Periodic Reporting for period 2 - GameofGates (Solute carrier proteins as the gates managing chemical access to cells)
Reporting period: 2018-04-01 to 2019-09-30
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. 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 proper protein processing in the ER.
Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far
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 but has not yet led to a systematic accompanying proteomics screen, only genomics. 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, allows for more screens with high statistical depth. The work over the period has produced evidence, that we have been able to start publishing, 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 are ongoing. 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 nine papers on transporters, partly sponsored by the GameofGates grant. With > 600 citations, they have been well received.
Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)
Already now, 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, the study is demonstrating that, irrespectively of charge and size, common drugs are requiring SLC transporters to exercise their function. By the end of the project, we will have created a first ever genetic interaction map for this large and important class of transporters. Together with the other screens we are doing on specific cellular process and drug transport, this overall genetic survey will generate some functional information on the majority of SLC transporters. Thus, with some luck, 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.