74 research outputs found
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
Crystallization and preliminary X-ray analysis of a D-alanyl-D-alanine ligase (EcDdlB) from Escherichia coli
A recombinant form of Escherichia coli DdlB (EcDdlB) has been prepared and cocrystallized with ADP and D-alanyl-D-alanine to represent the ternary complex of EcDdlB. Furthermore, EcDdlB has been cocrystallized under the same conditions with the ligands ATP and D-alanyl-D-alanine, representing the product-inhibited complex. The crystals belonged to space group P212121, with unit-cell parameters a = 53.0, b = 97.6, c = 109.5 Å and a = 51.2, b = 97.8, c = 110.1 Å, respectively, and both contained two molecules in the asymmetric unit. Complete data sets were collected to 1.5 and 1.4 Å resolution, respectively, from single crystals under cryogenic conditions using synchrotron radiation
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
Crystal structure of the di-haem cytochrome c peroxidase from Pseudomonas aeruginosa
AbstractBackground: Cytochrome c peroxidase from Pseudomonas aeruginosa (PsCCP) represents a new class of peroxidases which work without the need to create a semi-stable free radical for catalysis. The enzyme is located in the bacterial periplasm where its likely function is to provide protection against toxic peroxides. The soluble 323-residue single polypeptide chain contains two covalent c-type haems with very different properties: one of them is a low-potential (–330 mV) centre where hydrogen peroxide is reduced (the peroxidatic site); the other is a high-potential (+320 mV) centre which feeds electrons to the peroxidatic site from soluble electron-shuttle proteins such as cytochrome c and azurin.Results The crystal structure of the oxidized form of PsCCP has been determined to 2.4 å resolution by multiple isomorphous replacement, and refined to an R-factor of 19.2%. PsCCP is organized into two domains, both of them containing a covalent c-haem in a structure reminiscent of class 1 cytochromes c. The domains are related by a quasi-twofold axis. The domain interface holds a newly discovered calcium-binding site with an unusual set of ligands.Conclusion The likely function of the calcium site is to maintain the structural integrity of the enzyme and/or to modulate electron transfer between the two haem domains. The low-potential haem has two histidine axial ligands (His55 and His71) and the high-potential haem is ligated by His201 and Met275. There are no polar residues at the peroxidatic site in the inactive oxidized enzyme. The structure suggests that, in the half-reduced functional form of the enzyme, the low-potential haem has to shed His71 in order to make the enzyme catalytically competent. This process is likely to trigger a reorganization of the active site, and may introduce new residues into the haem pocket
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
A praenatalis és postnatalis mikrobiom jelentősége és hatásai a korai egyedfejlődés időszakában és az intervenciós kezelés lehetőségei
A gyulladásos és immunológiai folyamatok kapcsolata a várandósság alatt. Gyakorlati vonatkozások = The relationship between inflammatory and immunological processes during pregnancy. Practical aspects
Absztrakt:
A közlemény célja, hogy a biológiai háttér feltárásán túlmenően olyan új elvi és
kezelési megközelítéseket tárjon fel a reprodukcióval foglalkozó klinikusok
számára, amelyek a meddő és infertilis párok jobb és eredményesebb ellátását
szolgálják. A humán vonatkozásban a sikertelen terhességek 75%-a eredménytelen
beágyazódás következménye, és a beágyazódási kudarc a korlátja az IVF-kezelések
eredményességének is. A beágyazódáshoz és a szüléshez ún. „jó”, módosított
gyulladás szükséges, de a terhesség legnagyobb részében a gyulladás fenyegeti a
terhesség megtartását. Ekkor a gyulladásmentes állapotnak a fenntartása
rendkívül fontos, ezáltal lehetővé téve a magzaton az anyai epigenetikai hatások
érvényesülését, ami az extrauterin élethez való minél jobb alkalmazkodást teszi
lehetővé az utódok számára. A terhesség gyulladásmentes állapotának
fenntartásában a lepény által termelt (a luteoplacentaris shift után) nagy
mennyiségű progeszteron hormonnak döntő szerepe van. Többen leírták, hogy a
beágyazódás alatti gyulladás az embrióra mint egy idegen testre adott ősi
válaszként értelmezhető. A normálterhesség során ezt a gyulladást a
trophoblastok indítják el, és magában foglalja egyrészt a természetes ölősejtek
összetoborzását az implantáció helyére, a neutrofil beszűrődés gátlását,
másrészt egy sor gyulladásos citokin termelését. A „beágyazódási ablak” idején a
méh feltöltött állapotba kerülve több gyulladásos jelet, így prosztaglandin
E2-t és számos gyulladásos citokint, köztük a TNF-t, IL6-ot és
IFNγ-t termel. A fetoplacentaris egység egy félig idegennek tekinthető oltvány,
ún. „szemiallograft”, és az anyai gazdaszervezet (host) részéről a terhesség
felismerése, a következményes anyai immuntolerancia kialakulása elengedhetetlen
része a terhesség sikeres kiviselésének és az egészséges magzat megszületésének.
