Structure and mechanism of bile acid transporters

Bile acids are important detergent like substances produced in our body for emulsifying fat in our gut. They are made in the liver and are stored in the gall-bladder until they are needed to help break down the food we consume. The body has its own re-cycling system, which means that the bile acid is re-used. This re-cycling process requires that the bile acids are taken back up into the blood stream by the aid of some nifty protein machines. These protein machines, so-called transporters, recognize and are specific to the shape of bile acid. What is interesting is that if we can understand this process in greater detail, we may be able to stop this transporter from working. The result of doing this would mean that the bile acid usually kept in our body would be eliminated instead. Although this sounds wasteful, it could be very beneficial, as removing bile acid is one of the best ways of lowering cholesterol levels in the body. How do we do this? The best way is first determine what the transporter looks likes at the molecular detail. Just as a key is designed to fit a certain key-hole, so bile acid is made to fit its own transporter. So how do we decipher the three-dimensional shape of these proteins? To achieve this, we need to use X-ray protein crystallography. Unfortunately, X-ray crystallography requires the production of crystals from high concentrations of purified proteins. The production of protein crystals of this type is very difficult, and the scientific community has struggled over many years. These molecules are present in an oil-like environment and we need to use specific chemicals, detergents, to isolate them. In fact, we only know the shape of 3 of the 2,000 or so transporters in our body. However, our laboratory has developed new tools, which has made it possible to tackle some of these main hurdles. We can now isolate the bile acid transporters for the first time. Furthermore, we have managed to produce small crystals and so are well on the road to obtaining their 3D-shape. As bacteria, like us, also use a machine to transport bile acid we will also study this protein. In general bacterial proteins are rather easier to work with than their mammalian equivalents. This study will be useful, not only for drug design but also to understand the mechanisms of transporters in general.

Cholesterol homeostasis is achieved by coordinated regulation of cholesterol dietary absorption, de novo synthesis, and removal in the form of bile acid. Bile acid removal is a major factor in cholesterol elimination from the body with 0.5g per day being converted to bile acids in the liver. The bile acids go through a cycling process passing from the liver to the gut where they play a vital role in the solubilisation of lipids and lipid soluble vitamins. They are then reabsorbed in the ileum and transported back to the liver through the portal system. Only 5% of the bile acids are actually excreted. The system of recycling involves a number of key transporters that actively move the bile acids across the cell membrane against a concentration gradient. Two of the most important transporters belong to the solute carrier 10 family of transporters, Na+ dependent Taurocholic Cotransporting Polypeptide (NTCP) and Apical Na+ Dependent Bile Acid Transporter (ASBT). Since the reduction of bile acid reabsorption from the intestine has been shown to cause an increase in bile acid synthesis from cholesterol, ASBT is currently being used as a target for producing cholesterol lowering drugs with one class of compounds reported to cause a 10% reduction in levels of LDL cholesterol in phase II clinical trials. Both proteins have also been suggested as routes for delivering drugs to specific cells.
This proposal concerns the biochemical characterisation and solution of the structures of these mammalian secondary transporters. In addition we propose to solve the structure and investigate the mechanism of a bacterial homologue of these proteins for which we already have diffraction data extending to 3.2A. These structures will be invaluable in designing inhibitors of these proteins for use as specific drugs. At present there is no structural information available and the proteins do not show any significant homology to proteins of known structure. The research will be predominantly carried out at the Research Complex at Harwell.