Lysosomal function ranges from simple macromolecule digestion via autophagy to various cell-mediated death processes. Beyond these canonical tasks, lysosomes have also recently identified non-canonical roles in cell differentiation, extracellular matrix reorganization, cancer cell development, and immunological responses. Lysosomes are single membrane-bounded organelles whose dysfunction yields a variety of diseases due to the release of lysosomal proteins into the cytoplasm because of the damage of the lysosomal membrane – called lysosomal membrane permeabilization (LMP). Lysosome-targeted photodynamic therapy causing LMP and inducing immunological cell death can be exploited clinically, such as in cancer therapy, to eradicate cancerous cells. To monitor LMP in live cells, we can apply Acridin Orange (AO) staining. Applying precise light-induced membrane injury in AO-stained cells, a single-cell photodynamic therapy model triggers LMP. We can control which cells’ lysosomes get damaged using this model system in an image cytometric approach. In our measurements, we first monitored the change in AO fluorescence over several consecutive days with flow cytometry. The cells were labeled with different concentrations of AO to identify which dye concentration was the best for the sub-vital staining. With this labeling, the cells luminesce green, while the lysosomes specifically luminesce red. Before the daily flow cytometry measurements, the cells were labeled with the Hoechst DNA dye to identify the type of cell death. The cell line used was Jurkat cells, a line of CD4+/CD8- T lymphocytes. We also plan to induce LMP by applying LLOMe, a lysosomal poison leading to the release of lysosomal proteins. We want to identify the specific damage of the lysosomes, which causes the rearrangement of the galectin-1 and galectin-3, and what the galectin puncta assay will detect.

Témavezető: Zsolt Bacso

The HEK (Human Embryonic Kidney) cell line is often used to generate stable cell lines due to their high transfection efficiency. Stable expression of K⁺ channel genes has multiple benefits over transient expression, including controlled expression level and high transfection efficiency required for automated patch-clamp. CRISPR/Cas9 is an advanced method for creating stable cell lines by enabling precise transgene integration at specific genomic loci, providing better control over expression. Kv1.2 is a voltage-gated K⁺ channel that plays an essential role in the regulation of electrical activity in neurons and is one of the attractive pharmacological targets in epilepsy. The aim of this research was to generate a stable cell line expressing the Kv1.2 channel in HEK293T cells utilizing CRISPR/Cas9-mediated knock in. To integrate Kv1.2 under the promoter of the house keeping gene β actin two vectors were designed: 1) sgRNA/Cas9 vector that encodes guide RNA and the Cas9 protein, 2) destination vector that include the Kv1.2, green fluorescent protein (GFP), and Zeocin resistance genes. HEK293T cells were co-transfected with both vectors using Lipofectamine 2000 and cells were grown for 14 days in the presence of 100µg/ml zeocin to eliminate the non-transfected cells. Six single cell clones were selected using cloning disks and grown for 4-5 days. Flow cytometry was used to confirm the presence of single population of GFP-expressing cells. In order to characterize the stable cell line expressing Kv1.2, whole-cell patch-clamp electrophysiology was applied. Flow cytometric analysis revealed that clone 6 had the highest GFP signal with the narrowest variability so it was analyzed with patch- clamp. Kv1.2 currents were evoked by applying 15-500-ms-long depolarization pulses to +50 mV from a holding potential of -120 mV. Charybdotoxin at 14 nM inhibits 50% of Kv1.2 currents (as per literature) but in the generated cell line it only inhibited 23% of the current. Kv1.2 is insensitive to TEA (tetraethylammonium) but in the generated cell line 100 mM TEA inhibited 60% of the total current which confirms the presence of endogenous K⁺ current in HEK293T cells. We conclude that HEK293T is not a suitable candidate to generate a stable cell line expressing K⁺ channels as they have high endogenous K⁺ currents that contaminate the whole-cell currents. We are currently optimizing experiments to generate stable cell line using CHO (Chinese hamster ovary) cell line.

