Our research is centered around investigating the functional role of the non-coding genome in cancer development using colon cancer models. By employing epigenetic analysis, genome editing techniques, and computational approaches, we aim to unravel the functional significance of non-coding genetic variants in disease susceptibility. Additionally, we seek to identify and characterize functional regulatory elements that contribute to disease development. Through these efforts, our ultimate objective is to identify therapeutic vulnerabilities and leverage this knowledge for early disease detection.

Main research topics

1. Germline risk variants

We are dedicated to unraveling the role and impact of inherited non-coding mutations in cancer development, primarily identified through genome-wide association studies (GWAS). However, studying their influence on cancer risk poses significant challenges, including genetic correlation (linkage disequilibrium), cis-regulation (no genetic code), mild or weak phenotypes, complex genetic variants, limited model systems and methodological constraints. To overcome these hurdles, our primary objective is to identify and comprehensively investigate causal variants associated with cancer development. We employ precise genome and epigenome editing techniques, single-cell cloning, and cell-based assays to functionally annotate these causal variants and establish their connection to disease susceptibility genes (Spisak et al, Nat. Med, 2015 link). Through these approaches, we aim to deepen our understanding of their functional characteristics and their contribution to disease susceptibility. Furthermore, we strive to develop more sensitive systems and assays that leverage sophisticated phenotypes such as gene expression, cellular states, and morphology, with the ultimate goal of achieving scalability in our research endeavors.

2. Enhancer biology

Cis-regulatory elements, known as enhancers, are specific segments of the non-coding DNA that exert remote control over distant gene transcription. These elements interact with transcription factors (TFs), which are trans-acting proteins, to enhance the expression of target genes located on the same DNA molecule. Several large-scale genome-wide sequencing initiatives have provided evidence that enhancers are frequently transcribed into long non-coding RNA (lncRNA) or enhancer RNA (eRNA). The levels of these transcripts often exhibit a correlation with the expression levels of the target gene’s mRNA. Our research aims to identify cancer-specific enhancers and investigate their mechanisms of action, including TF binding, activation, interaction, and tissue-specific operation. Additionally, we seek to understand the consequences of altered target gene expression associated with cancer development. (Takeda and Spisak et al, Cell, 2018 link)

3. Early cancer detection

Epigenetic alterations, particularly DNA methylation and histone modifications, are known to exhibit tissue-specific and cancer-specific patterns. Investigating the rearrangement of the epigenetic landscape during cancer development, coupled with the utilization of liquid biopsy technology, holds great potential for advancing early cancer detection strategies. By integrating these approaches, we aim to enhance our understanding of epigenetic changes in cancer and develop effective methods for early detection. (Nuzzo, Berchuck, Korthauer and Spisak et al, Nat.Med. 2020 link)

Recent publications

Vízkeleti L., Spisák S., Rewired Metabolism Caused by the Oncogenic Deregulation of MYC as an Attractive Therapeutic Target in Cancers, Cells 2023, 12(13), 1745; link

Prosz A., Pipek O., Börcsök J., Palla G., Szallasi Z., Spisak S.*, Csabai I. Biologically informed deep learning for explainable epigenetic clocks.

Spisak S., Tisza V., Nuzzo PV., Seo JH., Pataki B., Ribli D., Sztupinszki Z., Bell C., Rohanizadegan M., Stillman DR., Alaiwi SA., Bartels AB., Papp M., Shetty A., Abbasi F., Lin X., Lawrenson K., Gayther AS., Pomerantz M., Baca S., Solymosi N., Csabai I., Szallasi Z., Gusev A., Freedman ML., A biallelic multiple nucleotide length polymorphism explains functional causality at the 5p15.33 prostate cancer risk locus.

Pre-prints

Vizkeleti L., Kiss C., Tisza V., Szigeti A., Gellert A., Csabai I., Pongor LS., Spisak S.,^# (2023) Epigenetic regulation explains the functionality behind colon cancer specific biomarker Septin9. BioRxiv

Prosz A., Duan H., Tisza V., Sahgal P., Topka S., Klus GT., Börcsök J., Sztupinszki Z., Hanlon T., Diossy M., Vizkeleti L., Stormoen DR., Csabai I., Pappot H., Vijay J., Offit K., Ried T., Sethi NS., Mouw KW, Spisak S^#, Pathania S^#, Szallasi Z^# (2023) Nucleotide excision repair deficiency is a targetable therapeutic vulnerability in clear cell renal cell carcinoma. BioRxiv

Gusev A.*, Spisak S.*, Fay AP., Carol H., Vavra KC., Signoretti S., Tisza V., Pomerantz M., Abbasi F., Seo JH., Choueiri TK., Lawrenson K., Freedman ML., Allelic imbalance reveals widespread germline-somatic regulatory differences and prioritizes risk loci in Renal Cell Carcinoma. BioRxiv

Spisak S., Chen D., Likasitwatanakul P., Doan P., Li X., Vizkeleti L., Tisza V., Silva PD., Giannakis M., Wolpin B., Qi J., Sethi NS., Utilizing a dual endogenous reporter system to identify functional regulators of aberrant stem cell and differentiation activity in colorectal cancer. BioRxiv

Group photo (2023)

Leader

Dr. Sándor Spisák (publications)

Members

The Systems Biology of Reproduction Research Group studies the obstetrical, biological, immunological, pathological and systems biological aspects of the ‘Great Obstetrical Syndromes’. The importance of the topic is supported by the fact that 70% of pregnancies and 15% of clinically recognized pregnancies end with miscarriage, and 25% of pregnant women have obstetrical syndromes that may have a severe impact on the health of both mother and child. The research group studies and characterizes signal transduction pathways that may play a key role in the development of miscarriages and obstetrical syndromes. The focus is on the systems biological investigation of the complex and overlapping dysregulation of maternal-fetal immune tolerance, trophoblast invasion and differentiation. Besides describing molecular pathways and signalling networks of pregnancy complications, our investigations may also reveal novel biomarkers and drug targets.

Scientific collaborations

• Research Centre for Natural Sciences
• University of Debrecen
• Eötvös Loránd University
• University of Pécs
• Semmelweis University
• Petz Aladár County Teaching Hospital (Győr)
• Biological Research Centre (Szeged)
• GraviDiagnostics Ltd.
• Maternity Clinic (Budapest)
• PentaCore Ltd.

• Perinatology Research Branch, National Institutes of Health (Detroit, MI, USA)
• Wayne State University (Detroit, MI, USA)
• University of Southern California (Los Angeles, CA, USA)
• Zymo Research Corporation (Irvine, CA, USA)
• Ben Gurion University (Beer Sheva, Israel)
• University of Basel (Basel, Switzerland)
• Medical University of Vienna (Vienna, Austria)
• Monash University (Clayton, Australia)
• Universitätsklinikum Hamburg-Eppendorf (Hamburg, Germany)
• Biognosys AG(Schlieren, Switzerland)
• University of Bologna (Bologna, Italy)
• Tampere University (Tampere, Finland)

Teaching activity

Postgradual training:

• Semmelweis University – Chapters from the immunobiology of pregnancy
• Eötvös Loránd University – The systems biological view of the immunology of pregnancy

PhD topic:

• Semmelweis University, Károly Rácz Doctoral School of Clinical Medicine – Pathomechanisms, early prediction and diagnosis of the Great Obstetrical Syndromes
• Eötvös Loránd University, Doctoral School of Biology – Cellular and molecular level immmune regulation of maternal-fetal attachment

Research news

• https://www.innoteka.hu/cikk/atuto_eredmenyek_a_preeclampsia_teren.2411.html
• http://mta.hu/tudomany_hirei/a-vilagon-elsokent-azonositottak-lenduletes-kutatok-egy-varandos-nok-es-magzataik-eletet-veszelyezteto-betegseg-korai-korfolyamatait-108944
• http://www.ttk.mta.hu/aktualis-hirek/a-vilagon-elsokent-azonositottak-lenduletes-kutatok-egy-a-varandos-nok-es-magzataik-eletet-veszelyezteto-betegseg-korai-korfolyamatait
• https://fiek.elte.hu/content/a-praeeclampsia-uj-korfolyamatainak-felfedezese.t.3913
• https://index.hu/techtud/egeszseg/2018/08/08/magyar_kutato_vezetesevel_fejtettek_meg_egy_veszelyes_terhessegi_betegseg_kialakulasat/

Current grant support

• NKFIH 2020-1.1.2-PIACI-KFI-2021-00273
• NKFIH 2019-2.1.7-ERA-NET-2020-00014

Perinatal Biobank news

• https://fiek.elte.hu/content/kutatasok-a-genanalitikai-laboratoriumban-es-perinatalis-biobankban.t.4816
• http://www.ttk.mta.hu/aktualis-hirek/a-magyar-tudomany-unnepe-alkalmabol-mutattak-be-az-mta-ttk-ban-kialakitott-nemzetkozileg-is-kiemelkedo-szinvonalu-genanalitikai-laboratoriumot-es-perinatalis-biobankot

Interviews, presentations

• http://novumtv.hu/adasaink/2015-12-26
• http://rtl.hu/rtlklub/fokusz/magyar-csapat-ert-el-attorest-a-hellp-szindroma-kutatasaban
• http://www.katolikusradio.hu/musoraink/adas/1/470526
• http://www.labroots.com/webinar/epigenetics-human-health-disease

Our research group on the cover

 

Leader

Nándor Gábor Than, MD PhD, senior research fellow

• MTMT: https://vm.mtmt.hu//search/slist.php?lang=0&AuthorID=10011696

• Google Scholar: https://scholar.google.hu/citations?user=ECdMsVAAAAAJ&hl=hu&oi=ao

• http://mta.hu/lendulet/than-nandor-gabor-lendulet-osztondijas-kutato-106439

Members

Munkatársak

1. Survival analysis in cancer. Our main goal is to develop tools to link cancer survival and gene expression or mutations based mono- or multigenic prognostic signatures. We develop online accessible tools for automated evaluation. We participate in international collaborations aiming the validation of identified prognostic and predictive signatures.

2. Cell culture studies. We strive to identify resistance biomarkers of targeted therapy agents used in oncology. We utilize cell culture models to inhibit and/or silence target genes. Then, the magnitude of response to chemo- and targeted therapy is evaluated and the phenotype changes are characterized.

3. Linking genotype to clinical response. We process next generation sequencing data generated by the TCGA consortia. We process the entire gene sequence and the expression of all genes in multiple thousand patients from various solid tumors. By linking different levels of data we can directly link a genotype change to genes with an effect on clinical outcome.

