Energy metabolism in cells with glycogen branching enzyme deficiency. Dominika Malinska, Malgorzata Bejtka,. Joanna Szczepanowska, Jerzy Duszynski.
Session 7: Protein structure and function Lecture
L7.2
L7.1
Hsp100 proteins — disaggregating chaperone machines
CG methylation and depletion Marek Wojciechowski, Honorata Czapinska, Matthias Bochtler International Institute of Molecular and Cell Biology (IIMCB), Warsaw, Poland e-mail: Matthias Bochtler
DNA methylation occurs in prokaryotes and eukaryotes, but in different forms and with different functions. In prokaryotes, methylation is very diverse. Mechanistically, the modification can affect the N4 or C5 of cytosine or N6 of adenine, in a wide variety of sequence contexts. Functionally, DNA methylation plays a role in restriction-modification systems for genome defense, in DNA repair for the distinction of parental and daughter strand, and also in the control of bacterial lifestyle. Some features of DNA methylation are conserved in primitive eukaryotes, but in higher eukaryotes, particularly vertebrates, methylation is predominantly reduced to the C5 methylation of cytosine in a single sequence context (CG, or more traditionally CpG). Its main role is the epigenetic control of DNA state, in crosstalk with appropriate histone modifications. For eukaryotic organisms that feature pronounced CG methylation, it is known that this sequence is not only important, but also rare: in fact, the actual number of CGs is about fourfold lower than statistically expected. In my talk, I will discuss the mechanistic connection between methylation and the depletion of target sequences and I will address the generality of the link across the kingdoms of life.
Krzysztof Liberek1, Szymon Ziętkiewicz1, Natalia Litwińczuk1, Alicja Kochanek1, Ewa Morawiec1, Wojciech Potocki1, Magdalena Ślusarz2, Sylwia Rodziewicz-Motowidło2 1Department of Molecular and Cellular Biology, Intercollegiate Faculty of Biotechnology, and 2Faculty of Chemistry, University of Gdańsk, Gdańsk, Poland
e-mail: Krzysztof Liberek
The chaperone protein network controls both initial protein folding and subsequent maintenance of proteins in the cell. Many factors leading to unfolding and misfolding of proteins eventually result in protein aggregation. Stress imposed by high temperature was one of the first aggregation-inducing factors studied, and remains one of the main models in this field. With protein aggregation occurring in response to heat exposure, the cell needs chaperones to control and counteract the aggregation process. Elimination of aggregates can be achieved by solubilization of aggregates and either refolding of the liberated polypeptides or their proteolysis. Here we focus on the molecular mechanisms by which Hsp100 chaperones in cooperation with Hsp70, Hsp40 and small Hsps chaperones liberate and refold polypeptides trapped in protein aggregates. Analysis of homology models obtained by us for Hsp100 chaperones (Hsp104 from S. cerevisiae and ClpB from E. coli) suggests the presence of characteristic network of ionic interactions between the first ATPase domain (NBD1) and the coiled-coil M subdomain that is characteristic for disaggregating chaperones. To test the importance of this structural aspect we have designed a series of point mutations. Mutants intended to disrupt ionic interactions resulted either in inactive proteins or in proteins that display unexpected biochemical properties. These include increase of ATPase activity, significant increase of in vitro substrate renaturation rate and partial independence on Hsp70 chaperone in refolding process. The increased activities paradoxically result in serious growth impediment and rapid loss of mutant Hsp104-encoding plasmids in yeast cells. We were able to suppress those effects by introducing complementary changes that restore particular salt bridges within suggested network. Therefore we propose the presence of novel structural aspect of disaggregating Hsp100’s that is responsible for regulation of the disaggregating/ refolding activity.
Parnas Conference Warsaw 2011
L7.3
Oral presentations
Structural aspects of PR10 protein — ligand interactions
O7.1
Mariusz Jaskolski Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University and Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland
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The effect of macromolecular crowding on DNA replication: A structure-function study Barak Akabayov, Charles C. Richardson
e-mail: Mariusz Jaskolski
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA
Pathogenesis-related proteins of class 10 (PR10) are uniquely found in plants where they are associated with stress-related processes. Despite their abundance and existence in many variants, their precise biochemical role remains unknown. However, their structure is well studied and defines a canonical PR10 fold consisting of a sevenstranded antiparallel β-sheet gripped over a long C-terminal α-helix, with a huge cavity (up to 4500 Å3) enclosed between these main structural elements. On the other hand, we have shown that plant proteins identified as specific binders of plant hormones from the cytokinin group (CSBP) belong to the same structural class despite very low sequence similarity. Cytokinins regulate plant development and senescence processes and are also involved in plant response to stress. We were able to show that trans-zeatin, a classic cytokinin hormone, is bound in the internal cavity of mung bean CSBP not in one but in at least three different, highly specific ways. Following this discovery, we have been able to show that also classic PR10 proteins (from yellow lupine) can bind both natural and synthetic cytokinin hormones. Others have shown that the proteins are capable of binding a wide range of different ligands, including the non-physiological (in plants) deoxycholate, which however shows resemblance to plant hormones from the brassinosteroid group. We also found out that a PR10 protein from St John’s wort is most likely and contrary to expectations not a hypericin-synthesizing enzyme but that it can bind in its internal cavity such exotic ligands as PEG molecules. Recently we have made the most puzzling discovery, showing that CSBP from lucerne can bind with very high specificity a completely unrelated hormone, gibberellic acid, from a diterpenoid group. Gibberellins are extremely important plant hormones regulating processes of cell growth and division. We have thus a very perplexing situation, where proteins that are regarded to be specific binders of specific ligands (CSBP) bind also other physiological ligands with what can be called structural specificity, and that structurally related proteins with unknown function bind a broad range of diversified ligands. These observations force us to rethink the notion of molecular recognition between a ligand and its binding protein.
e-mail: Barak Akabayov
Enzymatic activities in vivo occur in a highly crowded and confined environment mainly composed of protein and nucleic acid macromolecules. A crowded environment: 1) enhances the apparent concentration of the measured macromolecule, 2) leads to desolvation of water molecules around the proteins, 3) decrease degrees of freedom for diffusion and molecular hopping of proteins on their DNA substrates, and 4) enhances binding equilibria and catalytic activities of those macromolecules. However, the effect of macromolecular crowding on the structure of the proteins involved is poorly understood. We have characterized the effect of molecular crowding on DNA replication using the replication system of bacteriophage T7 as a model system. We probe the structural effects that are involved in a crowded environment using small angle X-ray scattering. Our results show the consequences of macromolecular crowding on DNA replication mediated by the T7 replisome.
