Institute of Biochemistry of the Romanian Academy

Bucharest, Romania

Institute of Biochemistry of the Romanian Academy

Bucharest, Romania

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Maekawa T.,Max Planck Institute For Pflanzenzuchtungsforschung | Cheng W.,Beijing Normal University | Cheng W.,China National Institute of Biological Sciences | Spiridon L.N.,Institute of Biochemistry of the Romanian Academy | And 12 more authors.
Cell Host and Microbe | Year: 2011

Plants and animals have evolved structurally related innate immune sensors, designated NLRs, to detect intracellular nonself molecules. NLRs are modular, consisting of N-terminal coiled-coil (CC) or TOLL/interleukin-1 receptor (TIR) domains, a central nucleotide-binding (NB) domain, and C-terminal leucine-rich repeats (LRRs). The polymorphic barley mildew A (MLA) locus encodes CC-containing allelic immune receptors recognizing effectors of the pathogenic powdery mildew fungus. We report the crystal structure of an MLA receptor's invariant CC domain, which reveals a rod-shaped homodimer. MLA receptors also self-associate in vivo, but self-association appears to be independent of effector-triggered receptor activation. MLA CC mutants that fail to self-interact impair in planta cell death activity triggered by the CC domain alone and by an autoactive full-length MLA receptor that mimics its ATP-bound state. Thus, CC domain-dependent dimerization of the immune sensor defines a minimal functional unit and implies a role for the dimeric CC module in downstream immune signaling. © 2011 Elsevier Inc.

Avram S.,University of Bucharest | Milac A.L.,Institute of Biochemistry of the Romanian Academy | Carta F.,University of Florence | Supuran C.T.,University of Florence
Journal of Enzyme Inhibition and Medicinal Chemistry | Year: 2012

Dithiocarbamates (DTC) are promising compounds with potential applications in antitumoral and glaucoma therapy. Our aim is to understand molecular features affecting DTC interaction with carbonic anhydrases (CAs), zinc-containing enzymes maintaining acid-base balance in blood and other tissues. To this end, we generate QSAR models based on a compound series containing 25 DTC, inhibitors of four human (h) CAs isoforms: hCA I, II, IX and XII. We establish that critical physicochemical parameters for DTC inhibitory activity are: hydrophobic, electronic, steric, topological and shape. The predictive power of our QSAR models is indicated by significant values of statistical coefficients: cross-validated correlation q2 (0.55-0.73), fitted correlation r2 (0.75-0.84) and standard error of prediction (0.47-0.23). Based on the established QSAR equations, we analyse 22 new DTC derivatives and identify DTC dicarboxilic acids derivatives and their esters as potentially improved inhibitors of CA I, II, IX and XII. © 2013 Informa UK, Ltd.

Anishkin A.,University of Maryland University College | Milac A.L.,U.S. National Institutes of Health | Milac A.L.,Institute of Biochemistry of the Romanian Academy | Robert Guy H.,U.S. National Institutes of Health
Proteins: Structure, Function and Bioinformatics | Year: 2010

Most crystallized homo-oligomeric ion channels are highly symmetric, which dramatically decreases conformational space and facilitates building homology models (HMs). However, in molecular dynamics (MD) simulations channels deviate from ideal symmetry and accumulate thermal defects, which complicate the refinement of HMs using MD. In this work we evaluate the ability of symmetry constrained MD simulations to improve HMs accuracy, using an approach conceptually similar to Critical Assessment of techniques for protein Structure Prediction (CASP) competition: build HMs of channels with known structure and evaluate the efficiency of proposed methods in improving HMs accuracy (measured as deviation from experimental structure). Results indicate that unrestrained MD does not improve the accuracy of HMs, instantaneous symmetrization improves accuracy but not stability of HMs during subsequent unrestrained MD, while gradually imposing symmetry constraints improves both accuracy (by 5-50%) and stability of HMs. Moreover, accuracy and stability are strongly correlated, making stability a reliable criterion in predicting the accuracy of new HMs. © 2009 Wiley-Liss, Inc.

Popescu C.-I.,University of Lille Nord de France | Popescu C.-I.,Institute of Biochemistry of the Romanian Academy | Callens N.,University of Lille Nord de France | Trinel D.,Lille University of Science and Technology | And 8 more authors.
PLoS Pathogens | Year: 2011

Growing experimental evidence indicates that, in addition to the physical virion components, the non-structural proteins of hepatitis C virus (HCV) are intimately involved in orchestrating morphogenesis. Since it is dispensable for HCV RNA replication, the non-structural viral protein NS2 is suggested to play a central role in HCV particle assembly. However, despite genetic evidences, we have almost no understanding about NS2 protein-protein interactions and their role in the production of infectious particles. Here, we used co-immunoprecipitation and/or fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy analyses to study the interactions between NS2 and the viroporin p7 and the HCV glycoprotein E2. In addition, we used alanine scanning insertion mutagenesis as well as other mutations in the context of an infectious virus to investigate the functional role of NS2 in HCV assembly. Finally, the subcellular localization of NS2 and several mutants was analyzed by confocal microscopy. Our data demonstrate molecular interactions between NS2 and p7 and E2. Furthermore, we show that, in the context of an infectious virus, NS2 accumulates over time in endoplasmic reticulum-derived dotted structures and colocalizes with both the envelope glycoproteins and components of the replication complex in close proximity to the HCV core protein and lipid droplets, a location that has been shown to be essential for virus assembly. We show that NS2 transmembrane region is crucial for both E2 interaction and subcellular localization. Moreover, specific mutations in core, envelope proteins, p7 and NS5A reported to abolish viral assembly changed the subcellular localization of NS2 protein. Together, these observations indicate that NS2 protein attracts the envelope proteins at the assembly site and it crosstalks with non-structural proteins for virus assembly. © 2011 Popescu et al.

