Crystal structures of trypanosoma brucei oligopeptidase B broaden the paradigm of catalytic regulation in prolyl oligopeptidase family enzymes

Oligopeptidase B cleaves after basic amino acids in peptides up to 30 residues. As a virulence factor in bacteria and trypanosomatid pathogens that is absent in higher eukaryotes, this is a promising drug target. Here we present ligand-free open state and inhibitor-bound closed state crystal structures of oligopeptidase B from Trypanosoma brucei, the causative agent of African sleeping sickness. These (and related) structures show the importance of structural dynamics, governed by a fine enthalpic and entropic balance, in substrate size selectivity and catalysis. Peptides over 30 residues cannot fit the enzyme cavity, preventing the complete domain closure required for a key propeller Asp/Glu to fix the catalytic His and Arg in the catalytically competent conformation. This size exclusion mechanism protects larger peptides and proteins from degradation. Similar bacterial prolyl endopeptidase and archael acylaminoacyl peptidase structures demonstrate this mechanism is conserved among oligopeptidase family enzymes across all three domains of life

SILA-PHARMACA. II STRUCTURE OF METHYL-IODIDE SALTS OF DIMETHYLPHENYLPIPERIDINOMETHYLSILANE AND N-(,6-PHENYLETHYL)PIPERIDINE

X-ray structures of the methyliodide salts of (N-(,B-phenylethyl)piperidine (N-(,B-phenyl- ethyl )-N-methyl-piperidiniumiodide) and dimethylphenyl-piperidinomethyl-silane (N- methyl-N-(phenyldimethylsilyl )-methyl- piperidinium iodide) are reported

Az onkogének és funkcionális fehérjék korszerű módszerekkel történő tanulmányozása trofoblaszt betegségekben = Studying oncogenes and functional proteins with up-to-date methods in trophoblastic diseases

A kutatási program számos eredménnyel zárult, melynek során jobban megértettük az anyai immunrendszer, a decidua extracelluláris mátrix és az placentaágy angiogenezisének szerepét a trofoblaszt betegségek biológiájában. Elsőként írtuk le az anyai immunrendszer apai antigénekre specifikus reakcióját, valamint demonstráltuk a HCG angiogenetikus hatását humán mintákban és az érkeresztmetszet vizsgálatok esetleges alkalmazhatóságát a perzisztáló esetek előrejelzésére és megelőzésére. Ezen túl kimutattuk, hogy a regulator T sejtek, melyeknek ugyan fontos szerepet tulajdonítanak a perifériás terhességi immunszupresszió kialakításában, a placentaágy területén nem játszanak szerepet az immunrendszer szabályozásában. Choriocarcinoma eseteken demostráltuk az immunsejtek inváziójának blokkolását a trofoblaszt sejthatáron, valamint a placenta ágy további vizsgálatainak során jellemeztük a decidua sejtek Laminin Receptor 1 (LR1) molekula expresszióját. A vizsgálatok során kimutattuk hogy mola terhesség decidua sejtjeiben az LR1 kifejeződése szignifikánsan magasabb, amely hozzájárulhat a trophoblaszt sejtek agrasszivabb viselkedéséhez. | The research project concluded with several results which helped us to better understand the role of the maternal immune system, the decidual extracellular matrix, and the placental angiogenesis in the biology of trophoblastic diseases. We demonstrated first that in pregnancy the maternal immune system is activated against a few specific paternal antigens. We also first showed that HCG has angigenetic potential in human pregnancy and that microvessel density study might be useful in predicting and preventing persistent trophoblastic diseases. Furthermore, we demonstrated that regulatory T cells, which are considered as important immunosuppressive cells in the peripheral blood, do not play role in the regulation of the local immune response at the implantation site. On choriocarcinoma cases we demonstrated the unusual invasion blockade at the border of the trophoblastic cells and by studying the extracellular matrix proteins at the implantation site, we demonstrated a higher expression of Laminin Receptor 1 molecule in molar decidual cells which might play an important role in the regulation of the trophoblast invasion

Structure of thermobifida fusca DyP-type peroxidase and activity towards kraft lignin and lignin model compounds

A Dyp-type peroxidase enzyme from thermophilic cellulose degrader Thermobifida fusca (TfuDyP) was investigated for catalytic ability towards lignin oxidation. TfuDyP was characterised kinetically against a range of phenolic substrates, and a compound I reaction intermediate was observed via pre-steady state kinetic analysis at max 404 nm. TfuDyP showed reactivity towards Kraft lignin, and was found to oxidise a -aryl ether lignin model compound, forming an oxidised dimer. A crystal structure of TfuDyP was determined, to 1.8Å resolution, which was found to contain a diatomic oxygen ligand bound to the heme centre, positioned close to active site residues Asp-203 and Arg-315. The structure contains two channels providing access to the heme cofactor for organic substrates and hydrogen peroxide. Site-directed mutant D203A showed no activity towards phenolic substrates, but reduced activity towards ABTS, while mutant R315Q showed no activity towards phenolic substrates, nor ABTS