International collaborations

• Igor Roninson, University of South Carolina, USA
• Saraswati Sukumar, John Hopkins, Baltimore, USA
• Pusztai Lajos, Yale Cancer Center, New Haven, USA
• Sophia Chernikova, Stanford University, USA
• Yatrik Shah, University of Michigan, USA
• Mathieu Lupien, University of Toronto, Canada
• Libero Santarpia, Humanitas Clinical and Research Center, Italy
• Reinhold Schäfer, Charité Berlin, Germany
• Jan Budczies, Charité Berlin, Germany
• Thomas Karn, University of Frankfurt, Germany
• Irina Nazarenko, University of Freiburg, Germany
• Antonio Postigo, ICREA, Spain
• Luca Magnani, Imperial College, London, UK
• Michael Lisanti, University of Manchester, UK
• Simak Ali, Imperial College, London, UK
• Casanova Emilio, Medical University of Vienna, Austria
• Baharia Mograbi, University of Nice-Sophia Antipolis, France
• Pascale Cohen, University of Lyon, France
• Khalid Khabar, King Faisal Research Centre, Riyadh, Saudi Arabia
• Takayuki Iwamoto, Okayama University, Japan
• Yong Han, Peking University, China

Website

http://gyorffy.semmelweis.hu

Publications

Group Leader

Members

Many undesired side-effects or therapeutic failure of drugs are the results of differences or alterations of drug metabolism. Our team deals with interindividual differences in drug metabolism and elimination for more than 20 years. Our research activity focuses on the function and regulation of cytochrome P450 (CYP) enzymes, primarily involved in the metabolism of xenobiotics.

Biochemical, molecular biological and mass spectrometric approaches are applied for studying I) metabolism and pharmacokinetic interactions of drugs and drug-candidates under development, II) factors influencing the expression and function of CYP enzymes (hormonal status, disease, drug therapy), III) moreover, diagnostic approaches for patients’ drug metabolism capacity provide tools for personalized medication.

Main research projects:

1. Genetic and non-genetic factors influencing the activity of CYP enzymes

Pharmacogenetic analysis of CYP enzymes involved in drug metabolism contributes to personalized drug therapy. Phenotypes of drug-metabolizing enzymes are primarily influenced by genetic polymorphisms (loss-of-function or gain-of-function mutations, copy number variations) resulting in decreased or increased enzyme activity or complete loss of enzyme function. However, non-genetic factors (e.g. age, sex, enzyme inhibition and induction caused by drug interactions as well as diseases) can transiently decrease or increase metabolic activity leading to the phenotype different from that predicted from genotype. This phenoconversion can substantially modify the genotype based estimation of the activities of drug-metabolizing enzymes.

Role of genetic and non-genetic factors in the development of individual drug metabolism capacity.

The effects of clinically relevant polymorphic CYP allele-variants were investigated using liver tissues of human organ donors. Gene specific and accurate methods for genotyping and haplotype determination are crucial in estimation of the impact of polymorphic CYP alleles that required development of PCR-based methods for identification of CYP1A2, CYP2B6 and CYP2D6 variants. Besides genetic polymorphisms, several non-genetic factors including sex, drug therapies, chronic alcohol consumption were proved to significantly alter the phenotype predicted from CYP genotypes, for example in case of CYP2B6, CYP2C9, CYP2C19, CYP2D6 enzymes.

Hepatic CYP2C9 activity (tolbutamide 4′-hydroxylation) in subjects carrying various CYP2C9 genotypes. The activity predicted from CYP2C9 genotype was modified by non-genetic factors (CYP2C9 inducer and inhibitor therapy, amoxicillin + clavulanic acid treatment, chronic alcohol consumption), while it was not affected by the CYP2C8 genotype. The median CYP2C9 activity (dotted line) is for the cutoff value between high and low intermediate metabolizers. PM poor metabolizer, IM intermediate metabolizer, EM extensive metabolizer. * P < 0.05; ** P < 0.001

Modelling of cross-talk between sterol homeostasis and drug metabolism

The natural changes in hormonal status, or therapeutic steroids can cause substantial alteration in CYP gene expression and in CYP enzyme protein levels. The cross-talk between drug-metabolizing CYP enzymes and steroids such as dehydroepiandrosterone (DHEA), dexamethasone and cholesterol is investigated.

Dehydroepiandrosterone activates the hepatic CAR (constitutive androstane receptor) nuclear receptor, which enters the nucleus and induces the transcriptions of CYP2B6, CYP2C9, CYP2C19 and CYP3A4 genes.

2.    Strategy for personalized medication adjusted to patients’ drug-metabolizing capacity

CYPtest^TM, the multi-step diagnostic system for the estimation of patients’ drug-metabolizing capacity, identifies defective CYP alleles by DNA analysis (CYP-genotyping) and provides information about the current expression of key drug-metabolizing CYP enzymes by CYP-phenotyping. CYPtest^TM has been introduced for patients who are on multi-drug therapy or for those who particularly benefit from tailored medication by increasing drug efficacy and by significantly decreasing the risk of the toxicity. The main focus is on the patients’ reduced or even extensive drug-metabolizing capacity (transplant recipients, psychiatric and neurologic patients as well as in patients suffering from liver dysfunction and cardiovascular diseases) which may potentially lead to therapeutic failure and severe adverse drug reactions. By recognizing poor or extensive drug metabolism, tailored medication adjusted to the patients’ drug-metabolizing capacity (by the optimization of the most appropriate drug and dosage) can minimize harmful side effects and ensure a more rational drug therapy and a more successful outcome.

Possibilities of personalized drug therapy

Systematic evaluation of the role of drug-metabolizing CYP enzymes in the metabolism of the antipsychotics, mood-stabilizers and antiepileptics frequently applied in treatment of psychiatric and neurologic patients as well as of immunosuppressant drugs, the main components of transplant patients’ therapy. The contribution of CYP polymorphisms (genetic and non-genetic variations) to inter-individual differences in the efficacy and toxicity of these drugs are estimated.

2.1. CYP3A-status, taking both CYP3A4 expression and CYP3A5 genotype into account, influences recipients’ calcineurin inhibitor therapy after transplantation. In liver transplant patients, CYP3A-status of the donor liver contributes to the recipient’s blood concentrations of ciclosporin and tacrolimus. It has been reported that patients transplanted with liver grafts from low or high expressers or with grafts carrying functional CYP3A5*1 allele required substantial modification of the initial calcineurin inhibitor dose. Donor livers’ CYP3A-status can better identify the risk of calcineurin inhibitor over- or underexposure, and may contribute to the avoidance of misdosing-induced graft injury in the early postoperative period.

The time for achieving therapeutic tacrolimus concentration was significantly reduced, confirming potential benefit of initial tacrolimus therapy adjusted to donor’s CYP3A-status over classical clinical practice of tacrolimus concentration guided treatment (4 vs 8 days, P<0.0001). Acute rejection episodes (3.6% vs 23.8%, P<0.0001) and tacrolimus induced nephrotoxicity (8% vs 27%, P=0.0004) were less frequent in patients on CYP3A-status guided tacrolimus therapy.

A) Influence of the donors’ CYP3A status (CYP3A5 genotypes and CYP3A4 expression) on the blood concentrations of tacrolimus in liver transplant patients.
B) Dose requirements of recipients in the course of CYP3A status of the liver donors are presented. (Low, Normal, High: the levels of CYP3A4 expression; ns: not statistically significant)

Previous experience in liver transplant patients contributed to personalized immunosuppressive therapy in heart transplant patients. In the early postoperative immunosuppressive therapy, high-dose corticosteroid treatment affected drug metabolizing capacity of patients by regulating transcription of CYP3A enzymes. In the 15-month post-operative period, The research group investigated the alterations in heart transplant recipient’s drug metabolizing capacity and phenoconverting factors that modified pharmacokinetics of immunosuppressive agents (primarily tacrolimus).

The dose-corrected tacrolimus blood concentrations in heart transplant recipients with CYP3A5*3/*3 genotype (A) and recipients carrying CYP3A5*1 allele (B) in a 15-month period after transplantation. The dotted line represents the daily dose of corticosteroid. *P<0.05, **P<0.001, ***P<0.0001

2.2.  The clinical consequences of decreased CYP2C9 function were investigated in epileptic children. It has been established that valproic acid, one of the first choices of antiepileptic drugs, is metabolized primarily by CYP2C9 in pediatric patients. Identification of loss-of-function mutations in CYP2C9 may lead to false prediction of a patient’s valproate metabolizing capacity, since CYP2C9 expression highly influences blood concentrations of valproate.

Serum concentrations of valproic acid in epileptic children with different CYP2C9-statuses. (CYP2C9*1/mut: heterozygous CYP2C9 genotype (CYP2C9*1/*2 or CYP2C9*1/*3); normal: intermediate CYP2C9 expressions; Low: low CYP2C9 expressions; *P <0.0001)

Patients’ CYP2C9-status guided dosing strategy for achieving the optimal blood concentration has been suggested.

Valproic acid dose required for the therapeutic serum concentrations epileptic children with various CYP2C9-statuses.

CYP2C9-guided (CYP2C9 genotype and CYP2C9 expression) treatment significantly reduced the ratio of patients out of the range of target valproate blood concentrations, the ratio of patients with abnormal serum alkaline phosphatase levels and the incidence of serious side effects, notably hyperammonemia.

2.3.   The incidence of adverse reactions in the anticonvulsant clonazepam therapy is highly attributed to the inter-individual variability in clonazepam metabolism by CYP3A and NAT2 (N-acetyl transferase 2) enzymes. The patients’ CYP3A4 expression was found to be the major determinant of clonazepam plasma concentrations; whereas CYP3A5 genotype and NAT2 acetylator phenotype did not influence the steady state levels of clonazepam.

A) Clonazepam concentrations normalized by dose and bodyweight in the patients expressing CYP3A4 at low, normal, and high levels. Yellow points indicate patients carrying CYP3A5*1
B)7-aminoclonazepam/clonazepam concentrations in patients with various CYP3A4 expression and NAT2 acetylator phenotype, *P <0.0001

However, the normal CYP3A4 expression and slow NAT2 acetylation phenotype evoking high plasma concentration ratio of 7-amino-clonazepam and clonazepam, may account for low efficacy or withdrawal symptoms of clonazepam. Prospective assaying of CYP3A4 expression and NAT2 acetylation phenotype can better identify the patients with higher risk of adverse reactions and can facilitate the improvement of personalized clonazepam therapy and withdrawal regimen.

2.4.   The atypical antipsychotic clozapine is effective in treatment-resistant schizophrenia; however, the success or failure of clozapine therapy is substantially affected by the variables that impact the clozapine blood concentration. CYP3A4 expression was found to be the major determinant of normalized clozapine concentration, particularly in patients expressing CYP1A2 at relatively low level. The functional CYP3A5*1 allele seemed to influence clozapine concentrations in those patients who expressed CYP3A4 at low levels. Strong association was observed between the metabolite/clozapine ratios and CYP3A4 mRNA levels, which confirmed the primary role of CYP3A4 in clozapine metabolism.

3. Development of in vitro models

Various in vitro models have been developed for in vitro testing of drugs that are useful in pharmacological, pharmacokinetic, and toxicity studies.

3.1. Human induced pluripotent stem cell-derived brain organoids in pharmacology and toxicology studies

Aging societies face new challenges, one of the most serious being the increasing incidence of cancer and neurodegenerative diseases. In order to successfully combat these diseases, researchers are working to map the human body as accurately as possible when developing model systems. In the case of the brain, cerebral organoids are able to do this, properly reproducing not only the cell types that make up the cortical plate, but also their structural arrangement. From these organoids, slice cultures are created that can be sustained for up to one year when grown at the air-liquid interface. Thus, mature neuronal and glia cell types can develop that allow the study of specific pathological changes in neurodegenerative diseases such as amyotrophic lateral sclerosis with frontotemporal dementia (ALS / FTD) and screening of drug molecules that can potentialy prevent or slow down the disease (Szebényi et al., 2021).

