Deciphering the lipid code for lysosomal channels and transporters in inflammatory, metabolic, and neurological disorders

溶质载体转运体(SLC)是一种完整的膜蛋白，它介导小分子在生物膜上的摄取、排出和交换。SLCs是动植物基因组中第二大膜蛋白家族，与多种疾病有关。近年来，越来越多的证据表明，脂质在调节SLC蛋白的功能以及将疾病表型与细胞内脂质相互作用的失调联系起来方面具有重要但不清楚的作用。由UKRI拨款资助的我们团队最近的发现表明，磷脂可以调节寡聚状态，控制开/关状态，并调节细胞内的运输。然而，对SLC家族内脂质调节功能的机械性理解仍然是难以捉摸的。我们的项目与OMassTreateutics合作，将通过体外和体内生物化学的独特组合、使用冷冻EM和结合脂质组学的自然质谱学(MS)进行结构研究，直接解决这一问题。我们的目标是创建第一张植物和动物细胞中脂质-SLC相互作用的相互作用图。具体地说，学生将使用一系列针对驻留在细胞中特定位置的SLC蛋白质的合成纳米体，并使用这些粘合剂亲和纯化目标蛋白质，以便随后使用天然MS进行脂质分析。这些蛋白质包括植物细胞中的液泡氨基酸转运体和动物细胞中溶酶体中的对应转运体。其目的是了解哪些脂类与这些蛋白质相关，以生成初步的脂类相互作用图。这些研究将与两种体内纳米盘重组(即无需事先纯化)相辅相成，这将使蛋白质能够以天然方式纯化，用于单颗粒冷冻-EM分析，随后使用分子动力学进行计算分析。预计通过与学生的讨论，实验室和计算工作之间的工作量平衡将随着项目的进展而确定。从天然MS和计算数据中获得的见解将被用于在冷冻-EM图中指定脂类密度，同时还用于通知和指导特定组成的脂质体中的生化转运分析。脂质体分析已经建立起来了，胆固醇的分子动力学管道也已经建立起来了，这是由一位DTP学生完成的。总之，所得到的数据将被用来为理解SLC生物学中特定的类脂类型(磷脂与胆固醇)对植物和动物细胞的重要性而制定蓝图，并促进我们对分子膜生物学这一被忽视的方面的理解。

Solute carrier transporters (SLC) are integral membrane proteins that mediate the uptake, extrusion and exchange of small molecules across biological membranes. SLCs represent the second largest family of membrane proteins in animal and plant genomes and are linked to numerous diseases. In recent years mounting evidence has indicated an important but unclear role for lipids in mediating the function of SLC proteins and linking disease phenotypes to dysregulation of lipid interactions in the cell. Recent discoveries from our group, funded through UKRI grants, have revealed that phospholipids can regulate oligomeric state, control on/off states and regulate trafficking in the cell. However, a mechanistic understanding of lipid regulated functions within the SLC family remain elusive. Our project, in partnership with OMass therapeutics, will directly address this question through a unique combination of in vitro and in vivo biochemistry, structural studies using cryo-EM and native mass spectrometry (MS) coupled with lipidomics. Our aim is to create the first interaction map for lipid-SLC interactions in plant and animal cells. Specifically, the student will use a range of synthetic nanobodies targeted to SLC proteins that reside in specific locations in the cell and use these binders to affinity purify the target proteins for subsequent lipid analysis using native MS. These include vacuolar amino acid transporters from plant cells and their counterparts in the lysosome in animal cells. The aim is to understand which lipids are associated with these proteins to generate an initial lipid interaction map. These studies will be complemented with both in vivo nanodisc reconstitutions (i.e., without prior purification), which will enable native-like purification of the proteins for single particle cryo-EM analysis, followed up with computational analyses using molecular dynamics. It is expected that through discussion with the student the balance of workload between laboratory and computational work will be decided as the project progresses. Insights gained from the native MS and computational data will be used to assign lipid densities in the cryo-EM maps, whilst also serving to inform and direct biochemical transport assays in liposomes of defined composition. The liposome assays have already been established, as have the molecular dynamics pipelines for cholesterol by a current DTP student. In summary, the resulting data will be used to develop a blueprint for understanding the importance of specific lipid types (phospholipid vs. cholesterol) in SLC biology for plant and animal cells and advance our understanding of this neglected aspect of molecular membrane biology.BBSRC theme - Mechanistic Molecular and Cellular Bioscience