The role of regulatory proteins in ageing processes
Researchers: Tibor Vellai, Gábor Bánhegyi, Csaba Sőti
The research area of the ER redox lab at the Department of Medical Chemistry, Molecular Biology and Pathobiochemistry is the study of the redox homeostasis of the endoplasmic reticulum. The MEDinPROT project (“The role of regulatory proteins in ageing processes”) focuses on the functional links between the proteins regulating mobile genetic elements, (Vellai’s team), autophagy and ER stress response (Bánhegyi’s team) and the thermal shock protein systems. While monitoring the activity of certain age dependent elements of autophagy, ER stress and thermal shock systems, a reduction can be observed while advancing in age. We presume that the gradually increasing activities of the mobile genetic elements in the somatic genome (taking into consideration that almost half of the human genome is made up of such mobile genetic elements) are responsible for the ageing process through transposone mutagenesis.
The quest for a recently discovered inflammatory process
Researchers: Péter Gál, László Cervenak, Gábor Pál
Inflammatory diseases cause severe health problems and in many cases there are no appropriate therapies for them. This is caused by the lack of knowledge regarding the exact mechanism of the inflammation and the molecules potentially targeted by medicine. The cooperation between the Hungarian Academy of Sciences’ Research Centre of Natural Sciences and the Research Lab of Semmelweis University’s 3rd Department of Internal Medicine has resulted in the discovery of a new and potentially significant inflammatory process. It was detected that the protease MASP-1, the enzyme in the blood responsible for the initiation of the complement system’s lectin pathway, is able to activate endothelial cells. In this case MASP-1 functions as a hormone; it transmits external signals (eg. a wound, the presence of extraneous cells) towards the core of the cell via its interaction with cell surface receptors (G-protein coupled). Along with this research selective inhibitors not present in nature were developed against the enzymes of the lectine pathway. Three research groups work together within the Synergy programme to better explore and understand the newly identified inflammatory process. The main focus of the research is to determine whether the adhesion molecule pattern of endothelial cells is changed by the aforementioned signal transmitting process. This is a prerequisite for white blood cells taking part in the inflammation to exit the blood vessel. It is also studied, whether a sufficiently strong adhesion enabling extravasation is created between the endothelial cells activated by MASP-1 and the neutrophil granulocytes.
Interaction between extracellular vesicles and the complement system
Researchers: Edit Irén Buzás, Mihály Józsi
Extracellular vesicles are submicron particles secreted by cells in an evolutionally conserved manner and are surrounded by a phospholipid bilayer. Extracellular vesicles (exosomes, microvesicles and apoptotic bodies) may pass blood tissue barriers and may play a significant role in the formation of several diseases. The vesicles entering the extracellular space may interact with the organism’s various cells and molecules, including the so-called complement system. The complement system is the organism’s fundamental defense system, being part of the innate immune system. It has been known for several decades that complement proteins have an important part in the development of several diseases. Our studies aim to answer the following questions. Do certain types of extracellular vesicles activate complements? What type of complement proteins do they bind to? What are the functional consequences of the binding of complement and complement inhibitory proteins to the surface of the extracellular vesicles in case of the formation of inflammatory symptoms? The research group lead by Edit Buzás (Semmelweis University) are in possession of the state-of the-art equipment to study and isolate extracellular vesicles. Combining their methodology and equipment with those of the complement research group lead by Mihály Józsi (Hungarian Academy of Sciences-Eötvös Loránd University) the opportunity of a systematic study of the interaction between complement proteins and extracellular vesicles has been set up.
The role of changes in signal transmission pathways in the energy retention of malign tumour cells
Researchers: Attila Ambrus, Balázs Győrffy, Péter Hauser, László Tretter
Normal cells among anaerobic conditions initiate glycolysis – in 1930 Warburg observed that tumour cells are able to do the same among aerobic conditions. This so-called “aerobic glycolysis” enables the immediate availability of macro molecules required for quick cell division. A similar phenomenon has also been observed in the rapidly dividing embryonic cells. One of the “driver” genes behind this process is GLUT1, which boosts the cell’s extra glucose intake required for the process and the HIF1a and HIF2a transcription factors responsible for the initiation of glycolysis through the regulation of several genes. The metabolic transformation of energy is in closely linked to the six distinct characteristics of the formation of malign tumours (oncogenic activation, turning off tumour suppressors, replicative immortality, apoptosis avoidance, neovascularisation, metastatisation) which implies that genes present in the aforementioned characteristics are the drivers of the process instead of genes independent of it. The basis of the study is the hypothesis that the driver mutations most frequently occurring in the signal transmission pathways determine what role aerobic glycosys and the citric acid cycle will have in the energy retention of the given tumour. Therefore, we apply a novel approach which involves the study of the genes linked to glycosys in clinical groups involving various mutations. Thus the subject of our studies are not glycosys/mitochondrial oxidation or mutation but the combination of the two. This project exceeds the previous descriptive analysis of bioinfomratics by enabling the identification of not only the changing genes, and by the more thorough understanding of the molecular basis of tumour progression through the study of anaerobic glycosys.
