Session 7. Protein Functions in Cellular Signaling

Lectures L7.2 L7.1 Neutrophil extracellular traps (NETs) in autoinflammatory responses Molecular basis of macrophage Joanna Cichy activation by lipopolysaccharide Jagiellonian University, Faculty of Biochemistry, Biophysics & Katarzyna Kwiatkowska Biotechnology, Department of Immunology, Kraków, Poland e-mail: Joanna Cichy Nnencki Institute of Experimental Biology, Department of Cell Biology, Warsaw, Poland e-mail: Katarzyna Kwiatkowska Neutrophils also referred to as polymorphonuclear leuko- cytes (PMNs) form a part of the front line of host defense Lipopolysaccharide (LPS, endotoxin) is a component of against pathogens and are key component of tissue infil- the outer membrane of Gram-negative bacteria. LPS is an trates in autoinflammatory diseases such as systemic lupus archetypical pathogen-associated molecular pattern which erythematosus (SLE) and psoriasis. Activated neutrophils activates of one of Toll-like receptors (TLR), TLR4, of im- can eliminate microbes by the formation of neutrophil ex- mune cells. Upon infection it strongly stimulates production tracellular traps (NETs), extracellular decondensed chro- of pro-inflammatory mediators by macrophages. Excessive matin networks decorated with microbicidal agents. NETs host responses to LPS can lead to a systemic inflammatory were first reported to limit the growth and spread of mi- condition named septic shock. A relatively high incidence croorganisms through mechanically trapping pathogens and mortality of the disease drive studies on molecular and facilitating interaction of anti-microbial agents that are mechanisms of activation of cells by LPS. displayed on NETs with bacteria or fungi. More recently, In a typical scenario, activation of TLR4 is a multistep NETs were associated with tissue damage and autoinflam- process which begins in blood serum with binding of LPS matory diseases. NETs constituents, including self DNA, monomers to LPS-binding protein. The protein transfers histones and myeloperoxidase are potential autoantigens. LPS to CD14, a protein anchored within sphingolipid- Since in addition to their beneficial role in host defense, and cholesterol-rich microdomians (rafts) of the plasma NETs may be an underlying component of cell cytotoxic- membrane. CD14 transfers LPS to TLR4/MD-2 com- ity and autoimmunity, it is likely that specific counter-regu- plex which dimerizes and triggers two signaling pathways latory mechanisms evolved to constrain NETs formation. engaging MyD88 and TRIF adaptor proteins. The TRIF- Here we will discuss our data on regulatory mechanisms dependent signaling pathway requires endocytosis of acti- underlying NETs generation and discuss a contribution of vated TLR4. We study mechanisms which control fast and NETs to a skin disease-psoriasis. reliable organization of TLR4 signaling complexes in the plasma membrane addressing questions such as: Are raft lipids shaping macrophage responses to LPS? What is the role of raft accumulation of CD14 for LPS-triggered sign- aling? Can we identify other raft proteins controlling TLR4 signaling?

BIO2014 Congress Session 7. Protein Functions in Cellular Signaling 143 L7.3 Oral presentations NF-kappa B regulation in response O7.1 to LPS and poly(I:C) stimulation Maciej Czerkies1, Zbigniew Korwek1, Wiktor Heme oxygenase-1 deficiency triggers Prus1, Sławomir Błoński1, Bing Tian2, Marek monocyte mobilization and impairs heart Kimmel3, Allan Brasier2, Tomasz Lipniacki1 function after acute myocardial infarction 1Institute of Fundamental Technological Research, Laboratory of Mateusz Tomczyk, Karolina Bukowska-Strakova, Krzysztof 2 Modelling in Biology and Medicine, Warsaw, Poland; University of Szade, Alicja Jozkowicz, Jozef Dulak, Agnieszka Jazwa Texas Medical Branch (UTMB), Department of Internal Medicine, USA; 3Rice University, Department of Statistics, USA Department of Medical Biotechnology, Faculty of Biochemistry, e-mail: Tomasz Lipniacki Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland e-mail: Agnieszka Jazwa NF-κB, IRF3 and AP-1 are most potent transcription fac- tors controlling innate immune responses to pathogens. Coronary embolism resulting mostly from atherosclerosis, Combining single cell and population techniques with leads to ischæmic injury of myocardium, subsequent incor- mathematical modeling we analyzed crosstalk of these rect heart contractility and heart failure, the main cause of pathways in mouse embryonic fibroblasts in response to death in industrialized countries. Heme oxygenase-1 (HO- LPS and poly(I:C). 1) is an inducible form of heme-degrading enzyme, which We found that both LPS and poly(I:C) activate mediating possesses cytoprotective, antoxidative and proangiogenic kinases IKKα/β and TBK1; interestingly, only poly(I:C) properties. stimulation leads to activation of IRF3 (activated by TBK1) A role of HO-1 in acute myocardial infarction (AMI) was and triggering of transcription of IFNβ, IRF7, and RIG-1 investigated in HO-1 knock-out and wild type mice ex- and other interferon regulated genes. LPS stimulation leads posed to a permanent ligation of left anterior descending to transient or oscillatory (in some cells) responses of NF- (LAD) coronary artery. Cardiac function was monitored κB, in contrast to switch-like responses (preceded by one weekly for 3 weeks with high frequency ultrasonography or two pulses in a fraction of cells) to poly(I:C) stimula- (Vevo2100 platform) and revealed more potent reduction tion, with fraction of switched-on cells increasing with the of ejection fraction in HO-1 knock-out mice. Coronary stimulation dose. As suggested by the experiment in which artery occlusion in wild type mice lead to strong upregula- cells are costimulated by LPS and IFNβ, the difference in tion of HO-1 expression, with no changes in expression NF-κB responses is caused by IFNβ para- and autocrine of other genes related to iron metabolism (HO-2, ferri- regulation that leads to activation of Eif2ak2 and suppres- tin). Some genotype-dependent differences were observed sion of NF-κB inhibitors synthesis. Correspondingly the in the expression of antioxidant enzymes (superoxide dis- blockade of INFβ receptor causes attenuation of response mutase 1, glutathione peroxidase 1, catalase) and cardiac to poly(I:C) at latter time points. Overall this suggests that myocytes-associated miRNAs (miR-29a, miR-199) at 1, 7 autocrine regulation breaks the negative regulation of NF- and 21 days after AMI. Moreover, we found much higher κB (and IRF3) leading to build up of nuclear NF-κB and numbers of classical (Ly6C++CD43+) and intermediate IRF3, followed by apoptosis in a fraction of cells (not ob- (Ly6C++CD43++) monocytes, both basal and induced by served in the case of LPS stimulation). The IRF3 activation AMI, in peripheral blood of HO-1 deprived mice. Con- is stabilized by positive feedback involving strongly upregu- trary in spleen, we observed higher numbers of classical lated IRF7 and RIG-1 (which is IRF7 responsive). and intermediate monocytes in wild type animals when We confirmed by mathematical modeling the dynamically compared to HO-1 knock-outs. Finally, we noticed that divergent responses to LPS (mimicking bacterial infec- abundance of SKL cells (Sca-1+/c-kit+/Lin-) including hæ- tion) and to poly(I:C) (mimicking viral infection). NF-κB is matopoietic stem cells (HSCs, Sca-1+/c-kit+/Lin-/CD48-/ known for exhibiting oscillatory responses to TNFα, which CD150+/CD34-) populations in bone marrow of HO-1 are replaced by switch-like responses in A20-deficients deprived mice was larger. No differences were found in cells. Here, we found that activation of the IRF3 pathway, case of bone marrow hæmatopoietic progenitor cells (Sca- or INFβ stimulation leads to the similar effect on NF-κB 1+/c-kit+/Lin-/CD48-/CD150-/CD34+) between both signaling, possibly due to inhibition of translation of NF- genotypes. κB inhibitors. The switch-like behavior, frequently associ- Consequences of AMI seem to be more severe in HO-1 ated with cell fate decisions, is associated with bistability knock-out mice. Lack of HO-1 may influence early and late arising here due to the positive feedbacks in IRF3/IRF7 responses to AMI related to antioxidant genes expression regulation. and monocyte/macrophage-dependent cardiac remod- eling.

Warsaw, September 9th–12th 2014 144 Session 7. Protein Functions in Cellular Signaling O7.2 O7.3 Silencing of connective tissue growth Physiological and pharmacological factor by small interfering RNA consequences of 2-methoxyestradiol, prevents fibroblasts to myofibroblast a plausible novel hormone transition in bronchial asthma Magdalena Gorska1, Alicja Kuban Jankowska1, Monika Katarzyna Wójcik1,3, Marta Michalik2, Paulina Gorzynik1, Michal Aleksander Zmijewski2, Maciej Wnuk3, Koczurkiewicz3, Zbigniew Madeja2, Marek Sanak1 Iwona Rzeszutek3, Anna Lewinska3, Michal Wozniak1 1Jagiellonian University Medical College, Department of Medicine, 1Department of Medical Chemistry, Medical University of Gdansk, Laboratory of Molecular Biology and Clinical Genetics, Kraków, Gdańsk, Poland; 2Department of Histology, Medical University of Poland; 2Jagiellonian University, Faculty of Biochemistry, Biophysics Gdansk, Gdansk, Poland; 3Department of Genetics, University of and Biotechnology, Department of Cell Biology, Kraków, Poland; Rzeszow, Rzeszow, Poland 3 Jagiellonian University Medical College, Faculty of Pharmacy, e-mail: Michał Woźniak ; Magdalena Gorska e-mail: Katarzyna Anna Wójcik 2-methoxyestradiol is one of the principal physiological Bronchial asthma is one of the most common chronic lung 17β-estradiol derivatives, believed to be novel and poten- diseases. It is inflammatory disorder of the lower airways tially active anticancer agent evaluated in ongoing advanced manifested by reversible constriction of bronchi but also clinical trials. Preclinical research suggests a wide spectrum progressing with an irreversible bronchial wall modeling. of possible anticancer mechanisms of 2-ME action that Despite basic pathomechanism of asthma, characterized by seem to be directly associated with the inhibition of an- influx of blood cells, many evidences suggest that extracel- giogenesis and induction of apoptosis in tumorous and lular matrix thickening and increased number of smooth proliferating cells. According to Sutherland et al., 2-meth- muscle cells is an early event. Growth factors and cytokines oxyestradiol inhibits cell proliferation at threshold con- like transforming growth factor type β (TGF-β) and con- centrations of 0.1μM–0.3μM, reaching maximum effect at nective tissue growth factor (CTGF) are known to induce 10μM–20μM. fibroblast to myofibroblast transition (FMT) during the The aim of the study was to determine physiological and course of asthma. pharmacological anticancer and genotoxic properties of Our previous studies demonstrated that human bronchi- 2-methoxyestradiol. al fibroblasts (HBFs) cultured from patients with asthma We used highly metastatic osteosarcoma 143B cell line. have much higher FMT rate, both spontaneously and at the The cells were treated with physiological (1 nM,10 nM) and presence of TGF-β. Moreover, we demonstrated that asth- pharmacological (1 µM, 10 µM) concentrations of 2-meth- matic HBFs are capable of CTGF synthesis and secretion, oxyestradiol. Induction of cell death were measured by PI/ especially when stimulated with TGF-β. Annexin V staining, respectively. Inhibition of cell cycle, In the current study we show that CTGF silencing sig- level of reactive nitrogen and nitrogen species and neu- nificantly attenuated TGF-β induced FMT in HBFs from ronal nitric oxide synthase localization were determined by asthmatics. We used small interfering RNA specific for imaging cytometry. Comet assay was performed in order to CTGF transcripts, and noted marked inhibition of FMT in determine DNA damage. HBFs. This was accompanied by an attenuation of TGF-β Fundamental long-lasting viewpoint on 17β-estradiol me- ability to stimulate this process. In control experiments, tabolites as biologically inactive excretion products has we demonstrated that CTGF is able to enhance TGF-β- been refuted by various researchers (Yang et al., 2013; Yuan induced FMT in a synergistic manner. These results, es- et al., 2013). We evidenced here that 2-methoxyestradiol ex- pecially in the light of our previous reports, suggest that erts anticancer properties not only at pharmacological but CTGF may contribute to FMT phenomenon as positive also physiological concentrations. Due to that fact it could feedback stimulus, both by induction and boosting TGF-β be reported as a plausible novel hormone. triggered FMT. Otherwise, CTGF expression seems con- trolled by the presence of TGF-β. Specific mechanism of References: synergism observed between this cytokines is amplified in Sutherland TE et al (2007) 2-methoxyestradiol — a unique blend of activi- the asthmatic HBFs. The advantage of CTGF inhibition ties generating a new class of anti-tumour/anti-inflammatory agents.Drug over TGF-β targeting seems to be a more specific involve- Discovery Today 12: 577–584. Yang CY et al (2013) Osteoblastoma in the region of the hip. Journal of the ment of CTGF into mechanism of remodeling, whereas Chinese Medical Association 76: 115-120. TGF-β signaling is important also for immunoregulation. Yuan W et al (2013) Estrogen Metabolite 2-methoxyestradiol Prevents Hy- In conclusion a bronchial fibroblast CTGF biosynthesis in pertension in Deoxycorticosterone Acetate-salt Rats. Cardiovascular Drugs the asthmatic lung amplifies FMT and is a promising target 160 and Therapy 27: 17-22. for pharmacological intervention in order to prevent bron- Acknowledgments chial remodeling in asthma. The project was funded by grant No. 2012/07/B/NZ1/00010 from Na- tional Science Center resources. M. Wnuk, I. Rzeszutek, A. Lewinska are supported by the Operational Programme Development of Eastern Poland 2007-2013, NoPOPW.01.03.00-18-018/09, the project Centre of Applied Biotechnology and Basic Sciences.

BIO 2014 Congress Session 7. Protein Functions in Cellular Signaling 145 O7.4 O7.5 The role of AnxA2-S100A10 complex Overexpression of adaptor protein in intracellular translocation of Ruk/CIN85 acompanied by activation TRPV5/6 channels in osteoblast-like of signalling pathways, depending cells undergoing mineralization on the PI3-kinase and transcription 1 2 1 factor NF-κB, in human breast Anna Cmoch , Patrick Groves , Slawomir Pikula adenicarcinoma cell line MCF-7 1Nencki Institute of Experimental Biology, Department of Biochemistry, Warsaw, Poland; 2Instituto de Tecnologia Quimica e Biologica, Iryna Horak1,2, Ganna Pasichnyk1, Olga Povorozniuk1,3, Universidade Nova de Lisboa, Department of Biological Chemistry, Anatoliy Samoylenko1, Liudmila Drobot1 Portugal e-mail: Anna Ćmoch 1Palladin Institute of Biochemistry, NASU, Kyiv, Ukraine; 2Kyiv Mohyla National Academy, Kyiv, Ukraine; 3Kyiv National Taras Schevchenko University, Kyiv, Ukraine Calcium ions, aside from their role as a second messen- e-mail: Iryna Horak ger, are fundamental components of the vertebrate bone matrix. Ca2+ enter cells through the plasma membrane via Introduction: Molecular basis of cancer is the deviation channels of the transient receptor potential family, vanil- of certain signaling pathways, including PI3K/Akt/mTOR loid type (TRPV). Among them TRPV5 and TRPV6 are and NF-κB, which is accompanied by the emergence of involved in the efficient uptake and storage of calcium in resistance to chemotherapy and hormonal drugs, a viola- bone [1]. Trpv5 (-/-)-deficiency leads to an osteoporosis tion of the regulation of apoptosis and cell differentia- development [2]. The role of AnxA2-S100A10 complex in tion, increased their invasive properties. It was previously regulation of TRPV5/6 channels has been recently broadly shown that MCF- 7 cells with overexpression of the adapt- investigated. In this study we provide the first evidence er protein Ruk/CIN85 are more resistant to anoikis, have for the regulatory mechanism of the AnxA2-S100A10 increased transforming potential and resistance to drugs complexes in the intracellular translocation of TRPV5/6 compared to wild type cells. The aim of this study was to channels to the plasma membrane of differentiating osteo- investigate the features of functioning of dependent of blasts. To test this we used osteoblast-like cells stimulated PI3K and transcription factor NF-κB signaling pathways in for mineralization with 50 µg/ml ascorbic acid and 7.5 mM the Ruk/CIN85-overexpressing cell line MCF-7. β-glycerophosphate. Subcellular fractionation evidenced Methods: Activation of kinases mTOR, ERK1/2, Akt, the increased content of TRPV5/6, AnxA2 and S100A10 Src, IKK, inhibitor IκB and p65 subunit of transcription in plasma membranes of cells upon treatment with stimula- factor NF-κB in response to short-term stimulation of tors of mineralization. Then, a direct molecular interaction MCF-7 cells with overexpression Ruk/CIN85 epidermal of AnxA2 and TRPV5/6 channels mediated via S100A10 growth factor (EGF) was investigated by Western blot was confirmed by FRET confocal analysis. Obtained re- analysis. The activity of NF-κB additionally analyzed using sults were complementary to previous observation from the reporter luciferase system. our laboratory [3], that AnxA2-S100A10 complexes and Results: MCF-7 cells with overexpression Ruk/CIN85 TRPV5/6 channels are selectively translocated into ma- was found to be characterized with constitutive activation trix vesicles (centres of mineral formation) released from of signaling pathways PI3K/Akt/mTOR and NF-κB, but mineralizing Saos-2 cells. Here we demonstrate that knock- not ERK1/2. Using PP2, the nonreceptor kinase Src in- down of S100A10 in Saos-2 cells strongly diminishes the hibitor, we demonstrated Src-dependent phosphorylation distribution of TRPV5/6 channels to plasma membranes of kinase Akt. EGF stimulation of cells leads to increase in and has profound impact on mineralization. Herein, we the phosphorylation of kinases ERK1/2, Akt, mTOR, Src, postulate the role of AnxA2-S100A10 in the modulation IKK, inhibitor IκB and p65 subunit of NFκB transcription of TRPV5/6 function in osteoblast-like cells as a novel factor in Ruk/CIN85-overexpressing cells compared to regulatory mechanism in the mineralization of osteoblasts. control. Response of these cells to growth factor stimula- The identification of mechanisms contributing to the bone tion was developed earlier and lasted longer compared with formation represents a new strategy in screening for thera- wild type cells. It has been demonstrasted that the reporter peutic targets of osteoporosis. luciferase activity of NF-κB in wild type cells is significant- ly lower than in the subline with overexpression of Ruk/ References: CIN85. 1. Vanoevelen J et al (2011) FASEB J 25: 3197–3207. Conclusions: These data suggest that Ruk/CIN85 in- 2. Nijenhuis T et al (2008) J Bone Min Res 23: 1815–1824. volved in the regulation of signaling pathways PI3K/Akt/ 3. Cmoch A et al (2011) BBRC 412: 683–687. mTOR and NF-κB in cells of human breast adenocarci- Acknowledgements noma cell line MCF-7. The work was supported by grant 2012/05/N/NZ3/00330 from the Pol- ish National Science Centre.

Warsaw, September 9th–12th 2014 146 Session 7. Protein Functions in Cellular Signaling O7.6 Posters Changes in level, subcellular P7.1 localization and posttranslational modifications of CacyBP/SIP Comparison of protein disulfide phosphatase upon oxidative stress reducing agents – properties of Agnieszka M. Topolska-Woś1,2, Walter old and new reductants 2 1 J. Chazin , Anna Filipek Justyna Adamczyk1, Wojciech Bal2, Artur Krężel1 1 Nencki Institute of Experimental Biology, Department of Molecular 1 2 Laboratory of Chemical Biology, Faculty of Biotechnology, University and Cellular Neurobiology, Warsaw, Poland; Vanderbilt University, of Wrocław, Wrocław, Poland; 2Inistitute of Biochemistry and Biophysics, Center for Structural Biology, USA Polish Academy of Sciences, Warsaw, Poland e-mail: Agnieszka M. Topolska-Woś e-mail: Artur Krężel CacyBP/SIP binds several ligands such as S100 proteins, Siah-1, Skp1, actin, tubulin and tropomyosin (Schneider & Protection of thiol groups from oxidation is extremely im- Filipek, 2011). It has been suggested that, through the inter- portant for the maintaining the activity and structure of action with cytoskeletal proteins, CacyBP/SIP might play a many proteins. Approximately 20% of human proteins are role in various cellular processes, like cell differentiation or predicted to contain disulfide bonds between cysteine resi- cytoskeleton organization. Recently, it has been shown that dues. The most common reductant used in molecular biol- CacyBP/SIP exhibits phosphatase activity toward ERK1/2 ogy and biochemistry are β-mercaptoethanol (βME) and and down-regulates the MAP kinase cascade (Kilanczyk et ditihiotreitol (DTT), both posses unpleasant smell. These al., 2009). In this work we investigated the influence of agents are very basic and their usage is limited to basic pH. oxidative stress on the CacyBP/SIP phosphatase level, its Reduction of disulfides in neutral pH is rather slow and subcellular localization and posttranslational modifications usually requires their high excess over the protein. There of in neuroblastoma NB2a cells. is still a need for new and more efficient reductants. In By applying Western blot we have shown that the level of recently years TCEP (tris(2-carboxyethyl) phosphine) be- CacyBP/SIP increases in NB2a cells after H2O2 treatment. came very popular. TCEP is advantageous over DTT with Other methods, immunofluorescence staining and proxim- respect to the pH range of application. Its pKa value is ity ligation assay, show that in control NB2a cells CacyBP/ 7.5, while pKa value of βME and DTT (thiol) is 9-10 [1]. SIP is present in the cytoplasm and that dimer is the major TCEP compared to these two agents has also the advan- form of this protein. Moreover, we have found that upon tages of being odorless, a more powerful reducing agent, H2O2 treatment CacyBP/SIP translocates to the nucleus, more hydrophilic, and more resistant to oxidation in air [2]. where it exists mainly as a monomer. As it is shown by 2D In 2012 a new protein disulfide reducing agent appeared on electrophoresis, nuclear translocation of CacyBP/SIP is the market – DTBA (dithiobutylamine). DTBA is a dithiol accompanied by changes in posttranslational modifications reducing agent that overcomes basicity limitation of DTT. of this protein. Furthermore, after treatment of NB2a cells The presence of the primary amine group in DTBA, as with H2O2, the spots representing highly acidic forms of opposed to the two hydroxyl groups of DTT, leads to sig- CacyBP/SIP (pI 3-4) disappear in the cytoplasmic fraction, nificantly lower pKa values of thiols. Another advantage of which makes the pattern of the forms similar to that seen the primary amine group is that it allows for easy removal in the nuclear fraction. Interestingly, changes in posttrans- of DTBA from the system by ion-exchange methods [3]. lational modifications of CacyBP/SIP observed in NB2a Our study revealed that DTT and DTBA are a very efficient cells after oxidative stress are similar to those found in chelating agent for a range of biologically relevant metal brain of Alzheimer disease subjects (Wasik et al., 2013). As ions, while TCEP remains almost non-coordinating reduct- it is known, this pathology is characterized, among others, ant. Both reducing dithiols form complexes with transition by an increase in the amount of reactive oxygen species. metal ions (Zn(II), Co(II), Cd(II), Ni(II) and Cu(I)) with In conclusion, our results showing changes in CacyBP/ low solubility which significantly limit their usage in IMAC. SIP level, its subcellular localization and posttranslational We showed that application of DTT and DTBA to metal- modifications after oxidative stress suggest that the protein loproteins affect their function. Our main goal is to sum- follows to the nucleus its ligands/substrates, for example marize the knowledge of reducing agents properties (both ERK1/2, and that reactive oxygen species are involved in positives and drawbacks) regarding their usage in biochem- this process. istry and molecular biology.