Quantity and quality of retrograde menstruation: a case control study

<p>Abstract</p> <p>Background</p> <p>The purpose of this study was to test the hypothesis that menstruation is associated with a higher concentration of endometrial cells in peritoneal fluid(PF) and with increased white and red blood cell concentration in PF when compared to nonmenstrual phases of the cycle.</p> <p>Methods</p> <p>PF was obtained at laparoscopy from 107 women with endometriosis (n = 59) and controls with a normal pelvis (n = 48) during the luteal (n = 46), follicular (n = 38) or menstrual (n = 23) phase of the cycle. Endometriosis was classified according to the classification of the American Society for Reproductive Medicine (rAFS into minimal (n = 25), mild(n = 20), moderate(n = 6) and severe(n = 8) disease. Cell counts (leucocytes, erythrocytes, thrombocytes) were determined on a cell counter. In a subset of 32 patients (13 controls and 19 women with endometriosis), PF was fixed, processed and thinlayers were prepared and stained with Papanicolaou method and with immunocytochemistry using monoclonal antibodies against cytokeratin 7(CK 7), CK 8/18, Ber-Ep4, vimentin, calretinin and CD68. Ber-Ep4 is a marker for cells with epithelial origin (in some cases for mesothelial cells as well). CD68 is specific for cells from monocyte/macrophage lineage; CK7 and CK8/18 are markers for both endometrial epithelial and mesothelial cells, whereas calretinin and vimentin are markers for both endometrial stromal and mesothelial cells.</p> <p>Results</p> <p>In comparison with the nonmenstrual phase of the cycle, analysis of PF during menstruation showed an increased concentration of leucocytes (3.3 &#215; 10⁹/L vs 0.8 &#215; 10⁹/L, P = 0.03), erythrocytes (0.3 &#215; 10¹²/L vs 0.02 &#215; 10¹²/L, P = 0.006), hematocrit (0.03 L/L vs 0.003 L/L, P = 0.01) and hemoglobin (0.8 g/dL vs 0.1 g/dL, P = 0.01). Mesothelial cells stained positively with CK7, CK8/18, vimentin, and calretinin. Cells positive for Ber-Ep4 were not observed, except in 2 patients with endometriosis investigated during menses. In all patients 50-98% of single cells were strongly positive for both vimentin and CD68.</p> <p>Conclusion</p> <p>When compared to nonmenstrual phases of the cycle, menstruation is associated with an increased concentration of red and white blood cells in PF. However, the presence of EM cells that are detectable by immunohistochemistry in PF is low during all phases of the cycle, including menstruation.</p

Characterisation of thiamine diphosphate-dependent 4-hydroxybenzoylformate decarboxylase enzymes from Rhodococcus jostii RHA1 and Pseudomonas fluorescens Pf-5 involved in degradation of aryl-C2 lignin degradation fragments

A thiamine diphosphate-dependent enzyme annotated as a benzoylformate decarboxylase is encoded in gene cluster ro02984-ro02986 in Rhodococcus jostii RHA1 previously shown to generate vanillin and 4-hydroxybenzaldehyde from lignin oxidation, and a closely related gene cluster is also found in the genome of Pseudomonas fluorescens Pf-5. Two hypotheses for possible pathways involving a thiamine diphosphate-dependent cleavage, either C-C cleavage of a ketol or diketone aryl C3 substrate, or decarboxylation of an aryl C2 substrate, were investigated by expression and purification of the recombinant enzymes, and expression of dehydrogenase and oxidase enzymes also found in the gene clusters. The ThDP-dependent enzymes showed no activity for cleavage of aryl C3 ketol or diketone substrates, but showed activity for decarboxylation of benzoylformate and 4-hydroxybenzoylformate. A flavin-dependent oxidase encoded by gene ro02984 was found to oxidise either mandelic acid or phenylglyoxal. The crystal structure of the P. fluorescens decarboxylase enzyme was determined at 1.69 Å resolution, showing similarity to known benzoylformate decarboxylase enzymes. The P. fluorescens decarboxylase enzyme showed enhanced carboligase activity between vanillin and acetaldehyde, rationalised by the presence of alanine vs serine at residue 73 in the enzyme active site, which was investigated further by site-directed mutagenesis of this residue. A hypothesis for a pathway for degradation of aryl-C2 fragments arising from oxidative cleavage of phenylcoumaran and diarylpropane structures in lignin is proposed

Sphingobacterium sp. T2 manganese superoxide dismutase catalyses the oxidative demethylation of polymeric lignin via generation of hydroxyl radical

Sphingobacterium sp. T2 contains two extracellular manganese superoxide dismutase enzymes which exhibit unprecedented activity for lignin oxidation but via an unknown mechanism. Enzymatic treatment of lignin model compounds gave products whose structures were indicative of aryl–Cα oxidative cleavage and demethylation, as well as alkene dihydroxylation and alcohol oxidation. 18O labeling studies on the SpMnSOD-catalyzed oxidation of lignin model compound guiaiacylglycerol-β-guaiacyl ether indicated that the an oxygen atom inserted by the enzyme is derived from superoxide or peroxide. Analysis of an alkali lignin treated by SpMnSOD1 by quantitative 31P NMR spectroscopy demonstrated 20–40% increases in phenolic and aliphatic OH content, consistent with lignin demethylation and some internal oxidative cleavage reactions. Assay for hydroxyl radical generation using a fluorometric hydroxyphenylfluorescein assay revealed the release of 4.1 molar equivalents of hydroxyl radical by SpMnSOD1. Four amino acid replacements in SpMnSOD1 were investigated, and A31H or Y27H site-directed mutant enzymes were found to show no lignin demethylation activity according to 31P NMR analysis. Structure determination of the A31H and Y27H mutant enzymes reveals the repositioning of an N-terminal protein loop, leading to widening of a solvent channel at the dimer interface, which would provide increased solvent access to the Mn center for hydroxyl radical generation