Hub Molecules Of Life

In the initial phase of the project, we first established a detailed plan for the coordination of our joint activities during the ISGSB meeting in Innsbruck (Sep2022). We decided that, besides regular digital meetings, the members of the consortium should meet in person at least 1-2 times per year. This has been successfully implemented, in part, by combining HubMOL meetings with joint attendance of international conferences and our joint teaching activities. All four Pis met in February 2024 in Bergen and participated in teaching the course on mathematical modeling of metabolic pathways (conducted in collaboration with the NORBIS research school). Scientifically, during this period, we have focused on the development and implementation of technologies that will be crucial during the next phase of the project. We developed and extended high-precision analytical methods for the time-resolved measurement required to establish fluxes in metabolic pathways and turnover rates of posttranslational modifications. This has been a collaborative effort and has involved all four partners: the Migaud lab synthesizes stable isotope-labeled precursors, needed to measure incorporation rates into hub molecules; the Innsbruck group establishes LC-MS procedures for the detection and quantification of site-specific protein acetylation dynamics; the group in Bergen develops cell-based model systems and LC-MS techniques to measure NAD metabolites and their turnover in cells and mitochondria. The generated data are corrected for natural isotope abundance and subjected to bioinformatics analyses and mathematical modelling approaches by the Tromsø group. This “workflow” is now well established and provides a strong base for our ongoing joint research. The developed technologies have been applied and validated in several experimental approaches and model systems and are now being used to address the main objectives of the HubMOL project. Given the opposing roles of the hub molecules CoA and NAD in histone acetylation, an important technological advancement is the accurate quantitative description of acetylation/deacetylation events in specific histone modification sites. Using both metabolic and chemical stable isotope labeling, the dynamics in two nearby acceptor sites could be established. Thereby, it was possible to detect the interdependence and differences in the dynamics within the two sites indicating hitherto undiscovered regulatory mechanisms. We have also developed a variety of cellular models to modulate NAD concentrations or the subcellular availability of this coenzyme. One approach is the targeted overexpression of an NAD-consuming enzyme in different subcellular compartments. Thereby the cellular NAD concentration declines which has varying physiological effects, depending on the compartment that has been targeted. Using the obtained metabolic information and proteomics data, a genome-scale metabolic model was generated that, for the first time, takes into account the regulation of NAD-dependent enzymes by the NAD concentration. In upcoming experiments, we will use these model systems to study the consequences of modulated NAD levels on other hub molecules. Specifically, we will monitor the impact on acetyl-CoA levels and the impact on histone acetylation dynamics, thereby establishing the cross-influence of hub molecule-dependent metabolic and signaling functions.

Metabolic disorders are a major burden on the European population and health care systems. Moreover, metabolic perturbations contribute substantially to other pathologies such as neurodegenerative disorders and cancer. The causes of metabolic dysregulation are manifold and lead to pathological shifts in biochemical processes, often in response to imbalanced nutrition. Likewise, changes in metabolism affect signalling mechanisms and gene regulation, aggravating the pathology. The tight interconnection between metabolism and signalling is still not well understood. HubMOL will fill this knowledge gap and open new horizons by exploring the functional duality of a set of small molecules that are involved in all cellular functions - the Hub Molecules Of Life (HubMOLs) including ATP, SAM (S-adenosylmethionine) and the vitamin-derived cofactors, NAD, FAD, and CoA. They mediate both metabolic reactions, but also signalling, for example, through posttranslational protein modifications (PTMs). The complexity of this emerging area requires interdisciplinary scientists equipped with a comprehensive set of skills and competences covering synthetic and analytical chemistry, experimental, computational and systems biology as well as clinical medicine. In a highly interactive team of international experts in these areas, HubMOL will develop new chemical and analytical tools enabling experimental studies of the complex interplay of cofactor metabolism and the dynamics of the PTMs they mediate. Predictive mathematical models will be developed to capture this interconnectivity and duality of hub molecules. These models will be iteratively improved through experimental verification cycles and eventually validated based on clinical data and samples from patients with neurodegenerative disorders undergoing NAD supplementation therapy. Thereby, HubMOL will establish fundamentally new insights into cofactor biology and lay the ground for patient-tailored vitamin supplementation concepts.