Human cancer cells are characterised by rapid glucose consumption, with most of the glucose-derived carbon being secreted as lactate despite abundant oxygen availability. This deviant energetic metabolism known as the »Warburg effect« has been designated as one of the hallmarks of the cancer. We were first to show that 6-phosphofructo-1-kinase (PFK1), the key regulatory enzyme of glycolysis might be posttranslationally modified in cancers. After proteolytic cleavage of the C-terminal portion of the enzyme, an active, shorter fragment was formed that was insensitive to citrate and ATP inhibition and could cause un-restricted metabolic flow over glycolysis leading to lactate excretion. Excretion of a primary metabolite – ethanol under the aerobic conditions is characteristic also for the yeast cells during the growth at high specific growth rates. To study the role of human shorter PFK1 fragments on the deregulated glycolytic flow, yeast cells might be the most appropriate model organism. For this purpose HD114-8D pfk nul strain has been chosen as a recipient for the modified truncated pfkM gene encoding 47 kDa fragment of human muscle type PFK-M isoenzyme. Transformants enabling different levels of gene expression were designed and tested under different growth conditions. Initially no growth of transfromants could be detected in the supplemented minimal media (SSM) with different fermentative sugars as a sole C-source. Detailed analyses revealed that deregulated glycolytic flux caused by highly active shorter fragments led to unbalanced NADH/NADPH ratio in transformants growing on fermentative sugars. By optimising the growth conditions a medium was selected that enabled faster growth of the transformant with the truncated pfkM gene in respect to those with the native PFK-M enzyme. Recombinant yeast cells could be eventually used for high throughput screening of drugs that might inhibit the activity of shorter fragments in cancer cells and restrict their proliferation.
F.01 Acquisition of new practical knowledge, information and skills
COBISS.SI-ID: 5577498