3.2. Pharmacokinetic variations leading to therapy resistence in cancer

The research group in collaboration with National Korányi Institute of Pulmonology and Drug Resistance Research Group investigates the role of pharmacokinetic variability in drug resistance that highly impact clinical outcome of cancer therapy. The major aim is to adapt and further develop in vitro modell systems to reveal resistance mechanisms against the anticancer agent paclitaxel (adenocarcinoma cell lines overexpressing CYPs).

3.3. Development of biomarkers for toxicological and safety studies

One of the main goals of the research team’s work is to identify extracellular microRNA-based biomarkers that are able to detect organ-specific damage earlier than conventional methods. miRNAs are RNA molecules that do not encode a protein but can bind to the 3 ‘untranslated region of mRNAs and inhibit its transcription as well as induce its degradation. A functionally determined proportion of miRNAs are found in extracellular vesicles. Validation of novel biomarker candidates is performed in vitro in  rat cell model and in vivo in laboratory animal model.

3.4. In vitro model for testung immunosuppression efficacy

Ex vivo immunostimulation model has been developed using peripheral blood mononuclear cells to characterize immunosuppressive agents on the basis of their inhibitory effects on the expression of pro-inflammatory cytokines. The model is to be used to assaying the efficacy of immunosuppressive therapy after organ transplantation and the immunological background of acute rejection events.

Lab Equipments:

• Cell-culture laboratory equipped with CO₂ incubator, Esco Class II Biological Safety Cabinet, microscope
• HPLC UV-VIS, radiodetector
• Fluorimeter
• NanoDrop 1000 Spectrophotometer
• Real-time PCR and end-point PCR systems (BioRad)
• Western blot systems
• Fluidigm Biomark^TM HD high throughput PCR system
• Bio-Rad Droplet Digital PCR systems
• QuantStudio 5 real-time PCR systems
• Agilent 4200 TapeStation nucleic acid analyzer
• Ultracentrifuge

Collaborations:

• Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia – The cross-talk between cholesterol homeostasis and drug- metabolism
• Palacky University, Olomouc, Czech Republic – The effect of the steroid type compounds (dexamethasone, dehydroepiandrosterone) on drug-metabolizing cytochrome P450 enzymes
• INSERM (Institut National de la Santé et de la Recherche Médicale) U632, Montpellier, France – The role of nuclear receptors in the regulation of cytochrome P450 enzymes
• University of Cambridge, Department of Clinical Neurosciences, Lakatos Lab – Development of ALS/FTD organoid models
• Department of Transplantation and Surgery, Semmelweis University, Budapest – Drug metabolism in transplant patients; Prevention of toxicity resulted from ciclosporin metabolism
• Heart and Vascular Center, Semmelweis University, Budapest – Drug metabolism in transplant patients; Prevention of toxicity resulted from immunosuppressant metabolism
• Department of Psychiatry and Psychotherapy, Semmelweis University, – Possibilities of personalized antipsychotic therapy
• Madarász Hospital, Heim Pál Children’s Hospital , Budapest – Personalized antiepileptic therapy
• 2^nd Department of Pediatrics, Semmelweis University – CYP copy number variations in tumorous tissues
• St László Hospital, Budapest – Personalized therapy of bone-marrow transplant patients
• 1st Department of Surgery, Semmelweis University, Budapest – The effect of portal vein ligation on drug-metabolizing function of the liver
• National Korányi Institute of Pulmonology – The mechanisms of therapy resistance, designing personalized therapies
• Centre for Energy Research, Institute of Technical Physics and Materials Science, Microsystems Laboratory – Establishing physiological organoid culture technology

Education/training:

• Semmelweis University, Budapest
• Eötvös Loránd University, Budapest
• Budapest University of Technology and Economics, Budapest

Selected publications:

Szebényi K, Wenger LMD, Sun Y, Dunn AWE, Limegrover CA, Gibbons GM, Conci E, Paulsen O, Mierau SB, Balmus G and Lakatos A: Human ALS/FTD Brain Organoid Slice Cultures Display Distinct Early Astrocyte and Targetable Neuronal Pathology. Nature Neuroscience 24: 1542–1554, 2021

Déri M, Szakál-Tóth Zs, Fekete F, Mangó K, Incze E, Minus A, Merkely B, Sax B, Monostory K: CYP3A-status is associated with blood concentration and dose-requirement of tacrolimus in heart transplant recipients Scientific Reports 11: 21389, 2021

Fekete F, Mangó K, Déri M, Incze E, Minus A, Monostory K: Impact of genetic and non-genetic factors on hepatic CYP2C9 expression and activity in Hungarian subjects. Scientific Reports 11: 17081, 2021

Csikány N, Kiss Á, Déri M, Fekete F, Minus A, Tóth K, Temesvári M, Sárváry E, Bihari L, Gerlei Zs, Kóbori L, Monostory K: Clinical significance of personalized tacrolimus dosing by adjusting to donor CYP3A-status in liver transplant recipients. British Journal of Clinical Pharmacology 87: 1790–1800, 2021

Menus Á, Kiss Á, Tóth K, Sirok D, Déri M, Fekete F, Csukly G, és Monostory K: Association of clozapine-related metabolic disturbances with CYP3A4 expression in patients with schizophrenia. Scientific Reports 10: 212383, 2020

Déri M. T, Kiss Á. F, Tóth K, Paulik J, Sárváry E, Kóbori L, Monostory K: End-stage renal disease reduces the expression of drug-metabolizing cytochrome P450s. Pharmacological Reports 72: 1695–1705, 2020

Kiss Á, Menus Á, Tóth K, Déri M, Sirok D, Gabri E, Belic A, Csukly G, Bitter I, Monostory K: Phenoconversion of CYP2D6 by inhibitors modifies aripiprazole exposure. European Archives of Psychiatry and Clinical Neuroscience 270: 71–82, 2019

Monostory K, Nagy A, Tóth K, Bűdi T, Kiss Á, Déri M, Csukly G: Relevance of CYP2C9 function in valproate therapy. Current Neuropharmacology 17: 99–106, 2019

Kiss Á. F, Vaskó D, Déri M. T, Tóth K, Monostory K: Combination of CYP2C19 genotype with non-genetic factors evoking phenoconversion improves phenotype prediction. Pharmacological Reports 70: 525–532, 2018

Kiss Á, Tóth K, Juhász C, Temesvári M, Paulik J, Hirka G, Monostory K: Is CYP2D6 phenotype predictable from CYP2D6 genotype? Microchemical Journal 136: 209-214, 2018

Tóth K, Csukly G, Sirok D, Belic A, Kiss Á, Háfra E, Déri M, Menus Á, Bitter I, Monostory K: Potential role of patients’ CYP3A-status in clozapine pharmacokinetics. International Journal of Neuropsychopharmacology 20: 529-537, 2017

Tóth K, Csukly G, Sirok D, Belic A, Háfra E, Kiss Á, Déri M, Menus Á, Bitter I, Monostory K: Optimization of clonazepam therapy adjusted to patient’s CYP3A-status and NAT2 genotype. International Journal of Neuropsychopharmacology 19: 1-9, 2016

Bűdi T, Tóth K, Nagy A, Szever Z, Kiss Á, Temesvári M, Háfra E, Garami M, Tapodi A, Monostory K: Clinical significance of CYP2C9-status guided valproic acid therapy in children. Epilepsia 56: 849-855, 2015

Monostory K, Tóth K, Kiss Á, Háfra E, Csikány N, Paulik J, Sárváry E, Kóbori L: Personalizing calcineurin inhibitor therapy by adjusting to donor CYP3A-status in liver transplant patients British Journal of Clinical Pharmacology 80: 1429-1437, 2015

Tóth K, Bűdi T, Kiss Á, Temesvári M, Háfra E, Nagy A, Szever Z, Monostory K: Phenoconversion of CYP2C9 in epilepsy limits the predictive value of CYP2C9 genotype in optimizing valproate therapy. Personalized Medicine 12: 199-207, 2015

Szebényi K, Péntek A, Erdei Z, Várady G, Orbán TI, Sarkadi B, Apáti Á: Efficient generation of human embryonic stem cell-derived cardiac progenitors based on tissue-specific enhanced green fluorescence protein expression. Tissue Engineering, Part C: Methods 21: 35-45, 2015

Szebényi K, Füredi A, Kolacsek O, Csohány R, Prókai Á, Kis-Petik K, Szabó A, Bősze Z, Bender B, Tóvári J, Enyedi Á, Orbán TI, Apáti Á, Sarkadi B: Visualization of Calcium Dynamics in Kidney Proximal Tubules. Journal of the American Society of Nephrology 26: 2731-2740, 2015

Szebényi K, Füredi A, Kolacsek O, Pergel E, Bősze Z, Bender B, Vajdovich P, Tóvári J, Homolya L, Szakács G, Héja L, Enyedi Á, Sarkadi B, Apáti Á, Orbán TI: Generation of a Homozygous Transgenic Rat Strain Stably Expressing a Calcium Sensor Protein for Direct Examination of Calcium Signaling. Scientific Reports 5: 12645, 2015

Belic A, Tóth K, Vrzal R, Temesvári M, Porrogi P, Orbán E, Rozman D, Dvorak Z, Monostory K: Dehydroepiandrosterone post-transcriptionally modifies CYP1A2 induction involving androgen receptor. Chemico-Biological Interactions 203: 597-603, 2013

Temesvári M, Kóbori L, Paulik J, Sárváry E, Belic A, Monostory K: Estimation of drug-metabolizing capacity by cytochrome P450 genotyping and expression. Journal of Pharmacology and Experimental Therapeutics 341: 294-305, 2012

Temesvári M, Paulik J, Kóbori L, Monostory K: High-resolution melting curve analysis to establish CYP2C19*2 single nucleotide polymorphism: comparison with hydrolysis SNP analysis. Molecular and Cellular Probes 25: 130-133, 2011

Monostory K, Dvorak Z: Steroid regulation of drug-metabolizing cytochromes P450. Current Drug Metabolism 12: 154-172, 2011

Rezen T, Rozman D, Pascussi J-M, Monostory K: Interplay between cholesterol and drug metabolism. Biochim Biophys Acta – Proteins and Proteomics 1814: 146-160, 2011

Rozman D, Monostory K: Perspectives of the non-statin hypolipidemic agents. Pharmacology and Therapeutics 127: 19-40, 2010

Belic A, Temesvári M, Kőhalmy K, Vrzal R, Dvorak Z, Rozman D, Monostory K: Investigation of the CYP2C9 induction profile in human hepatocytes by combining experimental and modelling approaches. Current Drug Metabolism 10: 457-461, 2009

Monostory K, Pascussi J-M, Kóbori L, Dvorak Z: Hormonal regulation of CYP1A expression. Drug Metabolism Reviews 41: 547-572, 2009

