OETDK.

The anti-cancer effects of polyphenolic compounds, epigallocatechin gallate (EGCG) and procyanidin C1 (PC1), are mediated by the 67 kDa laminin receptor (67LR). The binding of EGCG or PC1 to 67LR activates protein kinase A (PKA), followed by an increase in protein phosphatase 2A (PP2A) activity, which ultimately leads to the inhibition of cell growth. Substrates dephosphorylated by PP2A have been identified (e.g., myosin phosphatase target subunit (MYPT1) or the C-kinase potentiated protein phosphatase-1 inhibitor protein of 17 kDa (CPI-17)). However, the B regulatory subunit, which confers substrate specificity to PP2A in this pathway, has not yet been identified. Previous data suggest that PP2A holoenzymes containing B56γ or B56δ regulatory subunits can be activated by PKA-catalyzed phosphorylation. Therefore, our goal was to study the role of B56γ/δ phosphorylation in 67LR signaling using breast cancer cells as a model. We analyzed B56γ and B56δ expression in a selection of breast cancer cell lines with different subtypes. Based on our qPCR experiments, several cell lines showed significantly higher B56γ/δ mRNA levels compared to MCF-10A non-cancerous breast epithelial cells. At the protein level, B56γ and B56δ expression differed greatly between cell lines. We also tested for the presence of other components of the 67LR pathway, including 67LR, MYPT1, and CPI-17, using Western blotting. In our ongoing experiments, we treat MCF10A, MCF7, BT474, and MDA-MB231 cells with EGCG or PC1 and test B56 phosphorylation using anti-phospho-B56γ/δ antibody. In parallel, we conduct proliferation assays to test whether the growth-inhibitory effects of EGCG and PC1 on breast cancer cells are dependent on B56γ/δ expression and/or phosphorylation. Further experiments are needed to prove that B56γ/δ phosphorylation is necessary for the anticancer effects of EGCG and PC1. Our results may provide a better understanding of PP2A regulation in breast cancer cell signaling.

Témavezető: Dr. Kiss Andrea

Tankyrase 1 (TNKS1) and tankyrase 2 (TNKS2) are members of the PARP enzyme family, which consists of 17 members. PARP enzymes participate in poly-ADP-ribosylation of target proteins. Tankyrases (TNKSs) are involved in various cellular functions, of which telomere length maintenance was described first. TNKS enzymes have recently been found to play a role in metabolic regulation and energy homeostasis. The aim of this study is to better understand the TNKS1 and TNKS2 mediated effects governing the differentiation of white adipocytes. We would like to establish the exclusive TNKS mediated features in white adipose tissue and, subsequently, assess the applicability of TNKS inhibitors in regulating adipocyte differentiation.In the experiments, differentiated 3T3-L1 preadipocytes were applied as a model. We observed that the mRNA expression of TNKSs increased in the terminal differentiation phase. Our research group generated TNKS1 and TNKS2 single and TNKS1/TNKS2 double knockout 3T3-L1 cell lines using the CRISPR/Cas9 method. We characterised the knockout clones by qPCR. In prior experiments we performed an RNAseq analysis on the differentiated clones. During my research work, we performed functional analysis with Western blot method to confirm the RNAseq results. Through that, we showed changes in the expression of genes related to energy sensor pathways, such as AMPK, as well as protein degradation pathways such as ubiquitination. We have identified TNKS-related effects in adipocyte differentiation.Next, we began assessing the applicability of TNKS inhibitors in regulating adipocyte differentiation. TNKS inhibitors previously yielded contradicting results that we aim to clarify and determine their suitability. These studies will shed light on the role of the enzymatic activity of TNKSs in adipocyte differentiation.

Témavezető: Prof. Dr. Bay Peter és Rauch Boglarka

Introduction: Thrombospondin-1 (TSP1) is a multifunctional glycoprotein involved in cell-to-cell and cell-to-matrix communication. Its N-terminal domain contains a well-defined, high-affinity heparin-binding region that mediates interactions with heparan sulfate proteoglycans and other extracellular ligands. These interactions influence a wide range of cellular processes, including cell adhesion, spreading, proliferation, and migration. Previous structural and biochemical studies have identified a cluster of positively charged residues (most notably R29, R42, R76/R77) that form the core of the TSP1 heparin-binding surface. Our workgroup has identified Ser44 as a protein kinase A (PKA) phosphorylation site in TSP1, located within the heparin-binding region, suggesting that phosphorylation at this position may modulate heparin affinity through local structural or electrostatic effects.Aim: The aim of this study was to investigate whether post-translational modification at Ser44 influences the ability of the TSP1 N-terminal domain to bind heparin.Material and methods: Site-directed mutagenesis was used to introduce substitutions at Ser44, generating Ser44Ala (phosphonull) and Ser44Asp (phosphomimetic) variants of TSP1. Recombinant TSP1 N-terminal fragments were then expressed in BL21(DE3) E. coli cells. For the heparin-binding assay, equal amounts of each recombinant protein were incubated with heparin-agarose beads. The input, unbound, and bound samples were analyzed by SDS-PAGE, followed by gel staining or Western blotting.Results: Sequencing confirmed the successful introduction of the Ser44Ala and Ser44Asp mutations. All recombinant TSP1 N-terminal proteins were successfully expressed at comparable levels. In the heparin-agarose assay, both the wild-type and the phosphonull mutant bound efficiently to heparin. In contrast, the phosphomimetic mutant exhibited markedly reduced binding.Conclusion: Our findings support the concept that phosphorylation of Ser44 plays a regulatory role in modulating heparin binding by the TSP1 N-terminal domain. This project was funded by the National Research, Development and Innovation Fund of Hungary (FK135384 (A.B.).

