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

每个人类细胞都有一个外部防水外壳，由分子组成，分子头上有一个亲水(亲水)的基团，尾巴上有一个长长的憎水(疏水)尾巴。这些分子被称为脂类，包括饱和/不饱和脂肪和胆固醇等常见分子。一个特殊的脂质亚家族被称为神经鞘脂脂(SLS)及其更复杂的神经酰胺衍生物(有两个脂肪尾巴)。SLS不仅在结构上发挥作用，维持细胞膜的完整性，抵抗水分，让营养物质进入和排出；它们已经被发现是人类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水平上升或下降，这是一个不对劲的迹象。许多与老年相关的疾病现在都与SL水平的高低有关，如阿尔茨海默氏症、糖尿病、哮喘、癌症、多发性硬化症和神经衰竭性疾病。人类细胞必须产生足够的SLS才能保持细胞的正常工作，但当SLS很高时，SLS必须被降解，或者SL制造机器不得不关闭。制造SLS的分子机器是一种名为丝氨酸棕榈酰转移酶(SPT)的酶。它使用基本的构件--一种名为L的氨基酸--丝氨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，这两个亚基由两个基因编码-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，似乎LCB2是主力，而LCB1起着调节作用。这种SPT复合体(LCB1/LCB2)被认为是核心，但最近发现了更小的亚基(SsSPT)，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复合体相关，可以开启和关闭酶。我们想知道这台SPT机器是如何在分子水平上工作的，这样我们就可以了解如何增加或降低细胞SLS水平。这就是本研究项目的目标。有了这些知识，我们也许能够设计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，我们必须能够提纯SPT酶，我们通过在酵母中生产它来做到这一点(比如酿造)。人体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起作用的速度有多快，以及为什么它喜欢它所做的积木，例如它喜欢16或18个碳链长的脂肪链，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(头到尾)--这种“融合”起作用，使研究SPT变得更容易，而不是让片段不连接在一起。我们还将使用尖端技术将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回片段，并测量片段的质量。这将告诉我们在小组委员会复合体中连接了什么，并将所有这些信息聚集在一起将使我们能够对小组委员会进行分子拼图。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手臂和腿上的神经在大约30岁时崩溃。他们的SPT蛋白有特定的突变-LCB1和LCB2-他们仍然可以从L-丝氨酸制造SLS，但他们也使用甘氨酸和L-丙氨酸，产生的SLS对细胞有毒--人们认为这些不良的SLS会积聚并导致神经损伤。因此，我们还将制作突变的SPT来模仿这种疾病，并试图了解哪里出了问题。我们将是一个拥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，他们将共同构建关键机器的分子图景，该机器负责在每个人类细胞中制造适量的必要脂质。

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.