Molecular species variants of phospholipids: a code through which cells distinguish phosphoinositide signals and their synthetic intermediates

磷脂是细胞膜的组分，其保持内容物在细胞内和细胞之间分配。磷脂酰肌醇（PIPn）是磷脂的一个特定家族，其研究彻底改变了我们现在对这些分子的看法;它们不仅代表生物物理屏障，而且还具有一系列重要的调节作用，其中它们在特定膜位置的存在触发了具有特定工作的蛋白质的活性。这些工作包括在细胞表面传达激素和生长因子的存在，并协调适当的细胞反应，如生长和分泌（称为“PLC”和“PI 3 K”信号通路）。细胞需要解决的主要问题之一是如何将PIPn合成中使用的常见生物合成中间体与其他脂质合成中使用的中间体分开。我们的初步数据表明，他们可能做到这一点的方法之一是根据酰基链的组成来区分PIPn和由它们衍生的分子。磷脂通常有两个酰基链;这些是分子的疏水部分，将磷脂锚在生物膜中。大多数类型的磷脂包含各种不同的酰基链，但在大多数情况下，这种多样性的功能是未知的。然而，PIPn，特别是在哺乳动物细胞中，是相对分子均质的，具有一个硬脂酰基链（缩写为C18：0）和一个花生四烯酰基链（C20：4）的特征组成。当PLC信号通路被质膜（包围细胞的外膜）上的激素激活时，PIPn被转化为一种称为二酰基甘油的分子，然后转化为磷脂酸（PA）。我们假设磷脂“转移蛋白”基于其C18：0/C20：4组成从质膜选择性地提取该PA，然后将其递送到设计用于PIPn再合成的单独膜系统（“PM/ER接触位点”）的邻近区域。然后，这些转运蛋白将新产生的PIPn转运回质膜，形成“PI循环”。我们假设，一个有效的PI周期是必要的，以保持在质膜上的PIPn水平和维持慢性信号。我们计划通过化学合成具有不同酰基链的PIPn来测试这些假设，然后通过一种称为质谱的技术跟踪它们被传递到细胞时发生的事情。质谱法使我们能够区分我们已经传递到细胞的分子和已经存在的分子（基于用重同位素标记它们）。我们还计划使用几种尖端类型的显微镜来跟踪荧光标记的PA和PIPn以及使它们产生的酶，看看我们是否可以区分细胞中PLC刺激的磷酸肌醇合成发生的区域。我们还将直接测试的假设，选择的转移蛋白可以区分的PA和PIPn的酰基链组成。最后，我们将尝试干扰细胞的能力，以丰富其PIPn与C18：0/C20：4，看看这是否改变了效率的PI周期和慢性信号通过PLC和PI 3 K pathways.The结果这个项目将大大有助于我们的理解的具体功能的酰基链在PIPn和突出的潜在广泛作用的酰基链作为分子特征，以区分密切相关的脂质池。我们的研究结果还可能揭示PLC和PI 3 K信号通路慢性调节的潜在新点，具有治疗意义。过度活跃的PLC信号传导被假设为躁狂症的原因（基于锂治疗双相情感障碍），并且有压倒性的证据表明高水平的PI 3 K信号传导驱动许多癌症

Phospholipids are components of cellular membranes that keep contents partitioned within and between cells. Phosphoinositides (PIPn) are a specific family of phospholipids whose study has revolutionised the way in which we now think about these molecules; they do not merely represent a biophysical barrier, but also adopt a range of important regulatory roles in which their presence at a particular membrane location triggers the activity of proteins with a specific job to perform. These jobs include communicating the presence of hormones and growth factors at the cell surface and co-ordinating appropriate cellular responses, such as growth and secretion (called the 'PLC' and 'PI3K' signalling pathways). The levels of PIPn must therefore be tightly and independently regulated.One of the major problems the cell needs to solve is how to segregate common biosynthetic intermediates used in the synthesis of PIPn from those that are also used in the synthesis of other lipids. Our preliminary data suggests one of the ways they might do this is to differentiate PIPn and molecules derived from them on the basis of their acyl chain composition. Phospholipids typically have two acyl chains; these are the hydrophobic parts of the molecule that anchor a phospholipid in a biological membrane. Most types of phospholipids comprise a wide variety of different acyl chains but for the most part the function of this diversity is unknown. PIPn however, particularly in mammalian cells, are relatively molecularly homogeneous, with a characteristic composition of one stearoyl chain (abbreviated C18:0) and one arachidonoyl chain (C20:4). When the PLC signalling pathway is activated by hormones at the plasma membrane (the outer membrane that surrounds a cell), PIPn are converted into a molecule called diacylglycerol and then into phosphatidic acid (PA). We hypothesise that phospholipid 'transfer proteins' selectively extract this PA from the plasma membrane on the basis of its C18:0/C20:4 composition and then deliver it to a neighbouring region of a separate membrane system (a 'PM/ER contact site') which is designed for PIPn resynthesis. These transfer proteins then transport the newly made PIPn back into the plasma membrane, forming a 'PI cycle'. We hypothesise that an efficient PI cycle is necessary to preserve the levels of PIPn at the plasma membrane and sustain chronic signalling. We plan to test these hypotheses by chemically synthesising PIPn with different acyl chains and then tracking what happens to them when they are delivered to cells by a technique called mass spectrometry. Mass spectrometry allows us to distinguish between the molecules we have delivered to the cells and the ones that are already there (on the basis of labelling them with heavy isotopes). We also plan to use several cutting-edge types of microscopy to track fluorescently tagged PAs and PIPn and the enzymes which make them to see if we can distinguish areas in the cell where PLC-stimulated phosphoinositide synthesis takes place. We will also directly test the hypothesis that selected transfer proteins can distinguish the acyl chain composition of PAs and PIPn. Finally, we will attempt to interfere with the cell's ability to enrich its PIPn with C18:0/C20:4 to see if this alters the efficiency of a PI cycle and chronic signalling through PLC and PI3K pathways.The results of this project will contribute greatly to our understanding of the specific function of acyl chains in PIPn and highlight a potentially widespread role for acyl chains as molecular signatures to distinguish closely related lipid pools. Our results may also uncover a potentially novel point in the chronic regulation of PLC and PI3K signalling pathways, with therapeutic implications. Overactive PLC signalling has been hypothesised as a cause of mania (based on the treatment of bipolar disorders with lithium) and there is overwhelming evidence that high levels of PI3K signalling drive many cancers