Acknowledgements References: This work was supported by the European Union through the Europe- 1. Krężel A et al (2001) J Inorg Biochem 84: 77–88. an Regional Developmental Fund within the scope of the International 2. Krężel A et al (2003) Inorg Chem 42: 1994–2003. Ph.D. Studies in Neurobiology (MPD4-502) and by statutory funds from 3. Lukesh JL et al (2012) J Am Chem Soc 134: 4057–4059. the Nencki Institute of Experimental Biology. Acknowledgements This work was supported by the Ministry of Science and Higher Edu- cation (IP2012 018272). Instrumentation were supported by the Polish Foundation for Science (F1/2010/P/2013).

BIO 2014 Congress Session 7. Protein Functions in Cellular Signaling 147 P7.2 P7.3 Modulation of enzymes involved in A dramatic conformational synthesis of estrogens by methoxy change is involved in allosteric derivatives of resveratrol in non- regulation of muscle FBPase tumorigenic MCF10A breast epithelial cells Jakub Barciszewski1, Janusz Wiśniewski2, Dariusz Wanda Baer-Dubowska, Hanna Szaefer, Barbara Licznerska Rakus2, Andrzej Dżugaj2, Mariusz Jaskólski1,3 Poznan University of Medical Sciences, Department of Pharmaceutical 1Institute of Bioorganic Chemistry PAS, Center for Biocrystallographic Biochemistry, Poznań, Poland Research, Poznań, Poland; 2Wroclaw University, Department of Animal 3 e-mail: Wanda Baer-Dubowska Molecular Physiology, Wrocław, Poland; Adam Mickiewicz University, Faculty of Chemistry, Department of Crystallography, Poznań, Poland Resveratrol and some of its natural derivatives are con- e-mail: Jakub Barciszewski sidered as promising chemoprevetive and/or therapeu- tic agents interacting in pleotropic way with carcinogenic Until recently, it has been accepted that in vertebrate mus- process. Because resveratrol has several pharmacokinetic cles the glycolysis process dominates. Lactate, which is the and bioavailability limitations searching for its new syn- anaerobic product of glucose catabolism, is transferred to thetic analogues with higher anticancer activity, especially the liver where it is converted to glucose, which is finally re- in breast cancer, is reasonable. In postmenopausal women turned to the muscles. Out of the four regulatory enzymes when gonads stop to produced estrogens, active hormones that control glucose synthesis in human liver, only the are produced locally. Interference in the local production fructose-1,6-bisphosphatase isozyme (FBPase) is present and metabolism of estrogens, which are considered as ma- in muscles. Due to the absence of glucose-6-phosphatase jor breast cancer risk factor, seems to be a good alterna- in muscles, the synthesis of glucose is not possible there, tive for chemotherapy and/or chemoprotection of breast in contrast to glycogen synthesis. Interestingly, the activity carcinoma. of muscle FBPase differs from that of its liver counter- In this study we assessed the modulation of enzymes part, as it is 100 times more susceptible to AMP (adenosine involved in synthesis of estrogens: sulfatase (STS), sul- monophosphate) inhibition. FBPase is composed of four fotransferase (SULT1E1) and 17 beta-hydroxysteroid de- identical polypeptide chains (Mr 34,000Da/chain) and ex- hydrogenase 1 and 2 (17-beta HSD 1 and 2) by resveratrol ists as a dimer of dimers. The enzyme can assume at least and its three methoxy derivatives: 3,4,2’trimethoxy-trans- two distinct conformations, known as the R (relaxed) and stilbene (B), 3,4,2’,4’-tetramethoxy-trans-stilbene (D) and T (tense) states. Without effectors the enzyme exists in 3,4,2’,4’,6’-pentamethoxy-trans-stilbene (I) in breast non- the R state. AMP can induce the transition from the active tumorigenic epithelial cell line MCF10A. Cells were treated R-state to the inactive T-state. The allosteric site has been with the tested compounds for 72 hours and whole cell identified at the center of the tetrameric molecule where lysates were isolated. The level of enzymes was determined the four subunits converge. Recently, we have shown that by Western blot analysis. FBPase is active in muscles despite the presence of AMP. MCF10A cells showed low constitutive expression of 17- These findings offer a possibility to better understand the beta HSD 1, what is consistent with the literature data. mechanisms of activation and inhibition of this intriguing Methoxy derivatives and parent compound, resveratrol enzyme. In this work, we have crystallized human muscle were able to modulate the level of all enzymes involved FBPase with and without AMP. X-Ray diffraction data were in synthesis of estrogens, examined. The most significant collected and the two structures, of the R and T state, have changes were found in case of 17-beta HSD 2, which level been solved. Their analysis reveals a dramatic change of the was significantly increased as result of resveratrol or its conformation of the N-termini of all subunits upon AMP methoxy derivatives treatment. 17-beta HSD 2 is the domi- binding. Furthermore, these changes induce a rotation of nant form of this enzyme in normal epithelium of the 64° of the dimers with respect to each other. breast, and allows the epithelial cells to balance the amount of estrone against estradiol. Thus 17-beta HSD 2 induction by resveratrol and its methoxy derivatives may be consid- ered as important mechanism of breast cancer chemopre- vention by these compounds.

Warsaw, September 9th–12th 2014 148 Session 7. Protein Functions in Cellular Signaling P7.4 P7.5 Identification of palmitoylated Structural and functional properties of proteins involved in TLR4 signaling proteins involved in miRNA-mediated Justyna Dembińska, Paula Roszczenko, Kinga gene silencing revealed by hydrogen/ Borzęcka, Katarzyna Kwiatkowska deuterium exchange mass spectrometry 1 2 Nencki Institute of Experimental Biology, Warsaw, Poland Maja K. Cieplak , Krzysztof Tarnowski , Michal 2 3 4 e-mail: Katarzyna Kwiatkowska Dadlez , Marc R. Fabian , Nahum Sonenberg , Edward Darzynkiewicz1, Anna Niedzwiecka1,5 Palmitoylation is a post-translation modification of pro- teins via a covalently bound palmitic acid (C:16) which is 1Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Warsaw, Poland; 2 Institute of catalyzed by palmitoyl acyltransferase. In eukaryotic cells, Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, the acyl chain is transferred onto thiol group of cysteine Poland; 3 Department of Oncology, Lady Davis Institute for Medical residues (S-palmitoylation) or, less frequently, onto hydrox- Research, McGill University, Canada; 4 Department of Biochemistry yl group of serine and threonine residues (O-palmitoyla- and McGill Cancer Centre, McGill University, Canada; 5 Laboratory of Biological Physics, Institute of Physics, Polish Academy of Sciences, tion). This reversible modification controls translocation Warsaw, Poland of proteins to plasma membrane microdomains enriched e-mail: Anna Niedzwiecka in cholesterol and sphingolipids (rafts), their stability, cel- lular trafficking of proteins and protein-protein interac- miRNAs specifically regulate the expression of genes by a tions. We found that protein palmitoylation is crucial for network of protein-protein interactions. As they work on pro-inflammatory signaling triggered by Toll-like receptor the post-transcriptional level, they provide a much faster 4 (TLR4) in cells stimulated with lipopolysaccharide (LPS). way to react to changes in the cellular environment and can An exposure of RAW264 macrophage-like cells to 50-250 propagate the received signal by affecting multiple mRNAs µM of 2-bromopalmitic acid (BPA), an inhibitor of pal- containing the same miRNA-binding site. mitoyl acyltransferase, reduced in a dose dependent man- miRNAs interact with Argonaute proteins, which in turn ner the production of pro-inflammatory cytokines, TNF-α bind GW182 proteins (Glycine-Tryptophan proteins of and RANTES, stimulated with 100 ng/ml LPS. Therefore, 182 kDa). Artificial tethering of GW182 proteins to target we aimed to identify proteins which are palmitoylated in mRNAs has shown that GW182 proteins act downstream LPS-stimulated RAW264 cells. Traditional methods to of Argonaute proteins and are able to silence mRNAs in study protein S-palmitoylation rely on isotopic labeling of their absence. GW182 proteins act by recruiting the CCR4- cells, which requires special safety precautions and is time- NOT deadenylase complex via interactions with the scaf- consuming. We have applied a recently developed non- folding CNOT1 subunit [1, 2] of the complex. radioactive technique based on a “click chemistry” in two The GW182 proteins are predicted to be mostly disor- parallel approaches. In the first one, living RAW264 cells dered, excluding the Ubiquitin Binding Domain and the were labeled with ω-alkynyl-palmitate analog of palmitic acid RNA Recognition Motif (RRM). The NMR structure of (17-ODYA), and after lysis, subjected to a “click-it” reac- the RRM of the Drosophila melanogaster GW182 protein was tion with azido-azo-biotin catalyzed by Ca2+. Subsequently, published by Eulalio et al. in 2009 [3]. labeled proteins were bound to streptavidin beads and elut- Here, we study the structure and CNOT1 binding prop- ed with sodium dithionite which cleaved the azido-azo-bi- erties of the silencing domain of TNRC6C, one of the otin, and analyzed by immunoblotting. With this technique human paralogs of GW182, using hydrogen/deuterium it was found that LPS induced palmitoylation of several exchange (HDX) coupled with ion mobility mass spec- proteins, including Lyn tyrosine kinase of the Src family. trometry (IMS). HDX-IMS is a powerful method to study Palmitoylation of these proteins was inhibited in the pres- solvent accessibility of particular protein regions, which in ence of BPA. The second approach of detection of palmi- turn depends on the structural properties of a protein. toylated protein in LPS-stimulated cells was based on the fact that the activation of TLR4 by LPS occurs via CD14 References: located in plasma membrane rafts. Therefore, we decided 1. Fabian MR et al (2011) Nat Struct Mol Biol 18: 1211–1217. to perform immunoprecipitation of CD14, preceded by la- 2. Braun JE, Huntzinger E, Fauser M, Izaurralde E (2011) Mol Cell 44: belling of RAW264 cells with 17-ODYA and followed by 120–133. “click chemistry” in order to detect palmitoylated proteins 3. Eulalio, A. et al. (2009) Nucleic Acids Res 37: 2974–2983. which associate with CD14 in LPS-treated cells. Identifica- Acknowledgements tion of these proteins will involve the mass spectrometry This work was supported by the European Regional Development Fund methodology. through the Foundation for Polish Science International PhD Projects Programme and the Innovative Economy Operational Programme POIG.02.02.00-00-025/09 NanoFun.

BIO 2014 Congress Session 7. Protein Functions in Cellular Signaling 149 P7.6 P7.7 GPER/GPR30 estrogen receptor Identification of palmitoylated dysregulation in neoplastic transformation proteins involved in TLR4 signaling Adam I. Cygankiewicz1, Damian Jacenik1, Andrzej Justyna Dembińska, Paula Roszczenko, Kinga Semczuk2, Tomasz Rechberger2, Radzisław Borzęcka, Katarzyna Kwiatkowska 3 3 1 Trzciński , Adam Dziki , Wanda M. Krajewska Nencki Institute of Experimental Biology, Warsaw, Poland 1Department of Cytobiochemistry, Faculty of Biology and e-mail: Katarzyna Kwiatkowska Environmental Protection, University of Lodz, Łódź, Poland; 2IInd Chair and Department of Gynecology, Medical University of Lublin, Lublin, Palmitoylation is a post-translation modification of pro- Poland; 3Department of General and Colorectal Surgery, Medical teins via a covalently bound palmitic acid (C:16) which is University of Lodz, Łódź, Poland catalyzed by palmitoyl acyltransferase. In eukaryotic cells, e-mail: Adam Cygankiewicz the acyl chain is transferred onto thiol group of cysteine residues (S-palmitoylation) or, less frequently, onto hydrox- Estrogens are sex hormones, which play central role in yl group of serine and threonine residues (O-palmitoyla- modulation of both physiological as well as pathological tion). This reversible modification controls translocation processes through the human body in both genders. The of proteins to plasma membrane microdomains enriched molecular mechanism of estrogen action on cell has been in cholesterol and sphingolipids (rafts), their stability, cel- slowly deciphered in previous decades. However, classical lular trafficking of proteins and protein-protein interac- signaling pathway does not explain rapid changes in the tions. We found that protein palmitoylation is crucial for metabolism of the cells after exposure to estrogens. In pro-inflammatory signaling triggered by Toll-like receptor 2005, it was demonstrated that estrogens are natural ligands 4 (TLR4) in cells stimulated with lipopolysaccharide (LPS). not only for the canonical receptors ERα and ERβ, but also An exposure of RAW264 macrophage-like cells to 50-250 for GPER/GPR30 receptor belonging to the family of G µM of 2-bromopalmitic acid (BPA), an inhibitor of pal- protein coupled receptors (GPCRs). mitoyl acyltransferase, reduced in a dose dependent man- GPER/GPR30 was found to transactivate EGFR recep- ner the production of pro-inflammatory cytokines, TNF-α tor and as a result, ERK 1/2 and JNK of the MAP kinase and RANTES, stimulated with 100 ng/ml LPS. Therefore, pathway. Two genes i.e., c-Fos and CTGF were identified as we aimed to identify proteins which are palmitoylated in being regulated by GPER/GPR30. Initial reports have de- LPS-stimulated RAW264 cells. Traditional methods to scribed GPER/GPR30 role in neoplastic transformation, study protein S-palmitoylation rely on isotopic labeling of blood pressure, bone metabolism and glucose homeostasis. cells, which requires special safety precautions and is time- The importance of GPER/GPR30 has been established consuming. We have applied a recently developed non- after publications described that tamoxifen and other clas- radioactive technique based on a “click chemistry” in two sical ER inhibitors are agonists for GPR30. Additionally, parallel approaches. In the first one, living RAW264 cells estrogen-like compounds like phyto- and xenoestrogens were labeled with ω-alkynyl-palmitateanalog of palmitic acid appeared to be the activators of GPER/GPR30, which (17-ODYA), and after lysis, subjected to a “click-it” reac- make this receptor a key factor in cellular metabolism mod- tion with azido-azo-biotin catalyzed by Ca2+. Subsequently, ulation by environmental factors. labeled proteins were bound to streptavidin beads and elut- The aim of the work was to assess the expression of ed with sodium dithionite which cleaved the azido-azo-bi- GPER/GPR30 as compared to classical estrogen ERα and otin, and analyzed by immunoblotting. With this technique ERβ as well as EGFR receptors in endometrial and colo- it was found that LPS induced palmitoylation of several rectal cancers of different degree of neoplasia and to find proteins, including Lyn tyrosine kinase of the Src family. out if methylation of promoter regions has impact on the Palmitoylation of these proteins was inhibited in the pres- GPER/GPR30 expression. ence of BPA. The second approach of detection of palmi- In endometrial cancer as compared to normal endome- toylated protein in LPS-stimulated cells was based on the trium up-regulation of not only canonical estrogen recep- fact that the activation of TLR4 by LPS occurs via CD14 tors ERα and ERβ, but also GPER/GPR30 receptor was located in plasma membrane rafts. Therefore, we decided shown. It was also noticed that GPER/GPR30 expression to perform immunoprecipitation of CD14, preceded by la- increase is associated with tumor grade. Interestingly, simi- belling of RAW264 cells with 17-ODYA and followed by lar results, although to lesser extent were noticed in the case “click chemistry” in order to detect palmitoylated proteins of colorectal cancer. which associate with CD14 in LPS-treated cells. Identifica- Lower level of methylation of two CpG islands in the tion of these proteins will involve the mass spectrometry promoter region of the GPER/GPR30 receptor gene methodology. in endometrial cancer comparing to normal material was found. In the case of colorectal cancer, only one of two identified CPG islands was found to be hypomethylated. It suggests that one of the mechanisms responsible for the up-regulation of GPR30 receptor expression during neo- plastic transformation may be the decrease of methylation of CpG islands in its promoter region.

Warsaw, September 9th–12th 2014 150 Session 7. Protein Functions in Cellular Signaling P7.8 P7.9 Hansenula polymorpha glucose sensor Gcr1 Catabolite degradation of fructose-1,6- functions as a putative bisphosphatase in methylotrophic yeasts + pH-dependent glucose H -symporter Olena Dmytruk1, Nina Bulbotka1, Andriy Sibirny1,2 1, 2 1, 2 Iryna O. Denega , Olena G. Stasyk , 1Institute of Cell Biology, National Academy of Science of Ukraine, Lviv, Natalia O. Sybirna1, Oleh V. Stasyk2 Ukraine; 2Department of Biotechnology and Microbiology, University of 1 Rzeszow, Rzeszów, Poland Ivan Franko National University of Lviv, Department of Biochemistry, e-mail: Olena Dmytruk Ukraine; 2Institute of Cell Biology NAS of Ukraine, Department of Cell Signaling, Ukraine e-mail: Iryna Denega Fructose-1,6-bisphosphatase (FBP) is a key gluconeogenic enzyme, it catalyses the conversion of fructose-1,6-bispho- Several proteins involved in glucose sensing, signaling and sphate to fructose-6-phosphate, which later metabolizes transport have been described for methylotrophic yeast to glucose-6-phosphate. FBP is synthesized when cells are Hansenula polymorpha. One of them is Gcr1, the close hom- grown on non-fermentable carbon sources like ethanol. ologue of MstA, a functional high-affinity H+-dependent When yeast cells are subsequently shifted to a glucose-con- monosaccharide symporter from fungus Aspergillus niger. taining medium, FBP is rapidly inactivated and degraded. Gcr1 is a peculiar C-tale-less glucose sensor, required for This process is called catabolite degradation. glucose repression and regulation of hexose transport in In the methylotrophic yeasts, such as Pichia pastoris and H. polymorpha. Hansenula polymorpha, FBP also participates in methanol uti- It was previously demonstrated with bakers’ yeast ScSnf3 lization pathway. Therefore the investigation of catabolite and ScRgt2 glucose non-transporting sensors that substitu- degradation of FBP in methylotrophic yeastshas a great tion of one of the conserved arginine residues by a lysine scientific interest. residue in the hexose sensor’s core sequence converts them The wild type strains of P. pastoris and H. polymorpha were into constitutively signalling form. We demonstrated that analyzed by Western blot analysis. It was shown that FBP expression of the GCR1 allele encoding protein with the is mostly degraded after 5 hours of incubation of cells pre- corresponding R165K substitution in H. polymorpha gcr1∆ grown in gluconeogenic substrates in glucose-containing mutant cells did not complement their growth defect on medium. After shifting the cells from methanol or etha- glucose, but additionally impaired growth on fructose-, su- nol-containing media on glucose medium, FBP activities crose-, xylose-, ethanol- and methanol-containing media of P. pastoris and H. polymorpha wild-type strains decreased relative to gcr1∆ recipient strain. Remarkably, production of 4.5 and 2.5 times, respectively. FBP degradation of the Gcr1R165K protein also did not restore the normal catabo- P. pastoris mutants defective in autophagy (∆mon1, ∆ypt7, lite repression of AOX synthesis in cells grown on glucose. ∆ccz1) was also investigated and compared with the wild We analyzed the growth of the strains with overexpression type strain. of GCR1 and HXT1 on different carbon substrates un- The recombinant strains of P. pastoris with the deletion of der presence of glucose antimetabolite 2-deoxy-D-glucose FBP1 gene were constructed and analyzed. Δfbp strains (2-DOG) at different pH values. Strain NCYC 495/GAP- did not grow in the medium with non-fermentable car- GCR1 with constitutive expression of GCR1 was more bon sources, such as methanol and ethanol, and revealed sensitive to the exogenous 2-DOG relative to the control impaired growth in glycerol-containing medium. To study strains. Such sensitivity was pH dependent, since we ob- the mechanisms of catabolite degradation, recombinant served the difference in growth of the strains at pH 3.8, strains with the expression of FB1P fused with GFP and while at pH 4.0 all strains were equally sensitive to 2-DOG FBP1 fused with Myc were constructed and analyzed. It was in glycerol-containing medium. Sensitivity of all investi- shown that two different ways of FBP degradation can oc- gated strains to 2-DOG on pH 4.0 could depend on ex- cur in P. pastoris, but ubiquitin-proteasome dependent deg- pression of other genes encoding high-affinity glucose radation pathway is more preferred. transporters. Therefore, glucose sensor Gcr1 can most probably perform the H+-symport function in its native host, since 2-DOG sensitivity was dependent on pH (H+ concentrartion). To our knowledge, if confirmed, this is a first example of functional glucose-transporting hexose sensor in yeasts.

BIO 2014 Congress Session 7. Protein Functions in Cellular Signaling 151 P7.10 P7.11 Changes in mRNA and protein Diversity of human metallothionein expression levels of key components isoforms — new light on protein of Renin-Angiotensin System and stability and cellular functions Relaxin Family Peptide System under the influence of Angiotensin II and Agnieszka Drozd, Artur Krężel Relaxin 2 in prostate cancer cells University of Wrocław, Laboratory of Chemical Biology, Wrocław, Poland e-mail: Artur Krężel Kamila Domińska, Agnieszka W. Piastowska-Ciesielska, Karolina Kowalska, Zuzanna E. Matysiak, Tomasz Ochędalski Human metallothioneins are encoded by 17 genes located Department of Comparative Endocrinology, Medical University of Lodz, on chromosome 16q13. They consist of 61–68 amino acid Łódź, Poland residues, with 25–30% cysteine content. MTs exist in four e-mail: Kamila Domińska isoforms: MT1, MT2, MT3 and MT4, whereof the MT1 is present in seven sub-isoforms: MT1A, B, E, F, G, H, X. Several groups, including ours, have demonstrated that An- Differences between isoforms arise mainly from changes in giotensin II and Relaxin 2 play a important role in pros- the amino acid sequence, despite the sequence homology tate carcinogenesis.It has been shown that these peptides reaches ca. 90%. MT1 and MT2 are present in every tissue can modulate cell proliferation and apoptosis, invasion and type but MT3 and MT4 are found only in nervous system spread of many types of cells. High expression of Relaxin and squamous epithelium, respectively. Metallothioneins 2 and relaxin receptor RXFP1 as well as Angiotensin II and are involved in numerous cellular processes, they are im- angiotensin receptor type 1 (AT1) was observed in tumor portant regulators of metal ion homeostasis and protectors tissue compared to normal prostate tissue. Studies suggest against oxidative damage [1]. Their altered mRNA expres- that receptors expression: RXFP1, AT1 and also these pep- sion has been correlated with variety of cancers [2]. The tide hormones levels in blood can be new early markers of understanding of metallothionein functions is hampered metastatic potential in primary tumors. It is worth noting due to its high diversity. that interactions between renin-angiotensin system (RAS) Our previous studies on MT2A showed that metallothio- and therelaxin family peptide systems (RFPS) have been nein differs in Zn(II) affinity (10-8–10-12 M) and demon- reported in the organ fibrosis, the cardiovascular system strated high coordination dynamic of metal clusters [3]. So associated with blood-pressure and central nervous system far, not all metallothionein isoforms were characterized in associated with osmoregulation but so far, never in the car- terms of metal ion binding and their thermodynamic sta- cinogenesis of reproductive tissues. In the present study, bility. Here, we present the procedure of expression and we investigated the effects of Angiotensin II on some ele- purification of ten metallothionein isoforms MT1-MT4 ments of Relaxin Family Peptide System, such as RXFP1 with special attention to their apo- and holo-forms prepa- and RXFP2. Furthermore we examined the influence of ration. All purified apo-isoforms were characterized in Relaxin 2 on key components of Renin-Angiotensin Sys- terms of Zn(II) and Cd(II) binding using UV-Vis and CD tem for instance AT1, AT2, ACE. Changes in mRNA and spectroscopy. Zn(II)-saturated proteins were analyzed in protein expression levels were measured by Real-time RT- detail in terms of the number of metal ions bound per pro- PCR and Western blot, respectively. The fluorescent im- tein molecule using UV-Vis spectroscopy, fluorimetry and munocytochemical localization of angiotensin and relaxin ICP-MS. We found that all metallothionein isoforms bind receptors in prostate cancer cells was achieved using Alexa seven Zn(II) ions per molecule (Zn7T) in a tetrathiolate Fluor. The changes in local RA and RFP systems were ex- coordination environment. Our studies uncovered that all amined in two prostate cancer cell lines (LNCaP and PC3) metallothionein isoforms demonstrate similar Zn(II) affini- which are characterized by different invasive potential, as ties compared to MT2A, varying from nanomolar to low well as sensitivity to androgen. It seems that Rel-2 may picomolar range. This unusual properties of metallothio- influence on function and efficiency of renin-angiotensin neins are responsible for controlling of free Zn(II) ions system, while Ang II may have an impact on relaxin family under cellular conditions. peptide systems in prostate cancer cells. The results suggest that deregulation of the locally secreted peptide hormones, References: such as Rel-2 or Ang II can increase the risk of prostate 1. Krężel A et al (2007) Arch Biochem Biophys 463: 188–200. cancer development and progression. 2. Mehus AA et al (2014) Mol Cell Proteomics 4: 1020–1033. 3. Krężel A, Maret W (2007) JACS 129: 10911–10921. Acknowledgements Acknowledgements This workwas financially supported by the National Science Center, re- This work was supported by the Ministry of Science and Higher Educa- search grant: NN403 2081 39. tion (IP2012 018272). Instrumentation used in this study was founded by the Polish Foundation for Science (F1/2010/P/2013).

