Functional characterization of the putative lysosomal transporter protein Major facilitator superfamily domain containing 1 (Mfsd1) and its role in sinusoidal obstruction syndrome

溶酶体是膜结合的细胞器，其通过酸水解酶的协同作用介导来自细胞内或细胞外来源的大多数类别的大分子的降解。在这些大分子酶促降解为低分子量单体如氨基酸或单糖后，后者从溶酶体腔输出到细胞溶质，用于通过特定的跨膜蛋白（所谓的输出蛋白）进行代谢再利用。尽管一些代谢物的溶酶体转运蛋白是已知的，但绝大多数代谢物的输出蛋白（包括大多数氨基酸和单糖）目前尚未鉴定。一些溶酶体氨基酸输出蛋白在溶酶体膜上的雷帕霉素复合物（mTORC 1）介导的营养信号传导机制中发挥关键作用。本申请的最终目标是破译来自溶质载体超家族的溶酶体膜蛋白（称为Mfsd 1）的功能和底物。该蛋白质被认为是小溶质的转运蛋白，并在我们的初步实验中显示定位于溶酶体，但其底物至今未知。我们设想破译其生理作用和运输功能的蛋白质的详细特征，产生的功能细胞系的损失和收益，并详细分析了建立Mfsd1基因敲除小鼠模型。还应评估Mfsd1在mTORC 1介导的溶酶体膜营养感应中的可能贡献。我们对Mfsd1基因敲除小鼠的初步分析揭示了一种早期肝脏表型，其特征是肝窦阻塞和肝窦内皮细胞死亡，反映了一种称为肝窦阻塞综合征的人类肝脏疾病的情况。因此，应研究Mfsd1在窦阻塞综合征发病机制中的作用。此外，将检查Mfsd 1与另一种称为Glmp的溶酶体膜蛋白的相互作用，因为我们的初步数据表明两种蛋白质的直接相互作用，并且两种蛋白质的敲除小鼠具有惊人相似的表型。总之，我们的研究结果将大大加深对溶酶体膜转运蛋白功能的理解。我们将确定Mfsd1转运的代谢产物，并确定该蛋白在基因敲除小鼠中的生理功能，特别是在窦阻塞综合征中。

Lysosomes are membrane bound organelles which mediate the degradation of most classes of macromolecules from intracellular or extracellular sources by a concerted action of acid hydrolases. Upon enzymatic degradation of these macromolecules to low molecular weight monomers like amino acids or monosaccharides, the latter become exported from the lysosomal lumen to the cytosol for metabolic reuse by specific transmembrane proteins, so called exporter proteins. Even though lysosomal transporters for a few metabolites are known, the exporter proteins for the great majority including most amino acids and monosaccharides are currently unidentified. Some lysosomal amino acid exporters were shown to play a pivotal role in mechanistic Target of Rapamycin complex (mTORC1)-mediated nutrient signalling at the lysosomal membrane. The ultimate goal of this application is to decipher the function and the substrate(s) of a lysosomal membrane protein from the solute carrier superfamily, called Mfsd1. The protein is assumed to be a transporter for small solutes and was shown in our preliminary experiments to localize to lysosomes, but its substrate(s) is (are) unknown so far. We envisage deciphering its physiological role and transporting function by characterizing the protein in detail, generating loss and gain of function cell lines and analyse an established Mfsd1-knockout mouse model in detail. A possible contribution of Mfsd1 in mTORC1-mediated nutrient-sensing at the lysosomal membrane should also be evaluated. Our preliminary analyses of Mfsd1-knockout mice revealed an early liver phenotype characterised by sinusoidal obstruction and liver sinusoidal endothelial cell death, reflecting the situation in a human hepatic disease called sinusoidal obstruction syndrome. The role of Mfsd1 in the pathogenesis of sinusoidal obstruction syndrome should therefore be investigated. Furthermore the interaction of Mfsd1 with another lysosomal membrane protein called Glmp, will be examined since our preliminary data point to a direct interaction of both proteins and knockout mice of both proteins have a strikingly similar phenotype. In summary, our findings will significantly gain deeper insight into the understanding of transporter functions at the lysosomal membrane. We will identify the metabolites which are transported by Mfsd1 and will determine the physiological function of the protein in knockout mice and particularly in sinusoidal obstruction syndrome.