Periodic Reporting for period 4 - GameofGates (Solute carrier proteins as the gates managing chemical access to cells)
Reporting period: 2021-04-01 to 2021-09-30
Every biological system depends on access to energy and building blocks to survive, thrive and, if necessary, grow and divide. Biological systems are defined by the nucleic acids that determine their entities and a biological membrane that confines their identity and allows to create an own chemical and metabolic environment. Safeguarding this “biological” space vis-à-vis the external environment is essential for life and is managed by dedicated membrane transporters that broker the chemical exchange across the largely impermeable lipid bilayer membrane. In humans, the largest group of transporters in the genome are part of the “Solute Carrier Proteins” (SLC) superfamily. One would imagine that by managing the chemistry/biology interface and being essential for access to nutrients and life, these transporter class would be well characterized. Yet, paradoxically, this is not the case. Thus, there are a number of highly important challenges that GameofGates set out to tackle: 1) what is the specificity of the individual human SLCs? 2) Which nutrients and drugs do they transport? 3) What is the level of functional overlap? 4) How do they act in concert? The GameofGates project has as overall objective to understand the functional roles and “division of labor” of this large class of cellular transporters. As they represent “gates to life”, the principles governing their cooperation and functional integration should be elucidated with urgency. These principles should be relevant to understand the role of nutrition and metabolism in human pathophysiology and to understand chemical integration of biological system with their environments. The overall objective is to elucidate these principles and to understand how to best use the information for targeting drugs better. Through GameofGates we have made significant progress in this direction and have obtained evidence that the function of SLCs is indeed necessary for a variety of cellular processes, from phagocytosis, to the chromatin state as well as from drug import and activity to serving as adapters in immune signalling pathways.
A considerable amount of ground-work had to be established, thought to be necessary to enable GameofGates. This had to do both with establishing the biological parameters as well as with the development of the technical tools necessary. In terms of biological parameters we thought to explore the level of expression plasticity of SLCs in response to changes in environment and metabolic conditions. In the initial year, a large proportion of energy was spent trying to learn how to change growth culture conditions without completely upsetting cellular growth. This turned out to be a difficult thing to do. Ultimately, we opted to remove one nutrient at the time and perform gain of function screens. This worked out very well for amino acids and eventually we found, via a genetic approach that the cellular milieu influences the cellular SLC profile. The most important work has been enabled by the creation of a SLC-focused CRISPR library that due to its reduced size compared to genome-wide, allowed for more screens with high statistical depth. The work over the period has produced evidence for a role of SLCs in cell fitness (“essentiality”), immune function, cell death, epigenetic regulation, pH homeostasis and drug transport. Moreover, we have been working on some new approaches to test engagement of chemical matter, drugs and metabolites, to SLC transporters. This has been achieved through the monitoring of thermal stabilization of proteins in cells. Though stabilization could also be the result of indirect effects, in the majority of cases, direct binding and alteration of thermodynamic properties through direct binding is likely to be the cause. Additional breakthrough in terms of technological approach to SLC function comes from experimental strategies we developed that allow screening for drug-gene and gene-gene relationships effectively. Both technologies have empowered studies that resulted in findings that allow to conclude that many drugs need (at least one) SLC transporters to unfold their action and via genetic interaction profiles we could identify new SLCs. On one hand, these results will allow us to create new assays that are functionally dependent on individual SLCs and thus exploitable for drug discovery and on the other hand they have provided the basis for an IMI 2 grant (RESOLUTE; https://re-solute.eu/) dedicated to scale up and broaden these approaches to more cell types. Our laboratory already has published more than 20 papers on transporters, partly sponsored by the GameofGates grant.
It is clear that GameofGates is taking the field of metabolism and the role of cellular transporters in its regulation, well beyond the current knowledge domain and, technologically, beyond state of the art. For every example, it has demonstrated that very different cellular processes, phagocytosis and necroptosis, require specific members of the SLC superfamily. Importantly, our studies further demonstrate that, irrespectively of charge and size, common drugs are requiring SLC transporters to exercise their function. We have also created a first ever genetic interaction map for this large and important class of transporters which already helped to identify a mitochondrial transporter for NAD import. Together with the other screens we were doing on specific cellular process and drug transport, this overall genetic survey has generated some functional information on the majority of SLC transporters. The hits can further be validated in the future. Through additional experiments performed and papers published we further showed evidence that SLCs play a role in immune signalling and the chromatin state. Thus, with some luck, and we will see the impact in a few years, GameofGates will represent the largest single-push on SLC knowledge ever. Besides prominent publications, dissemination at conferences such as the International Transmembrane Transporters Society, the Bioparadigm Transporter Conference, the RESOLUTE workshops and the Gordon and Keystone meetings, also the founding of a new enterprise will contribute to the efficient and successful dissemination of GameofGates-sponsored work.