2. Conference of MEDInPROT

Chair by Dr András Perczel, chairman of the board

9.30 Welcome
Introduction of The MedInProt Program’ result
Introduction of the upcoming specialist book of protein science, titled "Proteins with 1001 faces"
Dr. András Perczel, biochemist, Eötvös Loránd University

10.00-10.15 Questions

10.15 Introduction of the Albert Szent-Györgyi lecture
Dr. László Nyitray biochemist, Eötvös Loránd University

10.25 Influences on the Synergies of the MedInProt Protein Science Research Synergy Program - Measuring Cooperation with Network Analysis
András Vicsek Research Director, Máté Schnellbach Lead Analyst, maven 7 Network Research Plc.

10.40 – 11.00 Coffee break

11.10 The role of signal transduction driver genes in developing an altered energy metabolism in cancer cells
Attila Ambrus – Balázs Győrffy - Péter Hauser – László Tretter

In our research our conceptual idea is that we assume that the driver mutations will also influence alterations in glycolysis and citrate cycle in the energy metabolism of the cells. We aim to evaluate expression of genes involved in metabolic cycles within sub-cohorts designated by mutation state of key driver genes. Besides evaluation in cell culture models, our goal is also to assessment clinical relevance in samples with known follow-up.

11.20 A novel pathomechanism for initiation inflammation
László Cervenak – Péter Gál – Gábor Pál

We prepared a stock of recombinant MASP-1 fragment and its inhibitor SGMI-1, developed by directed protein evolution, which were sufficiently pure to be applied to cell culture. Using these recombinant proteins in an in vitro model system, we discovered that MASP-1 is able to induce E-selectin adhesion molecule in endothelial cells, at the levels of both mRNA and protein, which is a hitherto unknown mechanism of inflammatory regulation driven by the cooperation of complement, endothelium and leukocytes. SGMI-1 blocks MASP-1 induced Ca-mobilization in a dose-dependent manner, and SGMI-1 does not produce any sign of toxic effects in endothelial cells even at concentrations much higher than the effective dose.

11.30 The regulation of interactions between calmodulin and the important vasomodulator enzimes eNOS and MLCK via sphingolipid mediators
Zoltán Benyó – Károly Liliom

The results of preliminary experiments reviel that sphingosine inhibits endothelial nitric oxide synthetase (eNOS), a key enzyme regulating the tone and permeability of vasculature, due to its binding to calmodulin (CaM), thereby preventing the interaction of the two proteins, which is the basis for the activation of eNOS. Nitrogen monoxide (NO), the product of eNOS enzyme, under normal physiologic conditions is a vasorelaxant and attenuates inflammation and thrombus formation, while in certain pathologic conditions its increased production leads to the initiation and progression of inflammation. We assume, that a portion of the vascular effects of sphingosine and related sphingolipid mediators in inflammation is due to their direct influence on the activity of Ca2+-CaM-dependent enzymes. In the MedInProt project, we investigate the molecular mechanisms of regulations elicited by the sphingolipid mediators sphingosine, sphinganine, C2-cerimde, and C16-ceramide on two enzymes, eNOS and myosin light-chain kinase (MLCK), which controls the contraction of vascular smooth muscle cells.

11.40 Role of KRAS mutations in the collective motility and invasion of tumor cells
András Czirok - József Tímár

KRAS is one of the most frequent oncogenic mutation in human tumors. As RAS participates in several growth factor-induced signaling cascades, tumors carrying the KRAS mutation tend to be more aggressive and resistant to chemotherapy. In addition to the well described RAS-RAF-MAPK signaling, the mutant RAS also activates the PI3K-AKT, PLC-PKC and JAK-STAT pathways. We will investigate the functional role of KRAS mutations in collective motility and cell adhesivity using in vitro imaging and automatic cell tracking techniques. We will identify the contribution of various signaling pathways by specific pharmacological inhibitors.

11.50 Podicin - interdependece of pathogenicity, dimerization and molecular structure
Veronika Harmat – Dóra Karancsiné Menyhárd – Kálmán Tory

We formerly showed that specific C-terminal missense mutations of podocin form an abnormal dimer with R229Q podocin resulting in an altered subcellular localization and late-onset nephrotic syndrome. Recent results showed that a C-terminal truncating mutation (p.F344Lfs*4) is also pathogenic when associated with R229Q podocin. The aim of the present work is to understand how different C-terminal truncating mutations alter the dimerization and the subcellular localization of podocin. 

12.00 Organic Anion Transporting Polypeptides: potential new targets of anti-cancer and anti-inflammatory therapies
Tamás Hegedűs – Csilla Laczka

The in vivo fate of drugs is significantly influenced by the activity of uptake transporters like the Organic Anion Transporting Polypeptides (OATPs). The aim of our project is to provide a better understanding of the activity, substrate recognition and regulation of OATPs by combining in silico and in vitro methods. Our result may promote the development of new strategies in the treatment of cancer and inflammatory processes.

12.10 Protein kinases in 4D
Csaba Hetényi – Attila Reményi

Our collaboration will elucidate the molecular mechanisms underlying protein kinase function. We will use X-ray structures of protein kinases and their complexes determined by the Reményi Group as the starting point for molecular dynamics simulations performed by the Hetényi Group. These studies will enable us to see protein kinases in action: we theoretically calculate their movements in time as the fourth dimension complementing three-dimensional static spatial information.