Monostory K, Pascussi J-M, Szabó P, Temesvári M, Kőhalmy K, Acimovic J, Kocjan D, Kuzman D, Wilzewski B, Bernhardt R, Kóbori L, Rozman D: Drug-interaction potential of 2-((3,4-(dichlorophenethyl(propyl)amino)-1-(pyridin-3-yl)ethanol (LK-935), the novel non-statin type cholesterol lowering agent. Drug Metabolism and Disposition 37: 375-385, 2009

Kóbori L, Kőhalmy K, Porrogi P, Sárváry E, Gerlei Zs, Fazakas J, Nagy P, Járay J, Monostory K: Drug-induced liver graft toxicity caused by cytochrome P450 poor metabolism. British Journal of Clinical Pharmacology 65: 428-436, 2008

Kőhalmy K, Tamási V, Kóbori L, Sárváry E, Pascussi J-M, Porrogi P, Rozman D, Prough RA, Meyer UA, Monostory K: Dehydroepiandrosterone induces human CYP2B6 through the constitutive androstane receptor. Drug Metabolism and Disposition 35: 1495-1501, 2007

Monostory K, Kőhalmy K, Prough, RA, Kóbori L, Vereczkey L: The effect of synthetic glucocorticoid, dexamethasone on CYP1A1 inducibility in adult rat and human hepatocytes. FEBS Letters 579: 229-235, 2005

Monostory K, Hazai E, Vereczkey L: Inhibition of cytochrome P450 enzymes participating in p-nitrophenol hydroxylation by drugs known as CYP2E1 inhibitors. Chemico-Biological Interactions 147: 331-340, 2004

Szűcs G, Tamási V, Laczay P, Monostory K: Biochemical background of toxic interaction between tiamulin and monensin. Chemico-Biological Interactions 147: 151-161, 2004

Tamási V, Hazai E, Porsmyr-Palmertz M, Ingelman-Sundberg M, Vereczkey L, Monostory K: GYKI-47261, a new AMPA antagonist is a CYP2E1 inducer. Drug Metabolism and Disposition 31:1310-1314, 2003

Tamási V, Vereczkey L, Falus A, Monostory K: Some aspects of interindividual variations in the metabolism of xenobiotics. Inflammation Research 52:322-333, 2003

Hazai E, Vereczkey L, Monostory K: Reduction of toxic metabolite formation of acetaminophen. Biochemical Biophysical Research Communications 291: 1089-1094, 2002

Tamási V, Kiss Á, Dobozy O, Falus A, Vereczkey L, Monostory K: The effect of dexamethasone on P450 activities in regenerating liver. Biochemical Biophysical Research Communications 286: 239-242, 2001

Monostory K, Vereczkey L, Lévai F, Szatmári I: Iprifalvone as an inhibitor of human cytochrome P450 enzymes. British Journal of Pharmacology 123: 605-610, 1998

Monostory K, Jemnitz K, Vereczkey L, Czira G: Species differences in metabolism of panomifene, an analogue to tamoxifen. Drug Metabolism and Disposition 25: 1370-1378, 1997

Monostory K, Vereczkey L: The effect of phenobarbital and dexamethasone coadministration on the activity of rat liver P450 system. Biochemical Biophysical Research Commununications 203: 351-358, 1994

Leader

Katalin Monostory

Members

The group consists of three collaborating research laboratories, the Transposons and regulatory RNAs Laboratory (lead by Tamás Orbán), the CRISPR Laboratory (lead by Ervin Welker), and the Membrane Transporter Laboratory (lead by Balázs Sarkadi). Based on the research legacy of the former Biomembrane Research Group, the laboratories are currently investigating various aspects of the regulation of gene expression, which also involves the development of new molecular biology assays that could be applied in the establishment of human disease models.

Among other Groups in the Institute of Enzymology, our Research Group closely collaborates with the Molecular Cell Biology Research Group (group leader László Homolya), especially in the area of human stem cell biology (with the laboratory lead by Ágota Apáti). In addition to these close collaborations within the Institute of Enzymology, we attempt to take advantage of the unique possibilities of the RCNS-ELKH, bringing togeteher research groups in chemistry, biology and experimental psychology.

Transposons and regulatory RNAs Laboratory

Head of the laboratory: Dr. Tamás Orbán, Ph.D.

 One main research focus of our lab is the investigation of DNA transposition in mammalian cells. Apart from understanding the endogenous regulatory mechanisms, we are examining the molecular defense mechanisms against transposition, and the process how DNA transposons can become domesticated during evolution, especially focusing on the human genome. An additional important aspect is the development of transposon-based gene delivery methods: we are investigating the efficiency of the Sleeping Beauty and the piggyBac transposon systems in mammalian models, including applications in embryonic stem cells.

The other important research aspect of our lab is the investigation of RNA interference pathways, in particular, the molecular details of microRNA maturation. Our current focus is on the regulation of microRNA clusters,  as well as the molecular details of the mammalian mirtron pathway. In addition, we are aiming to develop artificial mirtron-derived microRNAs that could be used for future therapeutic applications.

Group photo (2018):

Selected publications:

Orbán TI: One locus, several functional RNAs-emerging roles of the mechanisms responsible for the sequence variability of microRNAs. Biologia Futura. 2023, online ahead of print: doi: 10.1007/s42977-023-00154-7.

Gyöngy Z, Mocsár G, Hegedűs É, Stockner T, Ritter Z, Homolya L, Schamberger A, Orbán TI, Remenyik J, Szakacs G, Goda K: Nucleotide binding is the critical regulator of ABCG2 conformational transitions. eLife. 2023, 12:e83976.

Reé D, Fóthi Á, Varga N, Kolacsek O, Orbán TI, Apáti Á: Partial disturbance of Microprocessor function in human stem cells carrying a heterozygous mutation in the DGCR8 gene. Genes. 2022, 13(11):1925. (cover page story)

Raskó T, Pande A, Radscheit K, Zink A, Singh M, Sommer C, Wachtl G, Kolacsek O, Inak G, Szvetnik A, Petrakis S, Bunse M, Bansal V, Selbach M, Orbán TI, Prigione A, Hurst LD, Izsvák Z: A novel gene controls a new structure: PiggyBac Transposable Element-Derived 1, unique to mammals, controls mammal-specific neuronal paraspeckles. Molecular Biology and Evolution. 2022, 39(10):msac175.

Wachtl G, Schád É, Huszár K, Palazzo A, Ivics Z, Tantos Á, Orbán TI: Functional characterization of the N-terminal disordered region of the piggyBac transposase. International Journal of Molecular Sciences. 2022, 23(18):10317.

Kolacsek O, Wachtl G, Fóthi Á, Schamberger A, Sándor S, Pergel E, Varga N, Raskó T, Izsvák Z, Apáti Á, Orbán TI: Functional indications for transposase domestications – characterization of the human piggyBac transposase derived (PGBD) activities. Gene. 2022,  834:146609.

Fóthi Á, Biró O, Erdei Z, Apáti Á, Orbán TI: Tissue-specific and transcription-dependent mechanisms regulate primary microRNA processing efficiency of the human chromosome 19 MicroRNA cluster. RNA Biology. 2021, 18(8):1170-1180.

Schamberger A, Várady G, Fóthi Á, Orbán TI: Posttranscriptional Regulation of the Human ABCG2 Multidrug Transporter Protein by Artificial Mirtrons. Genes. 2021, 12(7): 1068.

Reé D, Borsy A, Fóthi Á, Orbán TI, Várady G, Erdei Z, Sarkadi B, Réthelyi J, Varga N, Apáti Á: Establishing a human embryonic stem cell clone with a heterozygous mutation in the DGCR8 gene. Stem Cell Research. 2020, 50:102134.

Zámbó B, Mózner O, Bartos Z, Török G, Várady G, Telbisz Á, Homolya L, Orbán TI, Sarkadi B: Cellular expression and function of naturally occurring variants of the human ABCG2 multidrug transporter. Cellular and Molecular Life Sciences. 2020, 77(2):365-378.

Biró O, Fóthi Á, Alasztics B, Nagy B, Orbán TI, Rigó J Jr.: Circulating exosomal and Argonaute-bound microRNAs in preeclampsia. Gene. 2019, 692:138-144.

Kolacsek O, Orbán TI: Transcription activity of transposon sequence limits Sleeping Beauty transposition. Gene. 2018, 676:184-188.

Szádeczky-Kardoss I, Csorba T, Auber A, Schamberger A, Nyikó T, Taller J, Orbán TI, Burgyán J, Silhavy D: The nonstop decay and the RNA silencing systems operate cooperatively in plants. Nucleic Acids Research. 2018, 46(9):4632-4648.

Kolacsek O, Pergel E, Varga N, Apáti Á, Orbán TI: Ct shift: A novel and accurate real-time PCR quantification model for direct comparison of different nucleic acid sequences and its application for transposon quantifications. Gene. 2017, 598:43-49.

Sándor S, Jordanidisz T, Schamberger A, Várady G, Erdei Z, Apáti Á, Sarkadi B, Orbán TI: Functional characterization of the ABCG2 5′ non-coding exon variants: Stem cell specificity, translation efficiency and the influence of drug selection. Biochimica et Biophysica Acta – Gene Regulatory Mechanisms. 2016, 1859(7):943-51.

Szebényi K, Füredi A, Kolacsek O, Pergel E, Bősze Z, Bender B, Vajdovich P, Tóvári J, Homolya L, Szakács G, Héja L, Enyedi Á, Sarkadi B, Apáti Á, Orbán TI: Generation of a Homozygous Transgenic Rat Strain Stably Expressing a Calcium Sensor Protein for Direct Examination of Calcium Signaling. Scientific Reports. 2015, 5:12645.

Szebényi K, Füredi A, Kolacsek O, Csohány R, Prókai Á, Kis-Petik K, Szabó A, Bősze Z, Bender B, Tóvári J, Enyedi Á, Orbán TI, Apáti Á, Sarkadi B: Visualization of calcium dynamics in rat kidney proximal tubules. Journal of the American Society of Nephrology. 2015, 26(11):2731-40.

Schamberger A, Orbán TI: 3’ isomiR species and DNA contamination influence reliable quantification of microRNAs by stem-loop quantitative PCR. PLoS ONE. 2014, 9(8): e106315.

Kolacsek O, Erdei Z, Apáti A, Sándor S, Izsvák Z, Ivics Z, Sarkadi B, Orbán TI: Excision efficiency is not strongly coupled to transgenic rate: cell type dependent transposition efficiency of Sleeping Beauty and piggyBac DNA transposons. Human Gene Therapy Methods. 2014, 25(4):241-52.

Anita Schamberger and Tamás I. Orbán: Experimental validation of predicted mammalian miRNAs of mirtron origin. In: RNA Mapping, Methods and Protocols, Methods in Molecular Biology, Lucrecia Alvarez and Mahtab Nourbakhsh (Eds.). Springer Verlag, ISBN: 978-1-4939-1061-8, 2014; 1182:245-63.

Orsolya Kolacsek, Zsuzsanna Izsvák, Zoltán Ivics, Balázs Sarkadi, Tamás I. Orbán: Quantitative analysis of DNA transposon-mediated gene delivery: the Sleeping Beauty system as an example. In: Genomics III – Methods, Techniques and Applications, iConcept Press Ltd Book, ISBN: 978-1-922227-096, 2014, 97-123.