Avram S.,University of Bucharest | Mihailescu D.,University of Bucharest | Borcan F.,Victor Babes University of Medicine and Pharmacy Timisoara | Milac A.-L.,Institute of Biochemistry of the Romanian Academy
Monatshefte fur Chemie | Year: 2012

Antimicrobial peptides are an important class of therapeutic agents used against a wide range of pathogens such as gram-negative and -positive bacteria, fungi, and viruses. The minimal inhibitory concentration at the level of the pathogen membrane is a major determinant of the pharmacokinetic behavior and, consequently, it can affect their antimicrobial activity. Here we generated quantitative structure-activity relationship models (3DQSAR- comparative molecular field analysis/comparative molecular similarity indices analysis) using a database of 33 mastoparan analogs, antimicrobial peptides with known experimental activity, and further used these models to predict the minimal inhibitory concentration for 18 new mastoparan analogs, obtained by computational mutagenesis. We discuss two options for structural alignment of mastoparan analogs: superposition of Ca trace atoms or superposition of all backbone atoms. Significant values of the cross-validated correlation q 2 (higher than 0.60) and the fitted correlation r 2 (higher than 0.90) of our models indicate that they are reliable enough for activity prediction in the case of new derivatives. This allows us to identify compounds with possibly enhanced antimicrobial activity against Bacillus subtilis, which are suggested for further experimental studies. © Springer-Verlag 2012.

Ciubotaru M.,Yale University | Ciubotaru M.,Horia Hulubei National Institute of Physics and Nuclear Engineering | Surleac M.D.,Institute of Biochemistry of the Romanian Academy | Metskas L.A.,Yale University | And 5 more authors.
Nucleic Acids Research | Year: 2015

V(D)J recombination is initiated by RAG1 and RAG2, which together with HMGB1 bind to a recombination signal sequence (12RSS or 23RSS) to form the signal complex (SC) and then capture a complementary partner RSS, yielding the paired complex (PC). Little is known regarding the structural changes that accompany the SC to PC transition or the structural features that allow RAG to distinguish its two asymmetric substrates. To address these issues, we analyzed the structure of the 12RSS in the SC and PC using fluorescence resonance energy transfer (FRET) and molecular dynamics modeling. The resulting models indicate that the 12RSS adopts a strongly bent V-shaped structure upon RAG/HMGB1 binding and reveal structural differences, particularly near the heptamer, between the 12RSS in the SC and PC. Comparison of models of the 12RSS and 23RSS in the PC reveals broadly similar shapes but a distinct number and location of DNA bends as well as a smaller central cavity for the 12RSS. These findings provide the most detailed view yet of the 12RSS in RAG-DNA complexes and highlight structural features of the RSS that might underlie activation of RAG-mediated cleavage and substrate asymmetry important for the 12/23 rule of V(D)J recombination. © 2015 The Author(s).

Ciubotaru M.,Yale University | Ciubotaru M.,Institute of Biochemistry of the Romanian Academy | Ciubotaru M.,Horia Hulubei National Institute of Physics and Nuclear Engineering | Trexler A.J.,Yale University | And 6 more authors.
Nucleic Acids Research | Year: 2013

During V(D)J recombination, recombination activating gene proteins RAG1 and RAG2 generate DNA double strand breaks within a paired complex (PC) containing two complementary recombination signal sequences (RSSs), the 12RSS and 23RSS, which differ in the length of the spacer separating heptamer and nonamer elements. Despite the central role of the PC in V(D)J recombination, little is understood about its structure. Here, we use fluorescence resonance energy transfer to investigate the architecture of the 23RSS in the PC. Energy transfer was detected in 23RSS substrates in which the donor and acceptor fluorophores flanked the entire RSS, and was optimal under conditions that yield a cleavage-competent PC. The data are most easily explained by a dramatic bend in the 23RSS that reduces the distance between these flanking regions from >160A in the linear substrate to <80A in the PC. Analysis of multiple fluorescent substrates together with molecular dynamics modeling yielded a model in which the 23RSS adopts a U shape in the PC, with the spacer located centrally within the bend. We propose that this large bend facilitates simultaneous recognition of the heptamer and nonamer, is critical for proper positioning of the active site and contributes to the 12/23 rule. © The Author(s) 2013. Published by Oxford University Press.