Abstracts 56
O7.2
Posters
Changes in human ednothelial cell proteom caused by N-homocysteinylated proteins
P7.1
Joanna Suszynska-Zajczyk1, Magdalena Łuczak1, Łukasz Marczak1, Edyta Gendaszewska-Darmach2, Tomasz Twardowski1, Hieronim Jakubowski1,3 1Institute
of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland; 2Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Technical University of Lodz, Lodz, Poland; 3University of Medicine and Dentristy, Department of Microbiology and Molecular Genetics, International Center for Public Health, Newark, NJ, USA e-mail: Joanna Suszyńska-Zajczyk
Homocysteine (Hcy) is a focus of scientific inquiry in the context of human diseases. Elevated plasma Hcy, termed hyperhomocysteinemia, caused by genetic defects in Hcy metabolism, leads to pathological changes in many organs and premature death due to cardiovascular complications [1]. Hcy is formed from dietary protein methionine as a by-product of biological methylation reactions. If not removed by transsulfurattion and transmethylation reactions, Hcy becomes toxic to human cells. A mechanism involving the conversion to Hcy-thiolactone (HTL) in the error editing pathway catalyzed by methionyl-tRNA synthetase [2] and indirect incorporation into protein by a post-translational modification of protein lysine residues (N-homocysteinylation) [3] contributes to Hcy toxicity. Protein N-homocysteinylation affects protein structure and function [3], causes cellular toxicity, elicits autoimmune response leading to the generation of antibodies against N-Hcy-proteins[4], and prothrombotic N-Hcy-fibrinogen [5]. To determine how HTL affects human endothelium, we studied cultured human umbilical vein endothelial cells (HUVECs), which are frequently used as a model of vascular cells and are known to efficiently metabolize Hcy to HTL and generate N-homocysteinylated proteins (N-Hcyprotein). Protein extracts were analyzed using 2-dimensional IEF/SDS/PAGE electrophoresis, followed by MALDITOF analysis. In HUVECs treated with HTL (0.5-5mM) for 24h, we found 24 differential proteins. Expression of most of them decreased and only expression of vimentin, UCHL1 and TXNDC5 protein increased. In HUVECs incubated with N-Hcy-FBS we recognised 34 differential proteins. We observed changes in the levels of protein expression dependent on the extent of FBS modification. After incubation with N-Hcy-FBS modified in the highest extent, the protein expression level was similar to the control. Chlorine intracellular channel protein I and dnaK-type molecular chaperone demonstrated other pattern. Many of the proteins whose expression levels were affected by HTL or N-Hcy-FBS are known to play role in an apoptosis. This result suggest high toxicity of the protein N-homocysteinylation to the endothelium. References 1. Mudd SH et al. (2001) The metabolic and molecular bases of inherited disease, 8th ed. New York: McGraw Hill, 2007-2056. 2. Jakubowski H (1997) J Biol Chem 272: 1935-1942. 3. Jakubowski H (1999) FASEB J 13: 2277-2283. 4. Undas A et al. (2004) Stroke 35: 299-304. 5. Jakubowski H (2008) J Physiol Pharmacol 59 (Suppl 9): 155-167.
Characterization of the interaction between Hsp70’s SBD and a ligand Itzhaq Azoulay, Esther Nachliel, Yossi Tsfadia, Abdussalam Azem Tel Aviv University, Department of Biochemistry, Tel Aviv, Israel e-mail: Itzhaq Azoulay
Molecular chaperones of the 70 kDa Heat Shock Protein family (HSP70) are present in all organisms, and have an essential roles in a wide range of inter cellular functions such as folding of nascent proteins targeting into different compartments of the cell and preventing their aggregation, these actions are coupled with ATP binding and hydrolysis. Its two functional domains are the 44 kDa Nucleotide Binding Domain (NBD), which responsible for the binding of the ATP and its hydrolysis, and the 26 kDa Substrate Binding Domain (SBD) is responsible for the binding and the releasing of polypeptide chains. In the ATP state the peptide substrate exhibits low affinity to the HSP70, while in the ADP state the substrate has high affinity to the SBD. The chosen model in this study was the Escherichia coli Hsp70, the DnaK, focusing on the SBD domain (PDB entry 1dkx). The structure consists of two sub domains: a β sandwich domain containing the hydrophobic cavity to which the NRLLLTG heptapeptide is bound, and a α helical segments followed after, that sit on top of it, and also known as lid. By using Molecular dynamics we have characterized the properties of the interaction of the peptides with the protein. Several simulations with different initial conditions of the SBD complex were carried out, in order to investigate the effect of electrical charge upon binding, and determine the lid’s role of the binding. Our interesting findings would be shown in the poster.
Parnas Conference Warsaw 2011
P7.2
P7.3
Protein and carbohydrate composition of the exopolymeric matrix as the main factor in the thionic bacteria biofilm formation
Life and death of proteins destined to the intermembrane space of mitochondria
Mariia Boretska, Andryi Ostapchuk, Irina Kozlova Zabolotny Institute of the NAS of Ukraine, Ukraine e-mail: Maria Boretska
Microbial populations on the solid surfaces are capable to form highly organized structures — biofilms. Major role in these biofilms has the exopolymer matrix — the polymer structure in which cells are immersed. Studying the biofilms formed by corrosion bacteria is needed to develop ways to protect large-scale metal structures as the primary site of microbial influence corrosion. The objective of our study was to investigate the composition of exopolymer complex and structural features of biofilm formatted by acidophobic bacteria Thiobacillus thioparus on the mild steel surface. We used an Albersheim method for allocating monosaccharide composition (analysis was performed on GC-MS system Agilent 6890N/5973 inert), Bradford method, the method of confocal laser scanning microscopy (LSM Pascal 5, Carl Zeiss, Germany). The greatest variety of monosaccharide composition of the samples is presented in association matrix biofilms (rhamnose, xylose, ribose and galactose were present only in samples of biofilms associated culture) were showed that. To visualize the carbohydrate part of the matrix was chosen lectin ConA + FITC (Sigma) is specific to glucose (the predominant monosaccharide), for cells visualization — DAPI (Sigma). The biofilm formed by the associative culture in an average of 40 mkm thick, the surface is more uniform in comparison with the biofilm’s monocultures it was shown. Localization of cells for both mono- and for the associative cultures was above matrix, the localization of carbohydrates - from the bottom. Specific production of exopolymers associated culture was 2.5 times more than for the monoculture. The study of the matrix protein composition of mono- and associated cultures thionic bacteria showed that the method of Bradford supported by microscopic observations (Thiazine Red was used to visualize the protein components of the matrix). It is shown the number of protein components of associative biofilms more than in monoculture (almost than 20 times), the location of protein structures has been mainly in the center of the matrix as one of main factor of creating of biofilm’s structure. Obviously, associated culture is able to produce more exopolymers and its characterization such as quantity and localization of the proteins and carbohydrates provides a powerful, dangerous and corrosive biofilms, formed by acidophobic thiobacteria.
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Piotr Bragoszewski, Agnieszka Gornicka, Agnieszka Chacinska International Institute of Molecular and Cell Biology in Warsaw, Laboratory of Mitochondrial Biogenesis, Warsaw, Poland e-mail: Piotr Bragoszewski
Nearly all mitochondrial proteins are synthesized in the cytosol and translocated into the organelle. Many of them have signal presequences that are proteolytically removed by specific peptidases upon their selective import into one of mitochondrial compartments: the matrix, the inner membrane or the intermembrane space (IMS). Also various types of non-cleavable targeting and sorting signals exist. This variety of signals is matched by the specific import pathways and translocases that recognize the signals and direct the precursor proteins into a final destination within mitochondria. Many IMS proteins share a coiled coil-helixcoiled coil-helix (CHCH) domain, which is stabilized by the formation of disulfide bonds within characteristic cysteine motifs. Upon synthesis and release to the cytoplasm these proteins stay reduced. Once the IMS precursor proteins enter the IMS via the protein channel formed by the translocase of the outer mitochondrial membrane TOM, they are recognized and oxidized by the mitochondrial IMS import and assembly machinery MIA. The majority of the cysteine-containing proteins are relatively small, typically smaller than 20 kDa. It is likely that such small proteins can traverse freely through the TOM complex (or other pores) in the outer membrane of mitochondria. Subsequently, the oxidative folding catalyzed by MIA is required to trap them in the IMS. In our project, we aim to understand the fate of IMS proteins under the conditions of oxidative folding restrictions. The conditional mutants of two essential components, Mia40 and Erv1, do not accumulate the precursor proteins in mitochondria that results in their death under restrictive conditions. We undertook a systematic analysis of the MIA substrate proteins to categorize them into two major classes: the proteins found in the cytosol or destroyed under the conditions of oxidative folding failure. This study forms a basis for the identification of mechanisms involved in the biogenesis of mitochondrial proteins and their influence on cellular protein homeostasis.