Schamberger A, Sarkadi B, Orban TI: Human mirtrons can express functional microRNAs simultaneously from both arms in a flanking exon-independent manner. RNA Biology. 2012, 9(9):1177-85. (cover page story)

Kolacsek O, Krízsik V, Schamberger A, Erdei Z, Apáti Á, Várady G, Mátés L, Izsvák Z, Ivics Z, Sarkadi B, Orbán TI: Reliable transgene-independent method for determining Sleeping Beauty transposon copy numbers. Mobile DNA. 2011, 2(1):5.

Tamás I. Orbán, Ágota Apáti, Zsuzsanna Izsvák, Zoltán Ivics and Balázs Sarkadi: Use of Transposon-Transposase Systems for Stable Genetic Modification of Embryonic Stem Cells. In: Methodological Advances in the Culture, Manipulation and Utilization of Embryonic Stem Cells for Basic and Practical Applications, Craig Atwood (Ed.). InTech, ISBN: 978-953-307-197-8; 2011: 259-274

Sarkadi B, Orbán TI, Szakács G, Várady G, Schamberger A, Erdei Z, Szebényi K, Homolya L, Apáti A: Evaluation of ABCG2 expression in human embryonic stem cells: crossing the same river twice? Stem Cells. 2010, 28(1):174-6.

Orbán TI, Apáti A, Németh A, Varga N, Krizsik V, Schamberger A, Szebényi K, Erdei Z, Várady G, Karászi E, Homolya L, Német K, Gócza E, Miskey C, Mátés L, Ivics Z, Izsvák Z, Sarkadi B: Applying a “double-feature” promoter to identify cardiomyocytes differentiated from human embryonic stem cells following transposon-based gene delivery. Stem Cells. 2009, 27(5):1077-87.

CRISPR Laboratory

Head of the Laboratory: Dr. Ervin Welker, Ph.D., D.Sc.

The laboratory is investigating the mechanism of action of CRISPR nucleases, working to improve the efficiency and accuracy of genome editing. Our research is primarily aimed at understanding how the Cas9 and Cas12a proteins function, with a particular focus on their roles in base editors and prime editors. The group is also working on the development of preclinical procedures focused on the genetic therapy of genetic diseases.

Group photo:

Selected publications:

András Tálas, Dorottya A. Simon, Péter I. Kulcsár, Éva Varga, Sarah L. Krausz & Ervin Welker BEAR reveals that increased fidelity variants can successfully reduce the mismatch tolerance of adenine but not cytosine base editors NATURE COMMUNICATIONS volume 12, Article number: 6353 (2021)

Talas, Andras; Huszar, Krisztina; Péter István Kulcsár, Julia K Varga, Éva Varga, Eszter Tóth, Zsombor Welker, Gergely Erdős, Péter Ferenc Pach, Ágnes Welker, Zoltán Györgypál, Gábor E Tusnády, Ervin Welker A method for characterizing Cas9 variants via a one-million target sequence library of self-targeting sgRNAs NUCLEIC ACIDS RESEARCH gkaa1220 (2021)

Tóth, Eszter ; Varga, Éva ; Kulcsár, Péter István ; Kocsis-Jutka, Virág ; Krausz, Sarah Laura ; Nyeste, Antal ; Welker, Zsombor ; Huszár, Krisztina ; Ligeti, Zoltán ; Tálas, András ; Welker, Ervin Improved LbCas12a variants with altered PAM specificities further broaden the genome targeting range of Cas12a nucleases NUCLEIC ACIDS RESEARCH 2020 Paper: gkaa110 (2020)

Kulcsár, Péter István ; Tálas, András ; Tóth, Eszter ; Nyeste, Antal ; Ligeti, Zoltán ; Welker, Zsombor ; Welker, Ervin Blackjack mutations improve the on-target activities of increased fidelity variants of SpCas9 with 5′G-extended sgRNAs NATURE COMMUNICATIONS 11 : 1 Paper: 1223 (2020)

Tóth, Eszter ; Czene, Bernadett C ; Kulcsár, Péter I ; Krausz, Sarah L ; Tálas, András ; Nyeste, Antal ; Varga, Éva ; Huszár, Krisztina ; Weinhardt, Nóra ; Ligeti, Zoltán ; Adrienn É Borsy, Elfrieda Fodor, Ervin Welker Mb- and FnCpf1 nucleases are active in mammalian cells: activities and PAM preferences of four wild-type Cpf1 nucleases and of their altered PAM specificity variants. NUCLEIC ACIDS RESEARCH 46 : 19 pp. 10272-10285. , 14 p. (2018)

Tálas, András ; Péter, Istvan Kulcsár ; Nóra, Weinhardt ; Adrienn, Borsy ; Eszter, Tóth ; Kornélia, Szebényi ; Sarah, Laura Krausz ; Krisztina, Huszár ; István, Vida ; Ádám, Bianka Gordos, Orsolya Ivett Hoffman, Petra bencsura, Antal Nyeste, Zoltán Ligeti, Elfrieda Fodor, Ervin Welker A convenientmethod to pre-screen candidate guide RNAs for CRISPR/Cas9 gene editing by NHEJ-mediated integration of a ‘self-cleaving’ GFP-expression plasmid. DNA RESEARCH 24 : 6 pp. 609-621. , 13 p. (2017)

Kulcsar, PI ; Talas, A ; Huszar, K ; Ligeti, Z ; Toth, E ; Weinhardt, N ; Fodor, E ; Welker, E. Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage. GENOME BIOLOGY 18 Paper: 190, 17 p. (2017)

Toth, E ; Weinhardt, N ; Bencsura, P ; Huszar, K ; Kulcsar, PI ; Talas, A ; Fodor, E ; Welker, E. Cpf1 nucleases demonstrate robust activity to induce DNA modification by exploiting homology directed repair pathways in mammalian cells BIOLOGY DIRECT 11 Paper: 46 , 14 p. (2016)

Toth, E ; Huszar, K ; Bencsura, P ; Kulcsar, PI ; Vodicska, B ; Nyeste, A ; Welker, Z ; Toth, S ; Welker, E Restriction enzyme body doubles and PCR cloning: on the general use of type IIs restriction enzymes for cloning. PLOS ONE 9 : 3 Paper: e90896 , 12 p. (2014)

Membrane Transporter Laboratory

Head of the Laboratory: Prof. Dr. Sarkadi Balázs, professor emeritus, Member of the Hungarian Academy of Sciences

The research area of this laboratory is the investigation of structure-function relationships of membrane transporter proteins and the role of these transporters in drug metabolism and distribution. One of the major topics is studying the human „ATP Binding Cassette” (ABC) transporters, having important physiological and pathological functions. Multidrug resistance (MDR) ABC transporters prevent the clinical efficiency of antitumor agents by extruding these drugs from the tumor cells, while these proteins are important players in the protection of cells and tissue sanctuaries against toxic agents. Since the role of ABC multidrug transporters in the ADME-tox properties is well documented, in drug development the authorities require the investigation of potential drug-transporter interactions. In our laboratory the ABC transporters studied include ABCG2 (BCRP, MXR), ABCB1 (Pgp, MDR1), ABCB11 (BSEP), ABCB6, as well as several ABCC type transporters.

Members of the laboratory also study the cellular calcium transporter proteins as well as their potential role in diseases, and we have developed functional and high-throughput toxicity assay system for studying these transporters.  In the past years, in a wide range of collaborations, we have studied the regulation of the expression and the polymorphic variants of these proteins, as well as their role in stem cell differentiation and protection.  As a response to the COVID-19 pandemic, we have also initiated a research project to study the interactions of the SARS-CoV-2 virus protein and the potential anti-COVID drugs with various membrane proteins.

Group photo:

Selected publications:

1: Ambrus C, Bakos É, Sarkadi B, Özvegy-Laczka C, Telbisz Á. Interactions of anti-COVID-19 drug candidates with hepatic transporters may cause liver toxicity and affect pharmacokinetics. Sci Rep. 2021 Sep 8;11(1):17810. doi: 10.1038/s41598-021-97160-3. PMID: 34497279; PMCID: PMC8426393.

2: Mózner O, Zámbó B, Sarkadi B. Modulation of the Human Erythroid Plasma Membrane Calcium Pump (PMCA4b) Expression by Polymorphic Genetic Variants. Membranes (Basel). 2021 Jul 30;11(8):586. doi: 10.3390/membranes11080586. PMID: 34436349; PMCID: PMC8401972.

3: Nagy T, Tóth Á, Telbisz Á, Sarkadi B, Tordai H, Tordai A, Hegedűs T. The transport pathway in the ABCG2 protein and its regulation revealed by molecular dynamics simulations. Cell Mol Life Sci. 2021 Mar;78(5):2329-2339. doi: 10.1007/s00018-020-03651-3. Epub 2020 Sep 26. PMID: 32979053; PMCID: PMC7966132.

4: Szabó E, Kulin A, Korányi L, Literáti-Nagy B, Cserepes J, Somogyi A, Sarkadi B, Várady G. Alterations in erythrocyte membrane transporter expression levels in type 2 diabetic patients. Sci Rep. 2021 Feb 2;11(1):2765. doi: 10.1038/s41598-021-82417-8. PMID: 33531564; PMCID: PMC7854743.

5: Telbisz Á, Ambrus C, Mózner O, Szabó E, Várady G, Bakos É, Sarkadi B, Özvegy- Laczka C. Interactions of Potential Anti-COVID-19 Compounds with Multispecific ABC and OATP Drug Transporters. Pharmaceutics. 2021 Jan 9;13(1):81. doi: 10.3390/pharmaceutics13010081. PMID: 33435273; PMCID: PMC7827085.

6: Sarkadi B, Homolya L, Hegedűs T. The ABCG2/BCRP transporter and its variants – from structure to pathology. FEBS Lett. 2020 Dec;594(23):4012-4034. doi: 10.1002/1873-3468.13947. Epub 2020 Oct 16. PMID: 33015850.

7: Kovacsics D, Brózik A, Tihanyi B, Matula Z, Borsy A, Mészáros N, Szabó E, Németh E, Fóthi Á, Zámbó B, Szüts D, Várady G, Orbán TI, Apáti Á, Sarkadi B. Precision-engineered reporter cell lines reveal ABCG2 regulation in live lung cancer cells. Biochem Pharmacol. 2020 May;175:113865. doi: 10.1016/j.bcp.2020.113865. Epub 2020 Mar 4. PMID: 32142727.

8: Zámbó B, Mózner O, Bartos Z, Török G, Várady G, Telbisz Á, Homolya L, Orbán TI, Sarkadi B. Cellular expression and function of naturally occurring variants of the human ABCG2 multidrug transporter. Cell Mol Life Sci. 2020 Jan;77(2):365-378. doi: 10.1007/s00018-019-03186-2. Epub 2019 Jun 28. PMID:31254042; PMCID: PMC6971004.