Sokolowska I.,Clarkson University | Dorobantu C.,Institute of Biochemistry of the Romanian Academy | Woods A.G.,Clarkson University | Macovei A.,Institute of Biochemistry of the Romanian Academy | And 2 more authors.
Proteome Science | Year: 2012

Liver infection with hepatitis B virus (HBV), a DNA virus of the Hepadnaviridae family, leads to severe disease, such as fibrosis, cirrhosis and hepatocellular carcinoma. The early steps of the viral life cycle are largely obscure and the host cell plasma membrane receptors are not known. HepaRG is the only proliferating cell line supporting HBV infection in vitro, following specific differentiation, allowing for investigation of new host host-cell factors involved in viral entry, within a more robust and reproducible environment. Viral infection generally begins with receptor recognition at the host cell surface, following highly specific cell-virus interactions. Most of these interactions are expected to take place at the plasma membrane of the HepaRG cells. In the present study, we used this cell line to explore changes between the plasma membrane of undifferentiated (-) and differentiated (+) cells and to identify differentially-regulated proteins or signaling networks that might potentially be involved in HBV entry. Our initial study identified a series of proteins that are differentially expressed in the plasma membrane of (-) and (+) cells and are good candidates for potential cell-virus interactions. To our knowledge, this is the first study using functional proteomics to study plasma membrane proteins from HepaRG cells, providing a platform for future experiments that will allow us to understand the cell-virus interaction and mechanism of HBV viral infection. © 2012 Sokolowska et al.; licensee BioMed Central Ltd.

Lazar C.,Institute of Biochemistry of the Romanian Academy | Macovei A.,Institute of Biochemistry of the Romanian Academy | Petrescu S.,Institute of Biochemistry of the Romanian Academy | Branza-Nichita N.,Institute of Biochemistry of the Romanian Academy
PLoS ONE | Year: 2012

Hepatitis B virus (HBV) belongs to the Hepadnaviridae family of enveloped DNA viruses. It was previously shown that HBV can induce endoplasmic reticulum (ER) stress and activate the IRE1-XBP1 pathway of the unfolded protein response (UPR), through the expression of the viral regulatory protein X (HBx). However, it remained obscure whether or not this activation had any functional consequences on the target genes of the UPR pathway. Of these targets, the ER degradation-enhancing, mannosidase-like proteins (EDEMs) are thought to play an important role in relieving the ER stress during UPR, by recognizing terminally misfolded glycoproteins and delivering them to the ER-associated degradation (ERAD). In this study, we investigated the role of EDEMs in the HBV life-cycle. We found that synthesis of EDEMs (EDEM1 and its homologues, EDEM2 and EDEM3) is significantly up-regulated in cells with persistent or transient HBV replication. Co-expression of the wild-type HBV envelope proteins with EDEM1 resulted in their massive degradation, a process reversed by EDEM1 silencing. Surprisingly, the autophagy/lysosomes, rather than the proteasome were involved in disposal of the HBV envelope proteins. Importantly, inhibition of the endogenous EDEM1 expression in HBV replicating cells significantly increased secretion of both, enveloped virus and subviral particles. This is the first report showing that HBV activates the ERAD pathway, which, in turn, reduces the amount of envelope proteins, possibly as a mechanism to control the level of virus particles in infected cells and facilitate the establishment of chronic infections. © 2012 Lazar et al.

Popa I.,Institute of Biochemistry of the Romanian Academy | Ganea E.,Institute of Biochemistry of the Romanian Academy | Petrescu S.M.,Institute of Biochemistry of the Romanian Academy
Biochemistry and Cell Biology | Year: 2014

The receptor for advanced glycation end products (RAGE) is involved in multiple stages of tumor development and malignization. To gain further knowledge on the RAGE role in tumor progression, we investigated the receptor expression profile and its subcellular localization in melanoma cells at different stages of malignancy. We found that RAGE clustered at membrane ruffles and leading edges, and at sites of cell-to-cell contact in primary melanoma cells (e.g., MelJuSo), in contrast with a more dispersed localization in metastatic cells (e.g., SK-Mel28). RAGE silencing by RNAi selectively inhibited migration of MelJuSo cells, whilst having no influence on SK-Mel28 cell migration, in a "wound healing" assay. Western blot detection of RAGE showed a more complex RAGE oligomerization in MelJuSo cells compared to melanocytes and SK-Mel28 cells. By competing the binding of antibodies with recombinant soluble RAGE, an oligomeric form running at approximately 200 kDa was detected, as it was the monomeric RAGE of 55-60 kDa. SDS-PAGE electrophoresis under reducing versus nonreducing conditions indicated that the oligomer of about 200 kDa is formed by disulfide bonds, but other interactions are likely to be important for RAGE multimerization in melanoma cells. Immunofluorescence microscopy revealed that treatment with two cholesterol-chelating drugs, nystatin and filipin, significantly affected RAGE localization in MelJuSo cells. SK-Mel28 cells showed a reduced RAGE glycosylation and association with cholesterol-rich membranes and also a considerable downregulation of the soluble forms. Our results indicate that RAGE isoform expression and subcellular localization could be important determinants for the regulation of its function in tumor progression. © 2014 Published by NRC Research Press.

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