The human serine palmitoyltransferase (SPT) complex; specificity, structure, regulation and inhibition.

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Every human cell has an outer water-resistant shell composed of molecules with a water-loving (hydrophilic) head group and a long, water-hating (hydrophobic) tail. These molecules are called lipids and include common molecules like saturated/unsaturated fats and cholesterol. One particular sub-family of lipids is called sphingolipids (SLs) and their more complex ceramide derivatives (which have two fatty tails). The SLs not only play structural roles that maintain the integrity of the cell membrane to resist water and let nutrients in and waste out; they have been found to be potent activators of the human immune system. Their concentrations are tightly controlled and if there is an increase or decrease in cellular SL levels it is a sign that something is wrong. Many diseases associated with old age are now linked to high or low SL levels such as Alzheimer's, diabetes, asthma, cancer, MS and nerve-wasting diseases. The human cell has to make enough SLs to keep the cell functioning properly but when SLs are high, the SLs have to be degraded or the SL-making machinery has to be switched off. The molecular machine that makes SLs is an enzyme called serine palmitoyltransferase (SPT). It uses basic building blocks - an amino acid called L-serine and a long chain fatty acid to make the first recognisable SL intermediate. This SPT enzyme is made up of two protein subunits (LCB1 and LCB2) that are encoded by two genes - LCB1 and LCB2 look similar and may have evolved from a common ancestor and it appears LCB2 is the workhorse whereas LCB1 plays a regulatory role. This SPT complex (LCB1/LCB2) was thought to be the core but recently smaller subunits (ssSPTs) have been discovered that can make the SPT enzyme work 100 times faster. Recently even more subunits (ORMs) have been found to be associated with the SPT complex and can turn the enzyme on and off. We would like to know how this SPT machine works at the molecular level so that we can understand how to increase or decrease cellular SLs levels. This is the goal of this research project. With this knowledge we might be able to design a small molecule drug or dietary supplement that could prevent the diseases listed above. To do this we have to be able to purify the SPT enzyme and we do this by producing it in yeast (like brewing). The human SPT is membrane-bound so that makes it difficult to work with in pure water. We have to use detergents (soaps) to extract the enzyme, then we can measure how fast it works and why it prefers the building blocks it does e.g. it prefers fatty chains 16 or 18 carbons long and we don't know why. We have used clever protein technology to join the LCB2/LCB1/ssSPT subunits together (head-to-tail) - this "fusion" works and makes it easier to study the SPT rather than having the bits not joined together. We will also use sophisticated technology to chemically join the LCB1, LCB2 and ssSPT pieces together - we will cut them back into bits using molecular scissors and measure the mass of the bits. This will then tell us what was joined to what within the SPT complex and bringing all this information together will allow us to make a molecular jigsaw puzzle of the SPT. There are also ~1000 people in the world with a rare disease, HSN1, that causes their nerves in their arms and legs to break down aged from ~30. They have specific mutations in their SPT proteins - LCB1 and LCB2 - they can still make SLs from L-serine but they also use glycine and L-alanine and the SLs produced are toxic to cells - it is thought that these bad SLs build up and cause nerve damage. So, we will also make mutant SPTs to mimic the disease and try to understand what has gone wrong. We will be a team of scientists with complementary skills - in Edinburgh, St. Andrews, Oxford and Bethesda, USA that together will build up a molecular picture of the key machine that is responsible for making just the right amount of essential lipids in every human cell.