Warsaw, September 9th–12th 2014 152 Session 7. Protein Functions in Cellular Signaling P7.12 P7.13 Ribosomal L12 protein facilitates The role of the LD loop in activation maturation of ribosomal 60S process of the HtrA (DegP) subunit in yeast cells protease from Escherichia coli Leszek Wawiórka1, Barbara Michalec-Wawiórka1, Donata Figaj1, Artur Giełdoń2, Marlena Bartczak1, Jolanta Eliza Molestak1, Kamil Deryło 1, Monika Szajwaj1, Jurek1, Barbara Lipińska1, Joanna Skórko-Glonek1 1 1 Monika Dźwierzyńska , Przemysław Grela , Vladislava 1 2 2 1 Department of Biochemistry, University of Gdańsk, Gdańsk, Poland; Liudkowska , Joanna Kufel , Marek Tchorzewski 2Faculty of Chemistry, University of Gdańsk, Gdańsk, Poland 1Department of Molecular Biology, Maria Curie-Sklodowska University, e-mail: Donata Figaj Lublin, Poland; 2Institute of Genetics and Biotechnology, Faculty of Biology, Warsaw University, Warsaw, Poland Exposition of bacteria to stress agents frequently leads to e-mail: Marek Tchorzewski intracellular protein misfolding and aggregation. Aggre- gated proteins cannot fulfil their biological function and Eukaryotic Ribosomal T-site, especially GTPase Associated seriously endanger cell’s safety. The occurrence of the im- Centre (GAC), is one of the most intriguing part of trans- properly folded and aggregated proteins leads to excessive lational apparatus. This region is responsible for binding expression of genes involved in cell protection against de- and stimulation of the translational factors (TF’s) at every structive effect of stressful agents. One of them is htrA step of the protein synthesis cycle. Molecular activity of gene, whose product, the HtrA protein, plays a crucial role GAC is based on the orchestrated interplay between rRNA in the extracytoplasmic protein quality control. fragments (highly conserved sarcin-ricin loop) and several HtrA (DegP) protein form Escherichia coli isaconserved in protein components including heteropentameric P-stalk evolution serine protease, localized in the periplasm. Syn- structure consisting of three types of P-proteins uL10/ thesis of HtrA is increased under heat, oxidative or reduc- P1/P2 and single uL11 protein. The GAC involvement in ing stress. HtrA plays a dual role in protection of a bac- translational cycle has been extensively studied, however, terial cell- it prevents protein aggregation and degrades its direct involvement in other cellular processes extending incorrectly folded proteins, due to its chaperone and pro- beyond its canonical functions was also postulated. As it teolytic activity, respectively. Proteolytic activity is regulated was reported P-proteins, especially uL10, exhibit addition- in thermal and/or allosteric manner. Under physiological al, so called extraribosomal functions in respect to various conditions HtrA protein exists as a proteolytically inactive metabolic pathways. In this communication we show that hexamer. During cell exposure to stress agents, when in- uL11 activity also goes beyond translation per se. We show correctly folded proteins occurr in the periplasmic space, that uL11 protein is an active element contributing to the PDZ-1 domain of HtrA recognizes and binds the specific ribosomal biogenesis process in yeast cells. Mutant yeast amino acid sequence of a substrate. Binding of a peptide cells, totally depleted of both genomic copies of genes for to the PDZ1 domain leads to conformational changes uL11 protein, exhibit extremely slow growth phenotype, within this domain, which are sensed by the L3 loop. Al- accompanied with the severe defect in ribosomal forma- losteric signal is passed on the LD loop and next on the tion and pre-rRNA processing. The GAC was shown to L2/L1 loops. As a result the protease active center achieves be a docking platform for the translational GTPases (e.g. proper conformation. At the same time hexamer dissoci- eEF-2), so it is tempting to assume that this ribosomal ates into trimers, which can form a proteolytically active region may also interact with the biogenesis factor Efl1, higher order oligomers. The active HtrA ensures proper which shares high degree of homology with translation bacterial cell protection against stress conditions. Moreo- factor eEF2. Like other translational GTPases, Efl1’s activ- ver, in the pathogenic E. coli strains (UPEC, EPEC) HtrA ity is strictly dependent on ribosome. The most prominent is implicated in virulence and the htrA mutant strains are molecular function of Efl1 refers to Tif6 antiassociation non-pathogenic. factor release at the very late cytoplasmatic step of pre- The aim of our work was to examine the role of the LD 60S maturation. Consequently, lack of Efl1 causes accumu- loop in the activation process of HtrA. According to the lation of Tif6 protein on the pre-60S particles, observed crystal structure (pdb 3ou0) we selected amino residues as its re-localization from the nucleus into the cytoplasm. which seem to play an important role in maintenance of the The similar phenotype was observed in the uL11 depleted HtrA proteolytic activity. For this purpose we introduced a yeast cells which strongly suggest severe perturbations in set of point mutations in the region coding for the LD loop the cross-talk between ribosome and Efl1. Noteworthy, our to obtain the following amino acid substitutions: P170G, data suggest that the function of uL11 seems not to be rig- F171A, L173A, E175A, E175L, G174S. Almost all of the orously limited to the Efl1 stimulation. Instead, uL11 may introduced mutations disturbed the activity in comparison exert more broad effect, affecting early steps of ribosome to wild type HtrA. Obtained data confirm the importance biogenesis. Our results extend current understanding of of LD loop in the HtrA protease activation. In particular, the molecular basis of the eukaryotic ribosome maturation an LD shape (role of P170 residue) and interactions with process, bringing new insight into molecular functions of the L2 loop (role of F171, L173 residues), or the L3 loop the uL11 protein. (E175 residue) play crucial roles in the maintenance of the proteolytic actity.

BIO 2014 Congress Session 7. Protein Functions in Cellular Signaling 153 P7.14 P7.15 The lack of Nrf2 transcription factor Role of Amyloid Precursor protein and heme oxygenase-1 in murine and its processing in the regulation of bone marrow macrophages affects cellular Store Operated Calcium Entry osteoclasts differentiation Kinga Gazda1, Jacek Kuźnicki1,2, Tomasz Węgierski1 Urszula Florczyk, Monika Viscardi, Witold 1International Institute of Molecular and Cell Biology, Laboratory of Nowak, Alicja Józkowicz, Józef Dulak Neurodegeneration, Warsaw, Poland; 2Nencki Institute of Experimental Biology, Warsaw, Poland Department of Medical Biotechnology, Faculty of Biochemistry, e-mail: Kinga Gazda Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland e-mail: Urszula Florczyk Alzheimer’s disease (AD) is the most common form of de- Increased number and activity of osteoclasts (OCLs) are mentia. It is characterized by the extraneuronal deposition associated with intense bone demineralization in osteo- of amyloid–β–peptide (Aβ) plaques, products of amyloid porosis, many types of tumors or inflammatory disorders precursor protein (APP) proteolysis by β–secretase and such as arthritis. Nrf2 transcription factor and heme oxyge- γ–secretase. Although the β–amyloid-induced toxicity re- nase-1 (HO-1), its downstream target, have been suggested mains the prevailing hypothesis of AD pathogenesis, the to play a role in osteoclastogenesis. Nevertheless, the stud- research based on it has neither clarified the disease - eti ies investigating the effect of genetic manipulation of those ology nor brought the long-awaited cure. Altered calcium mediators are lacking. Therefore, we aimed to investigate homeostasis has been recently proposed as one of the early the effect of Nrf2 and HO-1 deficiency in murine bone events responsible for disease development, but its exact marrow macrophages (BMMs) on OCL differentiation. effect is poorly understood. Endoplasmic reticulum (ER) is Bone marrow cells were isolated from tibial and femoral the major store of intracellular Ca2+. The regulated release bones of Nrf2- or HO-1-deficient mice (Nrf2-/- or HO-1-/-, of calcium from the ER causes activation of ER-localized respectively) and appropriate wild type mice and stimulated calcium sensors STIM1/STIM2 and the subsequent influx for 3 days with macrophage colony-stimulating factor (M- of calcium through Orai1 plasma membrane channel. This CSF) to obtain BMMs. Analysis of surface markers expres- Store Operated Calcium Entry (SOCE) is crucial for ER- sion (F4/80, Mac1) confirmed macrophage phenotype of refilling and cell signaling. It has been well demonstrated cells irrespective of the genotype. For OCL differentiation that familial AD mutations in presenilin-1 (PS1), the enzy- BMMs were cultured further for 3 days in the presence matic component of γ–secretase complex, and APP affect of M-CSF and receptor activator of nuclear factor kB li- intracellular calcium concentration and dynamics, including gand (RANKL). Mature OCLs were identified at day 6 by SOCE. Several mechanisms have been proposed to explain tartrate-resistant acid phosphatase (TRAP) histochemical the observed effects of PS1 mutations. However, the role detection. of APP and its FAD-causing variants in calcium homeosta- We showed that Nrf2 deficiency resulted in higher number sis is controversial. At present, there is no clear and com- of OCLs. Reversely, after Nrf2 activation by sulforaphane pact explanation how APP influences cellular calcium. in RANKL-stimulated Nrf2+/+ BMMs no TRAP-positive The purpose of this study is to analyze how disturbances cells corresponding to OCLs were observed, confirming of APP processing pathway affect SOCE response. We inhibitory effect of Nrf2. In addition, osteoclasts-specif- characterized a human cell line expressing high endoge- ic genes, such as Nfat-c1, Calcr, Ctsk, Itgb3, tended to nous levels of APP, α- and γ–secretase components as well be upregulated in RANKL-stimulated Nrf2-/- BMMs in as proteins constituting SOCE machinery. We have stably comparison to Nrf2+/+ BMMs. In contrast, less TRAP+ silenced the expression of APP, PS1 and ADAM genes cells were detected when HO-1 was absent. Accordingly, by viral gene transfer of specific shRNA cassettes. We aim other osteoclasts-specific genes were all downregulated in to perform quantitative co-localization analysis to charac- RANKL-stimulated HO-1-/- BMMs (vs. HO-1+/+ BMMs). terize dynamic interaction between endogenous Orai1 and In summary, Nrf2 exerts inhibitory effect on osteoclas- STIM1/STIM2 proteins, and to measure the SOCE inten- togenesis, while in our experimental setting HO-1 has op- sity in cells with impaired APP processing pathway. The posite effect. It cannot be excluded that in case of the lat- results will give insight into mechanisms by which develop- ter the differences may come from different properties of ment of AD is connected to cellular calcium disturbances BMMs HO-1+/+ and HO-1-/-, however, such hypothesis and may help to elucidate the effective disease treatment. requires further investigation.

Acknowledgements Supported by Iuventus Plus grant from the Ministry of Science and High- er Education (0244/IP1/2013/72).

Warsaw, September 9th–12th 2014 154 Session 7. Protein Functions in Cellular Signaling P7.16 P7.17 Searching for the function of human Special features of B-lymphoblastoid Sgt1A and Sgt1B isoforms cell lines from patients with X-linked Agnieszka Góral1, Małgorzata Sierant2, lymphoproliferative disease type 1 Barbara Nawrot2, Anna Filipek1 Inna Gordiienko, Larysa Shlapatska, Larysa 1Nencki Institute of Experimental Biology PAS, Warsaw, Poland; 2Centre Kovalevska, Svetlana Sidorenko of Molecular and Macromolecular Studies PAS, Łódź, Poland R.E. Kavetsky Institute of Experimental Pathology, Oncology and e-mail: Agnieszka Góral Radiobiology NAS of Ukraine, Laboratory of Signal Transduction Pathways, Ukraine The Sgt1 protein, originally identified in yeast as a sup- e-mail: Inna Gordiienko pressor of G2 allele of Skp1, conservatively functions throughout eukaryotes as a Hsp90 co-chaperone in the X-linked lymphoproliferative disease type 1 (XLP1) be- kinetochore assembly and proper chromosome alignment longs to genetically determined primary immunodeficiency (Kitagawa et al., 1999; Davies & Kaplan, 2010). Moreover, syndromes and is characterized by mutations in SH2D1A/ in human cells, Sgt1 is required for activation of some NLR DSHP/SAP gene. SH2D1A gene encodes a small 128 aa proteins during cell response to pathogen attack (da Silva adaptor protein SH2 domain protein 1A (SH2D1A), also Correia et al., 2007; Mayor et al., 2007). In humans, con- called Duncan's disease SH2-protein (DSHP), or SLAM/ trary to the majority of organisms, two isoforms, Sgt1A CD150-associated protein (SAP). SAP is involved in regu- (37.8 kDa) and Sgt1B (41.0 kDa), are expressed (Niikura lation of development and functions of T lymphocytes, and Kitagawa, 2003; Zou et al., 2004). The Sgt1B isoform NKT and NK cells. A number of evidence support the idea contains a 33-amino acid insertion (Ile110-Gly142) instead of that SAP is also involved in B cell differentiation and func- the Ser110 residue characteristic for Sgt1A sequence. It has tion, so the absence of functional SAP in XLP1 may affect been shown that RNAi-mediated silencing of both Sgt1 the B cells as well, and contribute to the dramatic increase isoforms leads to a mitotic delay and eventually cell death of B-cell lymphoma development in XLP1 patients. The (Steensgaard et al., 2004). aim of our study was to analyze receptor-mediated Akt and The aim of this work is to establish the role of particular ERK1/2 activation, and expression of several transcription Sgt1 isoforms in human cells, and we mainly focus on anal- factors in EBV-transformed B-lymphoblastoid cell lines ysis of cell proliferation and on identification of protein from XLP1 patients (XLP B-LCLs) in comparison with ligands specific for Sgt1A and Sgt1B. In order to find out conventional B-lymphoblastoid cell lines (B-LCLs). Studies whether both isoforms are essential for cell cycle progres- were performed on EBV-transformed XLP B-LCLs IARC sion, we decided to silence separately each Sgt1 isoform. 739, SC-XLP and RP-XLP; SAP- B-LCLs T5-1 and SAP+ For that, siRNA molecules were designed and synthetized B-LCLs MP-1. Western blot analysis was used for evalua- and then used to transfect HEp-2 (Human Epidermal Car- tion of Akt and ERK1/2 phosphorylation in response to cinoma) cells. At different time points after transfection, ligation of CD150, CD40, and IgM cell surface receptors. the protein level of individual Sgt1 isoforms was analyzed The expression levels of transcription factors IRF4, IRF8, by Western blot. The most effective siRNA sequences were BCL6, BLIMP1, SPIB, PU.1, and MITF were assessed us- selected and cloned into pPURhU6 vectors in order to es- ing quantitative RT-PCR. It was shown that SAP deficiency tablish stably transfected cell lines with diminished level of in XLP B-LCL did not abrogate CD150-mediated Akt and a given Sgt1 isoform. In turn, to identify protein targets ERK1/2 phosphorylation. CD150 crosslinking on XLP specific for Sgt1A and Sgt1B, HEp-2 cells were transfected B-LCL IARC 739 led to two waves of ERK1/2 phospho- with plasmid encoding Sgt1A-3xFLAG or Sgt1B-3xFLAG. rylation and induced rapid and sustained Akt phosphoryla- Then, co-immunoprecipitation on anti-FLAG agarose was tion. IgM ligation on XLP B-LCL IARC 739 mediated very performed and proteins bound to this resin were analyzed rapid, but transient ERK1/2 activation. However, IgM and by mass spectrometry. The analysis of the cell cycle of CD150 coligation resulted in rapid intense and sustained HEp-2 cells with diminished level of a given Sgt1 isoform ERK1/2 phosphorylation. Kinetics of pAkt after CD150 as well as the analysis of the results from mass spectrom- and IgM coligation remained on the level of CD150 alone. etry is in progress and will be presented. Thus, in XLP B-LCL the CD150 signaling with IgM coliga- tion play the dominant role in both Akt and ERK1/2 phos- Acknowledgements phorylation. SAP+ and SAP– conventional B-LCLs have This work was supported by statutory funds from the Nencki Institute of differences in kinetics and amplitude of receptor-initiated Experimental Biology, Warsaw and from Centre of Molecular and Macro- Akt phosphorylation. We found that significantly reduced molecular Studies, Lodz. IRF4, IRF8 and PU.1 expression levels are the key features of XLP B-LCLs. Conventional SAP+ B-LCL MP-1 dem- onstrated elevated expression level of MITF, while SPIB was practically undetectable in this cell line. On the other hand, SAP- T5-1 and XLP B-LCL IARC 739 cell lines were characterized by highest level of SPIB expression. Taking together, we found that XLP and conventional B-LCL have differences in kinetics and amplitude of receptor-mediated Akt and ERK1/2 phosphorylation. Analysis of transcrip- tion factors profile revealed the distinguishing features of XLP B-LCLs with SAP deficiency that may impair B cell differentiation in patients with XLP1.

BIO 2014 Congress Session 7. Protein Functions in Cellular Signaling 155 P7.18 P7.19 Dual effects of 2-methoxyestradiol — Altered S100A6 level in keratinocytes anticancer agent and neurotoxin? affects EGFR signaling and Monika Gorzynik1 Alicja Kuban-Jankowska1, growth differentiation Michal A. Zmijewski2, Agnieszka Daca3,4, Agnieszka Graczyk1, Michał Mlącki2, Tomasz Michal Wozniak1, Magdalena Gorska1 Wilanowski2, Wiesława Leśniak1 1Department of Medical Chemistry, Medical University of Gdansk, 1Nencki Institute of Experimental Biology PAS, Department of Gdańsk, Poland 2Department of Histology, Medical University of Molecular and Cellular Neurobiology, Warsaw, Poland; 2Nencki Institute Gdansk, Gdańsk, Poland 3Department of Pathophysiology, Medical of Experimental Biology PAS, Department of Cell Biology, Warsaw, University of Gdansk, Gdańsk, Poland 4 Department of Pathology and Poland Experimental Rheumatology, Medical University of Gdansk, Gdańsk, e-mail: Agnieszka Graczyk Poland e-mail: Magdalena Gorska S100A6 is a signaling, calcium binding protein most Osteosarcoma is one of the most malignant bone tumors abundant in fibroblasts and epithelial cells including- ke of childhood and adolescence. So far there is no effective ratinocytes. Its postulated main role is regulation of cell treatment of OS. Constantly looking for new drugs that proliferation and, according to some reports, also cell dif- could effectively change the lives of patients. The existing ferentiation. Due to the lack of transgenic S100A6 knock- operating methods are limited due to the high chemore- out mouse model its role in tissue differentiation was never sistance and metastasis of cancer. The research represent examined. that carcinogenicity and angiogenesis can be influenced by In this study we have established stably transfected S100A6 17β-estradiol metabolites [1]. 2-methoxyestradiol (2-ME), knockdown (KD), overexpressing (OE) and control (C) one of the natural 17β-estradiol derivatives is a very prom- HaCaT keratinocytes and utilized them to investigate ising pharmaceuticals fulfilling the role of a potential anti- changes in epidermal growth and differentiation. We in- cancer drug over the still under investigation [2, 3]. duced differentiation of those cells either by increased Our studies have been conducted on highly metastatic os- [Ca2+] in 2-dimensional culture or by growing them in an teosarcoma 143B and rat immortalized hippocampal HT22 air-lifted 3-dimensional culture and monitored the levels cell lines. The cells were treated with pharmacological (1 of protein markers of epidermal differentiation using RT- µM, 10 µM) concentrations of 2-methoxyestradiol. Neu- PCR, Western blot and immunofluorescence techniques. ronal nitric oxide synthase and 3-nitrotyrosine protein level We observed that in 2D culture S100A6 OE keratinocytes was determined by western blotting. Cell viability, induc- had higher amount of keratin 10, the early differentiation tion of cell death were measured by MTT and PI/Annexin marker, in comparison to S100A6 KD and control cells V staining, respectively. 2-ME. Intracellular level of nitric while the synthesis of loricrin — the late differentiation oxide was determined by flow cytometry. marker was markedly disturbed. Immunofluorescence Pathway signaling of neurodegenerative diseases and analysis of 3D cell cultures revealed that there were minor cancer may be overlapped. Justified by the 2-methox- changes between control and S100A6 KD epidermis. How- yestradiol, one of the natural 17β-estradiol derivatives, in- ever, epidermis built of S100A6 OE HaCaT cells showed duces neuronal signaling not only in hippocampal HT22 striking differences such as: impaired keratin 10 staining, cells, but also in non-neuronal tissue- osteosarcoma can- present only in the uppermost epidermal layers, and no lo- cer. We observed that 2-methoxyestradiol, in contrast to ricrin staining. We also discovered that the staining of Ki67, 17β-estradiol, specifically affects neuronal nitric oxide syn- the proliferation marker, in S100A6 OE epidermis was not thase and augments 3-nitrotyrosine level leading to osteo- confined to the basal layer but could be detected through- sarcoma and hippocampal cell death. Thus, we present dual out all epidermal layers. The above characteristics suggest- face of 2-methoxyestradiol that leads to cancer cell death ed higher growth potential of S100A6 OE epidermis and but on the other side may play the key role as a neurotoxin therefore we decided to assay the activation of EGFR and in pathogenesis of neurodegenerative diseases. ERK1/2 kinase pathways. EGF receptor staining showed that, in S100A6 OE epidermis its expression was higher References: than in control and S100A6 KD epidermis and not restrict- 1. Yang CY et al (2013) Osteoblastoma in the region of the hip. J ed to the basal layer. Higher EGFR level in S100A6 OE Chinese Med Association 76: 115–120. HaCaT cells was subsequently confirmed at mRNA and 2. Peyrat JF, Brion J-D, Alami M (2012) Synthetic 2-Methoxyestra- protein level. diol Derivatives: Structure-Activity Relationships. Curr Med Chem 19: 4142–4156. Our study revealed that overexpression of S100A6 leads 3. Bruce JY et al (2012) A phase II study of 2-methoxyestradiol na- to higher cell proliferation as shown by Ki67 staining and nocrystal colloidal dispersion alone and in combination with sunitinib higher EGFR level and in this way stimulates epidermal malate in patients with metastatic renal cell carcinoma progressing on growth but delays differentiation. These results give us an sunitinib malate. Invest New Drugs 30: 794–802. insight into a novel S100A6 signaling pathway that may lead Acknowledgments to understanding of S100A6 function in tissue differentia- The project was funded by grant No. 2012/07/B/NZ1/00010 from Na- tion. tional Science Center resources. Acknowledgements Financial support: Grant no. 2012/05/N/NZ3/00905 to AG; EFS Schol- arships for PhD students to AG and MM.