12.20 Development of novel purification methods for disordered proteins playing significant role in cancer
László Poppe – Ágnes Tantos

Novel affinity materials will be prepared by fixing proper units chelating 14-18 various heavy metal or lanthanide ions to magnetic nanoparticles (MNPs). Expectedly, some of the novel affinity-MNPs could bind the His-tag fused proteins more efficiently than Ni-NTA agarose. In one of the two proposed affinity-MNPs series, the linker binding the trifunctional metal fixing part formed from EDTA anhydride is a short hydrophobic chain, while in the other series the linker is a longer, hydrophilic arm.
The purification and binding abilities of the new affinity-MNP series will be tested by three such disordered proteins/protein regions playing significant role in cancer which can bind only slightly to Ni-NTA agarose, namely the matrix metalloproteinase (MMP9), B-cell CLL/lymphoma 9 protein (BCL9) and DNA mismatch repair protein (hMLH1).

12.30 Folding and unfolding in human ileal bile acid-binding protein
László Biczók – Mihály Kovács – Orsolya Tőke

12.40-13.40 Coffee break

13.40-14.10 Questions

14.10 Investigating the adhesion of immune cells
Miklós Kellermayer – Noémi Sándor – Bálint Szabó – Inna Székács

We study human monocytes, macrophages and dendritic cells combining three techniques. These are the high-throughput label free optical biosensor, computer-controlled micropipette and the total internal reflection based microscope.

14.20 The effect of mitochondrial DNA mutations on oxidative protein folding and drug toxicity
József Mandl – András Szarka

The ratio of cells with mtDNA mutations is increasing by age. These mutations can lead to mitochondrial dysfunction. One of our main goal to prepare a cytoplasmic hybrid cell line with mitochondrial DNA mutations to study the effects of aging. The terminal electron acceptor of oxidative protein folding is the mitochondrial electron transfer chain that can function with lower efficiency in the case of aging/mtDNA mutations. The mitochondrial dysfunction accompanied by the aging can also influence the metabolism of different drugs.

14.30 The role of regulatory proteins in the aging process
Gábor Bánhegyi – Csaba Sőti – Tibor Vellai

The heat shock transcription factor HSF1 directly regulates the activity of four UPR (unfolded protein response) genes in the nematode Caenorhabditos elegans.
Heat shock provokes endoplasmic reticulum stress in mammalian cells; however, silencing of Hsf1 did not decrease the level of stress markers, indicating other indirect connections in the control network.
The DAF-21/Hsp90 heat shock protein is required for the function and life-span extending effect of the stress-inducible DAF-16/FOXO transcription factor in C. elegans.

14.40 The interaction of extracellular vesicles with the complement system
Edit Búzás – Mihály Józsi

Extracellular vesicles (exosomes, microvesicles and apoptotic bodies) are produced by cells constantly and in various amounts and composition upon diverse stimuli. When entering the extracellular space, extracellular vesicles come into contact with the complement system, a complex protein network that is present in body fluids. The project aims to study the capacity of extracellular vesicles to activate the complement system and to investigate the presence of complement activating and inhibiting molecules on the vesicles.

14.50 Pharmaceuticals Incorporating Proteins (PHARMinPROT)
Attila Bóta – György Marosi

Development and complex characterization of protein pharmaceuticals on fibrous polymer carriers and biomimetic vesicle nano-vehiches were carried out. State of art biotechnology was involved from protein production until product formulation. Critical details of technology were investigated with physico-chemical methods focusing on the structural and morphological features covering the range from the atomic scale up to the dimension of micrometers.

15.00 Deciphering the role of protein complexes in signal transduction and carcinogenesis
László Buday – László Nyitray – Beáta Vértessy 

As stated at the first MedinProt conference, relying on a wide network of protein scientists within Hungary, we have submitted and succesfully received the first Hungarian BAG project grant for synchrotron access – this will provide us with ample possibilities for data collection during 2015 (coordinator: Veronika Harmat, lead scientist: Beáta G. Vértessy). Analysing the structural and functional characteristics of several protein complexes with key roles in signal transduction and carconigenesis, we shed light on the mechanism of phosphorylation-driven regulation. We performed succesful crystallization and crystallography experiments, have deposited a new 3D protein structure in the PDB, have published four articles acknowledging MedinProt support (including key publications in Nucleic Acids Res and Angewandte Chemie).

15.10 Development of microfluidic system for tracking immune complex initiated inflammation
Péter Fürjes – Krisztián Papp 

The specific aim of cooperation is the development of a complex autonomous microfluidic device and implementation of an immunological process that can be suitable for the measurement and activation of neutrophil granulocytes playing central role in inflammation. The proposed teamwork combines engineering and material processing with biological knowledge: the workgroup of HAS – Research Center for Natural Sciences provides the micromachining and microfluidic background, while the MTA-ELTE Immunology Research Group contributes the immunological expertise in the cooperation. Innovative application and development of micro and biotechnology devices, emerging from conventional material structures we develop such a complex Lab-on-a-chip system, which integrates the sensing and sample preparation possibilities.

15.20 Function and inhibition of the metastasis and inflammation promoting protein, S100A4
Andrea Bodor – Lajos Kalmár – László Nyitray – Gábor Pál 

During the last months we focused on molecular level elucidation of myosin filament disruption caused mainly by phosphorylation. A combination of NMR, CD, MS-MS techniques helped in the description of structural and dynamical properties, as well as aggregation behavior of varying lengths phosphorylated and non-phosphorylated myosin fragments. We found significant and conserved differences between the physical/chemical properties in the disordered C-terminal tail of non-muscle myosin II isoforms using bioinformatics analysis. Using directed protein evolution we produced antagonist peptides that bind S100A4 with picomolar dissociation constant and can be further improved.

15.30 Closing remarks