Abstracts 58
P7.4
P7.5
Antistreptokinase antibodies prevent inhibition of platelet reactivity
Adaptor protein Ruk/cin85 regulates NGF-dependent neuronal differentiation
Nataliia Burlova-Vasilieva, Oleksiy Savchuk, Ludmila Ostapchenko
Nadiya Byts, Lyudmyla Drobot
Educational and Scientific Centre “Institute of Biology” of National Taras Shevchenko University of Kyiv, Department of Biochemistry, Kyiv, Ukraine e-mail: Nataliia Burlova
Objective: to investigate platelet reactivity under thrombolytic theraphy. Methods: ADP-inducible platelet aggregation, anti-Sk (streptokinase) antibodies and plasminogen activators inhibitor type 1 (PAI-1) were determined by light transmission aggregometry Elisa and Western Blot respectively. Results: Sk and heparin inhibited АDP-inducible platelet aggregation by 30% in AMI patients. Next day after heparin canceling the platelet aggregation returned to the initial high level. PAI-1 activity in blood plasma of AMI patients exceeded the rate of relatively healthy donors by 200%. Heparin canceling didn’t prevent normalization of inhibitor activity. Anti-Sk antibodies were not detected in rabbit study population before and 7 days after Sk administration. Sk infusion increased PAI-1 level in rabbit platelet fraction from 41±5 ng/ml (baseline) to 95.8±7.4 ng/ml. ADP-inducible platelet aggregation in rabbit platelet rich plasma did not increase the baseline level and was inhibited by Sk in dosedependent manner. Sk in a concentration of 16 mkg/ml was associated with significantly reduced aggregation rate. ADP-inducible platelet aggregation peaked at 7 weeks after intravenous administration of Sk and approached 81±5%, considerably exceeding the baseline value (46±7%). The titers of anti-Sk antibody were 1:100. Inhibition effect of Sk on ADP-dependent platelet aggregation was not observed. Sk in concentration of 16 mkg/ml and 32 mkg/ml caused insignificant increment of aggregation rate. Platelet fraction treated with Sk released free and complex forms of PAI-1. Conclusion: We suggest that Sk influence platelet reactivity not only through plasmin and Sk-antibody complex formation, but also interacting with Anti-Sk antibodies attached to the platelet surface.
Palladin Institute of Biochemistry, Laboratory of Cell Signalling, Kyiv, Ukraine e-mail: Nadiya Byts
Despite extensive investigations of Cbl-interacting protein of 85 kDa (Ruk/CIN85) in receptor trafficking and cytoskeletal dynamics, little is known about its functions in central nervous system. Previous studies showed that the specific isoforms of adaptor protein Ruk/CIN85 are expressed in central nervous system and reported their function in dopamine receptor endocytosis in striatal neurons. Here we investigated the role of Ruk/CIN85 in NGF-induced differentiation and neurite outgrowth. PC12 rat pheochromocytoma cells were used as a model. We found that upon chronic NGF treatment the pattern of Ruk/CIN85 isoforms expression is changed in wild type PC12 cells as well as in PC12 cells stably overexpressing GFP-tagged Ruk/CIN85. While the levels of Ruk/CIN85 150 kDa isoform were increased, the middle 56 kDa isoform was downregulated and no changes in full-length 85 kDa isoform levels were observed. It is known that the activation of NGF/TrkA pathway leads to the transient expression of inhibitor of differentiation (ID1) gene, which is necessary for axonal elongation of immature neuron. In PC12 cells that overexpress Ruk/CIN85 the elevation of ID1 upon NGF treatment was almost undetectable. Moreover, the overexpression of GFP-tagged Ruk/CIN85 in PC12 cells inhibited NGF-induced neurite outgrowth. In conclusion, our data indicate that adaptor protein Ruk/CIN85 is involved in neuronal differentiation and NGF-dependent signalling.
Parnas Conference Warsaw 2011
59
P7.6
P7.7
Differential ubiquitination and degradation of intersectin 1 isoforms
Shb scaffold mediates interaction between adaptor ITSN1 and viral protein LMP2A
Mykola Dergai, Oleksandr Dergai, Olga Novokhatska, Liudmila Tsyba, Inessa Skrypkina, Alla Rynditch
Oleksandr Dergai1 , Mykola Dergai1 , Inessa Skrypkina1, Liudmyla Matskova2, Liudmyla Tsyba1, Gosta Winberg2, Alla Rynditch1
Institute of Molecular Biology and Genetics, NASU, Kiev, Ukraine e-mail: Mykola Dergai
Intersectin 1 is an evolutionarily conserved adaptor protein implicated in endocytosis, exocytosis, propagation and attenuation of mitogenic signal and cell survival. Its role also was shown in nervous system functioning and development. Possible role of ITSN1 in Alzheimer and Huntington`s diseases and Down syndrome was demonstrated. Despite intense study of ITSN1 nothing is known about posttranslational regulation of this adaptor protein. Here we present investigation of ubiquitination of ITSN1 isoforms. We studied major intersectin isoform in nonneuronl cells — ITSN1-s and recently identified shortest known isoform ITSN1-22a. It was shown that ITSN1-s undergoes monoubiquitination whereas ITSN1-22a is multiply ubiquitinated. Ubiquitination of ITSN1 isoforms is dependent on clathrin-mediated endocytosis and is not affected by mitogen starvation/stimulation. AIP4 ubiquitin ligase provides posttranslational regulation of ITSN1 isoforms. Moreover, another ubiquitin ligase Cbl modifies ITSN1-22a additionally. It is noteworthy that ubiquitin ligase activity of Cbl impact on association of ITSN1-s and ITSN1-22a isoforms in vivo. ITSN1-22a isoform undergoes combined mode of ubiquitination. It is polyubiquitinated on its specific C-terminal domain (CTD) and is monoubiquitinated on the N-terminal part common for ITSN1-s and ITSN1-22a isoforms. Polyubiquitination results in instability of ITSN1-22a in vivo in comparison to ITSN1-s and causes proteosomal degradation of ITSN1-22a. Furthermore, overexpression of ITSN1-22a causes enhanced degradation of ITSN1-s in HEK293 cells. Thus, ITSN1 isoforms are differentially ubiquitinated and degraded.