Warsaw, September 9th–12th 2014 156 Session 7. Protein Functions in Cellular Signaling P7.20 P7.21 Design of substrates of human 20S The role of central carbon proteasome caspase-like subunit using metabolism in the control of DNA combinatorial chemistry approach replication in eukaryotic cells Natalia Gruba, Magdalena Wysocka, Adam Aleksandra Konieczna, Aneta Szczepańska, Karolina Lesner, Dawid Debowski, Krzysztof Rolka Sawiuk, Robert Łyżeń, Grzegorz Węgrzyn Institute of Biochemistry, University of Gdansk, Gdańsk, Poland University of Gdańsk, Depertment of Molecular Biology, Gdańsk, Poland e-mail: Natalia Gruba e-mail: Aleksandra Konieczna

The degradation of proteins has become one of the most Replication of genetic material is a basic biological pro- commonly studied processes in nowadays’ biology. Degra- cess, essential for each organism. Processes occurring dur- dation of proteins in eukaryotic cells is a rapid and irrevers- ing the synthesis of DNA are the subject of many works ible process that keeps the concentration of intracellular and have been relatively well studied. However, the con- proteins to a level that allows for precise regulation of cel- trol mechanism of the regulation of this process remains lular functions [1]. A proteolytic enzyme, which has great poorly understood. Disturbances in the process of DNA importance in the cells regulatory is a proteasome that is replication result in the accumulation of harmful muta- responsible for the removal of damaged, modified and tions, often leading to appearance of genetic diseases, and misfolded proteins or proteins that have become expend- development of cancer. Therefore, it seems important to able [2]. As proteasome is involved in many fundamental investigate the mechanisms that regulate this fundamental biological processes, like the cell cycle, gene expression, biological process. immune responses or carcinogenesis, even the smallest dis- The aim of this study is to understand the global regula- ruption in its activity may cause very serious physiological tory processes that control DNA replication in eukary- consequences [3, 4]. otic organisms, as well as to answer the question whether In this project we decided to investigate systematically the these mechanisms are universal. This work focus primarily substrate specificity of 20S protesaome in P nonprime and on glycolysis conjunction with DNA replication. The ex- P prime (based on the Schechter-Berger notation) posi- pression of selected isoforms of genes of the glycolysis tions. pathway was reduced at the level of up to 95 % by using Here we report the selection of internal quenched sub- siRNA. Then the cell cycle was analyzed with particular re- strates for caspase-like specificity applaying combinatorial gard to the transition of the cells into the S phase. Moreo- chemistry approach. The library used, consist of peptides ver, the analysis of viability and cell number was carried that on N- and C- termini have accordingly donor and ac- out. The most pronounced difference in the transition of ceptor fluorophore of electron excitation. The general for- cells treated with siRNA into the S phase was observed in mula of the peptide library for “non-prime” positions is the case of the gene encoding glyceraldehyde 3-phosphate presented below: dehydrogenase (GAPDH), it amounted to about 50%. Fur- thermore, silencing of the genes encoding the hexokinase ABZ-X4-X3-X2-X1-ANB-NH2 (HK), phosphofructokinase (PFK) and enolase (ENO) re- ANB-NH2 – amide of 5-amino-2-nitrobenzoic acid sulted in decreased number of cells in the S phase by 30% ABZ – 5-aminobenzoic acid and 15%, respectively. Surprisingly, silencing the expression X1 – Asp, Glu; of the gene encoding the enzyme GPI resulted in enhance- X2, X3, X4 – coded amino acids exept for Cys. ment of the replication phase. These results facilitate understanding of the correlation In the next step of our research the peptide resulting from between metabolic processes and DNA replication. This above described studies was incorporated into internal can be practically used in studies on carcinogenesis and its quenched library with general formula: ABZ-Ile-Leu-Met- prevention, as well as biotechnological use of cell cultures. Asp-X1’-X2’-X3’-Tyr(3-NO2)-NH2 where in position X1’, X2’, X3’ set of proteinogenic amino acid exept Cys. The libraries of fluorescent substrates were synthesized manu- ally on solid resin support using “split and mix” method, Fmoc/tBu procedure. Deconvolution against human 20S proteasome was carried out using iterative solution method.

References: 1. Mizushima T et al (2004) Viley-VCH: Weinheim. 2. Elofsson M et al (1999) Chem Biol 6: 811–822. 3. Borissenko L et al (2007) Biol Chem 388: 947–955. 4. Groll M et al (2005) ChemBioChem 6 : 222–256. Acknowledgements This work was supported by the project contributing to the development of young scientist and PhD students under grant No 538-8710-B324-14.

BIO 2014 Congress Session 7. Protein Functions in Cellular Signaling 157 P7.22 P7.23 ­Role of AMPK and HGF in epithelial HtrA protease from Escherichia to mesenchymal transition coli and its redox state: implication for substrate degradation/affinity Pawel Konieczny, Tomasz Adamus, Marcin Majka and the activation process Department of Transplantation, Jagiellonian University Medical College, Kraków, Poland Tomasz Koper1, Katarzyna Węgrzyn2, Igor e-mail: Paweł Konieczny Konieczny2, Bogdan Banecki2, Adam Lesner3, Barbara Lipińska1, Joanna Skórko-Glonek1 AMP-activated kinase (AMPK) is a metabolic sensor that helps maintain cellular energy homeostasis. It is also in- 1Department of Biochemistry, Faculty of Biology, University of Gdańsk, Gdańsk, Poland; 2Intercollegiate Faculty of Biotechnology UG-MUG, volved in tumorigenesis and metastatic progression, how- Gdańsk, Poland; 3Faculty of Chemistry, University of Gdańsk, Gdańsk, ever its role in these processes remains unclear. Evidence Poland that AMPK cellular level is adjusted in response to HGF e-mail: Tomasz Koper induced signaling is missing. However, it seems that AMPK level and state of its activation might influence the epithe- HtrA protease from the bacterium Escherichia coli is a cru- lial to mesenchymal transition (EMT) process and led to cial factor of the periplasmic protein quality control sys- tumor progression and development of metastasis. tem. HtrA recognizes and degrades irreversibly damaged The aim of this study was to investigate changes in expres- proteins that emerge under various stress conditions such sion and activation of AMPK as a result of HGF stimula- as heat shock, oxidative stress, reducing stress, non-physi- tion. We used three cervical carcinoma cell lines accord- ological pH, etc. ing to their different clinical stage and invasiveness. C-4I, HtrA exists in various oligomeric forms. In the inactive/ isolated from primary tumor mass, represented an early resting state it forms hexamers. Upon the activation pro- stage of cervical cancer. HTB-35 originated also from tu- cess triggered by the presence of misfolded/unfolded sub- mor in situ, however, cells acquired mesenchymal charac- strates it re-assembles into higher-order oligomers consist- teristic, indicating the implementation of EMT. The third ing mainly of 12- and 24-mers. cell line was HTB-34, derived from metastatic site in lymph The structure of the hexamer is stabilized by the LA loops node, regained their epithelial phenotype indicating the which interact with the regulatory loops L1 and L2 of the implementation of mesenchymal to epithelial transition opposite subunit. The LA-L1-L2 loops cover the active program. Thus, such cell lines represented comprehen- site. In the active conformation the LA-L1-L2 contacts sive model of tumorgenesis and metastatic progression are no longer present and the active site becomes available of cervical carcinoma. qRT-PCR analysis were performed for the substrate. This process is accompanied by hexamer to examine the level of AMPK catalytic subunits (α1 and dissociation into trimers and simultaneous assembly into α2) after stimulation with HGF. Simultaneously, Western higher order oligomers. Blotting was used to investigate total AMPK level and its The proteolytic activity of HtrA shows strong temperature- phosphorylation status. dependence and at temperatures below 30°C is hardly de- Our analysis showed immense differences in AMPK tran- tectable in vitro. Our previous studies showed that the redox script and protein levels between examined cell lines. C-4I state of Cys57 and Cys69 of the LA loop may influence cell line revealed the highest AMPK mRNA and protein the proteolytic activity of HtrA at low temperatures (20- levels, followed by HTB34 with moderate expression and 25°C): (1) HtrA deprived of the S-S bond more efficiently HTB 35 with lowest expression among cells lines. HGF degraded reduced alkaline phosphatase in vivo and (2) the stimulation resulted in decreased expression of AMPK in cysteineless LA loops were more exposed to the solvent C4I cell line. In other cell lines HGF did not induce any and had more flexible structure, that probably facilitated changes in AMPK mRNA or protein levels. activation process or substrate binding. Our results allow to trace the process of metabolic deregu- The aim of our work was to further examine the role of lation of cancer cell in metastatic progression. Morover, we the disulphide bridge in the activation process of HtrA. tried to examine the connection between EMT promoting For this purpose we checked for the kinetics of proteolysis, factors like HGF and tumor cell metabolism at different affinity to substrate and oligomeric state of HtrA variants clinical stages. containing or deprived of the disulphide bridge in the pres- ence of the model substrates/ligands. The HtrA deprived of the S-S bond: (i) digested a model substrate more efficiently at 20 and 37°C, (ii) exhibited higher affinity to immobilized substrate, (iii) required sig- nificantly lower concentration of model ligand to assemble into higher-order oligomers, (iv) showed altered oligomer equilibrium in the absence of substrate, in comparison to HtrA with the preserved S-S bond.

Warsaw, September 9th–12th 2014 158 Session 7. Protein Functions in Cellular Signaling P7.24 P7.25 Cx43 up-regulation contributes to Yeast YIL096C binds and TGF-β1-induced activation of Smad- methylates 25S rRNA dependent signaling during fibroblast- Jan Kutner1, Andrzej Dziembowski2,3, Maga to-myofibroblast transition of primary Rowicka4,5,6, Krzysztof Ginalski1 human bronchial fibroblasts 1Centre of New Technologies, Laboratory of Bioinformatics and Milena Kosińska,1 Izabela Borek,1 Damian Systems Biology, University of Warsaw, Warsaw, Poland, 2Institute of 1 1,2 Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Ryszawy, Katarzyna Anna Wójcik, Katarzyna 3 1 1 1 Warsaw, Poland, Institute of Biochemistry and Biophysics, Polish Piwowarczyk, Marta Michalik , Jarosław Czyż Academy of Sciences, Warsaw, Poland, 4Department of Biochemistry 1 and Molecular Biology, University of Texas Medical Branch, Galveston, Jagiellonian University, Faculty of Biochemistry, Biophysics and 5 Biotechnology, Department of Cell Biology, Kraków, Poland; Texas, United States of America, Institute for Translational Sciences, 2 University of Texas Medical Branch, Galveston, Texas, United States Jagiellonian University Medical School, Department of Medicine, 6 Kraków, Poland of America, Sealy Center for Molecular Medicine, University of Texas e-mail: Milena Kosińska Medical Branch, Galveston, Texas, United States of America e-mail: Jan Kutner

Fibroblast-to-myofibroblast transition (FMT) is 1TGFβ - dependent process which plays a pivotal role in the induc- The ribosomal RNA contains 109 specific modifications tion of airway wall remodeling during bronchial asthma that affect translation and ribosome biogenesis. Only few [1]. It is regulated by the activation of Smad signaling in of them correspond to base methylations catalyzed by human bronchial fibroblasts (HBF) and accompanied by methyltransferases (MTases). In eukaryotes, most of the up-regulation of a-SMA expression - a marker of myofi- rRNA modifications are maintained by enzymes that re- broblasts [2]. The involvement of Cx43, a protein which quire guide snoRNA. In this work we identify a specific constitutes gap junctional channels, in the regulation substrate for Saccharomyces cerevisiae YIL096C (BMT5) [1, 2], of FMT in cardiac tissue has recently been reported [3]. a snoRNA-independent MTase, using in vitro isotope de- It prompted us to estimate the role of Cx43 in TGFβ1- pendent assays. We show that YIL096C binds and meth- induced FMT undergone by primary HBFs derived from ylates yeast 25S rRNA and possesses substrate specificity asthmatic patients (AS HBFs). Prolonged exposure of AS to oligoRNA containing already known methylation site 3 HBFs to TGFβ1efficiently induced FMT, which was ac- m U2634. This is highly consistent with the recent study by companied by up-regulation of α-SMA and Cx43 levels as independent group showing that BMT5 is a new m3U2634 demonstrated by immunofluorescence, immunoblotting MTase [3]. Our results contribute to better understanding and FACS analyses. These effects were correlated with the of rRNA base methylation process, ribosome biogenesis induction of Smad signaling, illustrated by nuclear trans- and function. Presented approach should be helpful in fur- location and accumulation of p-Smad2 in TGFβ1-treated ther studies on substrate specificity of rRNA base MTases. AS HBFs. Inhibition of gap junctional intercellular cou- pling by 18-α-glycyrrhetinic acid considerably reduced the References: efficiency of FMT. However, it had no effect on TGFβ1- 1. Wlodarski T et al (2011) PLoS One 6: e23168. induced nuclear translocation of Smad2 and Cx43 expres- 2. Szczepinska T et al (2014) PLoS Comput Biol 10: e1003514. sion level in AS HBFs. On the other hand, transient inhibi- 3. Sharma S et al (2014) Nucleic Acids Res 42: 3246–3260. tion of Cx43 expression by siRNA attenuated the α-SMA transcript quantity, FMT and Smad signaling activity in these cells. Our observations indicate overlapping gap junc- tional channel-dependent/independent effects of Cx43 on the efficiency of FMT during airway wall remodeling. Gap junctional coupling may participate in FMT of AS HBFs through mediating by-stander effects in a Smad2-inde- pendent manner. On the other hand, Cx43 affects Smad activity in these cells in a channel-independent fashion.

References: 1. Makinde T Murphy RF Agrawal DK (2007) Immunol Cell Biol 85: 348– 356. 2. Evans et al (2003) Exp Cell Res 282: 90–100. 3. Asazuma-Nakamura Y et al (2009) Exp Cell Res 315: 1190–1199.

BIO 2014 Congress Session 7. Protein Functions in Cellular Signaling 159 P7.26 P7.27 TROSPA — an intrinsically Regulation of Nrf2-dependent pathway disordered protein involved in the in normal and tumor cells of the tick colonization by Borrelia digestive system by ITC 2-oxoheptyl Dominik Lewandowski1, Anna Urbanowicz1, Katarzyna Lubelska1,2, Katarzyna Wiktorska1, Lidia Kamil Szpotkowski1, Karol Kamel1, Mariusz Śliwka1,2, Małgorzata Milczarek1,3, Zdzisław Chilmonczyk1 1,2 1,3 Jaskólski , Marek Figlerowicz 1National Medicines Institute, Department of Cell Biology, Warsaw, 1 Poland, 2Medical University of Warsaw, Faculty of Pharmacy with the Institute of Bioorganic Chemistry, Polish Academy of Sciences, 3 Poznań, Poland; 2Faculty of Chemistry, Adam Mickiewicz University, Labolatory Medicine Division, Warsaw, Poland, University of Lublin, Poznań, Poland; 3Institute of Computing Science, Poznan University of Faculty of Pharmacy with Medical Analytics Division, Lublin, Poland Technology, Poznań, Poland e-mail: Katarzyna Wiktorska e-mail: Anna Urbanowicz The nuclear factor (erythroid-derived 2)-like 2) Nrf2 plays a Borreliosis (Lyme disease) caused by bacteria belonging to key role in regulation of detoxification enzyme NAD(P)H Borrelia genus is one of the most prevalent tick-borne dis- dehydrogenase (quinone) (NQO1) [1]. Increasing in NQO1 eases in North America and Europe. In the latter, Borrelia activity contributes in normal cells to the maintenance of spirochetes are transmitted by Ixodes ricinus (Castor bean cellular homeostasis, whereas in cancer cells it is unwanted tick). Earlier it has been demonstrated that a protein pro- phenomena, as it desensitizes cells to endogenous or exog- duced in tick’s epithelial cells called TROSPA is essential enous mechanisms of their elimination [2]. for the bacteria to colonize the vector. Bacterial surface The isothiocyanates (ITC) are compounds which induce protein OspA binds TROSPA and this way enables Borrelia NQO1 activity through Nrf2 in the cells of a gastrointes- to attach to tick gut. tinal tract in vitro [3]. Due to the deregulation of signaling Despite intensive studies conducted during the last decade, pathways in cancer cells it is not known whether the Nrf2 the structure of TROSPA and its basic functions remain induction in normal and cancer cells by the ITC influences unknown. In order to learn more about this protein we in a similar way individual molecular targets which could produced a recombinant TROSPA_NΔ44 deletion mu- result in increasing activity or lack thereof. tant. It lacked transmembrane domain but still was capable ITC 2-oxoheptyl was selected for the study. It is a selec- of binding OspA at the similar level to the wild type pro- tive compound with potential activity on the normal and tein. The structure of the highly purified TROSPA_NΔ44 cancer cells. The tests were performed on CRL1790 nor- mutant in a solution was examined by using a number of mal cells and HT29 colon cancer cells. The effects of ITC techniques, such as small-angle X-ray scattering (SAXS), 2-oxoheptyl on translocation of Nrf2 from the cytoplasm dynamic light scattering (DLS), size-exclusion chromatog- to the nucleus in both cell lines was examined and com- raphy (SEC) or circular dichroism spectroscopy (CD). The pared. Then, changes in the expression level of the gene results coming from our analyses clearly demonstrated that encoding NQO1 and changes in the level of NQO1 pro- TROSPA shows the features characteristic for intrinsically tein were examined. The final step was to compare the level disordered proteins (IDP). In addition, based on the data of induction of NQO1 activity in both types of cell lines. collected so far, we generated the computational model of NQO1 protein levels and the location of the transcription three dimensional structure of the TROSPA_NΔ44 mu- factor Nrf2 was examined by immunocytochemistry. Ex- tant. amination of the gene encoding the expression of NQO1 was performed using the method of quantitative real-time Acknowledgements PCR (qPCR). NQO1 enzyme activity was tested biochemi- This work was partially supported by the Polish Government through cally. grant N N302 041536. It was found that ITC 2-oxoheptyl induced Nrf2 transloca- tion from the cytoplasm to the nucleus in both cell lines, leading to an increase in the expression of the gene encod- ing NQO1. However, changes in the cellular localization of Nrf2 and increased expression of the gene encoding NQO1 resulted in the increase in the level and activity of NQO1 protein only in normal cells. In the tumor cells, in- stead of induction of translocation and increase in expres- sion of the gene encoding NQO1, reduced protein levels and NQO1 activity were observed. The results indicate a possible influence of ITC 2-oxohep- tyl subsequent post-translational modifications, which may play an essential role in the final level of NQO1 activity in tumor cells HT29.

References: Dinkova-Kostova AT et al (2010) Arch Biochem Biophys 501: 116–123. Lau A, Villeneuve NF et al (2008) Pharmacol Res 58: 262–270. Morimitsu Y et al (2002) J Biol Chem 277: 3456–3463. Acknowledgements The research was co-financed by the European Union within the Euro- pean Social Fund.

Warsaw, September 9th–12th 2014 160 Session 7. Protein Functions in Cellular Signaling P7.28 P7.29 The chiral Schiff base as the Novel mTOR isoform and tumorigenesis activators of adenylate kinase Oksana Malanchuk, Sergiy Palchevskyy, Oleg Garifulin 1 2 1 3 A. Ludwiczak , E. Błocka , A. Wojtczak , M. Komoszyński The Institute of Molecular Biology and Genetics of NAS of Ukraine, 1Faculty of Chemistry, Nicolaus Copernicus University, Department Department of Cell Signalling, Ukraine of Crystallochemistry and Biocrystallography, Poland; 2Faculty of e-mail: Oksana Malanchuk Chemistry, Nicolaus Copernicus University, Department of Organic Chemistry, Poland; 3Faculty of Biology and Environmental Protection, Background: The mammalian target of rapamycin Department of Biochemistry, Poland (mTOR) plays a striking role in signal transduction and reg- e-mail: Agnieszka Ludwiczak ulates cell growth, metabolism, proliferation and survival in response to nutrients and growth factors. Deregulation of Adenylate kinase (AK, EC 2.7.4.3) are the ATP: AMP mTOR signaling pathway has been associated with numer- phosphotransferases catalyzing the reaction of bidirection- ous pathologies, including many human cancers. al transfer of phosphate residues from adenosine triphos- There is only one gene encoding mTOR. Therefore the phate (ATP) to adenosine monophosphate (AMP). The diversity of the mTOR-mediated signaling is conferred substrates of the enzymatic reaction are two molecules of by two functionally and structurally distinct multiprotein adenosine diphosphate (ADP). The presence of ADP in complexes (mTORC1 and mTORC2) with the well known the blood is necessary for initiation of platelet aggrega- mTOR (mTORα) in their core. Previously in our Labora- tion and maintain the process of repairing damaged tissue. tory for a fist time mTOR splicing isoform (mTORβ) was However, uncontrolled and growing the ADP concentra- identified. This isoform differs from well known mTORα tion increases platelet aggregation and thrombus forma- leaking HEAT and partially FAT domains but carrying in- tion. On the other hand, increase in the concentration of tact Kinase domain. It has been shown that mTORβ is an ATP initiates the inflammatory processes and formation of active protein kinase that mediates downstream signaling an atherosclerotic plaque. In our opinion, the reduction of through complexing with Raptor (mTORC1) and Rictor non-physiological concentrations of ADP and ATP in the (mTORC2) proteins and, significantly, is a potential onco- blood by the activation of adenylate kinases is more favora- gene. ble than block platelet purinergic receptors. Understanding Recently, we have identified a novel mTOR isoform – the molecular basis for regulation of the activity of these mTORδ. Importantly, this isoform has another principle kinases may be a starting point for effective treatment of of isoform formation then mTORβ (unpublished data). cardiovascular diseases, including heart attacks. Deletion in kinase domain resulted in frame shift within 3' A completely new group of regulators of kinases belonging - region and finally translation of unique protein sequence to the Schiff bases was obtained and tested. The synthesis which is absent in mTORα. So, current research proposal of these compounds, including derivatives of norephed- is focused on clarifying the involvement of mTORδ in tu- rine was conducted for catalytic applications [1,2]. These morigenesis. Characterization of oncogenic and signaling compounds have structural similarity to the derivatives activities of mTORδ and its expression in cancer will offer of suramin or anthraquinone-2-sulfonic acid- inhibitors new perspective target for cancer therapy. of nucleotidase. Additionally, they are characterized by a Results: Early using RT-PCR of various cell lines we similar distribution of heteroatoms to the ATP molecule, have identified a novel splicing isoform of mTOR kinase which led us to research their effect on the activity of ade- – mTORδ. The aim of this study was to characterize the nylate kinase from Aquifex aeolicus and Bacillus stearothermo- functional role of mTORδ in tumorogenesis. So, initially, philus. The tested compounds showed a 40-60% activation we have generated polyclonal antibody against mTORδ and of kinase. Both tested regulators was crystallized and their specific antibodies against Raptor and Rictor which are ap- crystal structures was determined[3]. plicable in various immunoassays. Then we have shown by Western-blot analysis that mTORδ expressed in different References: cell lines in vivo. Next, HEK293 stable cell line overexpress- 1. Błocka E, Jaworska M, Kozakiewicz A, Wełniak M, Wojtczak A (2010) ing mTORδ has been generated. To examine the oncogenic Tetrahedron: Asymmetry 21: 571–577. potential of mTORδ the various assays have been done: 2. Hayashi M, Miyamoto Y, Inoue T, Oguni N (1993) J Org Chem 58: MTT assay to analyze the cellular activity and proliferation; 1515–1522. 3. łocka E, Bosiak MJ, Wełniak M, Ludwiczak A, Wojtczak A (2014) Tetra- Soft Agar Assay to measure cell anchorage-independent hedron: Asymmetry 25: 554–562. proliferation potential; Scratch Migration Assay to study the migration capacity of cells. In result, HEK293 stable cell line overexpressing mTORδ demonstrates a low prolif- erative potential and not considerable migration capacity in compare to control HEK293 cell. Conclusions: In contrast to mTORβ isoform, mTORδ can inhibit cell proliferation. To elucidate the mechanism by which cell proliferation may be inhibited by mTORδ now we are exploring its effect on the cell cycle progression by flow cytometric analysis. Besides we plan to investigate both the localization of mTORδ in cell and its co-localiza- tion with Rictor or Raptor proteins.

