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

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.

Sphingolipids (SL) are "long chain bases (LCB)" that play essential structural roles in membranes as well as in cell signalling and immunomodulation. SLs are biomarkers for human disease; cells tightly control SL concentrations to maintain a "healthy balance" which if perturbed, is indicative of many age-related diseases such as Alzheimer's, asthma, diabetes, cancer and many neuropathies. A BBSRC priority is to reduce the burden of such diseases and to do this we must first understand the underlying molecular details of SL metabolism. Our project is focused on the essential first enzyme, serine palmitoyltransferase (SPT), in the de novo human SL biosynthetic pathway. SPT is a pyridoxal 5'-phosphate (PLP) dependent enzyme that catalyses the condensation of L-serine with C16 or C18 acyl-CoA to give the first SL intermediate, ketosphingosine. SPT is found in the endoplasmic reticulum (ER) and is a multi-subunit complex composed of 3 structural gene products; LCB1 and LCB2 which are thought to form a heterodimer and a small subunit, ssSPT, that activates the enzyme activity of the complex up to 100 fold. A rare neuropathy (HSN1) is caused by mutations in LCB1 and LCB2 that result in promiscuous SPT mutants that accept L-Ala and Gly and form toxic deoxySLs. We have determined the structure and mechanism of the cytoplasmic, bacterial, SPT homodimer but the human SPT has not been isolated in quantities to allow similar studies. Now, with Teresa Dunn (USA), we have made a significant breakthrough in being able to purify mgs of active wild-type and HSN1 mutant SPT fusions (LCB2/ssSPT/LCB1). Using enzymology, chemical crosslinking, proteolysis, mass spectrometry, structural biology and lipid analysis techniques we will gain the first insight into:- the subunit interactions within the SPT complex, the impacts of the HSN1 mutations of SPT specificity and how the ssSPT, post-translational modifications (phosphorylation) and other proteins (ORMs) regulate the SPT complex.

Beneficiaries - who they are and how will they benefit?
Academic
We will assemble a team of UK scientists (a mix of PIs and PDRAs) with complementary expertise in protein chemistry. The team is enhanced by two world-leading project partners; Dame Carol Robinson (Oxford) and Prof. Teresa Dunn (USA). We have chosen a technically challenging target - the membrane-bound, multi-subunit human SPT complex. This project will train and develop the skills of the three hands-on researchers employed on this project. Their resultant improved, rare skill-set will significantly enhance the future employability of each PDRA within industry or enable them to step up to an independent academic career (e.g. DJ Clarke). The PIs have published ~250 papers (6 together on the bacterial SPT) but this multi-disciplinary, human SPT project will present new challenges and broaden their research skills, as well as enhancing their research project management and data analysis abilities. With the support of Teresa Dunn we are in pole position for a BBSRC-funded, UK-team to lead the field in this highly competitive international field.

Pharmaceutical/Clinical
Recent research has shown that sphingolipids are involved in the onset and progression of a number of diseases including asthma, cancer and the age-related diseases diabetes and Alzheimer's. Therefore understanding the molecular basis of regulation of SL biosynthesis and metabolism has become a key aim for pharmaceutical companies. For example, the immunomodulating drug Fingolimod (tradename Gilenya, which targets SL GPCR receptors) was derived from myriocin. Gilenya was launched by Novartis in 2011 and is the first oral drug to be approved for treating multiple sclerosis. It has sales of £320M/year and has been used to treat 25,000 patients. Also, Amgen recently published the results of their x-ray structural and medicinal chemistry studies of human sphingosine kinase (which makes S1P). This shows the great potential that pharma places on controlling SL metabolism. The molecular complexity of human SPT has hindered progress towards development of a therapeutic so the impact of our study will be to shine light on the enzyme for the first time and release this bottleneck.

Impact on Patients with Rare Diseases
One of the most interesting aspects of studying human SPT is the insight that has come from studies of a rare disease, hereditary sensory neuropathy type I (HSN1 or HSAN1). Patients suffering from HSN1 display progressive neuronal degradation, bone loss and recurring foot and hand ulcers. Independent studies of an American and Australian family in 2001 revealed that the most common mutation is caused by a lesion at position C133 in LCB1 (mutants C133W and C133Y). Further work revealed the build up of toxic deoxySLs in HSN1 patients whose formation is catalysed by mutant SPTs. These aberrant deoxySLs are thought to cause cell damage and are now used as biomarkers to identify and monitor the disease. The clinical impact of this discovery is that reducing/delaying the formation of these molecules should alleviate/delay the onset of the disease. In 2008, 2010, and 2013 DJC presented in Boston at the HSN1 meetings of patients, scientists and clinicians. In 2011, the combined knowledge from many SPT studies (clinical, genetic, biochemical, structural) and discussions at these HSN1 meetings led a team of clinicians in Boston to begin a NIH-funded clinical trial of dietary supplementation of high doses of L-serine to 24 HSN1 patients from one family (Deater family). It is exciting and encouraging to witness such a fast translation from basic research to the clinic - just 9 years from the discovery of the genetic mutations (in 2002) to clinical trial (in 2011). There are ~1000 HSN1 patients in the USA, UK and Australia - but this is an underestimate. Our work has the potential to benefit those patients and others who are diagnosed as well as those with other related rare neuropathies.