Témavezető: Dr. Boratkó Anita és Raya Assala

R-loops are RNA:DNA hybrid structures that regulate gene expression, DNA replication, and DNA repair. While physiological R-loops maintain genome stability, aberrant R-loop accumulation—caused by deficient or mutated regulatory enzymes—disrupts cellular homeostasis and drives cancer pathogenesis.This study investigates whether oncometabolite-producing enzymes alter R-loop levels in breast cancer models. We focused on indoleamine-2,3-dioxygenase 1 (IDO1), which catabolizes tryptophan to kynurenine and other immunosuppressive metabolites, facilitating tumor immune evasion. Although meta-analyses link high IDO1 expression to worse survival, substantial heterogeneity exists across tumor types. We compared R-loop formation between wild-type and IDO1-overexpressing cells in ER+ and triple-negative breast cancer (TNBC) models.R-loop levels were assessed using complementary global and locus-specific methods. Global R-loop content was measured by S9.6 antibody-based dot blot hybridization, while DNA-RNA immunoprecipitation followed by quantitative PCR (DRIP-qPCR) quantified R-loops at specific genomic loci. DRIP-qPCR primers targeted previously characterized R-loop-enriched and R-loop-depleted regions.Dot blot analysis revealed no significant differences in global R-loop levels between control and IDO1-overexpressing cells. DRIP-qPCR detected measurable enrichment at selected loci; however, additional replicates are required to establish reproducible patterns and statistical significance.This study establishes a methodological framework for investigating connections between cancer metabolism and R-loop dysregulation. Future experiments with increased statistical power will clarify whether metabolic reprogramming through IDO1 contributes to pathological R-loop accumulation in breast cancer.

Témavezető: Prof. Dr. Székvölgyi Lóránt és Dr. Kissné Boros-Oláh Beáta

Transglutaminase 2 (TG2) is a multifunctional enzyme with calcium-dependent transamidation activity that enables it to cross-link proteins, forming isopeptide bonds between Glutamine and Lysine residues, or to modify proteins, resulting in amine incorporation or deamidation, thereby influencing cellular processes. Endothelial cells, such as HUVECs, express TG2 at high levels, and mapping the proteins that interact with TG2 or serve as TG2 substrates in these cells could define its roles in endothelial adhesion, motility, angiogenesis, and NO regulation. Our study aimed to identify Glutamine and Lysine donor substrates of TG2 in immortalised HUVEC cells and to compare them with the recently identified TG2-associated proteins.First, a large amount of cell lysate was prepared from immortalised HUVEC cells. Endogenous TG2 was activated by the addition of 10 mM calcium-chloride to incorporate biotin-pentylamine (BPA) into Glutamine-donor protein substrates. BPA was applied at high concentration to enhance labelling efficiency and prevent crosslinking of substrate proteins. The non-incorporated BPA was removed by dialysis, and successful incorporation was verified by Western blot. Biotinylated proteins were enriched with High-Capacity Neutravidin agarose and analysed by LC-MS/MS. The proteomics results were extracted using Scaffold software.We have identified eight Glutamine donor protein substrates: enolase (ENO1), filamin (FLNA), heat shock protein (HSP90AB1), prelamin (LMNA), myosin (MYH9), pyruvate kinase (PKM), tubulin (TUBA1B), and vimentin (VIM). The TRANSDAB database identified all these proteins as TG2 substrates and except PKM. PKM and ENO1 were previously identified as TG2-associated proteins in HUVEC cells. ENO1 and PKM are key glycolysis enzymes, while FLNA, VIM, MYH9, and TUBA1B play crucial roles in cytoskeletal function.In parallel, we began identifying TG2 Lysine-donor substrates using a biotinylated glutamine-donor-containing peptide (Biotin-Ahx-QVPL), and mass spectrometry analysis is in progress.Our results help to clarify the therapeutic possibilities of TG2 inhibition in tumors, cardiovascular diseases, and processes involving unwanted angiogenesis.