Témavezető: Kashmala Shakeel és Dr. Gyorgy Panyi

The voltage-gated NaV1.5 sodium channel is found mostly in the heart where it initiates cardiac action potentials. Its gain-of-function and loss-of-function mutations often lead to arrhythmias such as long QT and Brugada syndromes. In addition, recently NaV1.5 has been identified as a potential contributor to breast cancer progression, invasiveness and metastasis formation, which makes it an attractive drug target not only for certain cardiac conditions but cancer treatment as well. This inspires a search for isoform specific molecules to modulate its function. Drug repurposing is a time- and cost-efficient way of obtaining novel treatment methods. While testing the selectivity of inhibitors of the voltage-gated proton channel Hv1, we have found that a small molecule synthetized by our chemist collaborators, named NZ-58, also blocks the NaV1.5 channel. We measured the effect of the drug by the whole-cell configuration of the patch-clamp technique on transiently transfected CHO cells. By determining the blocking effect at various concentrations we constructed the dose-response relationship, which yielded an IC50 value of ~10 microM. An important aspect of channel modulators is functional selectivity or state-dependence: they may preferentially bind to various gating states of the channel, such as the open or inactivated state, which can allow, for example, selective targeting of hyperactive channels. Therefore the mechanism of action must be investigated to comprehensively characterize the modulator molecule. Our results indicate that NZ-58 affects neither the activation threshold voltage nor the voltage-dependence of steady-state inactivation of NaV1.5. However, it drastically slows both the activation and inactivation kinetics of the channel. Since different voltage-sensor domains (VSD) are responsible for channel opening and inactivation in NaV channels, this suggests that NZ-58 can bind to multiple VSDs. Moreover, current block is delayed compared to the kinetic changes implying the existence of yet another binding site, which we plan to investigate with further experiments. Even though NaZ-58 is not selective, as it blocks both Hv1 and NaV1.5, this is not necessarily a disadvantage since both channels have been implicated in tumor progression and invasion and blocking both may have a synergistic effect. Many such “dirty drugs” are in everyday clinical use today, but to avoid potential side effects, the exact mechanisms must be clarified.

Témavezető: Prof. Dr. Varga Zoltán

Tumor cells adapt to harsh microenvironments characterized by low oxygen, limited nutrients, and acidic pH, often caused by increased glycolysis and lactate production. Acidic conditions drive these cells to change key proteins, including ion channels, which are significant for maintaining cellular homeostasis. This adaptation may lead to ion channel mutations that support tumor cells' survival, proliferation, and invasiveness. To identify such mutations, we collected ion channel sequences found in various cancers from public databases. By aligning related sequences, we identified mutation patterns and determined the frequent mutation sites, such as R367C in the Kv1.3 K+ channel, which affects its voltage sensor. Kv1.3 is present in many cell types; it is most common in immune cells like lymphocytes, but is also associated with tumor progression. In this study, we aimed to compare the biophysical properties of the R367C mutant channel to the wild type by measuring gating parameters in normal and acidic pH conditions using various voltage protocols. We generated the R367C mutant using site-directed mutagenesis and investigated the channels using whole-cell and outside-out patch-clamp experiments on Chinese Hamster Ovary (CHO) cells transfected with wild-type or mutant channel genes. The channels were labeled with Green Fluorescent Protein (GFP) so we could identify the successfully transfected cells under a fluorescence microscope. We analyzed the channels' activation and inactivation kinetics, as well as their voltage sensitivity, using various voltage protocols. Our results show that the R367C mutant Kv1.3 has slower activation and inactivation kinetics, increased pH- sensitivity, lower tendency to inactivate and a wider active membrane potential range at low pH compared to the wild type channel. These data suggest that the mutant channel may be more active than the wild type in the acidic microenvironment, thereby likely contributing to tumor cell survival and proliferation. We hypothesize that ion channel mutations in tumor cells, driven by selection pressure from their microenvironment, such as pH changes, represent an adaptation mechanism to provide an advantage. In future experiments, we will study the pharmacological properties of the mutant channel and explore its functional relevance, particularly concerning pH- sensitivity and its role in tumor progression.