BIO 2014 Congress Session 7. Protein Functions in Cellular Signaling 161 P7.30 P7.31 Transport of the opioid peptides Calix[4]arene C-90 as selective inhibitor across Caco-2 monolayer of plasma membrane calcium pump and Michał Matysiewicz, Beata Jarmołowska, Marta new myometrium contraction agent Bukało, Natalia Smulska, Ewa Fiedorowicz, Iuliia Mazur1, Tetyana Veklich1, Oleksandr Anna Cieślińska, Elżbieta Kostyra Shkrabak1, Olga Tsymbalyuk2 Medical Biochemistry Peptides and Proteins Research Team; Faculty of 1Palladin Institute of Biochemistry of the National Academy of Sciences Biology & Biotechnology; University of Warmia and Mazury in Olsztyn, of Ukraine, Department of Muscle Biochemistry, Ukraine; 2Taras Olsztyn, Poland Shevchenko National University of Kyiv, Institute of Higher Technology, e-mail: Elżbieta Kostyra Ukraine e-mail: Iuliia Mazur Milk is the source of β-casomorphins (BCMs) – biologi- cally active peptides with opioid activity – which are sus- Plasma membrane Mg2+,ATP-dependent Ca2+-pump eject pected to play various roles in the human body. The local Ca2+ from cytoplasm to extracellular space and control influence of exogenous opioid peptides on gastrointestinal local Ca2+-signal in intracellular membrane microdomen. functions has been widely reported. After passing the gut Nonetheless, prior to our research there are no low-molec- barrier, β-casomorphins may affect the functions of immu- ular, selective and affine inhibitors. The calix[4]arene C-90 nological system, as well as dopaminergic, serotoninergic (5,11,17,23-tetra(trifluoro)methyl(phenyl-sulfonylimino)- and GABA-ergic systems in brain, regulate the opioid re- methylamino-25,26,27,28-tetrapropoxycalix[4]arene) is ceptor development and elicit behavioral effects. However, shown to efficiently and selectively inhibit the ATP hydro- possibilities and mechanisms of the intestinal transport of lase activity of Ca2+,Mg2+-ATPase in the myometrium cell β-casomorphins in human body in vivo have not been accu- plasma membrane fraction. Calix[4]arene C-90 was synthe- rately described so far. However, it is known that in the gas- sized in Institute of organic chemistry NAS of Ukraine trointestinal tract as well as in the blood, β-casomorphins by prof. V.I. Kalchenko and characterized by methods of can be degraded by the dipeptidyl peptidase IV (DPPIV; infrared spectroscopy and nuclear magnetic resonance. EC 3.4.14.5), a highly specified aminopeptidase that cleaves Activity of Ca2+,Mg2+-ATPase was determined on swine dipeptides (X-Pro) from N-terminus fragments of peptide. myometrium plasma membrane fraction by measurement The aim of this research was to determine the perme- of Pi formed as a result of ATP hydrolysis. The inhibition ability coefficients of the µ-opioid agonists human coefficient 0,5I values was 20,2 ± 0,5 and calyx[4]arene C-90 β-casomorphin-5 and -7 (BCM5, BCM7) in time intervals, (100 µM) decrease Ca2+,Mg2+-ATPase velocity maximum and to compare them with the permeability coefficient of on 75% comparatively to control. The inhibitory effect of µ-opioid antagonist peptide, human lactoferroxin A (LCF calix[4]arene C-90 was selective comparatively to other AT- A) - using Caco-2 monolayer. In order to determine the Pases localized in the plasma membrane: Na+,K+-ATPase pathway of investigated peptide transport across Caco-2 and “basal” Mg2+-ATPase. The inhibitory effect of calix[4] monolayer, two directions of the transport (apical to baso- arene C-90 on the Ca2+,Mg2+-ATPase activity was associ- lateral and basolateral to apical) have been studied. All ated with the cooperative action of four trifluoromethyl- investigated peptides were transported across the human phenyl sulfonylimine (sulfonylamidine) groups spatially intestinal cell line Caco-2 and the curves of cumulative oriented on the upper rim of the calix[4]arene macrocycle amount of transported peptides in time were linear in each (the calix[4]arene “bowl”). case. In addition, the hydrolysis of β-casomorphins dur- Due to results of confocal microscopy study calix[4]arene ing 60 min of experiment by dipeptidyl peptidase IV was C-90 increased calcium concentration in cytoplasm of observed. myometrium smooth muscle cells in rest on 45±9 % com- Our studies have shown that the transport of opioid pep- paratively to control, however, during 1,5-2 minutes the cal- tides across epithelial cells may be more efficient in the cium concentration was turning to output level. It is mean case of lower DPPIV activity. These data have also con- that other calcium-control system (as Na+,Ca2+-exchanger, firmed that DPPIV is the main factor limiting the half-life mitochondria) became more actively involved in calcium of opioid peptides in the intestine. Intestinal transport of extrusion if plasma membrane Ca2+-pump is inhibited. m-opioid receptor agonist and antagonist peptide has the Additionally the method of laser correlation spectroscopy potential to regulate the access of those peptides to their was used to determine effective hydrodynamic diameter of target sites in the body. It prompts the need to investigate smooth muscle cells and inference about cell contractil- the transport abilities of opioid peptides, especially in the ity was made. Calix[4]arene C-90 (50 µM) caused decrease cases of diseases such as allergy, mucosal immunity, celiac of effective hydrodynamic diameter on 26% like as utero- disease, autism and schizophrenia. tonic oxytocin (100 nM). Thus, calyx[4]arene C-90 provoke smooth muscle cells contraction. Also calix[4]arene C-90 (10 µM) decrease relaxation velocity maximum of uterine smooth muscle normalized on contraction amplitude on 20% comparatively to control measured in spontaneous muscle activity. The experimental findings seem to be important for stud- ies of regulation of calcium homeostasis in smooth muscle cells and also for investigation of the participation of the plasma membrane Ca2+-pump in control of electro- and pharmacomechanical coupling in myocytes.

Warsaw, September 9th–12th 2014 162 Session 7. Protein Functions in Cellular Signaling P7.32 P7.33 New piroxicam derivatives with Induction of particulate guanylyl antitumor activity and their effect on cyclase type A in monocytes multidrug-resistant colon cancer cells Małgorzata Mitkiewicz, Jakub Siednienko, Joanna Zyzak, Kamila Środa-Pomianek1, Berenika Szczęśniak-Sięga2, Marianna Kuropatwa, Ewa Kurowska, Janusz Matuszyk 2 2 1 Wiesław Malinka , Jadwiga Maniewska , Krystyna Michalak Laboratory of Signaling Proteins, Ludwik Hirszfeld Institute of 1Department of Biophysics, Wroclaw Medical University, Wrocław, Immunology and Experimental Therapy, Polish Academy of Sciences, Poland; 2Department of Chemistry of Drugs, Wroclaw Medical Wrocław, Poland University, Wrocław, Poland e-mail: Malgorzata Mitkiewicz e-mail: Krystyna Michalak Cyclic GMP (cGMP) is a common secondary messenger Data on potential antitumor activity of commonly used involved in many cellular processes. Synthesis of this nu- non-oncological drugs, such as non-steroidal anti-inflam- cleotide is catalyzed by guanylyl cyclases (GCs), which exist matory drugs (NSAIDs) often appear in literature, recently. as cytosolic (sGC) or membrane-bound (particulate, pGC) Many studies have revealed that NSAIDs may decrease the proteins. The data show that in monocytes mainly sGC is risk of cancer incidence, including breast, colon, lung, and present and the NO/sGC/cGMP pathway in regulation stomach cancer. The challenge is to obtain new, more ac- of immune cells response is well known [1, 2]. However, tive compounds possessing anticancer and/or multidrug the role of particulate guanylyl cyclase A (GC-A) in im- reversing activity. Piroxicam is a NSAID that belongs to mune cells remains unknown. Our observations and other the oxicam class of drugs. It is used as an analgesic in case reports [3, 4] indicate that active GC-A appears during cul- of post-operative pain or in rheumatoid and osteoarthritis. ture of monocytes isolated from peripheral blood mononu- NSAIDs are inhibitors of cyclooxygenase-2 (COX-2), the clear cells (PBMCs). It is supposed that such change in the enzyme which is expressed in the most solid tumors. phenotype of cells may influence their function in certain The research goal was to design and synthesize com- diseases, i.a. in cirrhosis, hypertension and heart failure. In pounds that would be able both to inhibit COX-2 activ- the plasma of patients was observed even 30-fold increase ity and modulate multidrug resistance (MDR). The ques- in concentrations of natriuretic peptides (ANP and BNP), tion was which structural changes should be introduced the activators of GC-A. Due to the small amount of stud- into chemical structure of piroxicam to obtain derivatives ies on the molecular mechanism of GC-A expression, this with maximal anticancer activity. Two of the newly syn- process remains unclear. In this light the main aim of our thesized derivatives exhibit the ability to inhibit the growth studies was to examine what external factors influence the of colon cancer cells LoVo and LoVo/Dx, sensitive and expression of GC-A in monocytes and how this process resistant to doxorubicin, respectively The expression level is regulated. As a model we used the monocytic cell line of the multidrug resistance proteins and some proteins THP-1. It was found that among many extracellular factors associated with apoptosis, and their corresponding genes only phorbol ester (PMA) and all-trans retionic acid (much (mRNA determination) have been analyzed by immunode- more weakly) affect induction of GC-A in THP-1. Using tection and PCR, respectively. Analysis at the level of pro- real-time PCR, Western blotting and ELISA we found that tein has shown that new piroxicam derivatives decrease the in PMA-stimulated cells active form of GC-A is produced. expression of multidrug transporter ABCG2 (BCRP), as Subsequently it was shown that both protein kinase C well as an anti-apoptotic Bcl-2 protein and change expres- (PKC) and mitogen extracellular kinases (MEK1/2) play sion of pro-apoptotic Bax protein. It was observed that an important role in the activation of GC-A encoding gene both oxicam compounds were also able to reduce the level in response to PMA. The implementation of specific in- of COX-2 expression. The studies with use of confocal hibitors of these kinases resulted in significant decrease of microscopy have confirmed the influence of new deriva- GC-A expression and activity in PMA-differentiated cells. tives on cytoskeleton structure in studied cancer cell lines. The participation of PKC in GC-A induction was also In conclusion, the newly synthesized piroxicam derivatives shown in the cultured PBMCs. Preliminary data indicate show promising properties as potential anticancer agents in that Sp1 transcription factor may be involved in the regula- case of colon cancer. tion of GC-A gene expression in THP-1 cells and PBMCs.

References: 1. Mitkiewicz M et al (2011) Immunobiology 216: 423–430. 2. Siednienko J et al (2011) Cytokines 54: 282–288. 3. Bender AT et al (2004) Cellular Signalling 16: 365–374. 4. Morita R et al (2003) J Immunol 170: 5869–5875. Acknowledgements: This project was supported by National Science Centre, Poland (Project SONATA No. UMO-2011/01/D/NZ3/04249).

BIO 2014 Congress Session 7. Protein Functions in Cellular Signaling 163 P7.34 P7.35 Subcellular distribution of the Cytochrome c interaction with tRNA, the immunophilin FKBP39 in living cells new properties of the known molecules Marek Orłowski1, Katarzyna Popławska1, Joanna Roza Pawlowska, Dominika Jedrzejczyk, Magdalena Pieprzyk1, Aleksandra Szczygieł-Sommer1, Mirosław Janicka, Anna Graczyk, Arkadiusz Chworos 2 2 1 Zarębski , Jurek Dobrucki , Andrzej Ożyhar Department of Bioorganic Chemistry, Centre of Molecular and 1Department of Biochemistry, Faculty of Chemistry, Wrocław University Macromolecular Studies, Polish Academy of Sciences, Łódź, Poland of Technology, Wrocław, Poland; 2Division of Cell Biophysics, Faculty of e-mail: Roza Pawlowska Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland Transfer RNAs (tRNAs) are the key molecules involved e-mail: Marek Orłowski in protein biosynthesis, responsible for transport of spe- cific amino acids to the ribosome during translation. Some The FK506-binding proteins (FKBPs), a distinct class of recent data suggest, that tRNAs not only play role as key immunophilins, interact with immunosuppressants and elements in protein synthesis machinery, but also this class catalyze the cis-trans conversion of prolyl bonds in proteins of small RNAs is involved in regulation of programmed using their peptidyl-prolyl isomerase (PPIase) activity dur- cell death [1] and gene expression regulation in response to ing protein folding events. Immunophilins are intracellu- stress conditions [2]. lar receptors of the immunosuppressants FK506 and ra- The apoptosis regulation by tRNA is conducted by direct pamycin that block antigenic responses and/or cell cycle binding of tRNA molecule to cytochrome c and thereby progression. These proteins are also a unique group of interfering with the apoptosome formation [1]. This novel, chaperons found in a wide variety of known organisms. surprising discovery sheds a new light on possible role of FKBPs are composed of variable numbers and types of increasing expression of tRNA in cancer cells and opens domains, which enable them to perform many important possibility to study tRNA in this regard, as potential model cellular functions in addition to protein folding. The pepti- molecules for therapeutic agents design. dyl-prolyl cis-trans isomerase FK506-binding protein of 39 Since Mei and Yong discovery in 2010 [1], new findings kDa (FKBP39) from Drosophila melanogaster is an inhibitor show quantitatively how cytochrome c binds to tRNA [3], of autophagy in larval fat body and the protein is probably however the mechanism of this interaction is still unclear. involved in ecdysone and juvenile hormone signal trans- Another unexplored problem is the affinity of individual duction as a transcriptional modulator of gene expression. tRNAs to cytochrome c. Thus, the aim of this work was to study the subcellular dis- Cytochrome c is a well explored protein primarily responsi- tribution of FKBP39 fused to a yellow fluorescent protein ble for the electron transport in mitochondrial respiratory (YFP) in living cells. The experiments were carried out in system located in an inter-membrane of mitochondria and mammalian COS-7 and HEK293 cells to facilitate the in- their release into the cytoplasm is one of the earliest steps vestigation of the subcellular trafficking of YFP-FKBP39 of induction of intrinisic apoptosis pathway [4]. in the absence of its endogenous expression. The data In current studies, we have designed and synthetized, spe- revealed that the distribution of YFP-FKBP39 was exclu- cific RNA molecules imitating mitochondrialGly tRNA , sively nuclear and nucleolar in both cell types analyzed. In tRNAAla, tRNAHis and tRNAPhe and test their ability to order to determine the nuclear localization signal (NLS), bind to cytochrome c and possible influence on the apop- a series of FKBP39 mutants tagged with YFP were pre- tosis. The cytochrome c/tRNA complexes formation was pared and examined in cells. In a series of deletion mutants, investigated using electrophoretic methods (based on the a segment (residues 180–250) displayed constitutive nuclear mobility shift measurement) and spectroscopy techniques. import suggesting the presence of a novel complex NLS Obtained results show, that all tested tRNA molecules form signal in a basic domain in the middle of FKBP39. Detailed complexes with cytochrome c and thereby may influence mutagenesis studies revealed that residues K188 and K191 on induction of apoptosis in vitro. Simultaneously there are are crucial for nuclear targeting of FKBP39. The results some differences observed between individual tRNAs. show that the immunophilin FKBP39 possess an active NLS in close proximity to a putative helix-turn-helix motif References: responsible for binding of DNA. 1. Mei Y, Yong J et al (2010) Molecular Cell 37: 668–678. 2. Nawrot B et al (2011) Cellular and Molecular Life Sciences. Acknowledgements 3. Suryanarayana et al (2012) Mol Biol Rep 39: 9187–9191. 4. Yong-Ling P et al (2008) Nature 9: 532-542. This work was supported by the National Science Centre grant (2012/05/B/NZ1/00659) and partly by a statutory activity subsidy from Acknowledgements the Polish Ministry of Science and Higher Education for the Faculty of Presented research was financially supported by the National Science Chemistry of Wrocław University of Technology. Centre (NCN) in Poland, grant number 2011/01/B/NZ3/02090 and a statutory fund.

Warsaw, September 9th–12th 2014 164 Session 7. Protein Functions in Cellular Signaling P7.36 P7.37 BAL cellular analysis in the lung ISG’ylation increases stability of hundreds diseases diagnosis using new proteins including STAT1 that prevents intramolecular quenched substrates premature termination of immune for human PR3 and HNE responses in LPS stimulated microglia Jadwiga Popow-Stellmaszyk1, Beata Popławska- Piotr Przanowski1, Stefan Loska1, Dominik Cysewski2, Wiśniewska2, Magdalena Wysocka1, Joanna Chorostowska- Michal Dabrowski1, Bozena Kaminska1 2 1 1 Wynimko , Krzysztof Rolka , Adam Lesner 1Laboratory of Molecular Neurobiology, Nencki Institute of 2 1Department of Biochemistry, Faculty of Chemistry, University of Experimental Biology, Warsaw, Poland; Laboratory of RNA Biology and Gdansk, Gdańsk, Poland; 2Department of Genetics and Clinical Functional Genomics, Institute of Biochemistry and Biophysics, Warsaw, Immunology, National Institute of Tuberculosis and Lung Diseases, Poland Warsaw, Poland e-mail: Piotr Przanowski e-mail: Jadwiga Popow-Stellmaszyk The initiation, progression and termination of brain in- Bronchoalveolar lavage (BAL) still occupies a very impor- flammation requires changes in gene expression, posttrans- tant position in the diagnosis of primary lung diseases of lational protein modifications and regulation of protein various origins: infectious, interstitial, and neoplastic pul- degradation. Microglia are brain resident macrophages monary pathology occurring in the course of other dis- which become activated in most neurological diseases. In- eases [1]. Of these, interstitial lung diseases caused by hu- flammatory activation could be mimicked in primary mi- man neutrophil elastase (HNE) and proteinase 3 (PR3), is a croglial cultures by treatment with lipopolisacharide (LPS). hallmark of numerous pulmonary diseases [2, 3]. Optimal We demonstrated the increase of Uba7 expression – an E1 treatment of respiratory infections requires the establish- enzyme that is crucial for ISG’ylation – posttranslational ment of a pathogenic agent. Obtained during this proce- modification similar to ubiquitination in inflammatory mi- dure data (composition of cells, enzymes and mediators) croglia. ISG’ylation is an important part of anti-viral re- allowed at a large extent explain a number of pathophysi- sponse and inflammatory processes, but unlike ubiquitina- ological pulmonary diseases. However, the limitation of tion, ISG’ylation do not lead to protein degradation and this method and lack of standardization cause the difficulty its function remains unclear. We found the increased level in comparing the results. Thus, other quick, sensitive and of ISG’ylation after LPS stimulation. Silencing of UBA7 uncomplicated diagnostic features are still needed. expression in immortalized BV2 microglial cells led to de- This work is an attempt to design and obtain new, selec- crease in a steady state level of hundreds proteins as dem- tive intramolecular quenched substrates of HNE and PR3 onstrated by mass spectrometry. many of these proteins using combinatorial chemistry approach (split and mix have been described as ISG’ylated proteins in earlier stud- method and Fmoc chemistry) on TentaGel S RAM resin ies. One of such proteins is transcription factor STAT1, as a solid support. The iterative deconvolution in solution which is also a main activator of UBA7 expression. We of such libraries (parallel against both enzymes) allowed us demonstrated that in microglia stimulated by LPS STAT1 to obtain two substrates ABZ-Met-Pro-Val-Ala-Trp-Glu- is ISG’ylated and the levels of both total and phosphor- Tyr(3-NO2)-NH2 for HNE and ABZ-Tyr-Tyr-Abu-Asn- STAT1 decrease after UBA7 silencing. This is accompa- Glu-Pro-Tyr(3-NO2)-NH2 for PR3 hydrolyzed in the pres- nied by premature termination of the immune response, as ence of only one of the desired enzymes. The last step was shown by the reduction of iNos and Ccl5 expression.Those to apply these highly sensitive substrates, perform measure- observations suggest that ISG’ylationincreases stability of ment of concentration of these enzymes in BALF samples many proteins, including STAT1, that prevents premature and see if it is possible to discriminate between different termination of immune responses. lung disorders.

References: 1. Chciałowski A et al (2011) Pneumonol Alergol Pol 79: 2, 75. 2. Prok J-A et al (2005) AJP 167: 651. 3. Drent M et al (1993) Eur Respir J 6: 803. Acknowledgments This work was supported by National Council of Science (NCN) under grant number DEC-2012/07/N/ST5/00178.

