The regulation and role of NAD biosynthesis in yeast ageing

Various ageing factors in backer`s yeast (S.cerevisiae) indicate that the regulation of NAD metabolism has a strong effect on yeast lifespan. For example activity of histone deacetylase Sir2 is influenced by intracellular NAD:NADH ratios and inhibited by its product nicotinamide. Kynurenine, a precursor of NAD is involved in lifespan extension of D.melanogaster and C.elegans and into a series of ageing related disease. Despite the strong hints for these correlations, the underlying regulatory mechanisms are not known. During a two years post-doc stay in the group of Dr. Markus Ralser (Cambridge Systems Biology, Centre, University of Cambridge) we plan to answer the following questions: How is NAD synthesis regulated and sensed in yeast and how does this influence lifespan? This will be done in a system biology approach, by combining systematic genetics with sensitive LC-MS/MS (MRM) analytics to determine metabolite and protein profiles in long-lived yeast mutants. We expect that our results will give a detailed understanding for the role of the NAD de novo biosynthesis in ageing. This will be done 1) by identifying regulators of NAD metabolism within the pool of genes with reported lifespan phenotypes and 2) by investigating the role of NAD synthesis during yeast ageing.

Metabolism is a central part of every living organism. More precisely, it is a characteristic that defines life itself. Together the enzymatic reaction sequences that are active in cells in order to convert nutrition into energy and important cellular building blocks such as amino acids, nucleotides, and lipids are assembling the so called metabolic network. Importantly this metabolic network is not a rigid structure, but presents as a highly flexible and regulated entity, able to quickly respond to altered biosynthetic demands, environmental challenges, and many other perturbations. Although in the last decades many individual enzymatic reactions have been studied in detail, we still have only limited knowledge about how these reactions act in concert and are regulated in a living organism. This represents a major challenge in understanding metabolism on a system wide level and is as a consequence hampering for example the targeted development of therapeutic strategies against diseases or aging. By applying state-of-the-art liquid chromatography tandem mass spectrometry (LC-MS/MS) we were able to study the metabolic changes occurring in cells, that are happening as a consequence of knock-outs of potential regulatory genes. Quantifying the triggered reconfiguration of the metabolic network allows to evaluate the regulatory potential of respective genes. Surprisingly, when establishing the LC-MS/MS methodology for this purpose, we made an unexpected but exciting discovery that was presenting striking experimental evidence about the evolutionary origins of the metabolic network during the origin of life itself. We found that sugar phosphates - metabolites that constitute the central metabolic pathways glycolysis and pentose phosphate pathway - start to interconvert into each other in an entirely enzymes free environment at moderate temperatures (70C). These interconversions are happening in a metabolism-like manner and are facilitated by ferrous iron, a transition metal which is abundant in Achean Sediments. This indicates that ferrous iron was available at high concentrations in oceans about 3.8 billion years ago, when first live emerged on earth. The non-enzymatic reaction network observed under these conditions was efficient and largely superimposable with modern glycolysis and pentose phosphate pathway. Furthermore we could show that this network is conditionally activated by changes of pH and iron-availability, providing access to a central property of modern metabolic networks, which is the ability to switch parts of the network on and off. Extending from these observations to another metabolic pathway, we found that achieving a non-enzymatic version of the citric acid cycle is also possible, however requires a complementary enabling chemistry, which is based on sulfate radicals rather than iron. Thus our results suggest that the basic architecture of the modern metabolic network was likely shaped by the physical and chemical constraints of the early worlds environment. Simple inorganic molecules that were abundant in the Archean oceans can serve as facilitators of interconversion sequences resembling the routes of central carbon metabolism in modern organisms. Therefore our experimental observations are supporting the hypothesis that the origin of the core topology of extant metabolic networks could have been fundamentally shaped by prebiotic chemical reaction routes rather than being the result of natural selection during later stages of evolution.