Mechanisms of LAM-mediated intracellular sterol traffic and its regulation by conserved kinases

了解细胞如何控制胆固醇运输该项目的目的是提高对细胞内胆固醇运输的理解，这是一个对所有细胞（从人类到真菌和植物）的健康至关重要的知之甚少的过程。细胞通过质膜与外界隔开，质膜由脂肪分子（脂质）的双层（两个并列层）组成，防止细胞内的分子逃逸。细胞膜的关键脂质是胆固醇，胆固醇可以强化细胞膜，防止细胞膜分裂成不同的区域，这些区域是有毒的，可能会导致从动脉粥样硬化到阿尔茨海默氏症等各种社会重要疾病。当质膜的物理性质必须改变时，例如当体温升高时，质膜中的胆固醇水平必须改变，以使其物理性质与新的条件相匹配。胆固醇流入和流出质膜是由专门的固醇转移蛋白进行的，这些蛋白折叠成带有铰链盖的盒子的整体形状。它们内部容纳单个胆固醇分子，并且它们可以重复地将固醇从细胞内的一个位点运送到另一个位点。我们最近在人类、真菌和植物的所有细胞中发现了一个称为LAMs的固醇转移蛋白大家族。LAMs将多余的胆固醇从质膜转移到称为内质网的内部细胞器，在那里转化为油的形式，储存在脂肪体中供以后使用。细胞有几条途径来应对新的条件，每条途径都包含多种蛋白质。这些通路中的大多数都受到称为激酶的蛋白质活性的调节，这些蛋白质通过物理修饰下游通路组件来打开它们。在所有复杂生命形式的所有细胞中发现的主要激酶之一是TORC 2。就像一个抵消温度变化的恒温器一样，TORC 2通过修改几个通路中的成分来响应质膜的变化，这些通路都抵消了原始的变化。TORC 2已知影响质膜的许多不同脂质，但到目前为止，它还没有直接与胆固醇联系起来。这个为期三年的项目是基于我们的新证据，即细胞使用TORC 2来控制LAMs的活性。我们将发现LAMs的重要固醇运输功能是否如我们的数据所示由TORC 2控制。我们还将测试这样一种范式，即像TORC 2这样的主调节器具有多个输出，这些输出联合收割机结合在一起以产生协同效应。为此，我们将研究细胞如何联合收割机结合胆固醇的变化与其他变化的脂质的质膜，TORC 2制定。我们还发现，细胞有时会迅速破坏LAMs。靶向蛋白质破坏是一种非常常见的控制途径;事实上，TORC 2的主要工作方式是通过改变破坏特定蛋白质的机制的活性。因此，我们将发现LAM是如何被破坏的，以及LAM蛋白的破坏是否是TORC 2对胆固醇运输施加的第二层控制。我们的工作将在芽殖酵母中进行，在芽殖酵母中LAM和TORC 2途径比在人类细胞中更容易理解。然而，所有参与酵母的蛋白质和脂质在人类中都有对应物，我们将研究我们在酵母中发现的影响是否适用于人类细胞。我们的基础研究的实际应用将有助于设计出操纵胆固醇运动重要的许多过程的方法。由于LAM蛋白和TORC 2存在于所有复杂的生命形式中，我们的研究将产生适用于植物，真菌和动物的知识。我们的研究结果不仅将增加对健康老龄化（动脉粥样硬化，阿尔茨海默氏症，II型糖尿病）的理解，而且还将增加对粮食安全的理解，因为胆固醇运输对作物和破坏作物的真菌都至关重要。

Understanding how cells control cholesterol trafficThe purpose of this project is to improve understanding of traffic of cholesterol inside cells, a poorly understood process of vital importance to the health of all cells, from humans to fungi and plants. Cells are separated from the outside world by the plasma membrane, which consists of a bilayer (two apposed layers) of fat molecules (lipids) that prevent the escape of molecules inside the cell. The key lipid for the plasma membrane is cholesterol, which strengthens the membrane and prevents it from dividing up into different regions, which would be toxic and may contribute to a wide variety of societally important diseases from atherosclerosis to Alzheimer's. When the physical properties of the plasma membrane have to change, for example when body temperature increases, levels of cholesterol in the plasma membrane must change so that its physical properties match the new conditions. Cholesterol flow into and out of the plasma membrane is carried out by specialised sterol transfer proteins, which fold into the overall shape of a box with a hinged lid. They accommodate a single cholesterol molecule inside, and they can repeatedly shuttle sterol from one site inside cells to another. We recently discovered a large family of sterol transfer proteins called LAMs in all cells in humans, fungi and plants. LAMs transfer excess cholesterol away from the plasma membrane to an internal organelle called the endoplasmic reticulum, where is converted into an oil form that is stored in fat bodies for later use. Cells have several pathways that respond to new conditions, each pathway containing multiple proteins. Most of these pathways are regulated by the activity of proteins called kinases, which physically modify downstream pathway components to switch them on. Among the major kinases found in all cells in all complex life forms is TORC2. Like a thermostat counteracting temperature changes, TORC2 responds to changes in the plasma membrane by modifying components in several pathways that all counteract the original changes. TORC2 is known to affect many different lipids of the plasma membrane, but so far it has never been linked directly to cholesterol.This three year project is based on our new evidence that cells use TORC2 to control the activity of LAMs. We will find out if the important sterol trafficking function of LAMs is controlled by TORC2 as our data suggests. We will also test the paradigm that master regulators like TORC2 have multiple outputs that combine to create synergy. For this we will study how cells combine changes in cholesterol with other changes in lipids of the plasma membrane that TORC2 enacts. We have also found out that cells sometimes rapidly destroy LAMs. Targeted protein destruction is a very common way in which pathways are controlled; indeed a major way TORC2 works is by altering the activity of the machinery that destroys specific proteins. Therefore, we will find out how LAMs are destroyed and whether destruction of LAM proteins is a second layer of control imposed by TORC2 on cholesterol traffic.Our work will be carried out in budding yeast where the LAM and TORC2 pathways are better understood than in human cells. However, all the participating proteins and lipids involved in yeast have counterparts in humans, and we will examine if the effects we discover in yeast apply in human cells. The practical applications following on from our basic research will be to help devise ways to manipulate many processes where cholesterol movement is important. Because LAM proteins and TORC2 are found in all complex life forms, our research will produce knowledge that is applicable to plants, fungi, and animals. Our results will add to the understanding not only of healthy ageing (atherosclerosis, Alzheimer's, type II diabetes), but also for food security since cholesterol traffic is vital both to crop plants and to fungi that destroy crops.