1Institute of Molecular Biology and Genetics NAS of Ukraine, Kiev, Ukraine; 2Karolinska Institute, Stockholm, Sweden
e-mail: Oleksandr Dergai
Epstein-Barr virus (EBV) is a member of the herpesvirus familyp. EBV is associated with a number of human malignancies, such as Burkitt’s lymphoma, Hodgkin’s lymphoma and the epithelial cell malignancy nasopharyngeal carcinoma (NPC). Only restricted set of viral genes is expressed within latent phase: LMP1, LMP2A, LMP2B, EBNAs and EBERs. Latent membrane proteins are key players in infected cells transformation. But little is known about mechanism governing internalization of LMP2A from plasma membrane and trafficking through cells compartment. The aim of current work is to identify protein-protein interaction that allows latent membrane proteins to get access to the host endocytic machinery. Here we report interaction between viral protein LMP2A and endocytic adaptor intersectin 1. Our immunoprecipitation data evidenced about complex formation between LMP2A and ITSN1 in vivo in different cell types. SH3-domains of ITSN1 were sufficient to precipitate LMP2A in vitro. Mutational analysis of LMP2A was undertaken to map sites of interaction with ITSN1. Interaction between LMP2A and ITSN1 was found to be a superposition of interaction of SH3domains of ITSN1 with -PXXP- motives of LMP2A and tyrosin-phosphorylated ITAM-motives of LMP2A with SH2-domain of adaptor protein Shb that binds simultaneously ITSN1 and LMP2A. Shb was shown to be phosphorylated and activated by Syk kinase in this complex. Moreover it was found negative cooperativity of Syk engagement to complex of ITSN1 and LMP2A. Current findings provide new data about LMP2A-driving signalosome assembly and functioning.
Abstracts 60
P7.8
P7.9
Expression, purification and evaluation ATPase activity of four essential tellurite resistance proteins
SCA14 mutations affect the intramolecular clamp between the c1 domain and carboxyl-terminus of pkc-gamma
Vinh Phu Hoang, Silvia Vávrová, Lenka Valkovičová, Mahesh Madyagol, Jan Turňa
Justyna Jezierska1, Joachim Goedhart2, Jan T. Westerink3, Eric A. Reits3, Dineke S. Verbeek1
Department of Molecular Biology, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynská dolina, 842 15 Bratislava, Slovak Republic
1Department of Genetics, University Medical Center Groningen, Groningen, Netherlands; 2Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, Netherlands; 3Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands
e-mail: Vinh Phu Hoang
This work is focused on many years of tellurite operon study in clinical strain Escherichia coli KL53. The main objective of our work is to study tellurite operon at the the protein level. In our previous work we studied this at the DNA level. We created a precise bioinformatics analysis of all essential Ter proteins (TerBCDE) to find their cell localization. We focused mainly on protein TerC as only putative trans membrane protein with regard to analysis of its membrane segments. The TerC protein has been predicted as innermembrane protein with 9 transmembrane domains. The BLAST search of this protein sequence showed that 80–85% of homology was assessed with the H(+)-transporting ATPase Lactobacillus johnsonii FI9785, cation-transporting ATPase Lactobacillus johnsonii ATCC 33200 and sodium/proton antiporter, CPA1 family Sulfurimonas autotrophica DSM 16294. We prepared recombinant plasmids harboring cloned ter genes to express and isolate proteins. We found out that C terminus of these proteins is crucial to maintain their original function. We also determined ATPase activity of Ter proteins. Activity of purified TerC protein was 30 times higher than ATPase activity in cell lysate of E. coli K12 and 65 times higher than activity in B. subtilis and 51 times higher than in Pseudomonas FH2. We also revealed ATPase activity of TerB protein which was 2.4 times lower than ATPase activity of TerC. To compare with ATPase activity in E. coli K12 it is 12 times higher and 27 times higher than B. subtilis and 21 times higher than in Pseudomonas FH2. Proteins TerDE have no ATPase activity.
e-mail: Justyna Jezierska
Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neurodegenerative disease, caused by mutations in PRKCG encoding for protein kinase C gamma (PKCγ). Most of the known mutations are missense mutations and are concentrated in the diacylglycerol (DAG)-binding C1B subdomain, which is important for PKCγ protein translocation and proper function. PKCγ is abundantly expressed in Purkinje cells (PCs) and has been shown to regulate the morphological development of PCs and motor learning functions and participates in various cell functions, including cell growth, differentiation and maintaining oxidative homeostasis. PKCγ undergoes a complicated activation cycle to be able to perform its kinase function. In order to effectively transduce extracellular signals, PKCγ proteins must be properly primed and positioned for optimal downstream signalling. Perturbations in their phosphorylation state, conformation or localization can disrupt kinase signalling and lead to altered physiological states, such as ataxia. We previously showed that SCA14 mutations in the C1B subdomain of PKCγ enhance translocation to the cell membrane upon phorbol ester stimulation due to an open protein conformation. However, the increased translocation kinetics did not result in an increased kinase activity, but the mutant PKCγ proteins showed decreased kinase function causing aberrant MAPK signalling. Our recent findings reveal that SCA14 mutant PKCγ has an open Cterminus, which causes the enhanced accessibility of C1B for DAG. As a consequence, the C-terminus of mutant PKCγ was shown to bind more efficiently to the kinase PDK1. The mutant PKCγ proteins are not efficiently degraded, and therefore subsequently accumulate in the insoluble fraction of cells. In conclusion, we show that SCA14 mutations affect the intra-molecular clamp between the C1 domain and the C-terminus of PKCγ leading to altered protein conformation affecting its degradation.
Parnas Conference Warsaw 2011
P7.10
P7.11
Calcium binding protein S100A6 (calcyclin) in Wharton’s jelly – localization and interacting partners
Does the lack of GRHL1 activity increase the chance of skin cancer development?
Ewelina Jurewicz1, Irena Kasacka2, Edward Bańkowski2, Anna Filipek1 1Nencki Institute of Experimental Biology, Warsaw, Poland; 2Medical University of Bialystok, Białystok, Poland
e-mail: Ewelina Jurewicz
Wharton’s jelly, a connective tissue in the umbilical cord, is composed of high amount of extracellular matrix and of a very low number of cells (myofibroblasts mainly) (Ferguson & Dodson, 2009). The mechanical properties of Wharton’s jelly, mainly elasticity, protect the umbilical vessels from compression and absence of this tissue around the umbilical arteries is associated with fetal death (Kulkarni et al., 2007). The S100A6 protein binds calcium ions and belongs to the S100 protein family (Leśniak et al., 2009). Originally S100A6 was isolated from Ehrlich ascites tumor cells in our laboratory (Kuźnicki & Filipek, 1987). Under normal conditions high expression of S100A6 is found in fibroblasts and epithelial cells (Kuźnicki et al., 1992) and interestingly its expression is characteristic for tissues which are specific for pregnancy such as deciduas (Thordarson et al., 1991). In the present work we show that S100A6 is present in human umbilical cord. The use of Phenyl-Sepharose chromatography served to obtain a fraction enriched in S100A6 in relation to the bulk of proteins contained in this tissue. Further investigation revealed that S100A6 was present both in the walls of the umbilical cord vessels (artery and vein) and in the surrounding Wharton’s jelly. Immunohistochemical studies confirmed the presence of S100A6 in the umbilical cord. An intensive immunoreaction for S100A6 was observed in Wharton’s jelly, both in the cytoplasm of myofibroblasts and in the extracellular matrix. Affinity chromatography performed on S100A6 resin indicated that Wharton’s jelly contains some proteins which could bind to S100A6. Thus, our results might suggest the involvement of the S100A6 protein in intra- and extracellular signaling pathways in this tissue. Acknowledgements This work was supported by grant N N301 293637 from the Ministry of Science and Higher Education of Poland to A.F. and by statutory funds from the Nencki Institute of Experimental Biology.