BIO 2014 Congress Session 7. Protein Functions in Cellular Signaling 165 P7.38 P7.39 Bacteriocins produced by The crucial role of sphingosine kinases in lactic acid bacteria regulation of dopaminergic cell survival Anna Pyra1, Natalia Jazienicka2, Krzysztof exposed to alfa-synuclein oligomers. J. Pawlik3, Ewa Brzozowska3 Implication in Parkinson's disease 1University of Wroclaw, Faculty of Chemistry, Department Joanna Pyszko, Anna Wilkaniec, Agata of Crystallogrzphy, Wrocław, Poland; 2Wroclaw University of Adamczyk, Joanna B. Strosznajder Environmental and Life Sciences, Faculty of Food Science, Department of Biotechnology and Food Microbiology, Wrocław, Poland; 3Polish Mossakowski Medical Research Centre, Polish Academy of Sciences, Academy of Sciences, Institute of Immunology and Experimental Department of Cellular Signalling, Warsaw, Poland Therapy, Wrocław, Poland e-mail: Joanna Pyszko e-mail: Anna Pyra Alpha-synuclein (ASN) is a presynaptic terminal protein, Bacteriocins are ribosomally synthesized antibacterial mol- involved in the regulation of neuronal plasticity. In oligo- ecules lethal to bacteria other than the producing strain meric form ASN promotes oxidative stress and leads to (Galvez et al., 2007, Joerger 2003). Purified bacteriocins neuronal cell death in Parkinson’s disease (PD). Our previ- could be used as preservatives or for reduction or elimi- ous data indicated that oxidative stress activates ASN re- nation of certain pathogens. Bacteriocins produced by lease from brain synaptoneurosomes. The aim of the cur- lactic acid bacteria (LAB) gained interest because they are rent study was to examine the effect of extracellular ASN in produced by bacteria largely considered beneficial to hu- the native and mutated forms (E46K,A53T, A30P) on gene man health and food production. Bacteriocins produced by expression and activity of sphingosine kinase 1 (Sphk1), LAB have relatively broad spectrum of antimicrobial activ- a key enzyme regulating the balance of the sphingolipids ity and they are not active and not toxic to eukaryotic cells mediators and cell survival/death signaling. This study was (Klaenhammer 1993). They are usually plasmid-encoded carried out using dopaminergic cells: PC12 and human which allows genetic manipulation. The mechanism of ac- SH-SY5Y. The immunochemical, spectrofluorometrical tion is not well known for bacteriocins but the most of the and Real Time PCR methods were applied. Our data indi- low molecular weight bacteriocins appear to interact with cated that extracellular native and mutated ASN (0.5 μM) the bacterial membrane. They display activity or their ac- in monomeric/oligomeric forms down-regulated Sphk1 tivity increases after complexation with other bacteriocins. gene expression/protein level and significantly reduced its The final goal of our research is determination ofthe activity. ASN and its mutated forms decreased dopamin- three-dimensional structures of low-molecular weight bac- ergic cell survival in a concentration dependent manner. teriocins produced by LAB. It helps to know the mode of Subsequently, pharmacological Sphks inhibition, by using action of these small proteins. The genes of selected bacte- SKI II (5 µM) evoked enhancement of ASN secretion, ac- riocins were cloned into several types of expression vectors tivation of gene expression for pro-apoptotic Bcl-2 pro- and try to overexpressed using different strains of bacterial teins: Bax and BH3-only protein Hrk, cytochrome c release cells. These genes were modified by cutting of the short from mitochondria and caspase-dependent degradation of nucleotide sequences corresponding to signal-peptides in poly(ADP-ribose) polymerase (PARP-1). Reduced Sphk1 protein. gene expression and activity was observed in our previ- For our experiments, we chose bacteriocins completely ous study, using 1-methyl-4-phenylpyridinium PD model. unknown in terms of 3D structure. It is worth to note In this stress exogenous S1P (1 µM) exerted neuroprotec- that there is no X-ray structure of bacteriocin produced tive effect, which was dependent on the S1P1 receptor by LAB in Protein Data Bank (PDB). There are only 23 stimulation. Summarizing, extracellular ASN in oligomeric bacteriocin’s NMR structures synthesized by LAB. In those form decrease gene expression and activity of Sphk1. On NMR structures the N-terminal region is usually followed the other hand, Sphks inhibition leads to enhancement of by a well-defined central amphiphilic a-helix followed by ASN secretion and to caspase-dependent apoptotic neu- less well defined a-helix in the C-terminal. ronal death. Therefore, our results show the close relation- Here we present the results of cloning and overexpression ship between extracellular ASN and Sphks inhibition and of selected bacteriocins produced by Lactobacillus sp. indicate Sphk1 as a molecular switch of neuronal cell sur- vival and death. S1P and agonists of S1P receptor offer References: the promising strategy against ASN toxicity and should be Galvez A et al (2007) Int J Food Microbiol 120: 51–70. considered in PD therapy. Joerger RD (2003 ) Poult Sci. 82: 640–647. Klaenhammer TR (1993) FEMS Microbiol Rev. 12: 39–85. Acknowledgements Supported by NCN grant 5870/B/PO1/2011/40 and by The European Union under The European Social Fund.

Warsaw, September 9th–12th 2014 166 Session 7. Protein Functions in Cellular Signaling P7.40 P7.41 Redistribution of CD14 in LPS- Do LTA, UEA, MAA and PNA lectins stimulated macrophages influence binding of Helicobacter Agnieszka Płóciennikowska, Mykola I. Zdioruk, Gabriela pylori to gastric mucins? Traczyk, Andrzej Sobota, Katarzyna Kwiatkowska Iwona Radziejewska1, Małgorzata Borzym- 2 3 Nencki Institute of Experimental Biology, Warsaw, Poland Kluczyk , Katarzyna Leszczyńska e-mail: Agnieszka Płóciennikowska 1Medical University of Białystok, Department of Medical Chemistry, Białystok, Poland; 2Medical University of Białystok, Department of CD14 is an abundant cell surface glycoprotein of mac- Pharmaceutical Biochemistry, Białystok, Poland; 3Medical University of rophages and other immune cells. It possesses a glyco- Białystok, Department of Microbiology, Białystok, Poland sylphosphatidylinositol (GPI) linker which anchors CD14 e-mail: Iwona Radziejewska within cholesterol- and sphingolipid-rich microdomains of the plasma membrane. CD14 serves as an acceptor of li- Helicobacter pylori colonizes the gastric mucosa of about half popolysaccharide (LPS), a major constituent of the outer of the world’s population and is responsible for gastroduo- membrane of Gram-negative bacteria. CD14 transfers denal diseases such as chronic gastritis, gastric and duo- LPS to a complex of MD-2 protein and toll-like receptor 4 denal ulcers and also gastric cancers. It seems to be clear (TLR4). Activated TLR4 triggers signaling pathways which that both bacterial virulence factors and host susceptibility engage MyD88 or TRIF adaptor proteins and lead to ex- features play a role in the development of infection. H. py- pression of pro-inflammatory cytokines. To reveal mech- lori colonizes the gastric mucosa by adhering to the mucous anisms which govern an involvement of CD14 in TLR4 epithelial cells and the mucous layer lining the epithelium. signaling, we followed dynamics of CD14 in LPS-stimulat- The best defined bacterial adhesins are the blood group ed macrophages. Laser scanning cytometry analysis showed binding adhesin (BabA) with affinity to Lewis b and H type that stimulation of cells with LPS led to a moderate eleva- 1 antigens and sialic acid binding adhesin (SabA) that binds tion of the amounts of CD14 on the surface of J774 cells. sialyl dimeric Lewis x. Human Lewis antigens represent ter- LPS is likely to up-regulate trafficking of CD14 toward the minal modifications on mucins. Lewis b structure on MUC plasma membrane from Golgi apparatus, as indicated by 5AC mucin seems to be the best proved receptor for H. confocal microscopy analysis. In agreement, biochemical pylori adhesins. Recently, the involvement of MUC 1 mucin analysis revealed that CD14 was enriched in raft fractions in the interaction with the bacterium has been proposed at the onset of cell stimulation with LPS. To get insight into [1, 2]. Lewis blood antigens are also expressed on the O- CD14 distribution in the plane of the plasma membrane, specific chains of the lipopolysaccharides (LPS) ofH.pylori . large sheets of the membrane were obtained by mechanical This phenomenon can be understood as a kind of molecu- cleavage of cells and subjected for immunogold labeling lar mimicry between bacteria and host. This, in turn, may and ultrastructural analysis. Prior to LPS stimulation, CD14 result in immune tolerance against antigens of the patho- was dispersed in the plane of the plasma membrane and gen and can facilitate colonization. Moreover, it is known occupied its invaginations where caveolin 1, but not clath- that H. pylori strains are able to adapt their outer membrane rin, was found. Stimulation of cells with 100 ng/ml LPS expression profile according to alterations in host environ- induced transient clustering of CD14 in the plasma mem- ment, including changes in mucosal glycosylation patterns, brane reflected by a shift of CD14-attributed gold labels by switching on and off specific gene expression [3]. from singlets to aggregates. CD14 clustering was fast and The main aim of our study was to assess the effect of ad- after 5 min of LPS action the amount of singular gold par- dition of four lectins on binding of H. pylori to gastric ticles marking CD14 was reduced by 42 % and more than mucins. The lectins used in our experiments have high 17% of CD14 was accumulated in clusters of at least 10 specificity to exact carbohydrate structures (that can be particles. CD14 clusters co-localized with PI(4,5)P2, a plas- present on both mucins and H. pylori lipopolysaccharides. ma membrane lipid recruiting adaptor proteins crucial for LTA (from Lotus tetragonolobus seeds) and UEA (from Ulex TLR4 signaling. Specificity of LPS action was confirmed europaeus seeds) bind αFuc, MAA (from Maakia amurensis using antibody which inhibited binding of LPS to CD14- seeds) binds Neu5Acα3Galβ4GalNAc and PNA (from clustering of CD14 was abolished in these conditions. No Arachis hypogaea peanuts) binds GalβGalNAc. clustering of GM1, raft-enriched ganglioside, was induced The study was carried on gastric juices taken from patients by LPS. Taken together, our studies indicate that clustering after eradication treatment and on babA2 genopositive and of CD14 is an initial step during stimulation of cells with babA2 genonegative H. pylori strains. To assess the binding LPS and CD14-enriched regions of the plasma membrane of bacterium to gastric mucins ELISA test was used. are involved in an initiation of signaling cascades of TLR4. We assumed that lectins used in the study could “block” potential carbohydrate receptors (on mucins) for bacterial adhesins. However, such inhibition was not observed in all examined samples.

References: Linden SK et al (2009) PLoS Pathog 5: e1000617. Guang W et al (2010) J Biol Chem 285: 20547–20557. Sheu SM et al (2007) Infect Immun 75: 2661–2667.

BIO 2014 Congress 167 Session 7. Protein Functions in Cellular Signaling 167 P7.42 P7.43 Personalized therapy of chronic Mechanism of inhibition of human platelet lymphocytic leukemia aggregation by Lys-plasminogen Małgorzata Rogalińska1, Paweł Góralski2, Jerzy Z. Yana M. Roka-Moiia, Dmytro D. Zhernossekov Błoński3, Jan Barciszewski4, Karolina Tarnowska1, Marta 1 5 6 Palladin Institute of Biochemistry of the National Academy of Sciences Wawro , Katarzyna Woźniak , Aneta Kocewa-Chyła , of Ukraine, Department of Enzyme Chemistry and Biochemistry, Henryk Piekarski2, Tadeusz Robak3, Zofia M. Kilianska1 Ukraine e-mail: Yana Roka-Moya 1University of Lodz, Faculty of Biology and Environmental Protection, Department of Cytobiochemistry, Łódź, Poland; 2University of Lodz, Faculty of Chemistry, Department of Physical Chemistry, Łódź, Poland; Plasminogen/plasmin system takes part not only in fi- 3Medical University of Lodz, Department of Hematology, Łódź, Poland; brinolysis but also in regulation of functional state of dif- 4Polish Academy of Science, Institute of Bioorganic Chemistry, Poznań, ferent cells. Platelet membrane can provide a surface for Poland; 5University of Lodz, Faculty of Biology and Environmental Protection, Department of Molecular Genetics Łódź, Poland; 6University assembly of plasminogen and its activators. On the surface of Lodz, Faculty of Biology and Environmental Protection, Department of certain blood cells Glu-plasminogen is transformed into of Thermobiology, Łódź, Poland Lys-form, which possesses open conformation and can e-mail: Małgorzata Rogalińska be more easily activated with the plasmin formation. We showed that exogenous Lys-plasminogen but not its native B-chronic lymphocytic leukemia (CLL) is a common type Glu-form inhibits platelet aggregation stimulated by ADP, of leukemia in Europe and North America with growing thrombin and collagen. The aim of this work is to define incidence of disease for younger people. the role of certain structural domains of plasminogen mol- The general goal of therapies directed towards lymphopro- ecule in the observed inhibitory effect.One of the possible lipherative disorders, like CLL, is elimination of leukemic mechanisms of plasminogen interaction with the surface cells by apoptosis induction. Due to unpredictable clinical receptors is the binding of carboxyl-terminal lysines by picture and personal patient’s differences in anti-cancer lysine-binding sites (LBS) of plasminogen molecule. In our treatment sensitivity, establishing optimal therapy for this experiments lysine analogue, 6-aminohexanoic acid abol- type of leukemia sometimes reflects difficulties. Therefore, ishes above mentioned inhibitory effect.Serine protease in- a special importance in case of patient’s resistance to ther- hibitor, aprotinin (5.5 IU/ml) does not make any influence apy is to search drug administration with potency to elimi- on the inhibitory effect of Lys-plasminogen during throm- nate in vitro leukemic cells from patient’s peripheral blood. bin-induced platelet aggregation. The used concentration The comparative analysis of CLL cells incubated with of aprotinin has no effect on platelet aggregation.Prein- anticancer agents (purine analogs combine with alkylat- cubation of washed platelets with K1-3, K4 and K5 taken ing agent mafosfamide or monoclonal antibody – rituxi- into equimolar concentration with Lys-plasminogen com- mab) by cytometric analysis (cell viability, apoptosis level), pletely abolishes inhibitory effect of the last one. The plas- thermal profiles by differential scanning calorimetry, and minogen kringle K5 added to the reaction mixture (washed protein expression related to apoptosis (PARP and Mcl-1), platelets and 1.2 µM Lys-plasminogen) even at concentra- could be useful in the most effective drug treatment choice tion 0.12 µM recovers platelet aggregation till 80%, where- or even monitoring patient’s treatment in vivo. as K1-3 and K4 at this concentration reach only near 60% Because of personal differences between patients in dis- as compared as control aggregation level. Plasminogen ease dynamics and the response to drugs registered to cure kringles K1-3, K4 and their combination have no influence of this type of leukemia, it seems to be important to per- on aggregation of washed platelets. There was no change sonalize therapy by choosing potentially effective type of in platelet aggregation in case of mini-plasminogen (K5 treatment with ability to induce apoptosis before its in vivo plus serine protease domain), where catalytic activity was administration to reduce ineffective patient’s response to inhibited by aprotinin or p-nitrophenyl-p'-guanidine ben- anticancer therapy. zoate.The observed effect of plasminogen kringles on the inhibition of platelet aggregation by Lys-plasminogen let us suggest that LBS of plasminogen kringles provide the plasminogen binding to the adhesive proteins of platelet surface (e.g. thrombospondin, vitronectin and fibrinogen). These proteins are secreted from α-granules and remain bound to platelet surface. The binding of plasminogen kringles to these proteins may lead to the disturbance of protein-protein interaction which is the necessary condi- tion for efficient platelet aggregation.

Warsaw, September 9th–12th 2014 168 Session 7. Protein Functions in Cellular Signaling 168 P7.44 P7.45 The Membrane-Type Matrix Does CacyBP/SIP influence the level Metalloproteinases MT3-MMP and MT5- of p-CREB via MAP kinase cascade? MMP in human umbilical cord blood Sara Rosińska, Wiesława Leśniak, Anna Filipek Lech Romanowicz, Zofia Galewska, Tomasz Nencki Institute of Experimental Biology PAS, Warsaw, Poland Gogiel, Krzysztof Sobolewski e-mail: Sara Rosinska Medical University of Białystok, Department of Medical Biochemistry, Białystok, Poland CacyBP/SIP has been shown to dephosphorylate ERK1/2 e-mail: Lech Romanowicz kinase which, in consequence, resulted in lower activity of the Elk-1 transcription factor (Kilianczyk E et al., 2011, The vascular system of mother and placenta plays an Biochem Biophys Res Commun). Since CacyBP/SIP is highly important role in the intrauterine development of the expressed in brain (Schneider & Filipek, 2011, Amino Ac- fetus. Preeclampsia is a conditions characterized by sys- ids) in the present work we examined the influence of temic vascular endothelial dysfunction. Metalloproteinases CacyBP/SIP overexpression on transcription factors en- (MMPs) are enzymes engaged in degradation of collagen gaged in different aspects of neuronal activity such as SRF, and protein cores of proteoglycans, including those which CREB, AP-1, STAT-3, NFĸB and NFAT. We used mouse bind peptide growth factors. The Membrane-Type Matrix neuroblastoma NB2a cells and applied two methods, dual Metalloproteinases (MT-MMPs) are extracellular matrix- luciferase reporter assay and Western blot analysis. Since degrading endopeptidases. Thus they exhibit a broad spec- preliminary data showed that CacyBP/SIP overexpression trum of substrate degradation including activation of some had a strongest effect on CREB (cAMP response element- secreted MMPs such as MMP-2 (gelatinase A). MMP-2 is binding) protein activity, we focused on analyzing this tran- the main collagenolytic enzyme of both umbilical artery scription factor in NB2a cells under normal conditions and and vein. The activation of proMMP-2 is mediated on the after induction of cells with KCl, agent which increases cell surface by MT1, 2, 3 and 5-MMP. We decided to evalu- the level of intracellular Ca2+ ions and the level of phos- ate the presence, content and activity of MT3-MMP and phorylated form of CREB (p-CREB). MT-5MMP in the umbilical cord blood of preeclamptic Using Western blot method we found that the level of p- patients. Since some MMPs may be released from blood CREB after CacyBP/SIP overexpression in NB2a cells was cells during blood clotting it was decided to compare these inhibited by about 50% and 75% in control and KCl treated proteinases in both the umbilical cord plasma and serum cells, respectively. Moreover, the luciferase reporter assay of healthy newborns and those delivered by mothers with revealed that the activity of CRE sequence was decreased preeclampsia. We used Western Immunoblot method and by 15% and 33% in control and KCl treated cells, respec- immunoenzymatic assay (ELISA) for detection of both tively. metalloproteinases. Since CacyBP/SIP is considered to be a member of mi- Umbilical cord blood plasma and serum of control and togen kinase phosphatase family (MKP), we analyzed its preeclamptic newborns contained MT3-MMP and MT5- influence on the level of phosphorylated ERK1/2 (p- MMP. Both enzymes existed in form of complexes with ERK1/2) and phosphorylated p38 (p-p38) kinases. The other extracellular matrix components and/or their tissue results showed that activity both kinases decreased after inhibitors in control and preeclamptic subjects. Free latent CacyBP/SIP overexpression. The activity of ERK1/2 was form of both MT-MMPs were observed after the action of diminished by about 55% and 45% in control and KCl in- reducing agent. Only trace of free active enzymes existed in duced cells, respectively, while the activity of p38 was lower control material. Furthermore, we found a huge increase in by about 75% and 56%, respectively. Therefore, our results the amount of MT3-MMP in preeclamptic umbilical cord indicate that CacyBP/SIP has an effect on CREB activity blood. MT5-MMP content increased also but in a lesser in neuroblastoma NB2a cells and suggest that the observed degree. Earlier studies showed no significant changes in effects are due to down-regulation of the MAP kinase cas- MMP-2 active form presence in preeclamptic umbilical cade. cord blood compared to control subjects. Simultaneous decrease in MT1-MMP observed earlier with an increase Acknowledgements in content of both investigated MT-MMPs suggested the This work was supported by grant from the National Science Center switch of main activating agent of MMP-2 between those (NZ3/00425) and by statutory funds from the Nencki Institute of Ex- membrane type metalloproteinases in preeclampsia. perimental Biology.

BIO 2014 Congress Session 7. Protein Functions in Cellular Signaling 169 P7.46 P7.47 Identification of palmitoylated The role of tissue-nonspecific proteins involved in TLR4 signaling alkaline phosphatase and Justyna Dembińska, Paula Roszczenko, Kinga phospholipase D in mineralization Borzęcka, Katarzyna Kwiatkowska Monika Roszkowska1, Agnieszka Strzelecka- 1 2 1 Nencki Institute of Experimental Biology, Warsaw, Poland Kiliszek , David Magne , Slawomir Pikula e-mail: Katarzyna Kwiatkowska 1Nencki Institute of Experimental Biology, Department of Biochemistry, Warsaw, Poland; 2Université Claude Bernard Lyon 1, Institut de Palmitoylation is a post-translation modification of pro- Chimie et Biochimie Moléculaires et Supramoléculaires UMR CNRS teins via a covalently bound palmitic acid (C:16) which is 5246, Villeurbanne, France catalyzed by palmitoyl acyltransferase. In eukaryotic cells, e-mail: Monika Roszkowska the acyl chain is transferred onto thiol group of cysteine residues (S-palmitoylation) or, less frequently, onto hydrox- Although it is currently accepted that tissue-nonspecific yl group of serine and threonine residues (O-palmitoyla- alkaline phosphatase (TNAP) is responsible for the initia- tion). This reversible modification controls translocation tion of mineralization [1], the function of phospholipase D of proteins to plasma membrane microdomains enriched (PLD) in this process is still unclear. PLD is able to release in cholesterol and sphingolipids (rafts), their stability, cel- phosphatidic acid which may promote budding of matrix lular trafficking of proteins and protein-protein interac- vesicles at the early stages of mineralization as well as sub- tions. We found that protein palmitoylation is crucial for sequent release of hydroxyapatite (HA) to the extracellular pro-inflammatory signaling triggered by Toll-like receptor matrix (ECM) [2]. 4 (TLR4) in cells stimulated with lipopolysaccharide (LPS). Physiological mineralization is a process by which HA is An exposure of RAW264 macrophage-like cells to 50-250 deposited in the ECM of bone tissue, whereas pathologi- µM of 2-bromopalmitic acid (BPA), an inhibitor of pal- cal mineralization occurs in soft tissues, especially in blood mitoyl acyltransferase, reduced in a dose dependent man- vessel walls. Vascular calcification, the most dangerous type ner the production of pro-inflammatory cytokines, TNF-α of pathological mineralization, is a common phenomenon and RANTES, stimulated with 100 ng/ml LPS. Therefore, in aging. However, excessive calcification of blood vessels we aimed to identify proteins which are palmitoylated in may also occur at earlier stages of life and have a dramatic LPS-stimulated RAW264 cells. Traditional methods to impact on function of cardiovascular system. It is likely study protein S-palmitoylation rely on isotopic labeling of that mechanisms of physiological and pathological miner- cells, which requires special safety precautions and is time- alization are similar. Numerous data suggest that vascular consuming. We have applied a recently developed non- smooth muscle cells (VSMCs) are able to “trans-differenti- radioactive technique based on a “click chemistry” in two ate” into mineralization-competent cells, to express TNAP parallel approaches. In the first one, living RAW264 cells and finally, to mineralize ECM [3]. Pathological mineraliza- were labeled with ω-alkynyl-palmitate analog of palmitic acid tion accompanies common diseases such as atherosclero- (17-ODYA), and after lysis, subjected to a “click-it” reac- sis, diabetes type 2, chronic kidney disease and bone tissue tion with azido-azo-biotin catalyzed by Ca2+. Subsequently, cancers. labeled proteins were bound to streptavidin beads and elut- The objective of this research was to compare mineraliza- ed with sodium dithionite which cleaved the azido-azo-bi- tion properties of two mineralizing cell lines: human fetal otin, and analyzed by immunoblotting. With this technique osteoblasts (hFOB 1.19) and human bone osteosarcoma it was found that LPS induced palmitoylation of several cells (Saos-2). Cells were stimulated for mineralization by proteins, including Lyn tyrosine kinase of the Src family. osteogenic factors, ascorbic acid and β-glycerophosphate. Palmitoylation of these proteins was inhibited in the pres- The amount of released HA was measured in stimulated ence of BPA. The second approach of detection of palmi- cells compared to resting cells by Alizarin Red Staining. The toylated protein in LPS-stimulated cells was based on the level of TNAP activity was determined by ALP ELISA As- fact that the activation of TLR4 by LPS occurs via CD14 say. In addition, a common inhibitor of TNAP – levami- located in plasma membrane rafts. Therefore, we decided sole - was applied. Furthermore, the role of phospholipase to perform immunoprecipitation of CD14, preceded by la- D (PLD) was examined in these cell lines. The activity of belling of RAW264 cells with 17-ODYA and followed by PLD was determined by use of Amplex Red PLD Assay “click chemistry” in order to detect palmitoylated proteins Kit. The two inhibitors of PLD were applied - ethanol and which associate with CD14 in LPS-treated cells. Identifica- 1-butanol. Our findings demonstrated that the activities of tion of these proteins will involve the mass spectrometry both enzymes, TNAP and PLD, are at a lower level in nor- methodology. mal cells compared to tumor cells.

References: 1. Hessle L et al (2002) Proc Natl Acad Sci USA 99: 9445–9449. 2. Mebarek S et al (2013) Int J Mol Sci 14: 5036–5129. 3. Shao J S et al (2006) Arterioscler Thromb Vasc Biol 26: 1423–1430. Acknowledgements The work was supported by a grant N401 140639 from Polish Ministry of Science and Higher Education.

