Global cellular proteo-lipidomic profiling of diverse lysosomal storage disease mutants using nMOST

Lysosomal storage diseases (LSDs) comprise ~50 monogenic disorders marked by the buildup of cellular material in lysosomes, yet systematic global molecular phenotyping of proteins and lipids is lacking. We present a nanoflow-based multi-omic single-shot technology (nMOST) workflow that quantifies HeLa cell proteomes and lipidomes from over two dozen LSD mutants. Global cross-correlation analysis between lipids and proteins identified autophagy defects, notably the accumulation of ferritinophagy substrates and receptors, especially in NPC1-/- and NPC2-/- mutants, where lysosomes accumulate cholesterol. Autophagic and endocytic cargo delivery failures correlated with elevated lyso-phosphatidylcholine species and multi-lamellar structures visualized by cryo-electron tomography. Loss of mitochondrial cristae, MICOS-complex components, and OXPHOS components rich in iron-sulfur cluster proteins in NPC2-/- cells was largely alleviated when iron was provided through the transferrin system. This study reveals how lysosomal dysfunction affects mitochondrial homeostasis and underscores nMOST as a valuable discovery tool for identifying molecular phenotypes across LSDs.

溶酶体贮积症（Lysosomal storage diseases, LSDs）包含约50种以细胞物质在溶酶体内蓄积为特征的单基因遗传病，但目前尚缺乏针对蛋白质与脂质的系统性全局分子表型分析。本研究开发了一种基于纳流的多组学单次检测技术（nanoflow-based multi-omic single-shot technology, nMOST）实验流程，可对二十余种溶酶体贮积症突变体的HeLa细胞蛋白质组与脂质组进行定量分析。通过对脂质与蛋白质进行全局互相关分析，本研究揭示了自噬缺陷现象，具体表现为铁蛋白自噬底物与受体的蓄积，尤以NPC1基因敲除（NPC1-/-）与NPC2基因敲除（NPC2-/-）突变体最为显著，这两类突变体的溶酶体均存在胆固醇蓄积。自噬与内吞底物递送失败现象，与溶血磷脂酰胆碱（lyso-phosphatidylcholine）水平升高以及冷冻电子断层扫描（cryo-electron tomography）所观测到的多层膜结构异常密切相关。在NPC2-/-细胞中，线粒体嵴、MICOS复合物组分以及富含铁硫簇蛋白的氧化磷酸化（OXPHOS）复合物组分出现缺失，而通过转铁蛋白系统补充铁离子后，该缺陷可得到显著缓解。本研究阐明了溶酶体功能异常如何影响线粒体稳态，并证实nMOST是用于挖掘各类溶酶体贮积症分子表型的高效发现工具。