References Ferguson VL, Dodson RB (2009) Eur J Obstet Gynecol Reprod Biol 144 (Suppl 1): S108-S113. Kulkarni ML, Matadh SP, Ashok C, Pradeep N, Avinash T, Kulkarni MA (2007) Ind J Ped 74: 787-789. Kuźnicki J, Filipek A (1987) Biochem J 247: 663-667. Kuźnicki J, Kordowska J, Puzianowska M, Woźniewicz BM (1992) Exp Cell Res 200: 425-430. Leśniak W, Słomnicki PŁ, Filipek A (2009) Biochem Biophys Res Commun 390: 1087-1092. Thordarson G, Southard JN, Talamantes F (1991) Endocrinology 129: 12571265.
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Agnieszka Kikulska, Michał Mlącki, Tomasz Wilanowski Nencki Institute of Experimental Biology PAS, Laboratory of Signal Transduction, Warsaw, Poland e-mail: Agnieszka Kikulska
Grainyhead-like (GRHL) family consists of highly conserved homologs of Drosophila Grainyhead (GRH) transcription factor. In mice, GRHL3 maintains the integrity of skin barrier formation through the regulation of transglutaminase 1. In Drosophila melanogaster, GRH controls the expression of DOPA decarboxylase which serves analogous function preserving the integrity of fly cuticle. Given the conserved role of GRHL in maintaining epithelial integrity, it is noteworthy that the evolutionary origin of GRHL family appears coincident with evolutionary origin of the epithelium. GRHL1 is normally active in the developing epidermis and hair follicles. Mice lacking this factor display an abnormal hair coat (defective hair anchoring) and reduced expression of desmoglein 1, a member of the desmosomal cadherin family and a direct target of GRHL1 regulation. It is still uncertain whether GRHL1 is involved in skin cancer development induced by UV radiation and what is the role of GRHL1 in skin barrier formation. The main goal of my research is to investigate skin cancer formation in Grhl1-deficient mice upon exposure to UV radiation. Experiments are performed on mice with three different Grhl1 genotypes: wild type, heterozygous and null. Grhl1 knock-out mice were generated in the Royal Melbourne Hospital (Australia) using genetic engineering methods. These animals were provided to us under a Material Transfer Agreement. Consensus DNA binding sequence of GRHL1 transcription factor is already known, which makes it possible to predict its putative targets genes that are linked to skin cancer development. My research is likely to discover novel signal transduction pathways that are relevant to skin cancer formation. I will utilize various molecular biology, histological and bioinformatics methods. References Wilanowski T et al. (2008) EMBO J 27: 886-897. Auden A. et al. (2006) Gene Expr Patterns 6: 964-970. Ting SB. et al. (2005) Science 308: 411-413. Boglev Y et al. (2011) Dev Biol 349: 512-522. Wilanowski T et al. (2002) Mech Dev 114: 37-50.
Abstracts 62
P7.12
P7.13
ERK1/2 is dephosphorylated by a novel phosphatase — CacyBP/SIP
Soluble form of heparin-binding EGFlike growth factor promotes EGF receptor intracellular traffic distinct from the lysosomal degradation and receptor recycling
Ewa Kilanczyk1, Slawomir Filipek2, Anna Filipek1 1Nencki Institute of Experimental Biology, Warsaw, Poland; 2Faculty of Chemistry, Warsaw University, Warsaw, Poland
e-mail: Ewa Kilanczyk
ERK1/2 kinase-dependent signaling pathways are involved in diverse cellular functions including gene expression, proliferation, differentiation etc. The activity of ERK1/2 kinase is regulated by phosphatases, among them there is MKP-3 phosphatase the association of which with ERK2 is highly specific and results in down-regulation of ERK2 kinase activity. Recently we have found that ERK1/2 binds to the CacyBP/SIP protein (Kilanczyk et al., 2009) and in this work we examined whether CacyBP/SIP might demonstrate phosphatase activity toward ERK1/2 kinase. We have found that CacyBP/SIP indeed dephosphorylates ERK1/2. The Km and Vmax values established for a standard phosphatase substrate, p-NPP, are 16.9±3.6 mM and 4.3±0.4 μmol/min, respectively. The CacyBP/SIP phosphatase activity was decreased by okadaic acid (IC50 = 45 nM). These experimental results were supported by a theoretical analysis which revealed important sequence similarities between CacyBP/SIP and the phosphatase-like proteins as well as certain MAP kinase phosphatases. We also found that decreased ERK1/2 activity in the nuclear fraction of NB2a cells after CacyBP/SIP overexpression results in a lower level of β-catenin which suggests that CacyBP/SIP, through dephosphorylation of ERK1/2, may affect β-catenin degradation and cell differentiation. Altogether our results show that CacyBP/SIP, as a phosphatase, might play a role as a new negative modulator of ERK1/2 kinase and that, in consequence, this protein might be implicated in differentiation of NB2a cells. Acknowledgements This work was supported by grants: N N401 057837 to E.K. from the Ministry of Science and Higher Education of Poland and by statutory funds from the Nencki Institute of Experimental Biology.
Natalia Korotkevich, Andrii Labyntsev, Alla Honcharenko, Denis Kolibo, Serhiy Komisarenko Palladin Institute of Biochemistry of the NASU, Molecular Immunology Department, Ukraine e-mail: Natalia Korotkevich
Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family growth factors. It has high affinity for heparin and heparan sulfate. HB-EGF precursor is synthesized as type I transmembrane protein (pro-HBEGF), which is cleaved by proteases resulting in shedding of soluble HB-EGF (sHB-EGF). sHB-EGF acts as mitogenic signal through the EGF receptor (EGFR). The ligand binding to EGFR results in EGFR activation and recycling or further lysosomal degradation of ligand-receptor complex. On the other hand, EGF binding to EGFR could promote EGFR nuclear translocation. In nucleus EGFR acts as transcription factor able to activate genes required for highly proliferating activities. But, the probable role of HB-EGF as a ligand of EGFR in this process is still unclear. HB-EGF has some peculiar properties comparing to other EGF family members. HB-EGF belongs to a heparin binding ligands, which implies that proteoglycans may also be involved in translocation complex. In addition, some other important questions remain to be answered, e.g. does the nuclear translocation complex consist of both ligand and receptor, or EGFR is active on its own, and whether EGFR kinase activity is required? In this work we focused on the investigation of the ability of sHB-EGF to induce EGFR nuclear importation. We have demonstrated that sHB-EGF induced EGFR internalization and its further intracellular importation into the Golgi apparatus and endoplasmic reticulum via retrograde movement. Increasing amount of EGFR in the nuclear fraction of sHB-EGF treated cells was observed, which implies that sHB-EGF could induce EGFR nuclear importation. In addition we demonstrated that the translocation complex consisted of both ligand and receptor. It has been also shown that addition of EGFR kinase inhibitor — AG1478 dramatically increased EGFR internalization. These data indicate that EGFR kinase plays an important role in induction of intracellular internalization process. We found here the new sHB-EGF activity, its ability to induce EGFR nuclear importation. We suggest that sHB-EGF is an essential element of translocation complex and is required for maintaining EGFR in active conformation.