Warsaw, September 9th–12th 2014 170 Session 7. Protein Functions in Cellular Signaling P7.48 P7.49 The effectiveness of chemotherapy and Effect of heme oxygenase-1 on electrochemotherapy in human ovarian C/EBPδ and C/EBPβ in myoblasts cell lines (OvBH-1 and SKOV-3) in vitro and rhabdomyosarcoma cell lines Jolanta Saczko1, Justyna Piłat1, Julia Bar2, Anna Marta Seczyńska, Maciej Cieśla, Mateusz Jeż, Choromańska1, Nina Rembiałkowska1, Aleksandra Kuzan1, Magdalena Kusior, Magdalena Kozakowska, Iwona Kamińska2, Małgorzata Kotulska3, Julita Kulbacka1 Paulina Marona, Józef Dulak, Alicja Józkowicz 1Department of Medical Biochemistry, Wroclaw Medical University, Department of Medical Biotechnology, Faculty of Biochemistry, Wrocław, Poland; 2Department of Pathomorphology and Clinical Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland Cytology, Medical University, Wrocław, Poland; 3Institute of Biomedical e-mail: Marta Seczyńska Engineering and Instrumentation, Wroclaw University of Technology, Wrocław, Poland Myogenic, adipogenic and osteogenic precursors originate e-mail: Jolanta Saczko from primitive mesenchymal stroma cells (MSCs). Muscle derived tumors, rhabdomyosarcoma(RMS), can exhibit Ovarian cancer is one of the common and most lethal traces of osteogenic and adipogenic differentiation in place cause of gynecological tumor. Currently the aggressive of proper myogenesis. A group of transcription activators surgery and standard chemotherapy are widely applied to regulating these processes are CCAAT enhancer binding improve the survival rate in patients with this cancer. The proteins (C/EBPs), with C/EBPb which may attenuate prognosis remains poor due to ovarian cancers are diag- myogenesis, and C/EBPd which may promote it. Recent- nosed in advanced stages. Thus there is a need for new ly we have reported that heme oxygenase-1 (HO-1), the therapeutic methods. Electrochemotherapy (ECT) is one cytoprotective enzyme which degrades heme to produce of the new technique based on combination of electropo- biliverdin, iron ions and carbon monoxide (CO), inhibits ration (EP) and chemotherapy. myoblast differentiation, and is elevated in RMS with less The aim of our investigation was the evaluation of the favorable outcome. Here we analyzed the effect of HO-1 electrochemotherapy effectiveness in comparison to stand- on C/EBP expression and activity. ard chemotherapy with bleomycin in two varian cancer cell All studies were performed in two in vitro models: in C2C12 lines. Two human ovarian cells lines were used: OvBH-1 murine myoblasts and in six RMS cell lines with ranging — human clear ovarian carcinoma with silent mutation of clinical aggressiveness. Some cell lines were genetically P53 gene and resistant to chemo- and radiotherapy and modified using retroviral or lentiviral vectors to overex- SKOV-3 line — human ovarian carcinoma cells resistant to press the enzymatically active or inactive form of HO-1. diphtheria toxin, cisplatin and adriamycin. In EP and ECT According to our findings, the C2C12 myoblasts with in- experiments different voltage values (from 0 to 1300 V/ creased level of HO-1 expression display the decrease in cm2), 8 pulse duration of 100μs, intervals between pulses nuclear C/EBPδ isoform as demonstrated by western blot- 1s were used. The chemotoxicity of applied treatments was ting, and decrease in binding of C/EBPδ to MyoD pro- determined by MTT assay. The expression of heat shock moter, as shown by chromatin immunoprecipitation. This protein — HSP27 was examined by immunocytochemistry phenomenon can be mimicked by incubation of cells with and by western blot method. carbon monoxide releasing molecule (CORM). Impor- The cytotoxicity with different concentrations of bleo- tantly, overexpression of C/EBPδ reverses the inhibitory mycin was not significant decrease in both cell lines. The effect of HO-1 on myoblast differentiation, resulting in highest decrease was observed after EP with bleomycin af- upregulation of MyoD and its downstream targets such as ter 72h of incubation for 1200 (V/cm2). The intensity of myogenin or myosin, and finally in formation of polynu- expression of small heat shock proteins HSP27 increased cleated myotubes (Kozakowska et al., 2012, Antioxid Redox after ECT in both treated cell lines. The results indicated Signal). that electroporation effectively supports chemotherapy In RMS we were not able to show differential expression with bleomycin on human ovarian cells in vitro. of C/EBPd between cell lines of different aggressiveness. Key words: ovarian cancer, bleomycin, electroporation, Instead, we found that C/EBPb isoform is upregulated in HSP27. the malignant RMS cell lines. Moreover, forced overexpres- sion of enzymatically active HO-1 in RMS led to upregula- tion of proproliferative C/EBPβ. This was accompanied by the decrease in levels of myosin, marker of myogenic differentiation. In contrast, forced overexpression of en- zymatically inactive HO-1 (H25A HO-1) was ineffective. To sum up, HO-1 regulates transcriptional activity of C/ EBPδ in myoblast cell line. In RMS the more important seems to be the effect of HO-1 on C/EBPβ isoform.

BIO 2014 Congress Session 7. Protein Functions in Cellular Signaling 171 P7.50 P7.51 Biophysical properties of hippocalcin Bovine β-casomorphin-5 levels signaling in rat hippocampal neurons determined in serum and urine of autistic Y. Y. Sheremet, V. P. Cherkas, A. V. Dovgan, children (autism spectrum disorders) N. I. Kononenko, T. M. Tsugorka, P. V. Belan Natalia Smulska1, Michał Matysiewicz1, 1 2 Bogomoletz Institute of Physiology, Department of General Physiology Marta Bukało , Barbara Chwała , Małgorzata of the Nervous System, Ukraine Moszyńska-Dumara2, Elżbieta Kostyra1 e-mail: Yevhenii Sheremet 1University of Warmia and Mazury, Faculty of Biochemistry, Department 2 2+ of Biology & Biotechnology, Olsztyn, Poland; Regional Children’s Hippocalcin is a neuronal Ca sensor protein that signals Specialized Hospital, Olsztyn, Poland in hippocampal neurons via translocation from the cyto- e-mail: Natalia Smulska sol to the plasma membrane. In spite of importance of this signalling for many different cellular functions precise Autism Spectrum Disorders (ASDs) are defined as a group biophysical mechanisms of hippocalcin signalling are still of developmental disabilities characterised by impairment unknown. In this study Lux-FRET approach was used in social interactions and communication, and repetitive, to measure spatio-temporal pattern of hippocalcin inser- stereotypical patterns of behaviour. ASDs, according to tion to the plasma membrane during translocation. We The Autism and Developmental Disabilities Monitoring found some specific sites on the dendritic plasma- mem (ADDM) Network, affect about 1 in 88 children in US brane, sized from diffractionally limited to several microns, (data for 2008). where the local hippocalcin insertion to the plasma mem- The opioid-excess hypothesis of ASDs suggests that this brane was higher than in neighbouring sites. We checked dysfunction is the consequence of the incomplete break- whether translocation at these specific sites are associated down and excessive absorption of peptides with opioid ac- with regions of higher intracellular free calcium concen- tivity causing disruption to neuroregulatory and biochemi- 2+ 2+ tration ([Ca ]i). Creating spatially uniform [Ca ]i tran- cal processes. Currently, scientist’s attention is drawn to the sients in dendritic segments, we showed that hippocalcin similarity between the action of the opiates and symptoms translocation was significantly different in neighbouring of the ASDs. It has been proven that children who suffer sites having the same (in terms of kinetics and amplitude) from autism have increased the levels of opioid peptides, patterns of [Ca2+] changes. Producing long-lasting eleva- which are occurred in the central nervous system in hu- 2+ i 2+ tions of [Ca ]i by activation of different Ca mobilizing mans. It was found that appearance of β-casomorphin-5 in mechanisms, we also demonstrated that hippocalcin trans- the diet is leads to development of many diseases and psy- location was observed in the same set of sites indepen- chological disorders. In significant number of people who 2+ 2+ dently of Ca sources. These results indicate that [Ca ]i suffer from autism is application of milk-free and gluten- is not the only determinant of hippocalcin translocation free diet lessens clinical symptoms of ASDs. and that local differences in the plasma membrane affinity According to one of theories disruptions in opioid system for hippocalcin are an important biophysical mechanism function may be possible cause of so called autistic behav- of hippocalcin signaling. Furthermore we have also de- iour. Elevated levels of both endogenous and exogenous veloped original approaches for quantitative separate and (β-casomorphins; BCMs) opioid peptides were reported in simultaneous measurement of hippocalcin concentration sera of ASDs patients. in cytosolic and membrane cellular fractions of single liv- All experiments were approved by the Bioethics Commit- ing hippocampal neurons. Based on these approaches and tee. For the research we used the serum and urine of the simulation of Ca2+ and hippocalcin diffusion in the den- children with diagnosed ASD. The samples were taken drites and spines of hippocampal neurons we have shown from Regional Children’s Specialized Hospital in Olsztyn. that hippocalcin concentration in dendritic membranes can The aim of this study was to determine the content of be many times locally increased during intrinsic patterns of β-casomorphin-5 in the serum and urine of children, who neuronal activity. Using FRAP-studies, we have obtained suffer from autism. diffusion coefficients of hippocalcin in the cytosol and in Samples of serum and urine were tested for the pres- the plasma membrane of dendrites, the kinetic constant of ence of β-casomorphin-5 with ELISA test. We observed hippocalcin insertion into the plasma membrane and other elevated levels of β-casomorphin-5 in serum in children parameters. These data allowed us to construct a quantita- with diagnosed ASD. We also demonstrated presence of tive biophysical model of hippocalcin cellular signaling in β-casomorphin-5 in urine of children with ASDs. Never- hippocampal neurons. We conclude that hippocalcin may theless, opiate dependence and autism disorders in children serve as a site specific messenger with a high dynamic range should be confirmed by further studies. allowing precise modulation of its targets.

Warsaw, September 9th–12th 2014 172 Session 7. Protein Functions in Cellular Signaling P7.52 P7.53 The role of apolipophorin III in insect Nucleic acid chaperone activity immune response to α-(1,3)-glucan of retroviral proteins Sylwia Stączek1, Agnieszka Zdybicka-Barabas1, Aneta Katarzyna Pachulska-Wieczorek1, Agnieszka K. Sowa-Jasiłek1, Adrian Wiater2, Małgorzata Pleszczyńska2, Stefaniak1, Katarzyna J. Purzycka1,Leszek Błaszczyk2, Jerzy Wydrych3, Paweł Mak4, Małgorzata Cytryńska1 Marcin Biesiada2, Ryszard W. Adamiak1,2 1Department of Immunobiology, 2Department of Industrial 1Institute of Bioorganic Chemistry Polish Academy of Sciences, Microbiology, 3Department of Comparative Anatomy and Departament of Structural Chemistry and Biology of Nucleic Acids, Anthropology, Faculty of Biology and Biotechnology, Maria Curie- Group of RNA Structure and Function, Poznań, Poland; 2Poznan Skłodowska University in Lublin, Lublin, Poland; 4Department of University of Technology, Institute of Computing Science, Poznań, Analytical Biochemistry, Faculty of Biochemistry, Biophysics and Poland Biotechnology, Jagiellonian University in Krakow, Kraków, Poland e-mail: Agnieszka Stefaniak e-mail: Sylwia Stączek Apolipophorin III (apoLp-III), an important component of Proteins with nucleic acid chaperone activity facilitate nu- insect hemolymph, is considered as insect homologue of hu- cleic acids folding by destabilizing misfolded, kinetically man apolipoprotein E. This multifunctional protein, involved trapped structures and enabling the formation of the ther- in lipid transport and immune response in insects plays i.a. modynamically most favored form. The HIV nucleocapsid a role of a patterns recognition receptor. The binding of domain of Gag and mature nucleocapsid protein (NC) act pathogen associated molecular patterns (e.g. β-glucan, LPS, as a nucleic acid chaperone and are involved in critical steps lipoteichoic acid) is followed by cellular and humoral response of HIV replication, such as primer tRNA annealing, reverse activation, where apoLp-III has documented contribution. transcription, vRNA dimerization and packaging, virion as- ApoLp-III acts also as a signalling molecule transmitting the sembly and proviral integration into host DNA. Also Ty1 information about the infection to the hemocytes. This pro- retrotransposon Gag protein lacking canonical NC domain tein may change the localization in the hemocytes in response is able to chaperone reverse transcription and dimerization. to the penetration of pathogens into the body of an insect. In addition, the transactivator of HIV transcription – Tat Despite the comprehensive knowledge of apoLp-III par- regulatory protein, may influence the remodelling and an- ticipation in immune response to different pathogens and nealing of nucleic acids during viral splicing and reverse PAMPs, there is no information about apoLp-III involve- transcription. However it remains unclear if Tat protein ment in immune response to α-(1,3)-glucan. From our displays nucleic acids chaperone activity. studies, it is known that α-(1,3)-glucan, an important com- We have comparatively investigated the in vitro nucleic acid ponent of fungal cell wall, is recognized by insect immune chaperone properties of diverse retroviral and retrotrans- system and triggers different types of immune reactions. poson proteins: NC, Gag, Tat. We determined the ability We investigated apoLp-III participation in insect immune of those proteins to chaperone nucleic acid aggregation, response to α-(1,3)-glucan using the greater wax moth Gal- annealing and strand exchange in duplex structures. Two leria mellonella as a model organism. The objectives of our different experimental models were used to assay the in vitro research were as follows: (i) testing ability of apoLp-III to chaperone activity of proteins. The first model mimics the binding α-(1,3)-glucan, (ii) checking the influence of α-(1,3)- annealing of the (-)ss DNA strand and acceptor RNA at glucan injection to insect hemocoel on apoLp-III level in the viral 3’ UTR during the first strand transfer of reverse hemolymph, (iii) determination of changes of apoLp-III transcription. The second corresponds to the placement of localization in the hemocytes in response to α-(1,3)-glucan. tRNALys3 on primer binding site (PBS) of HIV-2 RNA. In vitro incubation of G. mellonella hemolymph with α-(1,3)- glucan isolated from Aspergillus niger revealed binding of Acknowledgements apoLp-III to α-(1,3)-glucan molecules. This observation indi- This study was supported by grants from the National Science Center cated that apoLp-III acts as PRR recognizing fungal α-(1,3)- [2011/01/D/NZ1/03478] and Ministry of Science and Higher Educa- glucan. The changes in the level of apoLp-III after immuniza- tion [IP2012049272]. tion of G. mellonella larvae with α-(1,3)-glucan were examined using SDS-PAGE electrophoresis, as well as immunoblotting with anti-apoLp-III antibodies. The level of apoLp-III in the hemolymph of the α-glucan-challenged larvae changed de- pending on the dose of α-(1,3)-glucan and on the time after the immunization. For apoLp-III immunolocalization, the adherent hemocytes obtained at a different time after immunization of the larvae with α-(1,3)-glucan were incubated with anti-G. mellonella- apoLp-III antibodies and secondary FITC-labelled antibodies. The hemocytes were then imaged in a laser scanning confo- cal microscope. The observed changes in the localization of apoLp-III in the hemocytes after challenge of G. mellonella with α-(1,3)-glucan suggested apoLp-III involvement, as a sig- nalling molecule, in activation of immune reactions. All of these results indicate that apoLp-III actively partici- pates in insect immune response to fungal infections through recognizing and signalling the presence of α-(1,3)-glucan.

Acknowledgements The work was supported by grant 2013/09/N/NZ6/00838 (UMO- 2013/09/N/NZ6/00838) from the National Science Center, Poland.

BIO 2014 Congress Session 7. Protein Functions in Cellular Signaling 173 P7.54 P7.55 Distinct role of human annexin A6 isoforms Mechanism of interplay between human in vesicle dynamics during mineralization ribosomal P1-P2 dimer and RTA Lukasz Bozycki1, Agnieszka Strzelecka- Przemysław Grela1,2, Xiao-Ping Li1, Barbara Michalec- Kiliszek1, Rene Buchet2, Slawomir Pikula1 Wawiórka2, Leszek Wawiórka2, Monika Szajwaj2, Eliza 2 2 2 1 Molestak , Kamil Deryło , Monika Dźwierzyńska , Nencki Institute of Experimental Biology PAS, Department of 1 2 Biochemistry, Warsaw, Poland; 2University Lyon 1, ICBMS CNRS UMR Nilgun E. Tumer , Marek Tchórzewski 5246, Villeurbanne, France 1Department of Plant Biology and Pathology, School of Environmental e-mail: Agnieszka Strzelecka-Kiliszek and Biological Sciences, Rutgers University, New Brunswick, New Jersey, USA, 2 Department of Molecular Biology, Maria Curie-Skłodowska Bone is a dynamic form of connective tissue composed University, Lublin, Poland of cells (osteoblasts, osteocytes and osteoclasts), extracel- e-mail: Marek Tchórzewski lular matrix, providing tensile strength, and hydroxyapatite contributing to the mechanical resistance. Mineralization- The pentameric architecture of eukaryotic stalk represents competent cells release matrix vesicles (MVs) in which the a universally conserved entity across the entire domain initial steps of mineralization occur. It has been reported where two copies of ribosomal P1-P2 protein dimers are that the release of MVs is correlated with changes in cel- regarded as its main functional elements. All P-proteins lular actin distribution and tissue-nonspecific alkaline phos- possess universally conserved C-terminal fragment directly phatase (TNAP) activity. In addition, annexins, a family of involved in recruitment of external factors to the ribo- calcium- and membrane-binding proteins, were suggested some. To investigate the role of multiplication of highly to be involved in the Ca2+ homeostasis of mineralizing cells conserved motif present at the C-terminal domain of all and the Ca2+ influx into MVs. Due to an alternative splic- stalk P proteins, ricin A chain (RTA) which binds to the ing, human AnxA6 exists in two isoforms, AnxA6-1 and stalk to depurinate the sarcin/ricin loop (SRL), was used AnxA6-2, the latter of which carries the 524-VAAEIL-529 as a molecular probe. Using several structural forms of hu- deletion. The specific role of these two isoforms in miner- man P1/P2 protein dimers and their truncated variants we alization remains unclear. examined the interplay among individual C-terminal frag- Two human cell lines, osteoblastic hFOB1.19 and osteo- ments of both proteins within the form of dimer and RTA. sarcoma Saos-2, spontaneously releasing MVs, were used P1-P2 dimers bearing only one C-terminal domain on P1 to determine the participation of AnxA6 isoforms in protein were shown to have significantly higher level of mineralization. Transfection of both types of cells with interaction with RTA comparing to P1-P2 dimer where EGFP-AnxA6 isoform cDNA was performed and the C-terminal polypeptide from P1 protein was deleted. Our mineralization process, stimulated with ascorbic acid and results for the first time demonstrate, although the C-ter- b-glycerophosphate, was monitored. TNAP activity meas- minal polypeptide is absolutely required for proper binding ured by pNPP ELISA assay was 7-fold higher in osteo- of RTA to stalk proteins, the internal structural configu- sarcoma than in osteoblastic cells. Calcium nodule detec- ration of the intact P1-P2 hetero-dimer protein complex tion by Alizarin Red-S staining showed distinct influence represents crucial component in the web of interactions of AnxA6 isoforms on cellular morphology and mineral with external factors like RTA molecule. formation. Spectrophotometric determination of Ca2+ and Pi concentrations confirmed that AnxA6 isoforms were responsible for different chemical composition of the pro- duced minerals. Immunohistochemical analysis of AnxA6 distribution in stimulated cells revealed an enrichment of both AnxA6 isoforms in plasma membrane of Saos-2 cells, whereas in hFOB1.19 cells these isoforms had different lo- calizations: perinuclear for AnxA6-1 and cytoplasmic for AnxA6-2. F-actin, as determined by fluorescent microsco- py, accumulated in focal contacts or under the membrane of mineral containing MVs and its rearrangement was nec- essary for MV biogenesis and mineral formation. Cytocha- lasin D and levamisole inhibited the mineralization process in AnxA6-1- as compared to AnxA6-2-overexpressing cells. In conclusion, we assume that different targeting of AnxA6 isoforms to specific cellular compartments, points to their distinct role in vesicle dynamics during mineraliza- tion.

Acknowledgements This work was supported by a grant N N401 140639 from Polish Ministry of Science and Higher Education.

Warsaw, September 9th–12th 2014 174 Session 7. Protein Functions in Cellular Signaling P7.56 P7.57 Correlation between central Nucleolar localization of phosphogylcerate carbon metabolism and DNA mutase as a cell proliferation marker replication in eukaryotic cells J. Wiśniewski1, A. Gizak1, M. Grenda1, Karolina Sawiuk*, Aneta Szczepańska*, Aleksandra P. Mamczur1, J. Silberring2, D. Rakus1 Konieczna, Robert Łyżeń, Grzegorz Węgrzyn 1Department of Animal Molecular Physiology, University of Wroclaw, 2 University of Gdansk, Department Biology, Molecular Biology, Poland Wrocław, Poland; Department of Biochemistry and Neurobiology, AGH *both authors contributed equally University of Science and Technology, Kraków, Poland e-mail: Aneta Szczepańska e-mail: Janusz Wiśniewski Central carbon metabolism and DNA replication are crucial Over the last twenty years several lines of evidence has biochemical processes occurring in cells of all eukaryotic accumulated that that evolutionary primeval enzymes of organisms. Nevertheless, a very limited number of stud- carbohydrate metabolism may localize not only in cell cy- ies have been carried out to explain the cell growth regula- toplasm but also in other cellular structures such as mito- tion in relation to genetic material replication. We aimed to chondria, nuclei or nucleoli. It has been also demonstrated test a possible link between central carbon metabolism and that in these compartments, their function is not restricted DNA replication regulation in human fibroblasts. Expres- to the catalytic activity but they play a role in the regula- sion of selected genes, coding for enzymes involved in cen- tion of cell proliferation and differentiation. One of those tral carbon metabolism, were silenced by using siRNA, and enzymes is phosphoglycerate mutase (PGAM) – a glyco- kinetics of DNA replication was monitored in such cells lytic enzyme catalyzing the transformation of 3-phospho- relative to controls. We found significant differences in the glycerate into 2-phosphoglycerate. Proteomic studies has time of entering the S phase by control cells and fibroblasts demonstrated its presence in nucleoli, however its role in in which expression of the gene coding for either isoci- these compartments have not been established yet. Results trate dehydrogenase 2 or fumarase hydratase was silenced presented here shed light on the role of PGAM in nucleoli by specific siRNA. Namely, impairment of expression of and show that the enzyme may be engaged in ribosomal the isocitrate dehydrogenase 2 stimulated the cell cycle, RNA transcription. while inhibition of fumarase hydratase production had During our research we found that PGAM localized in an inhibitory effect. Moreover, silencing the gene coding nucleoli of proliferating cancer, the KLN-205, cells. With- for STAT3, which influenced expression of genes coding drawal of serum form the culture medium, which stops cell for p53 and p27 proteins, caused significant perturbations proliferation, caused removal of PGAM form the nucleoli. in DNA replication regulation. These results suggest that The re-supplementation of culture medium with serum or changes in central carbon metabolism, and in transcription addition of insulin and/or IGF-1 resulted in the returning of some genes, significantly influence regulation of DNA of PGAM to nucleoli. This nucleolar import was blocked replication in human fibroblasts. by inhibition of phosphatidyloinositol kinase – a protein engaged in transduction of signal in the insulin/IGF-1 pathway. This demonstrates that nucleolar localization of PGAM is connected with high metabolic/proliferatory ac- tivity of the cell. The results of immunocytochemical experiments showing nucleolar localization of PGAM were confirmed by MS analysis of proteins isolated form nucleoli of proliferat- ing cells. On the other hand, the MS experiments did not demonstrate the presence of PGAM in nucleoli of non- dividing cells. The results presented here show a relationship between the nucleolar localization of PGAM and rRNA synthesis and the capability of cells to divide and grow.

