Cells are made of membranes which are composed of chemicals called 'lipids' - these contain hydrophobic (water hating) and hydrophilic (water loving) parts. Membranes have to be strong to keep cell contents in but also be able to let molecules in (nutrients, metals, salts) - as well as keeping toxic materials out and expelling waste. They must also stop water flooding in and bursting the cell by increased osmotic pressure. Cells have evolved different membranes with different chemical composition. Mammals have complicated membranes and they generate 100s of different lipids. Similarly, yeast, plants and viruses have species-specific lipids. Bacteria too have unique and unusual lipids - they also play important roles in the immune response and inflammation. Mammals have evolved to recognise their own lipids as 'self' but can expertly detect foreign lipids from pathogenic bacteria, fungi and viruses. Once detected, the mammalian cell can mount an effective immune response to kill the invader. This then begs the question, if a bacterium has evolved to have lipids similar to a human's - how do we tell them apart? Looking more closely at the lipids themselves our project will focus on a special branch of interesting lipids called 'sphingolipids'. They were discovered >100 years ago in human brains by John Thudichum who knew that they played an important role in brain chemistry. It took until the 1930s for Herbert Carter to work out the chemistry of the sphingolipids - a polar, water soluble head and a fatty acid non-polar tail. They were found to be made from the common amino acid L-serine and a long carbon (>C16) chain. Scientists have long wondered about how sphingolipids are made inside the cell from common building blocks and then transported to the outside - this must happen very quickly when the cells are rapidly growing and dividing. Also, sphingolipids are dangerous - too many or too little in one cell can be lethal so the amounts are delicately controlled in a way we still don't fully understand. To uncover the chemical details and explore the enzymes involved we and other scientists are studying sphingolipid biosynthesis in humans, plants, yeast and bacteria. We have chosen an interesting bacterium Sphingomonas wittichii because it is not harmful to man - in fact it can degrade toxins to harmless molecules. These Sphingomonas are highly unusual because they make sphingolipids that resemble our own to some extent. We will explore how Sphingomonas makes sphingolipids by carefully characterising the genes that encode the enzymes that carry out the initial conversion of serine and the fatty acid, through the complex 2nd and 3rd steps, and beyond. We are helped because the Department of Energy (USA) have already sequenced the Sphingomonas wittichii genome and predict it to have >5000 genes. However, we do not know which ones are involved in sphingolipid biosynthesis. We will use chemical, biochemical, genetic and molecular biology methods to help us understand each step. We have already made a start and found an unusual small protein (~80 amino acids long) that we think links sphingolipid and fatty acid biosynthesis. Most of the work will be carried out in Edinburgh but we will also work with Jim Naismith in St.Andrews who can determine the 3D structure of a protein, as well as a genetics expert in the USA, Teresa Dunn. Our teamwork will put us ahead of our competitors. By the end of the grant we will have determined the basic roadmap of bacterial sphingolipid biosynthesis and be able to begin to compare it with the map in humans, plants and yeast. We'll obtain insight into how these species evolved to make the same sphingolipid and begin to understand how each controls the amount in each cell. Whilst we carry out the work we will make sure we give seminars to experts and the general public telling them what we've found out and will also publish in highly-rated international journals that will benefit UK science.

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.

Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.

Animal and bacterial cells have a protective, water-resistant outer shell that is composed of molecules with a water-loving (hydrophilic) head group and a long, water-hating (hydrophobic) tail. This large family of molecules are called lipids and include common things like saturated/unsaturated fats and cholesterol. One particular sub-family of lipids is called sphingolipids (SLs) and their more complex ceramide versions (which have two fatty tails). The SLs not only play structural roles in the outer shell that allow the cell membrane to resist water and let nutrients in and waste out; they are also able to stimulate the human immune system. SL levels are dynamic but also tightly controlled - any increase or decrease in the cellular SL levels is a sign that something has gone wrong. Changes in SL levels are strongly linked with old age and diseases such as Alzheimer's, diabetes, asthma, cancer and nerve-wasting diseases. An exciting area of research with direct implications for human health is the discovery that humans are hosts for many different types of bacteria - collectively these are known as the microbiota/microbiome. Current estimates are that for every human cell in our body, there is a bacterial one. These bacteria can be "bad" and cause disease (e.g. superbugs) but most are "good" bacteria and are beneficial to our well being. These bacteria live in our mouths, on our skin and in our gut and help us metabolise our food and are also thought to play protective roles. A surprising discovery was that the bacteria that live with us produce molecules that allow bacterial and human cells to communicate. One such family of molecules are the SLs - it is highly unusual that human and bacterial cells both make the same molecule and this suggests some sort of evolutionary link. Moreover, it has been calculated that we have several grams of SLs in our gut at any one time and they are making a vital contribution to our health. Recent studies have linked the microbiota to diseases such as diabetes, obesity and cancer. All cells make SLs by a multi-step pathway using simple building blocks - the steps are catalysed (sped up) by molecular machines called enzymes. Research has focussed on the enzymes involved in human SL biosynthesis but very little is known about SL biosynthesis in the microbiota. To fully understand the relationship between us and bacteria we must learn how bacteria make and transport such complex molecules as well as understanding how we metabolise them. We will study how gut and mouth bacteria make SLs with world experts in America and Germany with a collaborator from the UK. We will begin with a study of the enzyme serine palmitoyltransferase (SPT) that uses two main building blocks - an amino acid called L-serine and a long chain fatty acid, to make the first SL intermediate. We will determine the 3D structure of the SPT in each bacterium and compare their shapes and evolution. Of special interest, the structure of the bacterial SLs is unusual and contains distinctive chemical fingerprints and we will investigate their origins by feeding the bacteria heavy versions of the proposed building blocks and tracking their incorporation. Nothing is known about how the microbiota makes unusual branched chain SLs so we will study enzymes that convert can branch-chain amino acids into specific building blocks. Bacteria contain ceramides with an unsusual inositol sugar so we will purify and characterise the enzyme myo-inositol phosphate synthase (MIPS) that uses glucose phosphate as a substrate. At the end of our study we will have begun to define the biosynthetic blueprint of the microbiota. Our results will be of interest to academic microbiologists and chemists as well as those interested in human health. Moreover, a number of drug and healthcare companies are also interested in the microbiome and they could use our knowledge to develop therapies that may have impact on disease and long term well being.