Parnas Conference Warsaw 2011
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PPAR-gamma modulation influences EPC functions
Association of phosphoglycerate mutase with glycolytic complex regulates energy production in cancer cells
Jerzy Kotlinowski1, Anna Grochot-Przęczek1, Magdalena Kozakowska1, Ewa Zuba-Surma1, Rafał Derlacz2,3, Józef Dulak1, Alicja Józkowicz1
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Wojciech Kowalski1, Dominika Nocoń1, Andrzej Gamian2, Dariusz Rakus1
1Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Department of Medical Biotechnology, Krakow, Poland; 2Adamed Ltd, R&D Department, Pieńków, Poland; 3University of Warsaw, Faculty of Biology, Department of Metabolic Regulation, Warsaw, Poland
1Department of Animal Molecular Physiology, Wroclaw University, Wroclaw, Poland; 2Department of Medical Biochemistry, Wroclaw Medical University, Wroclaw, Poland
e-mail: Jerzy Kotlinowski
In contrast to normal, differentiated cells, cancer cells rely on glycolysis, even in the presence of oxygen. It has been hypothesized, based on in vitro experiments, that the association of the glycolytic enzymes, especially enzymes of triose phosphate metabolism, in a metabolic complex leads to efficient degradation of glucose to lactate. Here we provide a strong evidence that in cancer cells, all the enzymes of triose phosphate metabolism, from aldolase to pyruvate kinase consecutively, function as a macromolecular complex and that disruption of this complex inhibits lactate release and ATP synthesis in glycolytic pathway. Results of our experiments indicate that composition of the complex and modulation of glycolytic flux is affected by elevated lactate concentration and by glucose deprivation which regulates association of the phosphoglycerate mutase (PGAM) C-terminal region with the complex. High concentration of exogenous lactate not only disrupts the glycolytic complex and inhibits ATP synthesis, but also affects nuclear localization of PGAM and ceases cell proliferation. The studies unraveling the nuclear function of PGAM and the components of signaling pathway linking energetic metabolism with proliferative machinery are in progress. Our results open new insight into the role of metabolic complexes in the cancer cell biology and might provide new prospects for the cancer disease treatment using lowmolecular weight competitors which destabilize the glycolytic complex.
PPARγ nuclear receptor is a target for thiazolidinediones (TZD), the commonly used insulin sensitisers. Besides improving insulin action and normalising glycemia in diabetic patients TZD can also improve some functions of endothelial progenitor cells (EPC). Although activation of PPARγ was shown to be beneficial for EPC both in vitro and in vivo more experiments must be performed to demonstrate pathways involved in this actions. Our aim was to find expression changes in diabetic and healthy EPC and check how modulation of PPARγ activity affects EPC biology. Experiments were performed on the 12-weeks old wild type (wt) and diabetic (db/db) mice. For in vivo tests EPC were characterised as the CD45-/KDR+/Sca-1+ cells, whereas in vitro studies bone marrow population enriched in EPC (approx. 600 times) was used. Importantly, percentage of CD45-/KDR+/Sca-1+ cells was significantly reduced by 40–80% in the bone marrow of diabetic mice in comparison to wt (in db/db only 0.0003% of all cells were EPC). Reduction related to blood glucose levels suggested that diabetes influences the number of EPC according to its severity. These defect was partially or fully reversed by an oral application of rosiglitazone 10 mg/kg, for 14 or 28 days, respectively. We observed a similar trend for CXCR4+ cells in bone marrow: a reduction in db/db animals and stimulation upon rosiglitazone treatment. Although EPC number was reduced by 40% in blood of db/db mice the oral rosiglitazone administration did not change it. Furthermore, in in vitro assays, EPC isolated from db/db mice displayed impaired migratory and angiogenic potential. Migration of db/db cells was decreased by 50%, whereas ability to form tubes on matrigel by 40%. Importantly, in both tests functions of diabetic EPC were restored by rosiglitazone (10 μmol/L, 24 h) in a PPARγ-dependent manner. Next, based on the EPC transcriptome analysis we found that proteoglycan 4 (PRG4) was one of the strongest downregulated gene in db/db cells (7-fold decrease). Since PRG4 stimulates proliferation and survival of hemangioblasts we tried to induce its expression in vitro by rosiglitazone (10 μmol/L, 24 h) stimulation. We observed 2.5 fold PPARγ dependent induction in PRG4 expression both in wt and diabetic EPC. Thus, PPARγ is an important regulator of EPC biology and its induction may be used to improve the EPC functions.
e-mail: Wojciech Kowalski
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Actin polymerization by lysenin
Phosphorylation of p66Shc under oxidative stress in fibroblasts of NARP patients
Edward A. Czuryło, Natalia Kulikova, Andrzej Sobota Nencki Institute of Experimental Biology, Warsaw, Poland e-mail: Natalia Kulikova
Lysenin is a 33.5kDa protein isolated from coelomic fluid of an earthworm Eisenia foetida. It interacts with lipids, specifically recognizing sphingomyelin. It also causes longlasting (more than 30 min) contraction of isolated rat aorta strips. The mechanism underlying induction of contraction as well as the biological action of lysenin in animals has not been clarified. Our studies of the effect of lysenin on the ATPase activity of regulated actomyosin system has shown that addition of lysenin resulted in the abolishment of caldesmon inhibition and further activation of the ATPase activity up to the level of its activation by tropomyosin. This effect was observed at very low molar ratios of actin to lysenin. Pelleting of reconstituted smooth muscle actomyosin system with or without lysenin revealed that lysenin did not induce dissociation of the filament constituents. Moreover, previous electron microscopic observations have shown that addition of lysenin to F-actin resulted in filament cross-linking. Thus it may be assumed that the abolishment of caldesmon inhibitory properties is related to the suppression of caldesmon effect on the actin filament structure. It has been suggested that lysenin invokes structural changes in the F-actin helix favoring the ATP hydrolysis by myosin and suppressing the effect of caldesmon on the actin filament structure. To understand the process of G-actin polymerization at addition of lysenin we have performed studies of stoichiometric ratio of actin–lisenin interaction. Pelleting of actin with lysenin followed by the subsequent Western blotting analysis has shown that actin binds lysenin with the 1000:1 ratio. Therefore it may be suggested that lysenin changes actin conformation at the same time preventing the binding of next lysenin molecules.
Magdalena Lebiedzinska1, Agnieszka KarkucinskaWieckowska2, Jan M. Suski1,3, Gyorgy Szabadkai4, Grzegorz Wilczyński1, Jakub Wlodarczyk1, Maciej Pronicki2, Jerzy Duszynski1, Paolo Pinton3, Mariusz R. Wieckowski1 1Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland; 2Department of Pathology, The Children’s Memorial Health Institute, Warsaw, Poland; 3Department of Experimental and Diagnostic Medicine, Section of General Pathology, Interdisciplinary Center for the Study of Inflammation (ICSI) and LTTA Center, University of Ferrara, Ferrara; Italy; 4University College London, Department of Cell and Developmental Biology, Consortium for Mitochondrial Research, London, United Kingdom
e-mail: Magdalena Lebiedzińska
In our previous studies we have shown that p66Shc, the adaptor protein regulating mammalian lifespan is involved in pathology associated with oxidative stress observed in cases of mitochondrial disorders. Affected mitochondrial respiratory chain generates reactive oxygen species (ROS) which activates phosphorylation of p66Shc at serine 36, what in turn triggers further ROS production in mitochondria and may lead to serious cell damage or apoptosis. Activation of p66Shc phosphorylation also contributes to decrease of antioxidants enzymes levels. However, inhibition of p66Shc phosphorylation decreased ROS production and improved antioxidant defense [1]. Thus we examined whether p66Shc phosphorylation depends on the type of defect and if the decrease of ROS level also attenuates p66Shc phosphorylation. In fibroblasts of NARP patients, respiratory chain is working properly, and the oxidative stress results from hyperpolarization of inner mitochondrial membrane. In these fibroblasts abnormalities in bioenergetic parameters and antioxidant defense, together with high level of carbonylated proteins, lipid accumulation and affected mitochondrial structure and motility are observed. Interestingly, in cells of NARP patients we observe strong induction of p66Shc serine 36 phosphorylation pathway. Also in this cellular model, inhibition of p66Shc phosphorylation, similarly to antioxidants treatment, led to a decrease in superoxide anion production and to an increase in antioxidant enzymes levels. Our data suggest that serine 36 p66Shc phosphorylation is an important factor contributing to cell damage in cases of mitochondrial dysfunction (i.e. associated with ATP synthase defect). Both, inhibition of kinase phophorylating p66Shc (PKCβ) and antioxidants treatment result in decreased level of ROS, restrain phosphorylation of p66Shc and its pro-oxidative function.
