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

人丝氨酸棕榈酰转移酶（SPT）复合物；

基本信息

批准号：
BB/M003493/1
负责人：
Dominic Campopiano
金额：
$ 71.76万
依托单位：
University of Edinburgh
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2015
资助国家：
英国
起止时间：
2015 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FM003493%2F1
关键词：
human serine palmitoyltransferase SPT complex

项目摘要

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.

每个人体细胞都有一个防水外壳，由具有亲水（亲水）头基和长憎水（疏水）尾部的分子组成。这些分子称为脂质，包括饱和/不饱和脂肪和胆固醇等常见分子。脂质的一个特殊亚家族称为鞘脂 (SL) 及其更复杂的神经酰胺衍生物（具有两个脂肪尾）。 SL 不仅发挥结构作用，维持细胞膜的完整性，以防水、让营养物质进入和排出；它们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 水平升高或降低，则表明存在问题。许多与老年相关的疾病现在都与 SL 水平高或低有关，例如阿尔茨海默病、糖尿病、哮喘、癌症、多发性硬化症和神经消耗性疾病。人体细胞必须产生足够的 SL 才能保持细胞正常运作，但当 SL 较高时，必须降解 SL 或必须关闭 SL 制造机器。制造 SL 的分子机器是一种称为丝氨酸棕榈酰转移酶 (SPT) 的酶。它使用基本构建模块 - 一种名为 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，这两个亚基由两个基因编码 - LCB1 和 LCB2 看起来相似，可能是从共同的祖先进化而来的，看来 LCB2 是主力，而 LCB1 起着调节作用。这种 SPT 复合物 (LCB1/LCB2) 被认为是核心，但最近发现更小的亚基 (ssSPT) 可以使 SPT 酶的工作速度加快 100 倍。最近发现更多的亚基 (ORM) 与 SPT 复合物相关，并且可以打开和关闭该酶。我们想知道这个 SPT 机器如何在分子水平上发挥作用，以便我们了解如何增加或减少细胞 SL 水平。这是该研究项目的目标。有了这些知识，我们也许能够设计出可以预防上述疾病的小分子药物或膳食补充剂。为此，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（如酿造）。人类 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的构建模块，例如它更喜欢 16 或 18 个碳长的脂肪链，我们不知道为什么。我们使用巧妙的蛋白质技术将 LCB2/LCB1/ssSPT 亚基连接在一起（头对尾）——这种“融合”有效，使得研究 SPT 比不连接在一起更容易。我们还将使用先进的技术将 LCB1、LCB2 和 ssSPT 部件以化学方式连接在一起 - 我们将使用分子剪刀将它们切成小块并测量小块的质量。然后，这将告诉我们 SPT 复合体中的什么成分，并将所有这些信息结合在一起，使我们能够制作出 SPT 的分子拼图。世界上还有大约 1000 人患有一种罕见疾病 HSN1，这种疾病会导致他们的手臂和腿部神经从大约 30 岁开始就出现故障。它们的 SPT 蛋白（LCB1 和 LCB2）具有特定突变，它们仍然可以从 L-丝氨酸制造 SL，但它们也使用甘氨酸和 L-丙氨酸，并且产生的 SL 对细胞有毒——人们认为这些坏的 SL 会累积并造成神经损伤。因此，我们还将制作突变型 SPT 来模拟这种疾病，并尝试了解出了什么问题。我们将成为一支由技能互补的科学家组成的团队 - 位于美国爱丁堡、圣安德鲁斯、牛津和贝塞斯达，他们将共同构建关键机器的分子图谱，该机器负责为每个人制造适量的必需脂质细胞。