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

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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.

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.