Témavezető: Varga Zoltan

P-glycoprotein (Pgp) can export a broad range of anti-neoplastic drugs from tumour cells, resulting in chemotherapy resistance. During its transport cycle, Pgp alternates between conformations facing the cytoplasm (inward-facing, (IF)) and the extracellular side (outward-facing, (OF)). UIC2 is a conformation-selective antibody recognizing the IF Pgps. Previous research suggested that Pgp is present in raft and non-raft membrane microdomains. However, little is known about the functional significance of its dual localization. Confocal microscopy-based co-localization studies carried out with raft and non-raft markers demonstrated that Pgps spontaneously taking up the UIC2-reactive IF conformation are predominantly raft-localized (pool I), while those that take up this conformation in the presence of Pgp inhibitors, such as cyclosporine A (CsA), reside in the non-raft microdomains (pool II). Raft and non-raft membrane microdomains may each have a different melting temperature (Tm), which may affect the function of raft and non-raft resident Pgps differently. Therefore, we investigated how different temperatures (30 & 37°C) affect the function-dependent conformation changes of Pgp in the absence and presence of a known substrate, verapamil (Vp) by measuring the kinetics of UIC2 dissociation from the different Pgp pools. In the absence of CsA, when only pool I Pgps were visualized, we observed comparable UIC2 dissociation at 30 and 37°C supporting that this temperature decrease does not inhibit Pgp activity. 1 μM Vp strongly increased (8-fold) the rate of UIC2 dissociation at 37°C, while we observed only a 2-fold stimulation by Vp at 30°C. We assume that the temperature decrease reduces membrane fluidity, which affects the interaction of Pgp with Vp. We also noticed that large fractions of Pgps are active in both pools. CsA used in sequential labelling increased the active fraction of Pgps from ~40% to ~70% in pool I at 37°C, while decreasing it to ~10% at 30°C. In addition, CsA slowed down the UIC2 dissociation from pool I and reduced the extent of Vp stimulation. We hypothesize that CsA modulates the active fraction of Pgps by a membrane fluidity-dependent mechanism while slowing down the conformation changes of Pgps through its direct binding to the protein. According to our knowledge, this is the first study that could analyse the function raft and non-raft resident Pgps in the membrane of live cells.

Témavezető: Goda Katalin

Cariprazine (CP) is a novel antipsychotic drug used in the treatment of schizophrenia and manic or mixed episodes of bipolar disorder. It acts primarily as a D3 and D2 dopamine receptor partial agonist, with a preference for the D3 receptor. Certain members of the ABC transporter family including ABCB1, ABCG2 and ABCC1 are multidrug transporters that can export numerous lipophilic or amphiphilic drugs out of cells on the expense of ATP hydrolysis. Since the above-mentioned ABC transporters are present in pharmacologically relevant barrier tissues such as intestinal epithelium, blood-brain barrier, liver and kidneys, they are important determinants of the pharmacokinetics of their substrates. Inhibition or reduced expression of the transporters in response to other co-administered drugs may cause altered pharmacokinetics and unwanted side effects of chemotherapy. A recent study demonstrated that CP inhibits the transport activity of a mutant ABCG2 variant possessing altered substrate specificity compared to wild- type (Wt) ABCG2. Here we aimed to explore the interaction of CP with Wt ABC transporters including ABCG2 and ABCB1. We studied the effects of CP on the ATPase activity and on the transport activity of the transporters. In ATPase assays, we used membrane samples isolated from cells overexpressing one of the studied transporters. In transport assays, we used the same cell lines and studied the cellular accumulation of specific fluorescent substrates, such as calcein-AM for ABCB1 and mitoxantrone (MX) for ABCG2. We observed that CP reduced the ATPase activity of Wt ABCG2 and increased the cellular accumulation of MX in a concentration dependent manner supporting that it can act as an inhibitor of the Wt ABCG2. Since transporter ligands often regulate the cell surface expression of ABC transporters, we also examined this possibility by using a green fluorescent protein-based reporter assay system. We found that CP did not have any effect on the expression of ABCG2, while the ABCG2 substrate quercetin upregulated it in accordance with literature data. Interestingly enough, the inhibitory effect of CP appeared to be even stronger on ABCB1 supporting that CP is a dual ABC transporter inhibitor. Our observations call the attention to the risk of drug- drug interactions, when CP treatment is co- administered with other ABC transporter substrate drugs including certain anti-cancer drugs, digoxin or verapamil among others.

