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[
Structure,
2002]
The crystal structure of C. elegans Ap(4)A hydrolase has been determined for the free enzyme and a binary complex at 2.0 Angstrom. and 1.8 Angstrom, respectively. Ap(4)A hydrolase has a key role in regulating the intracellular Ap(4)A levels and hence potentially the cellular response to metabolic stress and/or differentiation and apoptosis via the Ap(3)A/Ap(4)A ratio. The structures reveal that the enzyme has the mixed alpha/beta fold of the Nudix family and also show how the enzyme binds and locates its substrate with respect to the catalytic machinery of the Nudix motif. These results suggest how the enzyme can catalyze the hydrolysis of a range of related dinucleoside tetraphosphate, but not triphosphate, compounds through precise orientation of key elements of the substrate.
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[
Acta Crystallogr D Biol Crystallogr,
2002]
The molecule diadenosine tetraphosphate (Ap(4)A) has been suggested to be a component of the cellular response to metabolic stress and/or, via the intracellular Ap(3)A/Ap(4)A ratio, to be involved in differentiation and apoptosis. Thus, the enzyme Ap(4)A hydrolase has a key metabolic role through regulation of the intracellular Ap(4)A levels. Crystals of this enzyme from the nematode Caenorhabditis elegans have been obtained in the presence of a non-hydrolysable substrate analogue, AppCH(2)ppA. The crystals belong to space group P2(1), unit-cell parameters a = 57.6, b = 36.8, c = 68.9 Angstrom, beta = 114.2degrees, and diffract to approximately 2.0 Angstrom. Determination of the structure of this complex will provide insights into the substrate specificity and catalytic activity of this class of enzymes.
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[
J Biol Chem,
2003]
The contributions to substrate binding and catalysis of 13 amino acid residues of the Caenorhabditis elegans diadenosine tetraphosphate pyrophosphohydrolase (Ap(4)A hydrolase) predicted from the crystal structure of an enzyme-inhibitor complex have been investigated by site-directed mutagenesis. Sixteen glutathione S-transferase-Ap(4)A hydrolase fusion proteins were expressed and their k(cat) and K-m values determined after removal of the glutathione S-transferase domain. As expected for a Nudix hydrolase, the wild type k(cat) of 23 s(-1) was reduced by 10(5)-, 10(3)-, and 30-fold, respectively, by replacement of the conserved P-phosphate-binding catalytic residues Glu(56), Glu(52), and Glu(103) by Gln. K-m values were not affected, indicating a lack of importance for substrate binding. In contrast, mutating His(31) to Val or Ala and Lys(83) to Met produced 10- and 16-fold increases in K. compared with the wild type value of 8.8 muM. These residues stabilize the P-1-phosphate. H31V and H31A had a normal kcat but K83M showed a 37-fold reduction in k(cat). Lys(36) also stabilizes the P-1-phosphate and a K36M mutant had a 10-fold reduced kcat but a relatively normal K-m. Thus both Lys(36) and Lys(83) may play a role in catalysis. The previously suggested roles of Tyr(27), His(38), Lys(79), and Lys(81) in stabilizing the P-2 and P-3-phosphates were not confirmed by mutagenesis, indicating the absence of phosphate-specific binding contacts in this region. Also, mutating both Tyr(76) and Tyr(121), which clamp one substrate adenosine moiety between them in the crystal structure, to Ala only increased K-m 4-fold. It is concluded that interactions with the P-1- and P-4-phosphates are minimum and sufficient requirements for substrate binding by this class of enzyme, indicating that
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[
J Biol Chem,
2007]
The biological methyl donor, S adenosylmethionine (AdoMet), can exist in two diastereoisomeric states with respect to its sulfonium ion. The "S" configuration, (S,S)AdoMet, is the only form that is produced enzymatically as well as the only form used in almost all biological methylation reactions. Under physiological conditions, however, the sulfonium ion can spontaneously racemize to the "R" form, producing (R,S)AdoMet. As of yet, (R,S)AdoMet has no known physiological function and may inhibit cellular reactions. In this study, two enzymes have been found in Saccharomyces cerevisiae that are capable of recognizing (R,S)AdoMet and using it to methylate homocysteine to form methionine. These enzymes are the products of the SAM4 and MHT1 genes, previously identified as homocysteine methyltransferases dependent upon AdoMet and S-methylmethionine respectively. We find here that Sam4 recognizes both (S,S) and (R,S)AdoMet, but its activity is much higher with the R,S form. Mht1 reacts with only the R,S form of AdoMet while no activity is seen with the S,S form. R,S-specific homocysteine methyltransferase activity is also shown here to occur in extracts of Arabidopsis thaliana, Drosophila melanogaster, and Caenorhabditis elegans, but has not been detected in several tissue extracts of Mus musculus. Such activity may function to prevent the accumulation of (R,S)AdoMet in these organisms.
