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

溶质载体转运蛋白（SLC）是一种完整的膜蛋白，介导小分子在生物膜上的摄取、挤压和交换。slc是动物和植物基因组中第二大膜蛋白家族，与许多疾病有关。近年来，越来越多的证据表明，脂质在介导SLC蛋白功能和将疾病表型与细胞中脂质相互作用失调联系起来方面发挥着重要但不明确的作用。在UKRI的资助下，我们小组最近的发现表明，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家族中脂质调节功能的机制理解仍然难以捉摸。我们的项目与OMass therapeutics合作，将通过体外和体内生物化学的独特组合，使用冷冻电镜和天然质谱（MS）结合脂质组学进行结构研究，直接解决这个问题。我们的目标是创建植物和动物细胞中脂质- slc相互作用的第一个相互作用图。具体来说，学生将使用一系列合成纳米体靶向细胞中特定位置的SLC蛋白，并使用这些结合物亲和纯化目标蛋白，以便随后使用天然质谱进行脂质分析。这些纳米体包括来自植物细胞的液泡氨基酸转运体及其在动物细胞溶酶体中的对应物。目的是了解哪些脂质与这些蛋白质相关，以生成初始脂质相互作用图。这些研究将辅以体内纳米盘重构（即，没有事先纯化），这将使蛋白质的原生纯化成为可能，用于单颗粒低温电镜分析，随后使用分子动力学进行计算分析。期望通过与学生的讨论，随着项目的进展，将决定实验室和计算工作之间的工作量平衡。从原生质谱和计算数据中获得的见解将用于在冷冻电镜图中分配脂质密度，同时也用于通知和指导定义组成的脂质体的生化运输分析。脂质体测定法已经建立，目前DTP学生已经建立了胆固醇的分子动力学管道。总之，所得数据将用于制定蓝图，以了解特定脂质类型（磷脂与胆固醇）在植物和动物细胞SLC生物学中的重要性，并促进我们对分子膜生物学中这一被忽视方面的理解。BBSRC主题-机械分子和细胞生物科学

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