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，通过酸水解酶的协同作用，介导细胞内或细胞外大多数大分子的降解。这些大分子被酶降解为低分子量的单体，如氨基酸或单糖，后者从溶酶体腔内出口到细胞质中，由特定的跨膜蛋白（即所谓的出口蛋白）进行代谢再利用。尽管已知一些代谢物的溶酶体转运蛋白，但包括大多数氨基酸和单糖在内的绝大多数蛋白的出口蛋白目前尚未确定。一些溶酶体氨基酸出口被证明在机制雷帕霉素靶复合物（mTORC1）介导的溶酶体膜上的营养信号传导中起关键作用。本应用程序的最终目标是破译溶质载体超家族中称为Mfsd1的溶酶体膜蛋白的功能和底物。该蛋白被认为是小溶质的转运体，并在我们的初步实验中显示可定位于溶酶体，但其底物目前尚不清楚。我们设想通过详细表征蛋白质，产生功能细胞系的损失和获得，并详细分析已建立的mfsd1敲除小鼠模型来破译其生理作用和运输功能。还应评估Mfsd1在mtorc1介导的溶酶体膜营养感应中的可能作用。我们对mfsd1基因敲除小鼠的初步分析揭示了以窦阻塞和肝窦内皮细胞死亡为特征的早期肝脏表型，反映了一种称为窦阻塞综合征的人类肝脏疾病的情况。因此，Mfsd1在窦状窦梗阻综合征发病机制中的作用应进一步研究。此外，Mfsd1与另一种称为Glmp的溶酶体膜蛋白的相互作用将被研究，因为我们的初步数据表明两种蛋白的直接相互作用和两种蛋白的敲除小鼠具有惊人相似的表型。总之，我们的发现将显著地加深对溶酶体膜转运蛋白功能的理解。我们将鉴定由Mfsd1运输的代谢物，并将确定该蛋白在敲除小鼠中的生理功能，特别是在鼻窦阻塞综合征中的功能。

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.