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

LAM介导的细胞内甾醇运输的机制及其保守激酶的调节

• 批准号: BB/P003818/1
• 负责人: Timothy Levine
• 金额: $ 49万
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FP003818%2F1
• 关键词: Mechanisms; LAM; mediated; intracellular; sterol

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.

了解细胞如何控制胆固醇运输这个项目的目的是提高对细胞内胆固醇运输的了解，这是一个知之甚少的过程，对从人类到真菌和植物的所有细胞的健康都是至关重要的。细胞通过质膜与外界隔开，质膜由一层(两层相对的)脂肪分子(脂)组成，防止分子在细胞内逃逸。质膜的关键脂类是胆固醇，它能加强质膜，防止它分裂成不同的区域，这将是有毒的，可能会导致从动脉粥样硬化到阿尔茨海默氏症的各种重要社会疾病。当质膜的物理性质必须改变时，例如当身体温度上升时，质膜中的胆固醇水平必须改变，以便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特殊的类固醇转移蛋白进行的，这些蛋白折叠成一个带有铰链盖子的盒子的整体形状。它们在细胞内容纳一种单一的胆固醇分子，它们可以重复地将类固醇从细胞内的一个位置运送到另一个位置。我们最近在人类、真菌和植物的所有细胞中发现了一个名为LAM的类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，在那里转化为脂肪形式，储存在脂肪体中供以后使用。细胞有几条对新条件做出反应的途径，每条途径都含有多种蛋白质。这些途径中的大多数都受称为蛋白激酶的蛋白质的活性调节，这些蛋白在物理上修改下游途径的成分以开启它们。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TORC2。就像恒温器抵消温度变化一样，TORC2通过改变几个途径中的成分来响应质膜的变化，这些途径都抵消了最初的变化。已知TORC2可以影响质膜的许多不同的脂质，但到目前为止，它从未直接与胆固醇联系在一起。这个为期三年的项目是基于我们的新证据，即细胞使用TORC2来控制LAM的活性。我们将发现LAM的重要的类固醇运输功能是否如我们的数据所表明的那样由TORC2控制。我们还将测试这样的范式，即像TORC2这样的主控监管机构拥有多个结合在一起创造协同效应的产出。为此，我们将研究细胞如何将胆固醇的变化与TORC2颁布的质膜脂类的其他变化结合起来。我们还发现，细胞有时会迅速摧毁LAM。靶向蛋白质破坏是控制通路的一种非常常见的方式；事实上，TORC2的一个主要工作方式是通过改变破坏特定蛋白质的机械的活动。因此，我们将了解LAM是如何被破坏的，以及LAM蛋白的破坏是否是TORC2对胆固醇运输施加的第二层控制。我们的工作将在萌芽酵母中进行，在那里LAM和TORC2途径比在人类细胞中更被理解。然而，酵母中涉及的所有参与蛋白质和脂类在人类中都有对应的作用，我们将检查我们在酵母中发现的影响是否适用于人类细胞。我们基础研究之后的实际应用将有助于设计方法来操纵许多胆固醇运动很重要的过程。由于LAM蛋白和TORC2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，也将增加对食品安全的理解，因为胆固醇运输对农作物和破坏作物的真菌都是至关重要的。

项目成果

Referee report. For: Structural bioinformatics predicts that the Retinitis Pigmentosa-28 protein of unknown function FAM161A is a homologue of the microtubule nucleation factor Tpx2 [version 1; peer review: 1 approved]

裁判报告。

• DOI: 10.5256/f1000research.28550.r70563
• 发表时间: 2020
• 作者: Alva V

Identification of seipin-linked factors that act as determinants of a lipid droplet subpopulation.

• DOI: 10.1083/jcb.201704122
• 发表时间: 2018-01-02
• 期刊: The Journal of cell biology
• 作者: Eisenberg-Bord M;Mari M;Weill U;Rosenfeld-Gur E;Moldavski O;Castro IG;Soni KG;Harpaz N;Levine TP;Futerman AH;Reggiori F;Bankaitis VA;Schuldiner M;Bohnert M
• 通讯作者: Bohnert M

FFAT motif phosphorylation controls formation and lipid transfer function of inter-organelle contacts.

• DOI: 10.15252/embj.2019104369
• 发表时间: 2020-12-01
• 期刊: The EMBO journal
• 作者: Di Mattia T;Martinet A;Ikhlef S;McEwen AG;Nominé Y;Wendling C;Poussin-Courmontagne P;Voilquin L;Eberling P;Ruffenach F;Cavarelli J;Slee J;Levine TP;Drin G;Tomasetto C;Alpy F
• 通讯作者: Alpy F