Retrovesical hydatidosis associated with urinary tract pathology — Case report

Cystic hydatidosis (CH) is a worldwide distributed parasitic zoonosis. It is considered one of the 17 neglected parasitic tropical diseases, among cysticercosis and soil transmitted helminthiases. CH is caused by the larval stage of Echinococcus granulosus, a tapeworm that usually infects dogs and other carnivorous animals as definitive hosts and herbivorous animals and rarely humans as intermediate hosts. Main primary localizations are the liver and the lung. In less than 3% they can primarily be present in the spleen. Treatment is mainly surgical, in some cases resulting in reoccurrence. In this paper we present the case of a male 55 years old patient who underwent a surgical intervention on his spleen for a solitary hydatid cyst as primary localization. Fifteen years after the operation the patient presented macroscopic haematuria; routine laboratory findings presented soft eosinophilia, 5%, without any other modification. There was found no palpable tumour in the pelvis by rectal examination. Abdominal ultrasound investigation revealed a 2×1 cm formation in the urinary bladder at the base of the left bladder-wall and a retrovesical, inhomogeneous 10×10 cm tumour with multiple septa and transonic zones. Computed tomography (CT) scan strongly suggested the presence of a bladder tumour and a hydatid cyst. The symptoms caused by the bladder tumour revealed the co-existing non-symptomatic retrovesical secondary CH, which is a rare complication of splenic Echinococcus granulosus infection. Close follow-up and a proper pre- and postoperative anti-parasitic medication of the patient could have prevented reoccurrence of CH

Humán dUTPáz: kooperativitás és sejtbeli kölcsönhatások = Human dUTPase: cooperativity and cellular interactions

Az enzimműködés mechanizmusának tisztázása és a katalitikus ciklus részletes leírása, valamint a humán dUTPáz sejtbeli siRNS csendesítése témákban jelentős cikkeket közöltünk, többek közt a JBC, Accounts Chem. Res és PNAS folyóiratokban. Fehérjekrisztallográfia, a 31P NMR, és a QM-MM számításos módszerek, valamint steady-state and tranziens enzimkinetikai módszerek együttes alkalmazása révén sikerült olyan egyedi intermediereket azonosítani, melyek hatékony gátlószerek tervezésében is szerepet játszhatnak. A humán dUTPáz gátlása rákterápiában lehet fontos. TAP-tag kísérleteinkben azonosítottuk az importin-alfa fehérjét, mint a humán dUTPáz egyik sejtbeli kölcsönható partnerét. Bizonyítottuk, hogy a nukleáris lokalizációs szignál (NLS) melletti foszforiláció kihat a dUTPáz intracelluláris lokalizációjára: a foszforilált fehérjét mimikáló mutáns kireked a sejtmagból. A vad típusú enzim, egy hiperfoszforilációt, valamint egy hipofoszforilációt mimikáló mutáns forma magi akkumulációját videomikroszkópos kísérletekkel hasonlítottuk össze. Megállapítottuk, hogy az utódsejtekbe jutó foszforilált dUTPáz pool a sejtciklus előrehaladtával, valószínűleg defoszforilációt követően újra akkumulálódhat a magban, de ez a folyamat lassú. Az NLS-környéki foszforiláció sejtciklushoz kötött mechanizmusát számos egyéb humán fehérjén is azonosítottuk – ez a folyamat általános jelentőségű lehet az utódsejtek magi proteómjának kialakításában. | Significant articles were published in journals such as JBC, Accounts Chem Res and PNAS, covering the topics of the molecular mechanism of action, distinct steps in the catalytic cycle, and siRNA silencing of human dUTPase. We identified multiple unique intermediary states using a combined approach of X-ray crystallography, 31P NMR, computational chemistry and steady-state/transient enzyme kinetics. These intermediates may form the basis of inhibitor design against human dUTPase, a potential for novel anticancer drugs. Using TAP-tag experiments, we identified importin-alfa as a cellular interacting partner of human dUTPase. We showed that a specific phosphorylation in the vicinity of the nuclear localization signal (NLS) impedes nuclear import. Videomicroscopy was used to compare nuclear accumulation kinetics in daughter cells following mitosis for wild type, as well as fór hypo- and hyperphosphorylation mimicking mutants. We showed that the phosphorylated dUTPase pool that is transmitted to the daughter cells may again accumulate in the cytoplasm, but only after dephosphorylation and in a rather slow manner. We identified similar mechanism for numerous other human proteins – this process may possess general significance in shaping the nuclear proteome of daughter cells