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Termine D, Becuwe M, Hofbauer HF, Barrasa MI, Pincus D, Imberdis T, Selkoe D, Freyzon Y, Srinivasan S, Soldner F, Nuber S, Sandoe J, Haque A, Welte MA, Clish CB, Terry-Kantor E, Jaenisch R, Kohlwein SD, Fanning S, Dettmer U, Walther TC, Kim TE, Farese RV, Landgraf D, Baru V, Noble T, Lou Y, Lindquist S, Newby G, Ho GPH, Ramalingam N
[
Mol Cell,
2018]
In Parkinson's disease (PD), -synuclein (S) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in S or lipid/fattyacid homeostasis affect each other. Lipidomic profiling of human S-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of S dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased S yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in S-overexpressing rat neurons. In a C.elegans model, SCD knockout prevented S-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on S homeostasis: in human neural cells, excess OA caused S inclusion formation, which was reversed by SCD inhibition. Thus, monounsaturated fatty acid metabolism is pivotal for S-induced neurotoxicity, and inhibiting SCD represents a novel PD therapeutic approach.
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[
PLoS One,
2017]
In this paper, the metabolic activity in single and dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus isolates was investigated. Our results demonstrated that there was less metabolic activity in dual species biofilms compared to S. aureus biofilms. However, this was not observed if S. aureus and S. epidermidis were obtained from the same sample. The largest effect on metabolic activity was observed in biofilms of S. aureus Mu50 and S. epidermidis ET-024. A transcriptomic analysis of these dual species biofilms showed that urease genes and genes encoding proteins involved in metabolism were downregulated in comparison to monospecies biofilms. These results were subsequently confirmed by phenotypic assays. As metabolic activity is related to acid production, the pH in dual species biofilms was slightly higher compared to S. aureus Mu50 biofilms. Our results showed that S. epidermidis ET-024 in dual species biofilms inhibits metabolic activity of S. aureus Mu50, leading to less acid production. As a consequence, less urease activity is required to compensate for low pH. Importantly, this effect was biofilm-specific. Also S. aureus Mu50 genes encoding virulence-associated proteins (Spa, SplF and Dps) were upregulated in dual species biofilms compared to monospecies biofilms and using Caenorhabditis elegans infection assays, we demonstrated that more nematodes survived when co-infected with S. epidermidis ET-024 and S. aureus mutants lacking functional spa, splF or dps genes, compared to nematodes infected with S. epidermidis ET-024 and wild- type S. aureus. Finally, S. epidermidis ET-024 genes encoding resistance to oxacillin, erythromycin and tobramycin were upregulated in dual species biofilms and increased resistance was subsequently confirmed. Our data indicate that both species in dual species biofilms of S. epidermidis and S. aureus influence each other's behavior, but additional studies are required necessary to elucidate the exact mechanism(s) involved.
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Kamp F, Winklhofer KF, Giese A, Lutz AK, Brunner B, Wender N, Hegermann J, Haass C, Eimer S, Bartels T, Beyer K, Exner N, Nuscher B
[
EMBO J,
2010]
Aggregation of -synuclein (S) is involved in the pathogenesis of Parkinson's disease (PD) and a variety of related neurodegenerative disorders. The physiological function of S is largely unknown. We demonstrate with in vitro vesicle fusion experiments that S has an inhibitory function on membrane fusion. Upon increased expression in cultured cells and in Caenorhabditis elegans, S binds to mitochondria and leads to mitochondrial fragmentation. In C. elegans age-dependent fragmentation of mitochondria is enhanced and shifted to an earlier time point upon expression of exogenous S. In contrast, siRNA-mediated downregulation of S results in elongated mitochondria in cell culture. S can act independently of mitochondrial fusion and fission proteins in shifting the dynamic morphologic equilibrium of mitochondria towards reduced fusion. Upon cellular fusion, S prevents fusion of differently labelled mitochondrial populations. Thus, S inhibits fusion due to its unique membrane interaction. Finally, mitochondrial fragmentation induced by expression of S is rescued by coexpression of PINK1, parkin or DJ-1 but not the PD-associated mutations PINK1 G309D and parkin 1-79 or by DJ-1 C106A.