Témavezető: Katalin Goda

Introduction: Astrocytes can critically affect neuronal excitability via eliciting tonic and phasic NMDA receptor dependent excitatory currents on them - known as ’slow inward currents’ (SICs). We plan to investigate the impact of the Piezo-1 mechanosensitive channel on the phasic NMDA receptor dependent neuronal currents, and the consequential changes of neuronal activity. Objective: The aim of the study was evaluating changes in (SIC) slow inward current activity with Piezo-1 channel agonist Yoda & channel antagonist Dooku. Methods: Recordings are carried out with mice of 2-3 weeks of age as brain specimens, on neocortical pyramidal neurons. The intracellular solution used was 120 ml KGlu and 3 mg/ml biocytin, and the extracellular solution used was normal artificial CSF. Recording solution included zero Mg solution, initially, Yoda (10 µm), Dooku (10 µm), and inhibitors of fast synaptic neurotransmission (10 µM NBQX, 20 µM D-AP5, 1 µM strychnine, 1 µM bicuculline). Results: The first experiments tested if activation of Piezo1 channels could change SIC activity on pyramidal neurons. Recordings in nominally magnesium-free showed that addition of the Piezo1 activator Yoda to a tendency of increase in SIC amplitude, charge transfer of the individual events, as well as rise time and decay. The SIC frequency showed a significant, more than twofold increase. With NMDA receptor inhibitor D-AP5 in the experimental preparation, SICs were abolished. Supplementation of the recording solution with Yoda did not cause any significant increase of SIC activity. When Dooku was applied alone, a decrease with no significant difference was seen in charge transfer of the individual events, the decay time, the frequency and the SIC activity with only a significant decrease of the rise time. When both Yoda and Dooku were applied the increase in SIC frequency and activity was reverted. Conclusion: With time, results have provided us with certainty that Yoda is proven activator of the Piezo1 channel, as multiple SICs seen after application and Dooku is an inhibitor, with significant reduction of SIC activity.

Témavezető: Dr. Pál Balázs Zoltán és Dr. Andrea Csemer

A sejtek membránjai háromféle elektromos tulajdonsággal jellemezhetőek, melyek együttesen alakítják ki a membránpotenciált. Ez magába foglalja a transzmembrán-, a felületi- és a dipólpotenciált. A legismertebb ezek közül a transzmembrán potenciál, amelyet a membrán két oldalán található ionok közötti koncentrációgradiens hoz létre. A felületi potenciál a membrán és víz közötti határfelületen jön létre a membránt alkotó foszfatidok feji végén található töltéssel rendelkező funkciós csoportok miatt. A dipólpotenciál a membránt alkotó molekulák és az azokhoz kapcsolódó vízmolekulák dipólmomentumából adódó elektrosztatikus potenciál. A becslések szerint a dipólpotenciál értéke a néhány száz millivoltos tartományba esik, ezáltal jelentősen meghaladja a transzmembrán és a felületi potenciál értékét. A dipólpotenciál változása ezáltal jelentősen befolyásolhatja olyan transzmembrán fehérjék térszerkezetét, amelyek töltéssel rendelkező aminosavat tartalmaznak. A dipólpotenciál értéke a membrán összetételétől függően megváltozhat. Vannak olyan anyagcserebetegségek (Gaucher-kór, SLO-szindróma), ahol a szervezet lipidháztartása felborul, ezáltal a sejtmembrán összetétele is megváltozhat, így a dipólpotenciál értéke is módosulhat. Ezekben a betegségekben emellett különböző immunológiai tünetek is megjelennek. Az interleukin-2 (IL-2), valamint ennek membránreceptora (IL-2R) az immunrendszer szabályozásában központi szerepet játszanak. Fontos szerepük van a T-sejtek osztódásában és differenciálódásában, ezáltal számos kórkép kialakulásában is (pl. sclerosis multiplex, T-sejtes leukémia). Korábban kutatócsoportunk megmutatta, hogy az IL-2R jelátviteli hatékonysága és mobilitása megváltozik, amennyiben a transzmembrán potenciál értékét változtatjuk (de- és hiperpolarizáció). Továbbá prof. Nagy Péter munkacsoportjában vizsgálták a dipólpotenciál hatását az ErbB receptorra, mely során az tapasztalták, hogy megváltozik a receptor asszociációja, ligand-kötő affinitása és jelátviteli hatékonysága. Kutatásunk célja olyan biofizikai mérések elvégzése, amelyek alapján összefüggést találhatunk a dipólpotenciál változása és az IL-2R jelátvitele között, ennek eredményeképpen az immunrendszer ezidáig ismeretlen szabályozó folyamataira deríthetünk fényt. Ezáltal mélyebben megérthetjük a szervezet lipidháztartásának és az immunrendszerrel összefüggő betegségek kapcsolatát.