Fémek szerepe a fehérjeszerkezetben és - működésben = The role of metals in protein structure and function

Fehérjekrisztallográfia, mágneses magrezonancia-spektroszkópia és molekulamodellezés segítségével vizsgáltuk az összefüggéseket néhány metalloprotein, valamint egy új típusú, rendezetlen fehérje szerkezete és működése között. Hatékony módszert fejlesztettünk ki a reakcióút kvantummechanikai számítására enzimekben. Tisztáztuk a DNS javításában fontos szerepet játszó dUTPáz által katalizált reakció legtöbb részletét. Meggyőző bizonyítékokat szolgáltattunk arra, hogy az enzimatikus foszfáthidrolízis során a dUTPázban nagy energiájú, trigonális bipiramisos elrendeződésű intermedier keletkezik. Kimutattuk, hogy a KAR-2 nevű molekula más, biszindol típusú ligandumoktól eltérő módon kötődik a kalmodulinhoz, ez magyarázza különleges fiziológiai hatását. Elvégeztük a hemoglobin hem-csoportjainak normál koordináták szerinti analízisét, amiből következtetéseket vontunk le a szerkezetre vonatkozóan. A deformációk azt mutatják, hogy a hem csoport szerkezete érzékeny a molekula távoli részében kötődő effektor jelenlétére, ami az allosztérikus szabályozás hatásmechanizmusának a tercier szerkezettel való kapcsolatát támasztja alá. A közelmúltban egy új agy-specifikus fehérjét izoláltunk, melynek átlagos rendezetlensége 46-47%, tehát szerkezet nélkülinek tekinthető. Részletes vizsgálatokat végeztünk e fehérje, illetve különböző fehérjékkel képezett komplexe szerkezetére vonatkozóan. | We investigated the relationship between the structure and activity of some metalloproteins and a new unfolded protein. We developed an efficient method for the quantum mechanical calculation of the reaction path in enzymes. Most details of the reaction catalysed by dUTPase, playing an important role in DNA repair, have been clarified. We provided convincing evidence that during enzymatic phosphate hydrolysis a high-energy, trigonal bipyramidal intermediate is formed. We have shown that the molecule KAR-2, in contrast to other bisindole-type ligands, has a different binding mode to calmodulin, which explains its special physiological effect. We performed the normal co-ordinate analysis of the hem groups of haemoglobin and derived conclusions on their structure. The deformations indicate that the structure of the hem group is sensitive to the presence of an effector bound in a distant region of the molecule. This finding supports the relation between the allosteric mechanism of action and the tertiary structure. Recently we isolated a new brain-specific protein, which is 46 to 47 per cent disordered, i.e. it can be considered as unfolded. We made detailed studies on the structure of this protein and its complex with others

Flexible segments modulate co-folding of dUTPase and nucleocapsid proteins

The homotrimeric fusion protein nucleocapsid (NC)-dUTPase combines domains that participate in RNA/DNA folding, reverse transcription, and DNA repair in Mason-Pfizer monkey betaretrovirus infected cells. The structural organization of the fusion protein remained obscured by the N- and C-terminal flexible segments of dUTPase and the linker region connecting the two domains that are invisible in electron density maps. Small-angle X-ray scattering reveals that upon oligonucleotide binding the NC domains adopt the trimeric symmetry of dUTPase. High-resolution X-ray structures together with molecular modeling indicate that fusion with NC domains dramatically alters the conformation of the flexible C-terminus by perturbing the orientation of a critical β-strand. Consequently, the C-terminal segment is capable of double backing upon the active site of its own monomer and stabilized by non-covalent interactions formed with the N-terminal segment. This co-folding of the dUTPase terminal segments, not observable in other homologous enzymes, is due to the presence of the fused NC domain. Structural and genomic advantages of fusing the NC domain to a shortened dUTPase in betaretroviruses and the possible physiological consequences are envisaged

Catalytic mechanism of alpha-phosphate attack in dUTPase is revealed by X-ray crystallographic snapshots of distinct intermediates, 31P-NMR spectroscopy and reaction path modelling.