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[
Pathog Dis,
2014]
Due to the resistance of Staphylococcus aureus to several antibiotics, treatment of S. aureus infections is often difficult. As an alternative to conventional antibiotics, the field of bacterial interference is investigated. Staphylococcus epidermidis produces a serine protease (Esp) which inhibits S. aureus biofilm formation and which degrades S. aureus biofilms. In this study, we investigated the protease production of 114 S. epidermidis isolates, obtained from biofilms on endotracheal tubes (ET). Most of the S. epidermidis isolates secreted a mixture of serine, cysteine and metalloproteases. We found a link between high protease production by S. epidermidis and the absence of S. aureus in ET biofilms obtained from the same patient. Treating S. aureus biofilms with the supernatant (SN) of the most active protease producing S. epidermidis isolates resulted in a significant biomass decrease compared to untreated controls, while the number of metabolically active cells was not affected. The effect on the biofilm biomass was mainly due to serine proteases. Staphylococcus aureus biofilms treated with the SN of protease producing S. epidermidis were thinner with almost no extracellular matrix. An increased survival of Caenorhabditis elegans, infected with S. aureus Mu50, was observed when the SN of protease positive S. epidermidis was added.
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[
Mol Cell Biol,
1997]
The cDNAs and genes encoding the intron lariat-debranching enzyme were isolated from the nematode Caenorhabditis elegans and the fission yeast Schizosaccharomyces pombe based on their homology with the Saccharomyces cerevisiae gene. The cDNAs were shown to be functional in an interspecific complementation experiment; they can complement an S. cerevisiae
dbr1 null mutant. About 2.5% of budding yeast S. cerevisiae genes have introns, and the accumulation of excised introns in a
dbr1 null mutant has little effect on cell growth. In contrast, many S. pombe genes contain introns, and often multiple introns per gene, so that S. pombe is estimated to contain approximately 40 times as many introns as S. cerevisiae. The S. pombe
dbr1 gene was disrupted and shown to be nonessential. Like the S. cerevisiae mutant, the S. pombe null mutant accumulated introns to high levels, indicating that intron lariat debranching represents a rate-limiting step in intron degradation in both species. Unlike the S. cerevisiae mutant, the S. pombe
dbr1::
leu1+ mutant had a severe growth defect and exhibited an aberrant elongated cell shape in addition to an intron accumulation phenotype. The growth defect of the S. pombe
dbr1::
leu1+ strain suggests that debranching activity is critical for efficient intron RNA degradation and that blocking this pathway interferes with cell growth.
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[
Parasitology,
1998]
In the savanna areas of tropical Africa, cattle are frequently infected with the filaria Onchocerca ochengi. This parasite is closely related to Onchocerca volvulus, the causative agent of human onchocerciasis (river blindness), and is capable of developing in the same vector, Simulium damnosum s.l. In North Cameroon, where both O. ochengi and O. volvulus are endemic, we carried out a field study (reported in this and 2 following papers) to examine to which extent the transmission of the 2 parasite species overlap and what influence this has on the epidemiology of human onchocerciasis. In this paper we report our experiments to determine which of the S. damnosum species in North Cameroon act as vectors of O. ochengi, how efficiently they do so and whether other Simulium species play a vector role. To this end, infected cattle were exposed near 5 rivers in different geographical areas. Among 14 Simulium species identified as aquatic and/or adult stages at these rivers, only 6 (S. squamosum, S. damnosum s.s., S. sirbanum, S. bovis, S. wellmanni and S. hargreavesi) were found to bite cattle in important numbers in at least 1 of the sites. The 3 species of the S. damnosum complex were all capable of ingesting microfilariae (mf) of O. ochengi and developing a proportion of them to infective larvae (L3). Whereas S. squamosum and S. damnosum s.s., the prevailing vectors in the Guinea and Sudan savanna respectively, showed a high vector competence (17% of ingested mf developed to L3), S. sirbanum, which was much rarer in both areas, appeared to have a much lower susceptibility (2%). Other boophilic Simulium species were only seen in certain sites and seasons, being either incapable of ingesting important numbers of O. ochengi mf from body regions where these mf were abundant (S. bovis, S. hargreavesi); not able to support the development of ingested mf to L3 (S. wellmanni), or bit cattle preferentially in the ears, where O. ochengi mf do not occur (S. hargreavesi). We conclude that in North Cameroon members of the S. damnosum complex are the only important vectors of O. ochengi, with S. squamosum and S. damnosum s.s. being the main vectors.