9: Mózner O, Bartos Z, Zámbó B, Homolya L, Hegedűs T, Sarkadi B. Cellular Processing of the ABCG2 Transporter-Potential Effects on Gout and Drug Metabolism. Cells. 2019 Oct 8;8(10):1215. doi: 10.3390/cells8101215. PMID: 31597297; PMCID: PMC6830335.

10: Oláh A, Ruppert M, Orbán TI, Apáti Á, Sarkadi B, Merkely B, Radovits T. Hemodynamic characterization of a transgenic rat strain stably expressing the calcium sensor protein GCaMP2. Am J Physiol Heart Circ Physiol. 2019 May 1;316(5):H1224-H1228. doi: 10.1152/ajpheart.00074.2019. Epub 2019 Mar 15. PMID: 30875251.

11: Apáti Á, Varga N, Berecz T, Erdei Z, Homolya L, Sarkadi B. Application of human pluripotent stem cells and pluripotent stem cell-derived cellular models for assessing drug toxicity. Expert Opin Drug Metab Toxicol. 2019 Jan;15(1):61-75. doi: 10.1080/17425255.2019.1558207. Epub 2018 Dec 17. PMID:30526128.

12: Zámbó B, Bartos Z, Mózner O, Szabó E, Várady G, Poór G, Pálinkás M, Andrikovics H, Hegedűs T, Homolya L, Sarkadi B. Clinically relevant mutations in the ABCG2 transporter uncovered by genetic analysis linked to erythrocyte membrane protein expression. Sci Rep. 2018 May 10;8(1):7487. doi:10.1038/s41598-018-25695-z. PMID: 29749379; PMCID: PMC5945641.

13: Erdei Z, Schamberger A, Török G, Szebényi K, Várady G, Orbán TI, Homolya L, Sarkadi B, Apáti Á. Generation of multidrug resistant human tissues by overexpression of the ABCG2 multidrug transporter in embryonic stem cells. PLoS One. 2018 Apr 12;13(4):e0194925. doi: 10.1371/journal.pone.0194925. PMID: 29649238; PMCID: PMC5896897.

14: Szabó E, Türk D, Telbisz Á, Kucsma N, Horváth T, Szakács G, Homolya L, Sarkadi B, Várady G. A new fluorescent dye accumulation assay for parallel measurements of the ABCG2, ABCB1 and ABCC1 multidrug transporter functions. PLoS One. 2018 Jan 17;13(1):e0190629. doi: 10.1371/journal.pone.0190629. PMID: 29342177; PMCID: PMC5771559.

15: Szabó Z, Héja L, Szalay G, Kékesi O, Füredi A, Szebényi K, Dobolyi Á, Orbán TI, Kolacsek O, Tompa T, Miskolczy Z, Biczók L, Rózsa B, Sarkadi B, Kardos J. Extensive astrocyte synchronization advances neuronal coupling in slow wave activity in vivo. Sci Rep. 2017 Jul 20;7(1):6018. doi: 10.1038/s41598-017-06073-7. PMID: 28729692; PMCID: PMC5519671.

16: Zámbó B, Várady G, Padányi R, Szabó E, Németh A, Langó T, Enyedi Á, Sarkadi B. Decreased calcium pump expression in human erythrocytes is connected to a minor haplotype in the ATP2B4 gene. Cell Calcium. 2017 Jul;65:73-79. doi: 10.1016/j.ceca.2017.02.001. Epub 2017 Feb 3. PMID: 28216081.

17: Apáti Á, Berecz T, Sarkadi B. Calcium signaling in human pluripotent stem cells. Cell Calcium. 2016 Mar;59(2-3):117-23. doi: 10.1016/j.ceca.2016.01.005. Epub 2016 Feb 17. PMID: 26922096.

18: Apáti Á, Szebényi K, Erdei Z, Várady G, Orbán TI, Sarkadi B. The importance of drug transporters in human pluripotent stem cells and in early tissue differentiation. Expert Opin Drug Metab Toxicol. 2016;12(1):77-92. doi: 10.1517/17425255.2016.1121382. Epub 2015 Dec 14. PMID: 26592535.

19: Szebényi K, Füredi A, Kolacsek O, Csohány R, Prókai Á, Kis-Petik K, Szabó A, Bősze Z, Bender B, Tóvári J, Enyedi Á, Orbán TI, Apáti Á, Sarkadi B. Visualization of Calcium Dynamics in Kidney Proximal Tubules. J Am Soc Nephrol. 2015 Nov;26(11):2731-40. doi: 10.1681/ASN.2014070705. Epub 2015 Mar 18. PMID: 25788535; PMCID: PMC4625667.

20: Várady G, Szabó E, Fehér Á, Németh A, Zámbó B, Pákáski M, Janka Z, Sarkadi B. Alterations of membrane protein expression in red blood cells of Alzheimer’s disease patients. Alzheimers Dement (Amst). 2015 Jul 21;1(3):334-8. doi: 10.1016/j.dadm.2015.06.007. PMID: 27239515; PMCID: PMC4878320.

21: Hegedüs C, Telbisz Á, Hegedűs T, Sarkadi B, Özvegy-Laczka C. Lipid regulation of the ABCB1 and ABCG2 multidrug transporters. Adv Cancer Res. 2015;125:97-137. doi: 10.1016/bs.acr.2014.10.004. Epub 2015 Jan 8. PMID: 25640268.

Leader

Tamás Orbán

Members

The Molecular Cell Biology Research Group investigates the biology of membrane transporter proteins, pluripotent stem cells, and stem cell differentiation; as well as explores their role in the pathophysiology of diseases by means of various cellular models. Using a variety of molecular biological, biochemical and cell biology approaches, we aim at establishing novel diagnostic methods, revealing novel therapeutic targets, as well as developing cellular assay systems and disease models. Our research group consists of three collaborating subunits: the Laboratory of Cell Polarity and Trafficking headed by László Homolya, the Human Pluripotent Stem Cell Lab headed by Ágota Apáti, and the Flow Cytometry Lab headed by György Várady.

Laboratory of Cell Polarity and Trafficking

Head of the laboratory: László Homolya, D.Sc.

The physiological barriers of our body are formed by polarized epithelial and endothelial cells. Membrane transporter proteins, localized in the membrane of these cells in a regulated fashion, control the translocation of various substances, such as nutrients, toxins, and drug molecules. Our research focuses on the better understanding of the establishment and maintenance of cell polarity; the targeting and trafficking of transporter proteins to the proper membrane compartment; and how the genetic alterations – especially disease-causing mutations – in the transporters affect their cellular routing and consequently their function.

Selected publications:

1. Lilienberg J, Hegyi Z, Szabó E, Hathy E, Málnási-Csizmadia A, Réthelyi JM, Apáti Á, and Homolya L. Pharmacological Modulation of Neurite Outgrowth in Human Neural Progenitor Cells by Inhibiting Non-muscle Myosin II. Front Cell Dev Biol. 9:719636. doi: 10.3389/fcell.2021.719636. eCollection (2021)
2. Bartos Z. and Homolya L.: Identification of specific trafficking defects of naturally occurring variants of the human ABCG2 transporter. Front Cell Dev Biol 9:615729. (2021)
3. Török G, Erdei Z, Lilienberg J, Apáti Á, and Homolya L. The importance of transporters and cell polarization for the evaluation of human stem cell-derived hepatic cells. PLoS One 15(1) e0227751 , 22 p. (2020)
4. Homolya L, Fu D, Sengupta P, Jarnik M, Gillet JP, Vitale-Cross L, Gutkind JS, Lippincott-Schwartz J, and Arias IM: LKB1/AMPK and PKA Control ABCB11 Trafficking and Polarization in Hepatocytes.PLOS ONE 9:(3) p. e91921. (2014)
5. Homolya L, Orbán TI, Csanády L, and Sarkadi B. Mitoxantrone is expelled by the ABCG2 multidrug transporter directly from the plasma membrane. Biochim Biophys Act. 1808(1): 154-63 (2011)
6. Sarkadi B, Homolya L, Szakács G, and Váradi A. Human multidrug resistance ABCB and ABCG transporters: participation in a chemoimmunity defense system. Physiol Reviews 86: 1179-1236, (2006)
7. Homolya L, Steinberg TH, and Boucher RC. Cell to cell communication in response to mechanical stress via bilateral release of ATP and UTP in polarized epithelia. Cell Biol., 150: 1349-1359, (2000)
8. Homolya L, Watt CW, Lazarowski ER, Koller BH, and Boucher RC. Nucleotide-regulated calcium signaling in lung fibroblasts and epithelial cells from normal and P2Y2 receptor (-/-) mice. Biol. Chem., 274: 26454-26460 (1999)
9. Lazarowski, E. R., Homolya, L., Boucher, R. C., and Harden, T. K.: Direct demonstration of mechanically induced release of UTP and its implication for uridine nucleotide receptor activation. Biol. Chem., 272: 24348-24354 (1997)
10. Homolya L, Holló Zs, Germann U, Pastan I, Gottesman MM, and Sarkadi B. Fluorescent cellular indicators are extruded by the multidrug resistance protein. Biol. Chem., 268: 21493-21496 (1993)

Human Pluripotent Stem Cell Lab

Head of the laboratory: Ágota Apáti, Ph.D.

The Human Pluripotent Stem Cell lab has strong experience in human pluripotent stem cell generation and differentiation (especially in the direction of cardiac, neuronal, endothelial and mesenchymal cell types), in studying membrane transporters as well as calcium signalling. Recently they develop induced pluripotent stem cell-based disease models (such as schizophrenia, DiGeorge syndrome, arteriosclerosis, and type II diabetes), as well as transgenic cellular reporter systems.