Témavezető: Dr. Király Róbert és Rahim Alaa Ahmed

Introduction: Calcification can be a physiological or pathophysiological process. The pathophysiological accumulation of calcium salts in soft tissues is known as ectopic calcification. A common cardiovascular ectopic calcification process is calcific aortic valve disease (CAVD). One of the inducers of CAVD is hyper-phosphatemia, a condition commonly found in chronic kidney disease (CKD) patients. Valve interstitial cells (VICs) are the most affected cell type in an aortic valve. Vitamin D, another known inducer of calcification, is generally used as a supplementation during CKD condition to maintain bone health. Vitamin D Receptor (VDR) needs to heterodimerize with Retinoid X Receptor (RXR) for its function like several other nuclear receptors (NRs) such as Peroxisome-Proliferator Activated Receptor gamma (PPARγ), Retinoic Acid Receptor (RAR), etc. It is also proven that nuclear receptors compete for heterodimerization with RXR in a ligand dependent manner.Hypothesis: Based on the above-mentioned information, we assume that the competition of nuclear receptors for RXR – induced by the application of different NR (RAR, PPARγ) agonists–may have a beneficial effect on aortic valve calcification.Methods: We cultured a primary human heart valve interstitial cell line (VIC) in 96 well plates as a model system to study valve calcification. The experiment took place in two types of media: growth medium (GM) needed for cell survival, and osteogenic medium (OM) containing 0.3 mM calcium and 2.5 mM phosphate. Cells were treated with calcitriol to enhance extracellular matrix calcification. We introduced two other nuclear receptor agonists: Rosiglitazone (RSG) for PPARγ and AM580 for RAR and checked their effect on calcification using alizarin red staining (ARS) and performed ELISA for Osteocalcin , a major marker of extracellular matrix mineralisation. Also, confocal microscopy was performed to quantify the nuclear translocation of VDR under different treatment conditions. Results: We found that calcitriol in the OM further increased calcification. There was a decreasing trend in VICs calcification when using AM580 and RSG together. Quantification of VDR nuclear translocation further confirmed that VDR-RXR heterodimerization decreased in the presence of calcitriol, AM580 and RSG together in comparison with calcitriol alone. These results suggest that ligand induced competition between RXR partners can beneficially influence the side effect of calcitriol treatment.

Témavezető: Dr. Arpan Chowdhury és Dr. György Vámosi

Vascular calcification (VC) is a progressive pathological process characterized by the deposition of hydroxyapatite within the arterial wall. It most commonly occurs in the intimal and medial layers of the arterial wall. Medial vascular calcification is an active, cell-regulated process, which is characterized by the differentiation of vascular smooth muscle cells (VSMCs) from a contractile to an osteo/chondrogenic phenotype. This phenomenon is frequently observed in patients with chronic kidney disease and type 2 diabetes, therefore, elevated serum phosphate level (Pi) and high glucose level are well-established risk factors and inducers of VC. Alpha-ketoglutarate (AKG) is a key player in cellular metabolism, mainly produced in the TCA cycle. AKG has been described as a potent anti-aging, antioxidant and anti-inflammatory agent. However, the potential anti-calcification feature of AKG and its underlying mechanism is not elucidated. Therefore, we aimed to investigate the effect of AKG on high Pi-induced osteochondrogenic reprogramming and calcification of VSMCs.We examined the effect of AKG on Pi-induced calcification in human aortic smooth muscle cells (HAoSMCs). Cells were treated with elevated Pi (2.5 mmol/L) and increasing dose of AKG (10-100 µmol/L). Calcium deposition was detected by Alizarin Red staining (day 4). Effect of AKG (5 mmol/L) was assessed in ex vivo aorta organ culture model and aortic Ca content was evaluated (day 7). The protein expression of osteogenic markers (Runx2, Sox9), and the contractile marker (SM22α) was determined by Western blot (48 hours).Our result show that AKG at 50 and 100 µmol/L concentrations completely inhibited calcification of HAoSMCs. In addition, AKG inhibited high Pi-induced calcification of mice aortic rings by 40%. Western Blot results indicate that all AKG doses reduced Runx2 protein level, while the protein level of Sox9 was attenuated in a dose-dependent manner. Furthermore, high Pi reduced the expression of SM22α by 35%, while the administration of AKG from 25 µmol/L markedly elevated the level of this contractile marker.The results suggest that AKG is a potent inhibitor of calcification by inhibiting osteochondrogenic phenotypic switch and calcification of VSMCs and by restoring contractile phenotype. However, further investigation is warranted to reveal the underlying mechanism of the anti-calcification property of AKG.

Témavezető: Dr. Jeney Viktória és Tóth Andrea