Témavezető: Dr. Vámosi György

A COVID-19 pandémia továbbra is globális kihívást jelent a vírus tüskefehérjéjének mutációja révén megjelenő új SARS-CoV-2 variánsok és a népesség heterogén immunitása miatt. A fertőzés első lépése a tüskefehérjék kölcsönhatása a célsejtek membránjának koleszterinben gazdag lipidtutajaiban dúsuló ACE2 receptorokkal, amelyet a vírus gazdasejtbe történő felvétele követ. Korábbi eredményeink alapján a fertőzés ezen kezdeti lépései erősen koleszterinfüggők, a célsejt membránjában található koleszterin ciklodextrinek általi depléciója hatékonyan gátolja azokat. A különböző variánsok általi fertőzés koleszterinfüggésének mértékét azonban nem vizsgálták. Hipotézisünk szerint a SARS-CoV-2 variánsok változó ACE2 affinitásuk révén eltérő mértékben érzékenyek a membrán koleszterintartalmában bekövetkező változásokra, amely terápiás relevanciával bírhat. Célunk, hogy összehasonlítsuk a WT, Alfa, Béta, Delta, Omikron Ba1 és Ba2 fenotípusú tüskefehérjék ACE2 affinitását, és annak kapcsolatát a receptorkötés és az internalizáció koleszterinkivonás iránti érzékenységének mértékével. Áramlási citometriával vizsgáltuk a fluorofórral konjugált tüskefehérje variánsok ACE2 kötődésének hatékonyságát ACE2-t és TMPRSS2-t stabilan expresszáló HEK293 sejtekben. Méréseinket az orvosi gyakorlatban is alkalmazott hidroxipropil-β-ciklodextrinnel (HPβCD) kezelt sejteken is elvégeztük, és összehasonlítottuk a koleszterin depléciójának hatását a variánsok esetén. Vizsgáltuk emellett a kötődés mértéke és a sejtmembrán lipidtutaj tartalma közötti korrelációt. 3D kvantitatív konfokális mikroszkópiával a tüskefehérje variánsok intracelluláris felvételét és a folyamat HPβCD-érzékenységét is jellemeztük. Kimutattuk, hogy a magas ACE2 affinitású variánsok (Béta, Delta) kötődése kisebb korrelációt mutat a sejtmembrán lipidtutaj tartalmával és kevésbé gátolható HPβCD-kezeléssel. Ezzel szemben az alacsony ACE2 affinitású variánsok (WT, Omikron Ba1) erősebb preferenciával rendelkeznek a magasabb tutajtartalommal rendelkező sejtek iránt és a CD-kezelés fokozottabban gátolja a tüskefehérjék kötődését és felvételét is. Ennek egyik magyarázata, hogy a variánsok eltérő preferenciát mutatnak a tutajon belül, illetve kívül elhelyezkedő ACE2 iránt, amely vizsgálatát megkezdtük. Eredményeink rávilágítanak a különböző SARS-CoV-2 tüskefehérjék és a sejtmembrán lipidösszetétele közötti dinamikus kapcsolatra, ami új terápiás megközelítést jelenthet. EKOP-24-2-DE-205, EKOP-24-4-II-DE-74, OTKA FK146740, OTKA FK143400

Témavezető: Dr. Zákány Florina és Dr. Kovács Tamás