Centralna tehniška knjižnica Univerze v Ljubljani (CTK)
  • In vitro modulation of O-GlcNAcylation and its impact on the function of selected immune and cancer cells = Vpliv modulacije N-acetilglukozaminilacije na delovanje izbranih imunskih in rakavih celic in vitro : doctoral dissertation
    Weiss, Matjaž
    O-GlcNAcylation is a common posttranslational modification that demands many components, such as glucose, glutamine, uridine, acetyl-CoA and cellular energy. O-β-N-acetylglucosaminyl transferase ... (OGT) is a transferase that catalyses the transfer of N-acetylglucosamine from UDP-GlcNAc to serine and threonine residues of nuclear and cytoplasmic proteins. O-GlcNAcase (OGA) is a reciprocal enzyme to OGT that removes O-GlcNAc moieties from glycosylated proteins. Therefore, O-GlcNAcylation is a dynamic process in physiological environments, regulated by OGT and OGA expression levels and the availability of UDP-GlcNAc components. The number of discovered proteins modified by O-GlcNAcylation is increasing and includes several transcription factors, epigenetic regulators, kinases, cytoplasmic enzymes and mitochondrial proteins. The complexity of this posttranslational process arises within the cellular context, where the protein kinases compete with OGT for the same serine or threonine hydroxyl groups on targeted proteins (e.g. AKT). Another possibility of crosstalk exists at neighbouring amino acids (e.g. c-Myc), where binding of the first moiety, being either GlcNAc or a phosphate, affects binding of the second. Dysregulated O-GlcNAcylation is associated with several pathologies including cancers, cardiovascular and neurodegenerative diseases. It is basically a metabolic gauge and thus the highest expression of OGT is found in ß pancreatic cells where chronically high O-GlcNAcylation results in insulin resistance, so that diabetes correlates with the high level of O-GlcNAcylation. Elevated expression of OGT and increased levels of O-GlcNAcylated proteins are characteristic for some types of cancer. In the fight against malignant transformation or pathogens, the immune system plays a crucial role. The role of O-GlcNAcylation in macrophages, neutrophils, T and B cells has been studied in recent years, but there were no studies done on human dendritic (DC) and natural killer cells. Most cells obligatorily require adequate O-GlcNAcylation for their proper development and function. However, the exact role of O-GlcNAcylation in many biological systems is still not well understood. In recent years, some new potential inhibitors have been synthesized, to be used as molecular probes to study the role of OGT in biological systems. The most potent and widely used OGT inhibitors are the so-called OSMIs that, however, still have some shortcomings, such us scarce selectivity. In an effort to overcome these, we synthesized new quinolinone-based OGT inhibitors, targeting the uridine diphosphate (UDP) binding OGT pocket. To further 2 improve their inhibitory potency, we designed peptidic conjugates by attaching peptide substrates to low molecular OGT inhibitors. Conjugated moieties were connected via selected linkers and the effect of linker length and flexibility on inhibitory potency was evaluated as well. Unfortunately, none of the synthesized peptidic conjugates or low molecular OGT inhibitors was more potent than those previously reported in the literature. The most potent inhibitor from our series was the fragment-based 3b with IC50 = 116.0 μM. Aberrant O-GlcNAcylation of some protein substrates has been also involved in inappropriate immune response, but this remained underexplored. In our study, we investigated the effect of OGT inhibition on human dendritic and natural killer cells. We showed the immunosuppressive effects of OGT inhibitor in NK cells, by confirming their decreased cytotoxicity against K562 target cells by 33.4 ± 10.9%, when they were pretreated with OSMI-1 (20 μM). The reason for this phenomenon could be in decreased number of degranulating NK cells and the reduced release of inflammatory molecules which has been also observed in our experiments, yet the mechanisms how OGT inhibitors altered cytotoxicity remains unknown. In addition, we showed that OGT also plays a role in DCs, as OGT inhibitor, OSMI-1, has affected the functionality and phenotype of dendritic cells. The presence of OSMI-1 during the monocyte to DC differentiation process modulated the mTOR/AKT and MEK/ERK pathways both in immature as in mature monocyte derived DCs (moDCs). OGT inhibition also altered relevant surface marker profiles, where reduced expression of CD80 and DC-SIGN, as well as increased expression of CD14, CD86 and HLA-DR in immature monocyte derived DCs were observed. This suggests that inhibition of O-GlcNAcylation hampers the transition of monocytes into immature moDCs. Moreover, the presence of OSMI-1 during the moDC differentiation and activation process led to a decrease in IL-10 and an increase in IL-6 secretion by immature and mature moDCs. Additionally, diminished endocytosis was observed in immature moDCs. Inhibition of OGT also disrupted the maturation process of moDCs and impaired the proliferation of allogeneic T cells induced by the OSMI-1-treated mature moDCs, whereas no changes in the polarization of T cells could be observed compared to control. Taken together, we confirm that the OGT inhibitor, OSMI-1, alters the differentiation and function of moDCs under in vitro conditions. OGT inhibitors could potentially be used as a supportive therapy to already known chemotherapeutics in the treatment of cancer. Namely, we have observed a significantly 3 enhanced effect of OSMI-1 and regorafenib on metabolic activity of selected human cancer cell lines. The IC50 value of regorafenib on AMO-1 human plasmacytoma cell line was decreased 3.68-fold, when these were co-treated with non-cytotoxic concentration of OSMI-1 (10 μM). Further studies revealed a synergistic cytotoxic effect of both substances on the AMO-1 cell line, in which the percentage of dead cells increased from 11% to 38% when they were co-treated with 5 μM regorafenib and 10 μM OSMI-1, where apoptosis was not the predominant type of cell death. The proliferation of AMO-1 cells was evidently inhibited after the use of both inhibitors, as we observed a decreased number of cell divisions and an increased population of cells in the G0/G1 phase of the cell cycle. These effects seem to be linked to the inhibition of cyclin D-dependent activation of cyclin-dependent kinase 4 (CDK4), the inhibition of the retinoblastoma protein and the inhibition of S-phase gene transcription.
    Vrsta gradiva - disertacija ; neleposlovje za odrasle
    Založništvo in izdelava - Ljubljana : [M. Weiss], 2022
    Jezik - angleški, slovenski
    COBISS.SI-ID - 97407491

    Povezava(-e):

    Repozitorij Univerze v Ljubljani – RUL
    Digitalna knjižnica Slovenije - dLib.si

    Dostop z namenskih računalnikov v prostorih NUK



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Centralna tehniška knjižnica Univerze v Ljubljani
prosto - na dom, čas izposoje: 14 dni
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0000052047/0000002609 Skladišče
IN: 320220174 52047/2609 Skladišče
IN: 320220174 		prosto - na dom, čas izposoje: 14 dni
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