Enzymatic synthesis and hydrolysis of nucleoside phosphate compounds play a key role in various biological pathways, like signal transduction, DNA synthesis and metabolism. Although these processes have been studied extensively, numerous key issues regarding the chemical pathway and atomic movements remain open for many enzymatic reactions. Here, using the Mason-Pfizer monkey retrovirus dUTPase, we study the dUTPase-catalyzed hydrolysis of dUTP, an incorrect DNA building block, to elaborate the mechanistic details at high resolution. Combining mass spectrometry analysis of the dUTPase-catalyzed reaction carried out in and quantum mechanics/molecular mechanics (QM/MM) simulation, we show that the nucleophilic attack occurs at the alpha-phosphate site. Phosphorus-31 NMR spectroscopy (31P-NMR) analysis confirms the site of attack and shows the capability of dUTPase to cleave the dUTP analogue alpha,beta-imido-dUTP, containing the imido linkage usually regarded to be non-hydrolyzable. We present numerous X-ray crystal structures of distinct dUTPase and nucleoside phosphate complexes, which report on the progress of the chemical reaction along the reaction coordinate. The presently used combination of diverse structural methods reveals details of the nucleophilic attack and identifies a novel enzyme-product complex structure

Glutamate Uptake Triggers Transporter-Mediated GABA Release from Astrocytes

Background: Glutamate (Glu) and c-aminobutyric acid (GABA) transporters play important roles in regulating neuronal activity. Glu is removed from the extracellular space dominantly by glial transporters. In contrast, GABA is mainly taken up by neurons. However, the glial GABA transporter subtypes share their localization with the Glu transporters and their expression is confined to the same subpopulation of astrocytes, raising the possibility of cooperation between Glu and GABA transport processes. Methodology/Principal Findings: Here we used diverse biological models both in vitro and in vivo to explore the interplay between these processes. We found that removal of Glu by astrocytic transporters triggers an elevation in the extracellular level of GABA. This coupling between excitatory and inhibitory signaling was found to be independent of Glu receptor-mediated depolarization, external presence of Ca2+ and glutamate decarboxylase activity. It was abolished in the presence of non-transportable blockers of glial Glu or GABA transporters, suggesting that the concerted action of these transporters underlies the process. Conclusions/Significance: Our results suggest that activation of Glu transporters results in GABA release through reversal of glial GABA transporters. This transporter-mediated interplay represents a direct link between inhibitory and excitatory neurotransmission and may function as a negative feedback combating intense excitation in pathological conditions such as epilepsy or ischemia

Structural determinants of Sleeping Beauty transposase activity

Transposases are important tools in genome engineering, and there is considerable interest in engineering more efficient ones. Here, we seek to understand the factors determining their activity using the Sleeping Beauty transposase. Recent work suggests that protein coevolutionary information can be used to classify groups of physically connected, coevolving residues into elements called "sectors", which have proven useful for understanding the folding, allosteric interactions, and enzymatic activity of proteins. Using extensive mutagenesis data, protein modeling and analysis of folding energies, we show that (i) The Sleeping Beauty transposase contains two sectors, which span across conserved domains, and are enriched in DNA-binding residues, indicating that the DNA binding and endonuclease functions of the transposase coevolve; (ii) Sector residues are highly sensitive to mutations, and most mutations of these residues strongly reduce transposition rate; (iii) Mutations with a strong effect on free energy of folding in the DDE domain of the transposase significantly reduce transposition rate. (iv) Mutations that influence DNA and protein-protein interactions generally reduce transposition rate, although most hyperactive mutants are also located on the protein surface, including residues with protein-protein interactions. This suggests that hyperactivity results from the modification of protein interactions, rather than the stabilization of protein fold.Molecular Therapy (2016); doi:10.1038/mt.2016.110

Crystallization and preliminary X-ray studies of dUTPase from Mason–Pfizer monkey retrovirus

Deoxyuridine 5′-triphosphate nucleotidohydrolase from Mason–Pfizer monkey retrovirus (M-PMV dUTPase) is a betaretroviral member of the dUTPase enzyme family. The nucleocapsid-free dUTPase (48426 Da) was co-crystallized with a dUTP substrate analogue using the hanging-drop vapour-diffusion method