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Study of de novo purine synthesis in model systems of Caenorhabditis elegans and HeLa cell lines
Součková, Olga ; Zikánová, Marie (advisor) ; Šebesta, Ivan (referee) ; Kučera, Lukáš (referee)
Purines are involved in many biologic processes and are required to maintain stable energy charge, for metabolic regulation, as cofactors in enzymatic reactions and as building blocks in DNA and RNA. Necessary role in purine synthesis is played by de novo purine synthesis (DNPS) that is highly active in developing cells. DNPS is complex pathway involving enzymes that assemble into multienzyme complex, purinosome, which facilitates flux of purine intermediates through sequence of ten enzymatically catalyzed reactions. Properly functioning enzymes and purinosome ensure the fast flux of unstable and potentially cytotoxic intermediates resulting in final product IMP, the branch point for AMP and GMP synthesis. The mutations in DNPS genes lead to inherited rare disorders that are accompanied by elevated concentrations of enzyme substrates in body fluids and cells. Lack of a suitable model system to study pathophysiology of DNPS raised the necessity to develop a system mimicking DNPS disorders. HeLa cells with malfunctioning DNPS were characterized on the level of genes, transcripts, proteins, metabolites, and for the presence of the purinosome. Homozygous or compound heterozygous mutations led to the absence of proteins, decreased enzymatic activities, accumulation of enzyme substrates, downregulation...
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Pathology and physiology of de novo purine synthesis.
Krijt, Matyáš ; Zikánová, Marie (advisor) ; Šebesta, Ivan (referee) ; Čajka, Tomáš (referee)
Purines are organic compounds with miscellaneous functions that are found in all living organisms in complex molecules such as nucleotides, nucleosides or as purine bases. The natural balance of purine levels is maintained by their synthesis, recycling and degradation. Excess purines are excreted in the urine as uric acid. Purine nucleotides may be recycled by salvage pathways catalysing the reaction of purine base with phosphoribosyl pyrophosphate. A completely new central molecule of purine metabolism, inosine monophosphate, can be synthesized from precursors during the de novo purine synthesis (DNPS). DNPS involves ten steps catalysed by six enzymes that form a multienzymatic complex, the purinosome, enabling substrate channelling through the pathway. DNPS is activated under conditions involving a high purine demand such as organism development. Currently, three DNPS-disrupting disorders have been described: ADSL deficiency, AICA-ribosiduria and PAICS deficiency. All three disorders are caused by genetic mutations leading to the impaired function of particular enzyme causing insufficient activity of respective DNPS step, manifested biochemically by accumulation of substrate of deficient enzyme, biologically by disruption of purinosome formation and clinically by unspecific neurological features,...
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