Understanding the role of the Lysosomal Integral Membrane Protein type 2 (LIMP-2/SCARB2) in lysosomal lipid transport

溶酶体中的脂肪堆积是脂肪沉积症的一个特征，它可导致许多溶酶体储存障碍，如Niemann-Pick病、Sandhoff病、Tay-Sachs病、Farber病和Gaucher病，具体取决于积累的脂质的类型。最近，我们提供的证据表明，丰富的溶酶体膜蛋白LIMP-2/SCARB2有助于沿着NPC1途径回收溶酶体低密度脂蛋白衍生的胆固醇。我们证明了属于CD36超家族清道夫受体的LIMP-2可以直接与胆固醇相互作用，并通过LIMP-2胞外结构域隧道将胆固醇转运到限制膜。由于LIMP-2还参与葡萄糖脑苷酶向溶酶体的转运，并参与磷脂转运事件，因此需要进一步阐明其在溶酶体脂转运过程中的分子作用。根据我们最近发表的工作和我们的初步数据，我们假设LIMP-2在溶酶体-内质网(ER)膜接触位置作为类固醇(脂)运输蛋白。在拟议的项目中，我们将通过生化分析利用LIMP-2隧道途径的额外脂肪底物来检验这一假设。在使用免疫共沉淀筛选的初步研究中，我们确定内体和内质网驻留的膜蛋白是LIMP-2的相互作用蛋白。我们计划通过免疫沉淀和显微镜定位实验进一步分析它们之间的相互作用以及它们在膜接触部位形成中的作用。过表达和丢失表达的分析将使我们能够破译这种LIMP-2复合体对脂质运输的重要性。我们还将研究LIMP-2的棕榈酰化在调节这种膜接触部位的形成和脂质运输过程中的作用。总之，在研究单位的框架内，我们的项目将增加对LIMP-2如何在将脂类从溶酶体腔转移到限制膜和其他细胞间隔中发挥基础作用的有价值的见解。

Lipid accumulation in lysosomes is a hallmark of lipidoses, which can lead to a number of lysosomal storage disorders such as Niemann-Pick disease, Sandhoff disease, Tay-Sachs disease, Farber disease, and Gaucher’s disease, depending on the type of accumulated lipid. Recently, we provided evidence that the abundant lysosomal membrane protein LIMP-2/SCARB2 contributes to the recycling of lysosomal LDL-derived cholesterol alongside the NPC1 pathway. We demonstrated that LIMP-2, which is belonging to the CD36 superfamily of scavenger receptors, directly interacts with cholesterol and can transport it to the limiting membrane via the LIMP-2 ectodomain tunnel. Since LIMP-2 also plays a role in the transport of glucocerebrosidase to lysosomes and is involved in phospholipid transfer events the further elucidation of its molecular role in lysosomal lipid transport processes is required. Based on our recently published work and our preliminary data we hypothesize that LIMP-2 serves as a sterol (lipid) transport protein at lysosome-endoplasmic reticulum (ER) membrane contact sites. Within the proposed project we will test this hypothesis by biochemically analyzing additional lipid substrates which utilize the LIMP-2 tunnel pathway. In preliminary studies using co-immunoprecipitation screens we identified endosomal and ER-resident membrane proteins as LIMP-2 interactors. We plan to further analyze their interaction and their role in the formation of membrane contact sites using immunoprecipitation and microscopy localization experiments. Overexpression and loss of expression analyses will allow us to decipher the importance of such LIMP-2 complexes for lipid transport. We will also study the role of palmitoylation of LIMP-2 to regulate such membrane contact site formation and lipid transport processes. In summary, within the framework of the research unit our project will add valuable insight in how LIMP-2 exerts fundamental roles in transferring lipids from the lysosomal lumen to the limiting membrane and to other cellular compartments.