Mass Spectrometric Profiling of Lysosomal Protein Interactions, Structures, and Half-lives via Cross-Linking and Pulse Stable Isotope Labeling

Abstract

In mammalian cells, the lysosome is the central organelle for the degradation of macromolecules and recycling of their building blocks. Mutations in almost every known lysosomal hydrolase lead to a group of rare but devastating diseases known as lysosomal storage diseases, demonstrating lysosomal protein's pathogenic significance. Besides, in recent years, it was reported that lysosomal proteins and such interacting with the lysosome play key roles in a variety of common diseases such as cancer and neurodegenerative disorders, leading to an increasing interest in the characterization of the lysosomal proteome. <br /> For the investigation of the lysosomal proteome on a large scale, lysosome enriched fractions were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Systematic comparisons of different lysosome enrichment methods revealed a significant increase of lysosomal proteins by up to 118-fold compared to whole-cell lysate using the superparamagnetic iron oxide nanoparticles method. An approach that was also found to be superior in terms of preserving intact lysosomes and enriching them in large quantities. The established enrichment methodology was then used to study lysosomal structures and their interactions using cross-linking MS. Moreover, it was used to determine protein half-lives by using pulse stable isotope labeling with amino acids in cell culture (pSILAC) in combination with data-independent acquisition (DIA) MS/MS. <br /> To further better characterize and validate cross-linking experiments, antibodies specific against commercially available cross-linkers were raised and affinity-purified from rabbits. Those were successfully applied in western blotting and immunocytochemistry experiments. The results further demonstrated the ability of the antibodies to retain peptides modified with water-quenched dead-links and cross-linker-modified proteins in immunoprecipitation assays (most likely also through water-quenched dead-links). <br /> The presented data cover the first large-scale cross-linking study of lysosome enriched fractions. Analysis of cross-links revealed a highly interconnected network of 847 proteins forming 1,024 protein-protein interactions (PPIs), including 67 % potentially novel PPIs, of which two were confirmed by co-immunoprecipitation (co-IP). Additionally, 161 cross-links mapped on 34 lysosomal proteins confirm known crystal structures in a physiological state, as well as validate predicated AlphaFold models and provide evidence for a novel tetrameric model of palmitoyl-protein thioesterase 1. For the lysosome-interacting proteins flotillin 1 and 2, the first heterodimeric structure, as well as a 38 oligomeric assembly were proposed. Further, the investigation of their putative vesicular cargo led to the identification of 328 proteins including 50 receptors. For instance, all three members of latrophilins (G-protein-coupled receptors) were identified to be endocytosed in a flotillin dependent manner. <br /> In addition, the pSILAC DIA MS/MS resulted in the identification of 33,968 half-lives for 6,915 unique proteins, presenting the most extensive turnover study so far. The study included a wild-type condition (control), representing the steady-state and different perturbational cellular states such as the impairment of autophagosome-lysosome fusion, proteasome activity, cathepsin activity, cholesterol homeostasis, and protein glycosylation. The results revealed a median half-life of 18 h in wild-type cells, with significant half-life differences of up to 30 h for specific proteins among the different perturbational cellular states. For example, a drastic increase of half-lives upon proteasomal inhibition or a decrease upon cathepsin D inhibition was observed. The data suggest that the regulation of cellular degradational processes are the primary driving factors influencing protein half-lives.