Selected publications:

1. Reé D, Borsy A, Fóthi Á, Orbán TI, Várady G, Erdei Z, Sarkadi B, Réthelyi J, Varga N, Apáti Á. Establishing a human embryonic stem cell clone with a heterozygous mutation in the DGCR8 gene. Stem Cell Res. 2020 Dec 22;50:102134. doi: 10.1016/j.scr.2020.102134. PMID: 33360445
2. Szabó E, Juhász F, Hathy E, Reé D, Homolya L, Erdei Z, Réthelyi JM, Apáti Á. Functional Comparison of Blood-Derived Human Neural Progenitor Cells. Int J Mol Sci. 2020 Nov 30;21(23):9118. doi: 10.3390/ijms21239118. PMID: 33266139
3. Hathy E, Szabó E, Varga N, Erdei Z, Tordai C, Czehlár B, Baradits M, Jezsó B, Koller J, Nagy L, Molnár MJ, Homolya L, Nemoda Z, Apáti Á, Réthelyi JM. Investigation of de novo mutations in a schizophrenia case-parent trio by induced pluripotent stem cell-based in vitro disease modeling: convergence of schizophrenia- and autism-related cellular phenotypes. Stem Cell Res Ther. 2020 Nov 27;11(1):504. doi: 10.1186/s13287-020-01980-5. PMID: 33246498
4. Szabó E, Reé D, Jezsó B, Vincze K, Földes G, Molnár AÁ, Réthelyi JM, Apáti Á. Generation of iPSC lines from peripheral blood mononuclear cells of identical twins both suffering from type 2 diabetes mellitus and one of them additionally diagnosed with atherosclerosis. Stem Cell Res. 2020 Dec;49:102051. doi: 10.1016/j.scr.2020.102051. PMID: 33099106
5. Berecz T, Husvéth-Tóth M, Mioulane M, Merkely B, Apáti Á, Földes G. Generation and Analysis of Pluripotent Stem Cell-Derived Cardiomyocytes and Endothelial Cells for High Content Screening Purposes. Methods Mol Biol. 2020;2150:57-77. doi: 10.1007/7651_2019_222. PMID: 30941720
6. Erdei Z, Schamberger A, Török G, Szebényi K, Várady G, Orbán TI, Homolya L, Sarkadi B, Apáti Á. Generation of multidrug resistant human tissues by overexpression of the ABCG2 multidrug transporter in embryonic stem cells. PLoS One. 2018 Apr 12;13(4):e0194925. doi: 10.1371/journal.pone.0194925. eCollection 2018. PMID: 29649238
7. Vőfély G, Berecz T, Szabó E, Szebényi K, Hathy E, Orbán TI, Sarkadi B, Homolya L, Marchetto MC, Réthelyi JM, Apáti Á. Characterization of calcium signals in human induced pluripotent stem cell-derived dentate gyrus neuronal progenitors and mature neurons, stably expressing an advanced calcium indicator protein. Mol Cell Neurosci. 2018 Apr;88:222-230. doi: 10.1016/j.mcn.2018.02.003. PMID: 29425968
8. Szebényi K, Füredi A, Kolacsek O, Csohány R, Prókai Á, Kis-Petik K, Szabó A, Bősze Z, Bender B, Tóvári J, Enyedi Á, Orbán TI, Apáti Á, Sarkadi B. Visualization of Calcium Dynamics in Kidney Proximal Tubules. J Am Soc Nephrol. 2015 Nov;26(11):2731-40. doi: 10.1681/ASN.2014070705. PMID: 25788535
9. Bacskai I, Mázló A, Kis-Tóth K, Szabó A, Panyi G, Sarkadi B, Apáti Á, Rajnavölgyi É. Mesenchymal Stromal Cell-Like Cells Set the Balance of Stimulatory and Inhibitory Signals in Monocyte-Derived Dendritic Cells. Stem Cells Dev. 2015 Aug 1;24(15):1805-16. doi: 10.1089/scd.2014.0509. Epub 2015 Apr 29. PMID: 25808140
10. Apáti A, Orbán TI, Varga N, Németh A, Schamberger A, Krizsik V, Erdélyi-Belle B, Homolya L, Várady G, Padányi R, Karászi E, Kemna EW, Német K, Sarkadi B. High level functional expression of the ABCG2 multidrug transporter in undifferentiated human embryonic stem cells. Biochim Biophys Acta. 2008 Dec;1778(12):2700-9. doi: 10.1016/j.bbamem.2008.08.010. PMID: 18793608

Flow Cytometry Lab

Head of the laboratory: György Várady, Ph.D.

The Flow Cytometry lab serves as a core facility and research laboratory. Our cell sorter allows us to generate single cell clones and homogeneous cell lines based on the cellular fluorescence intensities. In addition, cytometers are used for the assessment of protein levels, transfection efficiency, and multidrug resistance, as well as for cell cycle analysis. Our main research area includes the functional investigation of ABC transporters using fluorescent dyes and the characterization of multifactorial diseases (gout, type 2 diabetes) by the analysis of the membrane proteins in red blood cells.

Main instruments:

FACSAria III cell sorter;

Attune Nxt with 3 (viola, blue, red) and 4 (viola, blue, yellow, red) lasers and plate reader;

FACSCanto II with 3 (viola, blue, red) lasers with plate reader

Selected publications:

1. Szabó E, Kulin A, Korányi L; Literáti-Nagy B, Cserepes J, Somogyi A, Sarkadi B, and Várady G. Alterations in erythrocyte membrane transporter expression levels in type 2 diabetic patients Sci Rep. 11(1):2765 (2021)
2. Telbisz Á, Ambrus Cs, Mózner O, Szabó E, Várady Gy, Bakos É, Sarkadi B, Özvegy-Laczka Cs. Interactions of Potential Anti-COVID-19 Compounds with Multispecific ABC and OATP Drug Transporters Pharmaceutics 13 (1) 81 (2021)
3. Kovacsics D, Brózik A, Tihanyi B, Matula Zs, Borsy A, Mészáros N, Szabó E, Németh E, Fóthi Á, Zámbó B, Szüts D, Várady Gy, Orbán T I, Apáti Á, Sarkadi B. Precision-engineered reporter cell lines reveal ABCG2 regulation in live lung cancer cells Biochemical Pharmacology 175: 113865 (2020)
4. Zámbó B, Bartos Z, Mózner O, Szabó E, Várady Gy, Poór Gy, Pálinkás M, Andrikovics H, Hegedűs T, Homolya L, Sarkadi B. Clinically relevant mutations in the ABCG2 transporter uncovered by genetic analysis linked to erythrocyte membrane protein expression Sci Rep. 8(1): 7487 (2018)
5. Szabó E, Türk D, Telbisz Á, Kucsma N, Horváth T, Szakács G, Homolya L, Sarkadi B, Várady Gy. A new fluorescent dye accumulation assay for parallel measurements of the ABCG2, ABCB1 and ABCC1 multidrug transporter functions Plos One 13(1): e0190629 (2018)
6. Zámbó B, Várady Gy, Padányi R, Szabó E, Németh A, Langó T, Enyedi Á, Sarkadi B. Decreased calcium pump expression in human erythrocytes is connected to a minor haplotype in the ATP2B4 gene Cell Calcium 65. 73 (2017)
7. Várady Gy, Szabó E, Fehér Á, Németh A, Zámbó B, Pákáski M, Janka Z, Sarkadi B. Alterations of membrane protein expression in red blood cells of Alzheimer’s disease patients Alzheimers & Dementia: Diagnosis Assessment & Disease Monitoring 1(3) 334 (2015)

Leader

László Homolya

Members

The Protein Bioinformatics Research Group at the Institute of Enzymology, Research Centre of Natural Sciences is headed by Dr. Gabor Tusnady, and was founded in 2019, when the Protein Structure Research Group and the Transmembrane Protein Bioinformatics Research Group were fused.

The group’s main aim is to investigate membrane proteins topology and 3D structure by developing state-of-the-art computational techniques, like CCTOP or TMFoldRec algorithms, as well as to establish and maintain databases relating to transmembrane protein structure and topology. The group also investigates the disordered regions in transmembrane and globular proteins and the interactions of transmembrane proteins. Beyond bioinformatics studies, the group is involved in developing a high throughput experimental method for generating topology data of transmembrane proteins.

In addition to these recent achievements in the field of transmembrane proteins, the group was involved in several studies using next generation sequencing (NGS) ranging from genome sequencing of the chicken DT40 bursal lymphoma cell line and Mycoplasma sp. HU2014, through studying mutagenic impact of common cancer cytotoxics and clinical validation of TruSeq Custom Amplicon assay performed on formalin-fixed and paraffin-embedded tumor samples, to the identification of known and unknown plant viral pathogens.

Recent collaborators

• Prof. Zoltán Szentirmay, Surgical and Molecular Tumor Pathology Centre, National Institute of Oncology
• Dr. Erika Tóth Surgical and Molecular Tumor Pathology Centre, National Institute of Oncology
• Zoltán Szállási Zoltán, Pediatrics, Harvard Medical School; Pediatrics, Boston Children’s Hospital
• Éva Várallyay Éva, Diagnostic Group of Agricultural Biotechnology Institute

Main equipment

• Supermicro computer cluster (1024 CPU core, 4Gb RAM/CPU core (sum 2Tb RAM), 240 Tb redundans winchester capacity.
• Web server (20 CPU core, 160Gb RAM, 2x4Tb redundans Sata disk, 2x4Tb PCIexpress SSD disk)
• 60Tb QNAP NAS server

Webpage of the group:

http://mbk.enzim.ttk.mta.hu

Leader

Gábor Tusnády

Members

Our research activity is focused on the general aspects of protein structure, structural dynamics and conformational flexibility. We are particularly interested in the role of protein-protein interactions and allosteric conformational transitions in different biological processes. Our model proteins are mainly allosteric enzymes that can self organize to form sophisticated multi-molecular structures. In our research we apply both in silico modeling and wet laboratory experiments in a concerted manner.

Major research projects:

1.) Activation and regulation of the complement system

The complement system (CS) is a major effector arm of the innate immune system. The CS consists of about 40 protein molecules and it is capable of recognizing, labeling and eliminating dangerous structures such as pathogenmicroorganisms. The majonents of the CS are serine protease enzymes that activate each-other in a cascade-like manner. An intact complement system is essential for maintaining the immune homeostasis of the body; however, its uncontrolled, pathological activation may results in self-tissue damage.Recently it has been turned out that the pathological complement activation contributes to the development of numerous disease conditions.  Our research concentrates on the study of the early serine proteases that initiate the complement activation. We recombinantly express the proteases in different expression systems and characterize their structural, biochemical and enzymological properties. Special emphasis is laid on the study of the natural and artificial inhibitors of the complement serine proteases. During the recent years we have determined the 3D structure of several serine proteases, we have clarified the exact mechanism of the activation of the lectin pathway of CS, and we have discovered a novel mechanism for initiation of inflammation.

Fig. 1. Crystal structure of autoactivating MASP-2 moleculesment activation contributes to the development of numerous disease conditions.

2.) The functional landscape of proteins

The functional landscape of a protein is the part of its free energy landscape that corresponds to the native state. The functional motions of proteins (catalysis, ligand binding, allosteric regulation) occur on this hypersurface having a complex topology. We plan to reveal the general properties of this landscape (hierarchical structure, the nature of

dynamics, etc.) on a structurally representative subset of proteins, focusing on proteins that are studied in the institute by other methods. We determine the functional dynamics of each protein by performing simulations on models of various resolutions and applying a range of analysis methods. The functional landscape is particularly important for understanding the mechanisms of allostery and allosteric communication pathways. We construct structural models of protein-protein complexes, and describe allosteric communication within these complexes using network models.

Fig.2. Illustration of the free energy landscape of a protein. The functional landscape is the bottom part of the funnel-shaped surface, and it has a complex topology.

3.) The bacterial flagellar export system

The bacterial flagellum is the main locomotion organelle of bacteria. It is composed of three major parts: the basal body, the hook, and the filament. The filament is basically made of more than ten thousand copies of a single protein,

flagellin. Flagellin (FliC) monomers are transported through a narrow channel of about 20 Å to the tip of the filament where they self-assemble into the growing structure. Flagellin and several other components of the flagellum are transferred to the central channel by the flagellar type III secretion system, or in other words export system. The precise mechanism of this process is unknown. Our focus is to reveal the exact functions of the components of the export system, and furthermore to utilize the flagellar export system to produce recombinant proteins secreted into the medium.

Fig. 3. The bacterial flagellum

Fig. 4. Schematic structure and putative mechanism of the flagellar export system

4.) Identification and characterization of interactions between proteins participating in signal transduction processes leading to cancer or inflammation

The essential physiological processes of the living cells are based on the sophisticated network of biological macromolecules, mainly that of proteins. The signal transduction pathways are mediated by different protein-protein interactions (e.g. strong or weak; direct or indirect), that regulate the functions of the allosteric enzymes.

