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Impairment of lipid homeostasis causes lysosomal accumulation of endogenous protein aggregates through ESCRT disruption

ABSTRACT: Protein aggregation increases during aging and is a pathological hallmark of many age-related diseases. Protein homeostasis (proteostasis) depends on a core network of factors directly influencing protein production, folding, trafficking, and degradation. Cellular proteostasis also depends on the overall composition of the proteome and numerous environmental variables. Modulating this cellular proteostasis state can influence the stability of multiple endogenous proteins, yet the factors contributing to this state remain incompletely characterized. Here, we performed genome-wide CRISPRi screens to elucidate the modulators of proteostasis state in mammalian cells, using a fluorescent dye to monitor endogenous protein aggregation. These screens recovered components of the known proteostasis network, and uncovered a link between protein and lipid homeostasis. We showed that increased lipid uptake and/or disrupted lipid metabolism led to increased lysosomal, detergent-insoluble protein aggregates and, concomitantly, accumulation of sphingomyelins and cholesterol esters. Proteomic analysis of lysosomes revealed ESCRT accumulation at the lysosomes, suggesting disruption of ESCRT disassembly, lysosomal membrane repair, and microautophagy. Lipid dysregulation leads to lysosomal membrane permeabilization, but does not otherwise impact fundamental aspects of lysosomal and proteasomal functions. Together, these results demonstrate that lipid dysregulation impairs proteostasis via ESCRT disruption, potentially as a result of modified membrane properties.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Lymphoblast, Bone Marrow

DISEASE(S): Chronic Myeloid Leukemia

SUBMITTER: Niclas Olsson

LAB HEAD: Fiona McAllister

PROVIDER: PXD054648 | Pride | 2024-12-20

REPOSITORIES: Pride

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Source:

			Action	DRS
	Human_UP000005640_20596_one_prot_seq_per_gene_20231222.fasta	Fasta
	Mapping_files_TMTchannels_samples.csv	Csv
	Peptide_IDs.csv	Csv
	p011557_HEX_plex01_inj02.raw	Raw
	p011559_HEX_plex01_inj03.raw	Raw

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Project description:Lysosomes represent a central degradative compartment of eukaryotes, yet little is known about biogenesis and function of this organelle in parasitic protists. Whereas the mannose-6 phosphate (M6P)-dependent system is dominant for lysosomal targeting in metazoans, oligosaccharide independent sorting was reported in other eukaryotes. In this study, we investigated the phagolysosomal proteome of the human parasite Trichomonas vaginalis, protein targeting and involvement of lysosomes in hydrolase secretion. The organelles were purified using conventional Percoll and OptiPrep density gradient centrifugation, and a novel purification protocol based on phagocytosis of lactoferrin-covered magnetic nanoparticles. The analysis resulted in the lysosomal proteome of 462 proteins, which were sorted into 21 functional classes. Hydrolases represent the largest functional class and include proteases, lipases, phosphatases, glycosidases, and other hydrolases. Identification of a large set of proteins involved in vesicular trafficking (80) and turnover of actin cytoskeleton rearrangement (29) indicate high dynamics of phagolysosomal compartment. Several lysosomal proteins including the cysteine protease TvCP2 were previously shown to be secreted. Our experiment showed that secretion of TvCP2 was strongly inhibited by chloroquine that increased intralysosomal pH and thus indicated TvCP2 secretion through lysosomes rather than the classical secretory pathway. Unexpectedly, we identified in the phagolysosomal proteome divergent homologs of M6P receptor TvMPR, although T. vaginalis lacks enzymes for M6P formation. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine protease CLCP that possesses two glycosylation sites. Mutation of any of the sites redirected CLCP to the secretory pathway. Similarly, introduction of glycosylation sites to secreted β-amylase redirected this protein to lysosomes. Thus, unlike other parasitic protists, T. vaginalis seems to utilize glycosylation as recognition marker for lysosomal hydrolases. Whether TvMPR or other possible receptors are involved in this process and what is the precise structure of the lysosomal recognition marker need to be clarified in future studies.

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Impairment of lipid homeostasis causes lysosomal accumulation of endogenous protein aggregates through ESCRT disruption

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