The study of immune cell adhesion
Researchers: Miklós Kellermayer, Noémi Sándor, Inna Székács, Bálint Szabó
Three different but complementary technologies are used in the study of human monocytes, macrophages and dendritic cells. These are the following: label-free optical biosensor with high permeability (Corning EPIC), computer driven micropipette (CellSorter) and a fluorescent microscope operating on the basis of complete reflection (TIRFM). These technologies share the common characteristic of being able to study living cells on the optically available flat surface. In case of the EPIC and the computer driven micropipette protein specificity is provided by the biochemical treatment of the surface. The EPIC provides a high resolution monitoring of the kinetics of cell adhesion. The TIRF microscope enables the tracking of intracellular proteins taking part in cell adhesion by studying the cells fixated at different times after the immunocytochemical marking. The computer driven micropipette enables the precise identification of the singular cells’ adhesive force expressed in nN unit. The three technologies are also used after silencing the RNA coding integrin subunits and after blocking the receptor by antibody. By assessing the results obtained in such manner we aim to set up a new biophysics model, which provides information on the course of the integrin-dependent adhesion of leukocytes.
The regulation of the interaction between calmodulin and the enzymes eNOS and MLCK essential in blood vessel reactivity by sphingolipid mediators
Researchers: Zoltán Benyó, Károly Liliom
The objective of our research is the integrative (on subcellullar level, and on the levels of cells, tissue and organism) understanding of the circulatory system’s molecular regulatory mechanisms. We aim to study physiological processes in detail and to identify their operative disorders in certain medical conditions (dyslipidemia, diabetes, traumatic brain injury and stroke). Modern pharmacological and gene manipulation techniques are used in our studies in order to provide a theoretical basis for the development of new therapies for the circulatory disorders in the aforementioned conditions by identifying the normal and abnormal operation of the regulatory processes.
Main research methods:: Classic and tissue-specific conditional gene knockout animal models, tissue flowmetry by laser-speckle and laser Doppler-flowmetry, reactivity of isolated blood vessels, human and mouse endothelium cell culture, identification of the intracellular Ca2+-level by fluorescent dyes, gene expression analysis by immunohistochemistry, Western-blot and RT-PCR protocol.
The effect of mitochondrial DNA mutations on oxidative protein folding and drug toxicity
Researchers: József Mandl, András Szarka
The rate of mitochondrial DNA (mtDNA) mutations significantly increases with age progression that may lead to mitochondrial dysfunctions. Based on the mitochondrial cascade hypothesis, the individual’s genetic background determines the mitochondrial function and its durability. This determines the rate of the individual’s age dependent mitochondrial decline. When the mitochondrial function drops below a certain limit, several defects typical of old age appear. In the study of ageing cytoplasmatic hybrid cell lineages with mtDNA mutations must be created. With the help of the cell lineages we aim to study the process of oxidative protein folding. The final acceptor of the oxidative protein folding is the mitochondrial electron transfer chain, thus it may work with a reduced efficiency in old age/in case of mtDNA defect. Ageing is accompanied by mitochondrial functional change, which may affect drug biotransformation or the biotransformation of drug molecules may interfere with the ER and mitochondrial redox conditions. Therefore, we aim to study the impact of these processes exerted on each other on the cybrid lineages that we have created.
The impact of KRAS mutations on the collective migration and invasion of tumour cells
Researchers: András Czirok, József Tímár
One of the most common oncogenetic flaw of human malign tumours is the KRAS mutation. The RAS signal regulating/strengthening system has a key role in most of the growth factor signalling pathways, therefore tumours carrying KRAS mutations are more aggressive and more resistant to common therapies. Besides the RAS-RAF-MAPK pathway, the mutant RAS is able to control the PI3K-AKT, PLC-PKC and JAK-STAT signalling pathways. The cooperation between the research groups of Semmelweis University (SE) and Eötvös Loránd University (ELTE) in the field of tumour biology and signalling pathology are complementary: the SE group is working on the development of model systems and their molecular biological testing, while the ELTE group is conducting the functional studies of the signalling pathways’ activities and the identification of the parameters of collective cell movement. The aim of the studies is the precise characterisation of the mutations and the functional heterogeneity of the allele-specific KRAS. The impact of KRAS mutation on collective cell movement, cell adhesion and invasion is studied by in vitro imaging experiments and individual cell-tracking techniques. The role of the different signalling pathways is studied by specific inhibitors. The results of the projects have the potential to be clinically useful and enable us to design a substance correcting the impact of mutant RAS variations.
The connection between the pathogenicity, the dimerization and the structure of podocin
Researchers: Veronika Harmat, Dóra Menyhárd Karancsiné, Kálmán Tory
The steroid-resistant nephrotic syndrome (SRNS) is one of the main causes of chronic kidney failure in childhood. 12-18 percent of the SRNS cases derive from the mutations of the podocin coding gene, NPHS2. Podocin is the component of the podocyte filtration slits, and as the member of the stomatin family, it forms dimer oligomers. We have previously demonstrated that the polymorphism of R229Q, which is considered to be the hypomorphic variant of NPHS2, is pathogenetic only if is paired with certain 3’ missense mutations on the other parent allele. These trans-associations lead to pathological dimer formations and the intracellular dislocation of podocin. Based on epidemiological data, a 3’ truncating mutation (F344Lfs*4) also forms a pathogenetic structure with the R229Q variant. The aim of our current research is to understand the impact of this truncating mutation on podocin localisation compared with that of other 3’ truncating mutations. The impact of truncating mutations and their associations on podocin localisation is studied by transient expression on podocyte culture. The structure of podocin is simulated based on the crystal structure of the Pyrococcus horikoshii stomatin and theoretical methods are used for the study of the structure and dimerization ability of different variations. In order to support our hypothesis, we aim to investigate the behaviour of the different variations’ segments responsible for dimerization, in solution by chromatographic methods and CD and NMR spectroscopy. We plan the complete expression and cleaning of the C-terminal intracellular domain of podocin in order to obtain a sufficient amount of protein for the crystallization and structure definition of podocin. Based on the above we aim to understand the connection between dimerization, intracellular delocalisation and clinical practice.