Probing the interplay of substrate and lipid interactions in the mechanism of a transporter family linked to age-related metabolic diseases and cancer

Advances in healthcare practices and technology have increased human lifespan, but increased longevity correlates with an increase in age-related diseases, including diabetes, obesity and cancer. These chronic diseases have life-changing effects on patients and are an enormous burden on our health services.

Age-related diseases such as those named above are closely linked with cellular metabolism. In fact, symptoms can often be alleviated by reducing the number of calories consumed in the diet (termed caloric restriction). Caloric restriction has been shown to reverse age-related diseases and prolong the life of many different species, including primates, suggesting these benefits could also be extended to humans.

For the human body to use nutrients from the diet, the nutrients have to be transported from the bloodstream into cells, where they are processed for energy production and the synthesis of cellular components. Nutrient uptake is performed by proteins called transporters that exist in the cell membrane, which is the greasy, oil-like barrier between the inside of the cell and the environment. Transporters are miniature molecular machines that "pump" chemicals across the cell membrane using an energy source provided by the cell. As with any pump, cutting it off from its energy source or jamming up the mechanism will stop it from functioning.

Citrate is a key nutrient for normal metabolism and fat production in humans, and is transported into cells by a protein called INDY, which stands for I'm not dead yet because disruption of INDY function in fruitflies doubles their lifespan. In mice, disrupting INDY protects them from diabetes and obesity. Therefore, drugs that stop human INDY transporter proteins from functioning properly could be used to treat age-related metabolic diseases and promote healthy ageing.

The aim of this study is to understand how INDY transporters are able recognise different chemical compounds, and how they harness an energy source to power movement of these chemicals across the cell membrane. In understanding how they work, we can design chemical inhibitors that could cut off the energy supply or act as molecular spanners in the works of these molecular machines. To develop drugs that target INDY proteins we need to know what these proteins look like, how they recognise the nutrients they transport, how the energy source is harnessed, and how the natural environment, i.e. the oil-like membrane, influences the activity of these transporters.

Using a wide variety of complementary experimental approaches, we will address these gaps in our knowledge, which will give us a profound understanding of how these proteins work at a fundamental level, and will lay the foundation for the development of INDY specific drugs in the future.

In many organisms, caloric restriction has beneficial effects, from reversing age-related diseases such as diabetes and obesity, to extending lifespan. In eukaryotes, reducing the cytoplasmic concentration of citrate and dicarboxylates such as succinate and malate, modulates energy homeostasis and induces caloric restriction-like benefits. The divalent anion sodium symporter (DASS) family of membrane transporters are the primary uptake route of di- and tricarboxylates into the cytoplasm. Disruption of DASS function can increase lifespan (leading to the alternative name INDY, which stands for I'm Not Dead Yet), protect against insulin resistance and adiposity, and stall tumour development, making human DASS attractive drug targets. However, many fundamental details of the DASS mechanism remain unclear. This not only limits our understanding, but also our ability to design inhibitors targeting this family. Our overall aim is to illuminate the fundamental molecular mechanism and modes of regulation of DASS transporters, paving the way for DASS inhibitor development to treat age-related molecular disease and promote healthy ageing.

During transport, DASS transporters interact with three key components; the substrate (citrate, dicarboxylates), the coupling ion (Na+), and the lipid bilayer. These interactions and how their interplay influences overall activity is not understood. We have generated several testable hypotheses, such as novel substrate interaction sites, and that the lipid environment strongly influences substrate binding and transport. We propose to test these hypotheses using a range of experimental approaches, from high throughput and high resolution biochemical and biophysical assays, to lipidomics and molecular dynamic simulations.

Our findings will advance our fundamental understanding of DASS transporter function, illuminate membrane transport mechanisms in general, and provide a springboard for the future development of DASS inhibitors.