha and IFN-gamma in a nitric oxide-dependent manner. We examined signal transduction in NIT-1 cells in response to cytokines to determine the mechanism for TNF-alpha-induced apoptosis. We found that NIT-1 cells are defective in the activation of nuclear factor-kappaB (NFkappaB) as a result of functionally deficient RelA activity, because overexpression of RelA protected NIT-1 cells from apoptosis. TNF-alpha also did not induce phosphorylation of c-Jun N-terminal kinase in NIT-1 cells. Together, these defects prevent expression of anti-apoptotic genes in NIT-1 cells and make them susceptible to TNF-alpha. To determine whether similar defects in primary beta cells would induce the same effect, we examined TNF-alpha-induced apoptosis in islets isolated from mice deficient in NFkappaB p50. These islets were as susceptible as wild-type islets to TNF-alpha and IFN-gamma-induced cell death. In contrast to wild-type islets, cell death was not prevented by inhibition of nitric oxide in p50-deficient islets. Blocking NFkappaB has been proposed as a mechanism for protection of beta cells from cytokine-induced cell death in vivo. Our results suggest that this would make beta cells equally or more sensitive to cytokines.     

Lutein effect on retina and hippocampus of diabetic mice

Oxidative stress markers and functional tests were studied to confirm early biochemical and functional changes in retina and hippocampus of diabetic mice. The effects of lutein treatment were also tested. Mice were induced diabetic by alloxan injection and divided into subgroups: control, control+lutein, diabetic, diabetic+lutein, diabetic+insulin, and diabetic+insulin+lutein. Treatments started on Day 4 after alloxan injection and animals were sacrificed on Day 14. Malondialdehyde and glutathione concentrations and glutathione peroxidase activity were measured as oxidative stress markers. The following functional tests for retina and hippocampus were performed: electroretinogram and Morris water maze test. NFkappaB activity was also measured. Oxidative stress and NFkappaB activity increase in the retina and hippocampus after 15 days of diabetes. Impairment of the electroretinogram and a correlation between latencies of the water maze test and glycated hemoglobin (HbA1c) levels were observed. Lutein prevented all these changes even under hyperglycemic conditions. Retina appears to be affected earlier than hippocampus by diabetes-induced oxidative stress. Although a proper glycemic control is desirable in preventing the development of diabetic complications, it is not sufficient to prevent them completely. Lutein could be an appropriate coadjuvant treatment for the changes observed in this study.     

Saccharomyces cerevisiae strain expressing a plant fatty acid desaturase produces polyunsaturated fatty acids and is susceptible to oxidative stress induced by lipid peroxidation

Although oxygen is essential for aerobic organisms, it also forms potentially harmful reactive oxygen species. For its simplicity, easy manipulation, and cultivation conditions, yeast is used as an attractive model in oxidative stress research. However, lack of polyunsaturated fatty acids in yeast membranes makes yeast unsuitable for research in the field of lipid peroxidation. Therefore, we have constructed a yeast strain expressing a Delta12 desaturase gene from the tropical rubber tree, Hevea brasiliensis. This yeast strain expresses the heterologous desaturase in an active form and, consequently, produces Delta9/Delta12 polyunsaturated fatty acids under inducing conditions. The functional expression of the heterologous desaturase did not affect cellular morphology or growth, indicating no general adverse effect on cellular physiology. However, the presence of polyunsaturated fatty acids changed the yeast's sensitivity to oxidative stress induced by addition of paraquat, tert-butylhydroperoxide, and hydrogen peroxide. This difference in sensitivity to the latter was followed by the formation of 4-hydroxy-2-nonenal, one of the end products of linoleic fatty acid peroxidation, which is known to play a role in cell growth control and signaling. Here we show that this yeast strain conditionally expressing the Delta12 desaturase gene provides a novel and well-defined eukaryotic model in lipid peroxidation research. Its potential to investigate the molecular basis of responses to oxidative stress, in particular the involvement of reactive aldehydes derived from fatty acid peroxidation, especially 4-hydroxy-2-nonenal, will be addressed.     

Modulators of the human CCR5 receptor. Part 3: SAR of substituted 1-[3-(4-methanesulfonylphenyl)-3-phenylpropyl]-piperidinyl phenylacetamides

SAR and PK studies led to the identification of N-(1-{(3R)-3-(3,5-difluorophenyl)-3-[4-methanesulfonylphenyl] propyl}piperidin-4-yl)-N-ethyl-2-[4-methanesulfonylphenyl]acetamide as a highly potent and selective ligand for the human CCR5 chemokine receptor with good oral pharmacokinetic properties.