A keringő progeszteron mennyiségének funkcionális vagy abszolút csökkenése,
hiánya miatt (a 36. terhességi hét után a fiziológiásan is „öregedő” lepény
csökkenő hormontermelésének következtében) elégtelenné válnak a
progeszteronhatások, ami az IL8 és egyéb gyulladásos citokinek termelését és a
terminusra jellemző gyulladást már nem képes visszaszorítani, és ez a méhnyak
éréséhez, a fájások megindulásához és szüléshez vezet („jó” gyulladás). Orv
Hetil. 2019; 160(32): 1247–1259.
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Abstract:
The aim of this review is to explore, in addition to revealing the biological
background, new conceptual and therapeutic approaches for reproductive
clinicians to provide better and more effective care for sterile and infertile
couples. In humans, 75% of unsuccessful pregnancies are the result of failures
of implantation, and implantation failure is the limiting factor for in
vitro fertilization treatment. A modified “good” inflammation is
necessary for implantation and parturition, but for most of pregnancy,
inflammation threatens the continuation of pregnancy. During this period,
maintaining the non-inflammatory condition is extremely important, enabling the
maternal epigenetic effects to occur in the fetus, making it possible for the
offspring to adapt as much as possible to the extrauterine life. In the
maintenance of the non-inflammatory condition of pregnancy, a large amount of
progesterone hormone produced by the placenta (after the luteo-placental shift)
plays a crucial role. It has been reported that the role of inflammation during
implantation is an ancestral response to the embryo as a foreign body. During
normal pregnancy, this inflammation is initiated by the trophoblast and involves
the suppression of neutrophil infiltration, the recruitment of natural killer
cells to the site of implantation as well as the production of a range of
proinflammatory cytokines. During the “implantation window”, the uterus is
primed to produce several inflammatory signals such as prostaglandin
E2 and a range of proinflammatory cytokines, including TNF, IL6
and IFNγ. The feto-placental unit is a semi-foreign graft called a “semi
allograft”, and the recognition of pregnancy by the mother (host) and the
resulting maternal immune tolerance is an essential part of successful pregnancy
and the birth of a healthy fetus. Because of the functional or absolute
reduction of circulating progesterone (due to the decreasing hormone production
of the physiologically “aging” placenta after around the 36th week of pregnancy)
progesterone effects become insufficient. Therefore it is unable to suppress the
production of IL8 and other inflammatory cytokines and the term inflammation,
leading to cervical ripening, uterus contractions and parturition (“good”
inflammation). Orv Hetil. 2019; 160(32): 1247–1259
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 × 10<sup>9</sup>/L vs 0.8 × 10<sup>9</sup>/L, P = 0.03), erythrocytes (0.3 × 10<sup>12</sup>/L vs 0.02 × 10<sup>12</sup>/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
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