The allosteric regulation, based on the conformational flexibility of proteins, is of great significance in the function of enzymes; and we have previously gained insight into such regulatory mechanisms by using isolated proteins. Based on our knowledge, in the frame of the present project, we would like to identify and characterize enzyme-enzyme interactions at the molecular level that are involved in signal transduction processes having pathophysiological relevance. According to our hypothesis there is a relationship between chronic inflammation and cancer, and in this context we would like to study the role of protein-protein interactions in which certain kinases (e.g. Aurora, Rock1, Rock2 , phosphoglycerate kinase) and phosphodiesterases (e. g. PDE4, PDE5) might be involved. The molecular interactions will be studied in vivo using mammalian cell cultures and in vitro applying enzymological and biochemical methods.

Collaborations

Complement research:

Department of Biochemistry, Department of Chemistry, Eötvös University, Budapest

3^rd Department of Internal Medicine, Semmelweis University , Budapest

University of Aarhus, Denmark

University Hospital, Bern, Switzerland

University of British Columbia, Vancouver, Canada

Bioinformatics:

Department of Medical Chemistry, Molecular Biology and Pathobiochemistry , Semmelweis University, Budapest

University of Michigan, USA

Bacterial flagellins:

Pannon University, Veszprém

Instuments

FPLC for protein purification

QCM, DSC, FT IR

Teaching

Undergraduate courses: Pázmány Péter Catholic University, Faculty of Information Technology and Bionics: biophysics, biochemistry and molecular biology

Supervising B.Sc., M.Sc. and Ph.D. students at the Eötvös University and University of Technology

Leader

Péter Gál

Members

Conventionally, deoxyuridine incorporation into DNA is regarded to represent erroneous lesions, however chemical features of the uracil base does not show a remarkable difference from thymine except from a single methyl group at  the 5^th position of the pyrimidine ring. As the balance of the cellular nucleotide pool is deterministic in the quality of DNA synthesis, dUTPase catalyzing the hydrolysis of dUTP is a major player in the maintenance of this balance and uracil-free genome. Our research is focused on the genome metabolism of uracil substituted DNA in the framework of a new paradigm, suggesting that deoxyuridine lesions might assign unique fate for DNA in special cases. These cases are extensively studied recently by a set of research groups including ours.  The special instances in which the presence of uracil was verified to have a unique role are the following:

• Immunoglobuline gene diversification
• Transcriptional regulation
• HIV life cycle
• and developmental biology

We focus on some of the above mentioned or other examples of uracil-DNA mediated cellular events. We employ this paradigm on model organisms such as mammalian or tumor-derived cell lines and Drosophila melanogaster. The physiological role of uracil substituted DNA is studied either genome scale by methods of molecular and cell biology. In Drosophila, complex developmental consequences are also examined in our lab.

Muha et al, 2012 PLoS Genetics

Interconnections of thymine-less cell death with cellular signaling pathways

Almost all organisms employ a cluster of metabolic enzymes devoted for thymine biosynthesis in order to utilize thymine bases instead of uracil in their genome. The medical significance of this metabolic pathway is marked by the fact that nearly one-third of anti-cancer drugs used in clinics are targeted against thymidilate biosynthesis (such as fluoro-pyrimidines, anti folates) potentially inducing the so called thymine-less cell death. Personalized medicine aiming to minimize side effects and maximize the efficiency of chemotherapies requires prior knowledge about the characteristics of tumorous cells in order to predict the desired effect of a particular drug. Detailed mapping of protein networks participating or being affected by thymine-less cell death help us to estimate the receptivity of tumorous cells for drugs targeting thymidilate biosynthesis. We would like to contribute for this knowledge through our research that includes the characterization of thymine-less cell death. We especially aim to explore the involvement of dUTPase in the cellular process of thymine-less cell death.

Study of a molecular switch: Structural and molecular biological research on the Staphylococcus aureus pathogenicity island regulation

The bacterial genom frequently contains mobile genetic elements, which can replicate more or less independently from the bacterial chromosome. Some of these are phage related such as the pathogenicity islands (PIs), which have significant biomedical importance, since these are responsible for horizontal transfer of several toxins and virulence factors (for eg. the toxic shock syndrome).

Phage mediated mobilisation of some Staphylococcus aureus PIs are induced by formation of a repressor:derepressor complex of the Staphylococcal repressor protein (Stl) with a phage-related dUTPase. Studying the detailed mechanism of this interaction can provide much needed deep insight into bacterial gene expression regulation pathways, and potentially facilitates the design of new anti-bacterial compounds. In this project we investigate this system using various in vitro techniques (native gel electrophoresis, electrophoretic mobility shift assay, steady-state and transient kinetics, VIS and fluorescence spectroscopy, mass spectrometry and X-ray crystallography).

Studying regulation of macromolecular transport between nucleus and cytoplasm  in space and time

Nucleotide metabolism pathways important in so-called preventive DNA repair, among these nuclear and mitochondrial localization of dUTPase enzyme in eukaryotes, can universally be observed. Mechanism of the localization and regulation, we would like to explore, is questionable. Apropos of this, we found out the intriguing dynamic regulation role of cell cycle coupled (cdk1 kinase mediated) phosphorylation, that also applies to several proteins of human proteome. Cessation of phosphorylation potential and mimication of constitutive phosphorylation by mutation of serine position to glutamine or glutamic acid decisively affect dUTPase pool of daughter cells’ nuclear proteome. This observation likely reveals a general regulatory mechanism. According to our work hypothesis phosphorylation by cdk1 kinase near NLS significantly influance dynamic nuclear transport of several human proteins.

Thymidine biosynthesis is essential as dTTP is one of the nucleotide building blocks of DNA. Normally, three major pathways exist for dTTP synthesis, but only one of these is present in mycobacteria. This one involves the dUTPase reaction which also serves to eliminate excess dUTP to prevent DNA uracilation. Due to the exclusive biosynthetic role of dUTPase in mycobacteria, these organisms are ideal to investigate the physiology of the dUTPase reaction and the mechanisms underlying genom integrity in the living mycobacterial cell. The laboratory model Mycobacterium smegmatis is used for our experiments, where we have successfully rescued the dUTPase knock-out phenotype with several mutant dUTPases. Using a complex array of methods of cell and molecular biology, we are getting novel insights into a biological problem of potentially central interest of cell biology. This system provides unparalleled opportunity to study how mutant dUTPase enzymes perform under normal conditions as well as under diverse shock conditions in the mycobacterial cell.

Structural and mechanistic insights into antimalarial drug target CTP:phosphocholine cytidylyltransferase (CCT) from Plasmodium falciparum

Despite the intensive antimalarial research, malaria is still one of the deadliest  infectious diseases today. Amongthe causative agents, Plasmodium falciparum is responsible for the vast majority of mortal outcomes. The recent challenges include the spread of multidrug resistant strains, urging the discovery of novel antimalarial targets. CTP:phosphocholine cytidylyltransferase (CCT) catalyzes the rate-limiting step within de novo phosphatidylcholine biosynthesis, a novel validated antimalarial drug target. We perform biochemical and structural characterization of CCT from Plasmodium falciparum to gain insights in mechanism of action and characterize parasite-specific functional elements.

Current collaborations:

Buday László, MTA TTK

Liliom Károly, MTA TTK

Szüts Dávid, MTA TTK

Vékey Károly, MTA TTK

Perczel András, ELTE

Harmat Veronika, ELTE

Náray-Szabó Gábor, ELTE

Nyitray László, ELTE

Kovács Mihály, ELTE

Málnási-Csizmadia András, ELTE

Vellai Tibor, ELTE

Nyulászi László, BME

Poppe László, BME

Oláh Julianna, BME

Fésüs László, DE

Matthias Wilmanns, EMBL Hamburg

Barabás Orsolya, EMBL Heidelberg

Dolores Gonzalez-Pacanowska, Granada

Keith Wilson, Univ York

Iva Pichova, Czech Academy of Science, Prága

Henri Vial Univ Montpellier

Rachel Cerdan Univ Montpellier

Bostjan Kobe Univ Queensland

Leader

Beáta G. Vértessy

Members

Soft Tissue Calcification

Abnormal mineralization in the middle sized arteries and in other soft tissues can be due to mutations in the ABCC6 gene encoding an ABC transporter or in the ectonucleotide pyrophosphatase ENPP1 gene. Mutations in either gene can cause pseudoxanthoma elasticum (PXE, OMIM 264800) or generalized arterial calcification of infancy (GACI, OMIM 208000). The expression pattern of ENPP1 is wider than that of ABCC6, but both proteins are expressed in the liver at high levels and are localized to the basolateral plasma membrane of hepatocytes. Mendelian disorders are associated to these two genes. PXE is characterized by severe skin, eye and arterial manifestations, while the very rare GACI causes severe prenatal aortic calcification with a high mortality rate. GACI and PXE represent two extremes of clinical spectrum of calcification disorders. In spite of the intensive international collaborative research efforts in which our group plays an important role, currently there is no therapy for these conditions. These disorders serve as models for the understanding of the pathomechanism of arterial calcification for a wide range of distinct diseases including more frequent clinical phenotypes such as “acquired” macular degeneration, β-thalassemia, vascular calcification (sometimes leading to coronary artery disease or stroke), uremia, and other rare calcification disorders.

The focus of our research is to find and test future therapeutics for these rare diseases in preclinical mouse models.

Epigenetics

Covalent modifications of the chromatin are called epigenetic modifications. These modifications regulate the activity of the different parts of our genome and participate thereby to gene expression and cellular differentiation. Tamás Arányi is a senior research fellow (assoc. professor), who started research by studying the transcriptional regulation of genes of high medical relevance, tyrosine hydroxylase and later ABCC6, both associated with various diseases. He became immediately interested in the role of epigenetic modifications and their role in the regulation of the target genes. He also set up some tools used since in epigenetic research. Tamas demonstrated the functional role of epigenetic modifications in the regulation of the studied genes.

His research focus progressively turned recently to the study of the dynamic nature of epigenetic modifications (mainly DNA methylation) and the role of environmental stress in altering global epigenetic landscape. These environmental factors, for example viral infection or nutritional challenges are part of our everyday life. The discovery of their influence on our epigenetic profiles will participate in understanding the molecular mechanisms of the long-term effects of the various environmental stress factors often accompanied by major clinical relevance like obesity, hypertension or cancer.

Publications:

https://vm.mtmt.hu//search/slist.php?nwi=1&inited=1&ty_on=1&url_on=1&cite_type=2&orderby=3D1a&location=mtmt&stn=1&AuthorID=10000030&Scientific=1

and

http://www.ncbi.nlm.nih.gov/pubmed/?term=aranyi+t

Collaborations

• Department of Cell and Molecular Biology, University of Hawai’ (USA)
• Department of Deramatology, Th. Jefferson University, Philadelphia (USA)
• PXE International, Bethesda (USA)
• The Netherlands Cancer Institute (Holland)
• Angers University Hospital, Angers (France)

Leader

László Buday

Members