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

每个人体细胞都有一个防水外壳，由亲水的头基团和憎水的长尾组成。这些分子被称为脂质，包括饱和/不饱和脂肪和胆固醇等常见分子。一种特殊的脂类亚家族被称为鞘脂（SLs）及其更复杂的神经酰胺衍生物（有两条脂肪尾部）。SLs不仅发挥结构作用，维持细胞膜的完整性，抵抗水分，让营养物质进出；它们被发现是人体免疫系统的有效激活剂。它们的浓度受到严格控制，如果细胞SL水平增加或减少，这是出问题的迹象。许多与老年相关的疾病现在都与高或低的SL水平有关，如阿尔茨海默氏症、糖尿病、哮喘、癌症、多发性硬化症和神经消耗性疾病。人类细胞必须制造足够的SLs来保持细胞正常运作但是当SLs高的时候，SLs必须被降解或者制造SLs的机器必须被关闭。制造SLs的分子机器是一种叫做丝氨酸棕榈酰转移酶（SPT）的酶。它使用一种叫做l -丝氨酸的氨基酸和一种长链脂肪酸来制造第一个可识别的SL中间体。这种SPT酶由两个蛋白质亚基（LCB1和LCB2）组成，它们由两个基因编码——LCB1和LCB2看起来很相似，可能是从一个共同的祖先进化而来的，LCB2似乎是主力，而LCB1起调节作用。这种SPT复合物（LCB1/LCB2）被认为是核心，但最近发现了更小的亚基（ssSPTs），可以使SPT酶的工作速度提高100倍。最近，更多的亚基（orm）被发现与SPT复合物相关，并可以打开和关闭酶。我们想知道这个SPT机器在分子水平上是如何工作的，这样我们就可以了解如何增加或减少细胞的SLs水平。这就是这个研究项目的目标。有了这些知识，我们也许能够设计出一种小分子药物或膳食补充剂来预防上面列出的疾病。要做到这一点，我们必须能够纯化SPT酶，我们通过在酵母中生产它来实现这一点（比如酿造）。人类的SPT是膜结合的，因此很难在纯水中使用。我们必须使用洗涤剂（肥皂）来提取酶，然后我们可以测量它的工作速度，以及为什么它更喜欢它所做的构建块，例如，它更喜欢16或18个碳的脂肪链，我们不知道为什么。我们使用了聪明的蛋白质技术将LCB2/LCB1/ssSPT亚基连接在一起（从头到尾）——这种“融合”起作用，使研究SPT变得更容易，而不是让这些位不连接在一起。我们还将使用复杂的技术将LCB1、LCB2和ssSPT片段化学连接在一起——我们将使用分子剪刀将它们切成小块，并测量小块的质量。这将告诉我们在SPT复合体中什么与什么连接，把所有这些信息结合在一起将使我们能够制作出SPT的分子拼图。世界上还有大约1000人患有一种罕见的疾病，HSN1，这种疾病会导致他们的手臂和腿部神经在30岁左右崩溃。它们的SPT蛋白LCB1和LCB2有特定的突变，它们仍然可以用l -丝氨酸制造SLs，但它们也使用甘氨酸和l -丙氨酸，产生的SLs对细胞是有毒的，人们认为这些有害的SLs积累起来会导致神经损伤。因此，我们也将制造突变的spt来模拟疾病，并试图了解哪里出了问题。我们将是一个具有互补技能的科学家团队-在爱丁堡，圣安德鲁斯，牛津和美国贝塞斯达，他们将共同建立一个关键机器的分子图谱，该机器负责在每个人类细胞中制造适量的必需脂质。

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.