Reference 1. Lebiedzinska M et al. (2010) Biochim Biophys Acta – Bioenergetics 1797: 952-960.
Parnas Conference Warsaw 2011
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Dissecting the function of the ArfGAP2/3 proteins in the COPI trafficking machinery
Evaluation of horizontal gene transfer from genetically modified plants to bacteria
Lena Lifshitz1, Irit Pevzner1, Joelle Bigay2, Anna Parnis1, Dan Cassel1
Mahesh Madyagol, Vinh Phu Hoang, Stanislav Stuchlík, Jan Turňa
1Technion-Israel Institute of Technology, Department of Biology, Israel; 2Université de Nice Sophia-Antipolis et CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, France
Department of Molecular Biology, Faculty of Natural Sciences, Comenius University in Bratislava, Bratislava, Slovak Republic
e-mail: Lena Lifshitz
From yeast to mammals, two types of GTPase activating proteins, ArfGAP1 and ArfGAP2/3, control GTP hydrolysis on the small G protein Arf1 at the Golgi apparatus. Although functionally interchangeable, they display little similarity outside the catalytic GAP domain. ArfGAP1 is controlled by membrane curvature through its ALPS (amphipathic lipid packing sensor) motifs, whereas Golgi targeting of ArfGAP2 depends on heptameric protein complex named coatomer, the building block of the COPI coat. Using a reporter fusion approach and in vitro assays, we identified several functional elements in ArfGAP2/3. We show that the Golgi localization of ArfGAP3 depends on a central basic stretch. This region interacts directly with coatomer, which we found essential for the catalytic activity of ArfGAP3 on Arf1-GTP. In order to further understand the role of ArfGAPs in the COPI system we are performing detailed analysis of the interactions between the GAP2/3 and COPI constituents at the molecular and structural level.
e-mail: Mahesh Madyagol
Horizontal gene transfer (HGT) is today recognized as being the most important factor for the rapid global dissemination of antimicrobial resistance. One of the main concerns regarding the use of GM food in human and animal nutrition is the effect that newly introduced sequences may have on the organism. Public and scientific concerns about the environmental and food safety of genetically modified (GM) crops overshadow the potential benefits offered by crop biotechnology to improve food quality. The main objective of this work is to quantify the risk of horizontal gene transfer from from the GM plants to (GMOs), and food derived thereof, to the bacterial microflora. For studying potential gene transfer we have chosen CP4 EPSPS (5-enolpyruvylshikimate 3-phosphate synthase) gene from Round Ready maize (Monsanto) chromosome. The CP4 EPSPS enzyme is a key enzyme in the biosynthesis of aromatic amino acids in microorganisms and plants. It is the target of the broad-spectrum herbicide glyphosate. During our earlier studies of HGT of the CP4 EPSPS gene from GM maize through gastrointestinal tract to bacteria living in animal gut we have observed a functional truncated CP4 EPSPS gene form present in bacteria. This truncated CP4 EPSPS gene was cloned into the T7 expression vector pET-28b(+) along with native full-length CP4 EPSPS. We have tested the truncated form of CP4 EPSPS indetail and carried a comparative study with native full-length CP4 EPSPS. We have expressed, purifed and characterized both forms of CP4 EPSPS synthases. We have studied enzyme activity of both forms of purified CP4 EPSPS proteins. We have found that both enzymes shown positive for both forms of CP4 EPSPS. This will help to decide that some truncated forms of CP4 EPSPS could confer full function of native full-length CP4 EPSPS and these findings also should be taken into account in risk assessment of possible HGT from GM plants.
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Energy metabolism in cells with glycogen branching enzyme deficiency Dominika Malinska, Malgorzata Bejtka, Joanna Szczepanowska, Jerzy Duszynski
Cannabidiol inhibits expression and constitutive signaling of Kaposi’s sarcomaassociated G protein-coupled receptor and induces programmed cell death
Nencki Institute of Experimental Biology, Department of Biochemistry, Laboratory of Bioenergetics and Biomembranes, Warsaw, Poland
Yehoshua Maor1,3, Jinlong Yu1, Paula M. Kuzontkoski1, Bruce J. Dezube2, Xuefeng Zhang1, Jerome E. Groopman1
e-mail: Dominika Malinska
Glycogen storage disease type IV (GSD IV) results from mutation in the GBE1 gene encoding a glycogen branching enzyme. The disease manifests itself by dysfunctions of liver, skeletal muscle and nervous system. The hallmark of GSD IV are intracellular aggregates of linear glycogen molecules, which are characterized by poor water solubility and high damage potential towards intracellular structures. Additionally, the efficiency of glucose release from such abnormal glycogen is strongly impaired, which may lead to energetic imbalance in the cell. It is not clear to which extent the disruption of glucose metabolism contributes to the cell injuries occurring in GSD IV and, on the other hand, how much it results from a mechanical damage due to presence of intracellular glycogen aggregates. In our study we compare energy metabolism of fibroblasts with functional GBE and with GBE deficiency (cell lines derived from GSD IV patients as well as cells with suppressed expression of GBE). The preliminary data will be presented regarding mitochondrial morphology, respiratory parameters as well as growth and survival rate of the cells under different accessibility of substrates for glycolysis and for the oxidative phosphorylation.
1Division of Experimental Medicine and 2Department of Hematology and Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, USA; 3 Institute of Drug Research, Medical Faculty of the Hebrew University of Jerusalem, Jerusalem, Israel
e-mail: Yehoshua Maor
Kaposi’s sarcoma (KS) is the most common neoplasm caused by Kaposi’s sarcoma associated herpesvirus (KSHV), also termed human herpesvirus 8 (HHV-8). It is prevalent among the elderly in the Mediterranean, inhabitants of sub-Saharan Africa, and among immunocompromised individuals like organ transplant recipients and AIDS patients. The virus encodes for several gene products that function like cytokines and chemokines. Of special interest is the KSHV G protein-coupled receptor (vGPCR), which by its constitutive activity can alter key signaling pathways and cell functions, implicating these pathways in the initiation of the neoplasm. Cannabidiol (CBD), a plant-derived cannabinoid, exhibits promising anti-tumor effects without inducing psychoactive side effects. In this study we investigated the effects of CBD both on the infection of endothelial cells by KSHV, and on the growth of KSHV-infected endothelium, an in vitro model for the transformation of normal endothelium to Kaposi’s sarcoma. We found that while CBD did not affect the efficiency of KSHV infection of endothelium, this cannabinoid reduced proliferation and induced apoptosis in KSHV-infected endothelial cells. CBD appeared to have these effects by inhibiting the expression of KSHV viral G protein-coupled receptor (vGPCR), the chemokine growth-regulated protein alpha (GROα), vascular endothelial growth factor receptor 3 (VEGFR-3), and the VEGFR-3 ligand vascular endothelial growth factor C (VEGFC). Our study suggests that CBD may be a novel agent for the treatment of Kaposi’s sarcoma.