BIO 2014 Congress Session 7. Protein Functions in Cellular Signaling 175 P7.58 P7.59 The effect of the exogenous cholesterol Expression of VEGFs and its receptors on fibroblast-to-myofibroblast in abdominal aortic aneurysm transition in bronchial fibroblasts Malgorzata Wolanska1, Emilia Bankowska-Guszczyn1, derived from asthmatic patients Krzysztof Sobolewski1, Radoslaw Kowalewski2 1 1 1 Dawid Wnuk , Milena Kosińska , Monika Rak , Katarzyna A. 1Medical University of Bialystok, Department of Medical Biochemistry, Wójcik1,2, Zbigniew Madeja1, Jarosław Czyż1, Marta Michalik1 Białystok, Poland; 2Medical University of Bialystok, Department of 1 Vascular Surgery and Transplantology, Białystok, Poland Jagiellonian University, Faculty of Biophysics, Biochemistry and e-mail: Małgorzata Wolańska Biotechnology, Department of Cell Biology, Kraków, Poland; 2Jagiellonian University Medical School, Department of Medicine, Kraków, Poland Background: The development of abdominal aortic an- e-mail: Dawid Wnuk eurysm (AAA) is thought to be related to an imbalance of VEGF and its receptors. This study aims to evaluate the The development of bronchial asthma is determined by expression of VEGF family members and their receptors two processes: chronic inflammation and bronchial wall in AAA wall, AAA mural thrombus and normal aorta wall. remodeling in the respiratory tract resulting from local Materials and methods: AAA specimens (mural throm- overproduction of cytokines and growth factors, mainly bus — luminal layer, mural thrombus — abluminal layer, TGF-β1. Prolonged exposure of human bronchial fibro- AAA wall) were collected from 24 patients undergoing blasts (HBFs) to TGF-β1 leads to the surplus of extracel- elective open AAA repair. Abdominal aortas from 12 or- lular matrix (ECM) components and phenotypic shifts gan donors served as controls. The expression of VEGF towards myofibroblast phenotype resulting in subepithe- (VEGF-A, VEGF-B, VEGF-C and VEGF-D) and VEGF lial fibrosis [1]. Multiple studies have demonstrated the receptors (VEGFR-1, VEGFR-2 and VEGFR-3) was eval- increased propensity of “asthmatic” bronchial fibroblasts uated using Western blot. to myofibroblastic transition (FMT), in comparison to -fi Results: Increased expression of VEGF-B, VEGF-C and broblasts derived from non-asthmatic patients [2]. How- VEGF-D was found in the AAA wall compared to nor- ever, the mechanisms underlying these phenotypic shifts mal aorta. No significant difference in VEGF-A expres- still remain unclear. Previously, we have demonstrated that sion was demonstrated between aortic wall samples. The inhibitors of cholesterol biosynthesis pathway attenuate expression of all investigated VEGFs was increased in the TGF-β1-induced FMT in “asthmatic” HBFs in a manner mural thrombus of AAA compared to normal aorta. Fur- dependent on intracellular cholesterol level [3]. The intra- thermore, the expression of VEGF-C and VEGF-D was cellular cholesterol levels depend on the activity of choles- significantly increased in the abluminal layer of the mural terol biosynthesis and LDL-endocytosis. Consequently, we thrombus. Increased VEGFR-1 expression was found in examined the effect of the exogenous cholesterol on the the mural thrombus, particularly in the abluminal layer. All intensity of TGF-β1-induced FMT in “asthmatic” HBFs. aneurysm samples expressed higher levels of VEGFR-2 Our data show the correlation between the increased mag- and VEGFR-3. nitude of TGF-β1-induced phenotypic shifts undergone by Conclusions: Our study demonstrates that the ablumi- “asthmatic” HBFs and reduced levels of extracellular lipids. nal layer in AAA mural thrombus contains high levels of Moreover, TGF-β1-induced increase of α-SMA expression VEGFs and VEGF receptors, suggesting that this layer in HBFs cultured in the absence of lipids is independent may play a significant role in AAA disease pathogenesis. In- of the canonical TGF-β/Smad signaling. It may depend creased expressions of VEGF and their receptors in AAA on the non-canonical TGF-β pathways which involve lipid mural thrombus may impact different pathways involved in raft-dependent ERK or Akt signaling. AAA etiology.

References: 1. Makinde et al (2007) Immunol Cell Biol 85: 348–356. 2. Michalik et al (2009) Med Sci Monit 15: 194-201. 3. Michalik et al (2013) Europ J Pharmacol 704: 23–32.

Warsaw, September 9th–12th 2014 176 Session 7. Protein Functions in Cellular Signaling P7.60 P7.61 The oxidative stress-associated Src Role of mRNA binding proteins in BRCA1 activator and c-Src tyrosine kinase- mRNA translation in leukemia cells a novel targets in the mechanism Magdalena Wolczyk1*, Paulina Podszywalow-Bartnicka1*, of photodynamic therapy Tomasz Skorski2, Margaret Nieborowska-Skorska2, Marta Woźniak, Kamila Duś-Szachniewicz, Krzysztof Skowronek1,3, Katarzyna Piwocka1 Barbara Ziółkowska, Piotr Ziółkowski 1Nencki Institute of Experimental Biology PAS, Warsaw, Poland; 2Temple University, Philadelphia, United States; 3The International Institute of Department of Pathology, Wrocław Medical University, Wrocław, Poland Molecular and Cell Biology, Warsaw, Poland e-mail: Marta Woźniak *join the first authorship e-mail: Katarzyna Piwocka Photodynamic therapy is an effective method in the diag- nosis and treatment of various tumors, but still the detailed The BCR-ABL1 oncoprotein plays a major role in the de- molecular mechanisms and pathways are not well known. velopment and progression of chronic myeloid leukemia In response to the cytotoxic influence of PDT, cells acti- (CML). To study the role of BCR-ABL1, we employed vate and inhibit different proteins that prevent them from mouse progenitor cell line expressing high level of BCR- reactive oxygen species (ROS). It has been reported the ABL1 corresponding to drug-resistant cells from blast cri- oxidative stress-associated Src activator/ Homo sapiens chro- sis of the disease. We previously demonstrated that CML mosome 9 open reading frame 10 protein (Ossa/ C9orf10) progression correlates with increased aneuploidy resulting protects cancer cells from an oxidative stress-induced ap- from affected mitosis. This was caused by BRCA1 down- optosis by Src family kinases activation. The main aim of regulation leading to decreased expression of protein mem- the present study was investigating whether the PDT leads bers of spindle assemble checkpoint [1]. BRCA1 tumor to Ossa expression and if this may be connected with co- suppressor regulates crucial cellular processes involved in expression of c-Src tyrosine kinase. Additionally, apoptosis DNA damage repair and cell cycle control. In this study we and necrosis following an oxidative stress caused by PDT investigated the mechanisms responsible for BCR-ABL1- on cancer cells were analyzed. For these purpose, Ossa and mediated BRCA1 downregulation. Herein we found that c-Src proteins were examined in MCF-7 human breast downregulation of BRCA1 protein is associated with en- cancer cells after 5-aminilevulinic acid-mediated photody- hanced half-life and increased levels of BRCA1 mRNA in namic therapy. The effects of PDT on the expression of a BCR-ABL1 transformed cell line and in CML primary the above proteins were detected in different time points cells from patients. Lowered luciferase synthesis level under (0, 7, 18, 24 hours after irradiation) using immunohisto- BRCA1 3’UTR control indicated that BRCA1 mRNA trans- chemistry and Western blot analysis. Our results showed a lation is affected in BCR-ABL1 expressing cells. Silencing high expression of Ossa at early time interval after PDT, and overexpression studies suggested that mRNA binding which was accompanied by a low expression of c-Src tyros- proteins regulate BRCA1 mRNA translation and stability. ine kinase. This relation between expression of two above Studies based on site directed mutagenesis revealed that proteins indicate that Ossa could protect cancer cells from phosphorylation pattern plays a key role in BRCA1 mRNA PDT through activation of c-Src in response to an oxida- translation in BCR-ABL1 expressing cells. Currently we use tive stress. In conclusion, analyzed proteins may be targets BRCA1 Stellaris RNA FISH probes and SmartFare RNA to achieve better efficiency of PDT treatment. probes to visualize BRCA1 mRNA together with mRNA binding proteins. Altogether, we showed a novel mecha- nism affecting BRCA1-dependent signaling in CML, in which BCR-ABL1 expressing cells modulate translation of BRCA1 mRNA, leading to protein downregulation. This may ultimately contribute to genomic instability and pro- vide justification for targeting PARP1 and/or RAD52 to induce synthetic lethality in CML.

Reference: Wolanin K et al (2010) Mol Cancer Ther 9: 1328–1338.

Acknowledgements This work was supported by grants: IP2011 043071 from the Polish Min- istry of Science and Higher Education and 2011/01/B/NZ3/02145 from the National Science Centre.

BIO 2014 Congress Session 7. Protein Functions in Cellular Signaling 177 P7.62 P7.63 The new model of mRNA degradation Silencing of parafusin inhibits based on the recent advances in the DcpS the secretory pathway 7 enzyme specificity towards m GDP Elzbieta Wyroba1, Li Liu2, Birgit H. Satir2 1 2 Anna Wypijewska del Nogal , Marius D. Surleac , Joanna 1Nencki Institute of Experimental Biology, Warsaw, Poland; 2Anatomy Kowalska1, Maciej Lukaszewicz1, Janusz Stepinski1, Richard and Structural Biology Department, Albert Einstein College of E. Davis4, Martin Bisaillon5, Jacek Jemielity1,3, Edward Medicine, New York, USA Darzynkiewicz1,3, Adina L. Milac2, Elzbieta Bojarska1,3 e-mail: Elżbieta Wyroba 1University of Warsaw, Division of Biophysics, Warsaw, Poland; 2Institute of Biochemistry of the Romanian Academy, Department of Parafusin (PFUS) is an important signaling component Bioinformatics and Structural Biochemistry, Romania; 3University of for regulatory control of the secretory pathway and the Warsaw, Centre of New Technologies, Warsaw, Poland; 4University of new protein — cloned by us in 1994 (Subramianian SV Colorado, Department of Biochemistry and Molecular Genetics, United et al., 1994, Proc Natl Acad Sci USA 91: 9832–9836) — in States; 5University of Sherbrooke, Department of Biochemistry, Canada e-mail: Anna Wypijewska del Nogal the evolutionary conserved phosphoglucomutase (PGM) superfamily. Though it is 50.7% identical to rabbit muscle PGM has four insertions and two deletions and negligi- Decapping Scavenger (DcpS) enzyme was reported to hy- ble enzymatic activity. PFUS attaches to secretory vesicles drolyze the free 5’ mRNA cap (m7GpppN) in the 3’→5’ docked underneath cell surface and undergoesphosphoglu- mRNA decay pathway, following mRNA degradation by cosy- lation/dephosphorylation during exocytosis. Subse- exosome exonuclease. The turnover of m7GpppN by DcpS quently it relocates to the newly formed secretory vesicles generates 7-methylguanosine monophosphate (m7GMP) to enable a next Ca2+-triggered exocytic event. These cyclic and nucleoside diphosphate (NDP). DcpS was also shown post-translational modifications are essential for unique to play a role in the 5’→3’ mRNA decay pathway, where it PFUS function (Satir BH et al., 1989, Proc Natl Acad Sci facilitates the Xrn1 exonuclease activity. However, in this USA 86: 930–932; Andersen AP et al., 1994, Biophys Res case, the cap hydrolysis is performed by another pyroph- Commun 200: 1353–1358; Levin S et al., 1999, Protein Eng 12: osphatase, Dcp2, yielding 7-methylguanosine diphosphate 737–746) and are reflected in the results obtained after 2D (m7GDP) and monophosphate mRNA. electrophorsis of this protein upon immunoprecipitation Here, using HPLC and fluorescence titration assays, we with pan-PGM antibody. Six spots at Mr 63 kDa with pI’s show that DcpS is not able to hydrolyze m7GDP, although from 6.8 to 6.3 were detected whereas immunoblot analysis it binds m7GDP with a high association constant. This with a PFUS specific peptide antibody (to insertion 2 not finding is of biological significance. Therefore, we proved present in PGM) revealed only two spots of pI 6.7 and 6.5, it on DcpS enzymes from different species: humans, model respectively. organisms (C. elegans and S. cerevisiae) and a parasitic nema- To elucidate the details of PFUS intracellular targeting tode organism (A. suum). Moreover, we found that m7GDP three different gene constructs were created to perform is a competitive inhibitor of DcpS enzymes. Computa- gene silencing by feeding in model eukaryote Paramecium tional approaches such as molecular modeling and docking that produced different phenotypes. The effect of RNAi of m7GDP, m7GpppG and several other cap analogs were was followed by Western blot analysis and quantification used to explain m7GDP resistance to the DcpS-mediated of PFUS versus PGM combined with double-labeling ex- hydrolysis and to identify molecular determinants for its ef- periments in the confocal microscopy using two specific ficient binding. Furthermore, we show that DcpS enzymes antibodies: against PFUS and the content of the dense core are able to hydrolyze 7-methylguanosine triphosphate secretory vesicles (DCSVs). RNAi experiments proved that (m7GTP) in vitro, releasing m7GMP and a diphosphate. knockdown of PFUS inhibits dense core secretory vesicle This suggests that DcpS might possibly be involved in synthesis with minor or no effect on other PGM isoforms. m7GDP turnover, provided m7GDP is initially converted Thus a proper incorporation of PFUS into the scaffold to m7GTP. is indispensable for maintenance and maturation of the Overall, we postulate that DcpS specificity towards m7G- DCSVs and subsequently for Ca2+ regulated exocytosis DP may play a regulatory role in both the 3’→5’ and (Liu L et al., 2011, Eur J Cell Biol 90: 844–853) and isolated 5’→3’mRNA decay pathways, affecting activity of other DCSVs contain glycosylated PFUS in their scaffold (Liu cap-binding proteins, such as Dcp2 or eukaryotic transla- L et al., 2009, Eur J Cell Biol 88: 301–313). PFUS or its or- tion initiation factor, eIF4E. This is important for proper thologs are present in many eukaryotic cells (Wyroba E & understanding of DcpS function in norm and diseases Satir BH, 2000, Biochem Cell Biol 78: 683–690; Wyroba E such as spinal muscular atrophy or cancer. The new model et al., 1995, Eur J Cell Biol 68: 419–426) and its Toxoplasma of mRNA degradation will be presented, highlighting that counterpart was also identified (Matthiesen SH et al., 2001, DcpS enzyme is a regulator of the mRNA decay pathways Eur J Cell Biol 80: 775–783; Matthiesen SH et al., 2003, Cell at the level of m7GDP metabolism. Microbiol 5: 613–624). Based on the PFUS/PGM sequence analysis against archea and eubacteria sequences we postu- late that PFUS appeared early in evolution acquiring a new function apart from the glycolytic one represented in PGM superfamily of proteins.

Warsaw, September 9th–12th 2014 178 Session 7. Protein Functions in Cellular Signaling P7.64 P7.65 High-mannose type N-glycosylation Cact-2 and dorsal-2 mRNA expression of β integrins in human leukocytes in the development of Apis mellifera is influenced by rapamycin Ewa A. Zaobidna1, Krystyna Żółtowska1, Marta Ząbczyńska1, Ewa Pocheć1, Elżbieta Łopieńska-Biernat1 2 1 Katarzyna Bocian , Anna Lityńska University of Warmia and Mazury, Departament Biology and 1Institute of Zoology, Department of Glycoconjugate Biochemistry, Biotechnology, Poland Jagiellonian University, Kraków, Poland; 2Institute of Zoology, e-mail: Ewa Zaobidna Department of Immunology, Faculty of Biology, University of Warsaw, Warsaw, Poland Honeybees (Apis mellifera) are important ecosystem element e-mail: Marta Ząbczyńska as pollinators. They are also producers of several products valuable for humans. The global population of A. mellifera The interactions mediated by N-glycans are crucial in im- is decreasing rapidly. Numerous factors affect this phe- mune cells. Most surface glycoproteins exist in the fully pro- nomenon: viral, bacterial, fungal and parasitic diseases, cessed form, it means that they contain mainly the complex chemical-like pesticides (van Engelsdorp & Meixner, 2010). type N-glycans. Although more and more research have The immunological response of insects involves the Toll shown that the mannose-rich oligosaccharides remaining pathway. Insect Toll and the Toll-like receptors (TLRs) are at low levels in mammalian cell, also play an important role transmembrane signal transducing proteins that play critical in their physiology. Our previous results shown that immu- roles immunity. Following conformational changes of the nosuppressive drugs, rapamycin (RAPA) and cyclosporine activated receptor, several intracellular proteins are recruit- A (CsA) increase the high-mannose glycosylation in some ed to form a receptor complex. Activation of this complex proteins in allogeneic stimulated mixed leukocyte reaction leads to the degradation of the NF kappa B inhibitor (IκB) (MLR) (Pochec et al., 2012). The purpose of our present Cactus and subsequent nuclear translocation of the NF-κB study was to identify RAPA affected proteins using mass transcription factor Dorsal (Valanne et al., 2011). The genes spectrometry (MS). We focused on one of the band (102 responsible for the synthesis of these receptors are cact-2 kDa) from three with increased GNA-positive glycosylation (GB13520)and dorsal-2 (GB18032). in MLR culture. MS identified proteins for this band belong The aim of the study was analysis of expression of genes: to β integrin family; β3 subunit (score: 6137) and β1integrin cact-2 and dorsal-2, during development of A. mellifera. precursor subunit (score: 636). To confirm these results we The material included 5 larval stages of A. mellifera: lar- used endoglycosidase H which removes mannose-rich oli- vae (L8), pre-pupae (PP), two stages of pupae (P3, P4) and gosaccharides from proteins. The results demonstrated the newly emerged imago. RNA was isolated from tissues with high-mannose N-glycans are presented on the β3 subunits kit total miniRNA (A&A Biotechnology, Poland). cDNA in allogeneic stimulated and non-stimulated leukocytes, but synthesis and amplification was done with TranScriba Kit only in MLR cells we observed two forms of this integrin. and StartWarm 2xPCR Master Mix (A&A Biotechnology). What is interesting, only the mature protein is abundant in Quantitative real-time PCR was performed using SYBR- mannose-rich oligosaccharides, while the precursor variant Green PCR-MIX Taq™ (A&ABiotechnology) according does not show the presence of this type of glycans. The to the manufacturer's instructions. The mean value ± SD precursor and mature forms we found also in β1 integrin, was used for analysis of relative transcript’s levels for each but unlike to β3 subunit, their expression is not depend- time point using the ΔΔCt method. The data were ana- ent on allogeneic stimulation. Endoglycosidase digestion lyzed and normalized relative to rp49 transcript levels by showed that the mature β1 subunit did not expressed high- AB analysis software (7500v2.0). All samples were tested in mannose glycans in contrast to premature precursor glyco- triplicate with lightcycler (Applied Biosystem, FAST7500). protein, which is strongly high-mannose glycosylated. We demonstrated the first time, the presence of expres- To the best of our knowledge, this is the first work that sion of mRNA cact-2 and dorsal-2 during developmental shows the effect of immunosuppressive drugs on leuko- stages of A. mellifera. Up-regulated expression of cact-2 and cyte glycosylation. High-mannose structures are expressed dorsal-2 genes was observed in development in all studied on mature variant of β3 and precursor of β1 integrins. Tak- stages. The pattern of cact-2 and dorsal-2 genes expression ing into account the significant role of glycans in immune demonstrates the increase of immune activity during de- system there is no doubt that RAPA-dependent alterna- velopment of A. mellifera. The highest expression of these tions in high-mannose glycosylation of β integrins have the genes was detected in imago stage (cact-2: 83-times; dorsal-2: physiologic relevance in immunosuppressive state. 13,3-times).

References: References: Pochec E, Bocian K, Popek M, Litynska, A (2012) FEBS J 279: 249. Valanne S et al (2011) J Immunol 186: 649–656. Van Engelsdorp D, Meixner MD (2010) J Invertebrate Pathol 103: 80–95. Acknowledgments This work was supported by grants: K/ZDS/004193 (JU), IP2010003370 (UW) and 'Bratniak' Foundation of the JU Students and Graduates.

BIO 2014 Congress Session 7. Protein Functions in Cellular Signaling 179 P7.66 P7.67 Effect of TGFβ1 signaling pathway Effect of TGFβ1 signaling pathway inhibition on skeletal muscle myoblasts inhibition of MMP-9 and MMP-2 activity Paulina Kasprzycka, Marta Gawor, Katarzyna Ilach, during myoblasts differentiation Maria A. Ciemerych, Małgorzata Zimowska Paulina Kasprzycka, Marta Gawor, Katarzyna Ilach, University of Warsaw, Faculty of Biology, Department of Cytology, Maria A. Ciemerych, Małgorzata Zimowska Warsaw, Poland University of Warsaw, Faculty of Biology, Department of Cytology, e-mail: Małgorzata Zimowska Warsaw, Poland e-mail: Małgorzata Zimowska Matrix metalloproteinases (MMPs) are enzymes involved in many physiological and pathological processes. Their pro- Matrix metalloproteinases (MMPs) are enzymes involved in teolytic activity is essential during the activation and deacti- many physiological and pathological processes. Their pro- vation of signaling molecules. These enzymes affect tissue teolytic activity is essential during the activation and deacti- architecture through the proteolysis of extracellular matrix vation of signaling molecules. These enzymes affect tissue components, also in repair of damaged skeletal muscles. architecture through the proteolysis of extracellular matrix The crucial role in the regeneration of muscle tissue re- components, also in repair of damaged skeletal muscles. pair is played by two of metalloproteainases: MMP-9 and The crucial role in the regeneration of muscle tissue re- MMP-2. Their activity is regulated by TGFβ1 and increased pair is played by two of metalloproteainases: MMP-9 and during skeletal muscle regeneration. However, the interac- MMP-2. MMPs plays a role not only in the extracellular tions of these factors are not specified. Our recent studies matrix transformation, but also in the proliferation and dif- suggest that MMPs plays a role not only in the extracellular ferentiation regulation of myoblasts (cells responsible for matrix transformation, but also in the proliferation and dif- the reconstruction of damaged muscle fibers). MMP-9 and ferentiation regulation of myoblasts (cells responsible for MMP-2 activity is regulated by transforming growth fac- the reconstruction of damaged muscle fibers). TGFβ1 re- tor beta 1 (TGFβ1) and increased during skeletal muscle presses activity of myogenic regulatory factors, expression regeneration. of genes encoding proteins specific to the skeletal muscle, TGFβ1 is a member of the TGF-β superfamily, which and thus blocks differentiation of these cells. In our project exerts wide range of biologic effects. This cytokine is in- we inhibited TGFβ1 signaling pathway by reducing recep- volved in cell proliferation, differentiation, extracellular tor 1 transforming growth factor β (TGFβR1) biological matrix production, and cell migration. In skeletal muscle activity. Next, we identified the MMP-9 and MMP-2 ac- TGFβ1 promotes fibrosis during regeneration, represses tivity and also interactions between MMPs and TGFβ1 in expression of genes encoding proteins specific to the skel- myoblasts isolated from Soleus muscles. In our study we etal muscle, and thus blocks differentiation of myoblasts. also tested how the inhibition of TGFβR1 impacts on the However, the interactions of MMP-9, MMP-2 and TGFβ1 regulation of differentiation in skeletal muscle myoblasts. in myoblasts are not specified. We inhibited TGFβ1 signaling pathway by reducing bio- logical activity of receptor 1 transforming growth factor β (TGFβR1). Next, we identified the MMP-9 and MMP-2 activity and also interactions between MMPs and TGFβR1 in myoblasts isolated from Soleus muscles. In our study we also tested how the inhibition of TGFβR1 impacts on the regulation of differentiation in skeletal muscle myoblasts.

Warsaw, September 9th–12th 2014