Parnas Conference Warsaw 2011
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Recombinant perfringolysin O, a useful probe for cholesterol detection
The influence of elevated level of homocysteine on human placental function
Ewelina Marszałek, Piotr Koprowski, Andrzej Sobota
Olga P. Martsenyuk1, Berthold Huppertz2 , Kateryna L. Romanets1, Maria Y. Obolenskaya1
Nencki Institute of Experimental Biology, Department of Cell Biology, Warsaw, Poland e-mail: Ewelina Marszałek
Cholesterol is one of major lipids in mammalian cells and is essential for plasma membrane structure and function. Cholesterol content and distribution in cells are strongly regulated by de novo synthesis as well as by endocytic uptake from media. Disturbances in these processes affect cholesterol deposition in lysosomes, evoking neurodegenerative diseases, such as Niemann-Pick type C disease. To detect cholesterol deposits in lysosomes we prepared a recombinant toxin, perfringolysin O, which interacted specifically with the lipid. Perfringolysin O (PFO) from Clostridium perfringens is a member of cholesterol-dependent cytolysins. These toxins are responsible for life-threatening pathogenicity of a number of bacteria. The toxins bind to plasma membrane cholesterol and induce cell lysis. Due to selective interaction with cholesterol, PFO can be used as a probe to visualize or even quantitate cholesterol in cell membranes. We decided to optimize production of a fusion protein of PFO and glutathione transferase (GST). To avoid potential secretion of the toxin, the open reading frame of PFO was modified in such a way that it lacked sequence for first 29 amino acids that serve as a leader peptide. We mutated the only cystein, Cys459 to alanine, since this modification was shown to be responsible for toxin inactivation. On the other hand, we introduced new cystein in position 397 to facilitate fluorophore modification of PFO. The codon bias of PFO gene was optimized for Escherichia coli expression and the gene was synthesized by a commercial vendor. The gene was subcloned from pUC57 to pGEX4T vector for expression of N-terminal GST fusion protein. The protein was expressed in E. coli BL21 cells and purified by GSH-agarose. The produced PFO, when incubated with erythrocytes, induced hemolysis at 50 pg/ml. Incubated with liposomes composed of different lipids the recombinant protein bound to vesicles containing cholesterol. In the result of interaction with cholesterol-containing membranes oligomers of PFO were formed, as demonstrated by SDS/ PAGE analysis. Altogether, the presented data indicate that the prepared recombinant PFO can be used as a probe to detect and visualize cholesterol in cells. Reference Flanagan JJ et al. (2009) Biochemistry 48: 3977-3987.
1Institute of Molecular Biology and Genetics, National Academy of Science of Ukraine, Kyiv, Ukraine; 2Department of Cell Biology, Histology and Embryology, Medical University of Graz, Graz, Austria
e-mail: Olga Matsenyuk
Background: Elevated level of homocysteine (Hcy) increases the risk of placental insufficiency, spontaneous abortion, preeclampsia and developmental defects of the fetus. Hcy is an aminothiol which is remethylated to methionine by methionine synthase or transsulfurated to cysteine by cystathionine β-synthase (CBS). The lack of CBS makes tissues more sensitive to Hcy and causes its accumulation leading to hyperhomocysteinemia (hHcy). The goal of our study was to examine the influence of hHcy on the placental functions — proliferation and apoptosis and on the ability of transsulfuration pathway to protect the tissue from the adverse effect of Hcy. Objectives: Placental samples were obtained from the I (8–10 weeks) and the III (38–40 weeks) trimesters of gestation, three samples from each term. Explants were cultivated in DMEM/F12 in the presence of 20, 40, 80 µM Hcy with or without 20 nM folic acid for 48h at 37°C and 5% CO2, 5% oxygen. Ki-67 positive nuclei of cytotrophoblast cells and the square of M30 positive regions were counted and related to the villous circumference and the square, correspondingly. The expression, localization and enzymatic activity of CBS were examined by RT-PCR, WB, IHC and incorporation of [14C]serine residue into cystathionine. Results: The proliferation index is higher (4.2 vs 0.7) and the apoptotic index is lower (0.8 vs 1.5) in the explants from the I trimester in comparison with the III one. Hcy induces concentration-dependent down-regulation of proliferation index and up-regulation of the apoptotic one. The apoptotic changes are more pronounced in the explants from the III trimester. The highest concentration of Hcy (80 mkM) induces the separation of syncytiotrophoblast from cytotrophoblast. The expression of CBS gene at RNA and protein levels and the catalytic acidity of the protein (~50 mU/mg protein) were ascertained for the first time. The CBS protein was localized in trophoblast and stroma cells of villous chorion. The elevated concentrations of Hcy induce upregulation of CBS content. Conclusion: The elevated level of homocysteine provokes the disbalance between the proliferation and apoptotic processes in human placenta. We speculate that the presence of CBS and its activation under mild hHcy provides the evidence that placenta has metabolic resources to withstand the elevation of Hcy and maintain redox homeostasis via transsulfuration pathway.
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The activity of adenosine deaminase in erythrocytes and leukocytes is normal even in cases of neoplastic pathology
Grainyhead-like 1 (GRHL1) transcription factor in development of skin cancers
Elena M. Bakurova, Kseniya O. Mironova, Tatiyana V. Moroz, Berta G. Borzenko National Medical University of M. Gorkiy, Department of Biochemistry, Ukraine e-mail: Ksenia Mironova
Biochemist Otto Varburg concluded that tumours are more frequent in the tissues poorly supplied with oxygen, and actually, normal cells are transformed into malignant ones due to the oxygen lack. It has been proved at present that the duration of the relapse-free period depends on the oxygen starvation of the tumor while cancers characterized with pO less than 10 mm Hg are expected to relapse earlier, it also depends, to great extent, on the severity of anemia. Dysmetabolic processes inside the blood cells lead to the cells dysfunction, they also impact cancer capacity to progress. Our task was to determine the activity of adenosine deaminase (ADA), it being the enzyme of adenosine catabolism in erythrocytes and leukocytes in patients with stomach cancer, lung cancer. ADA deficiency in lymphocytes is known to lead to the development of severe combined immunodeficiency. Under simultaneous estimation there was also a sorption capacity of the erythrocytes membranes as well as the reaction of the blast transformation (stimulation index) of lymphocytes. The examination included 15 patients with lung cancer T3-4N1-nM0, 30 patients with stomach cancer T3-4N1-nM0, 40 healthy volunteers. Spectrophotometric method has been applied in the process of the study. Decrease of ADA activity in lymphocytes to 19.02±2.33 nmol/min·mg in stomach cancer and 26.93+5.89 nmol/min·mg in lung cancer to be compared to 51.79±8.09 nmol/min·mg in control (p
