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LYSET/TMEM251 KO secretome from MIA PaCa-2 cells

ABSTRACT: Analysis of proteins secreted over 24 h in OptiMEM by LYSET/TMEM251 knockout and control MIA PaCa-2 cells.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Pancreas

DISEASE(S): Pancreas Adenocarcinoma

SUBMITTER: Wilhelm Palm

LAB HEAD: Wilhelm Palm

PROVIDER: PXD029658 | Pride | 2022-09-29

REPOSITORIES: Pride

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			Action	DRS
	CP0480-1.zip	Other
	CP480_RawFile2Sample.txt	Txt
	checksum.txt	Txt
	oecf3-CP0480-01.raw	Raw
	oecf3-CP0480-02.raw	Raw

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LYSET/TMEM251 SILAC Co-IP from MIA PaCA-2

Project description:Endogenous LYSET/TMEM251 was isolated via Co-IP from MIA PaCa-2 cells. LYSET KO cells were used as controls. Cells were grown in SILAC medium for 3 passages

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LYSET/TMEM251 KO cellular and lysosomal proteomes from MIA PaCa-2 cells

Project description:Proteome analysis of post-nuclear supernatants (PNS) and enriched lysosomal fractions from LYSET/TMEM251 knockout and control MIA PaCa-2 cells. Lysosomes were loaded with iron nanoparticles (DexoMAG C, Liquids Research). PNS were generated from cell homogenates and used as input to enrich lysosomal fractions over magnetic LS MACS columns (Miltenyi).

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LYSET/TMEM251 KO and GNPTAB KO secretomes from MEFs

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GCAF(TMEM251) regulates lysosome biogenesis by activating the mannose-6-phosphate pathway

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mRNA expression data from RalGAPβ-deficient human pancreatic ductal adenocarcinoma cells (MIA PaCa-2), MIA PaCa-2 parental cells, and MIA PaCa-2 control cells

Project description:Pancreatic ductal adenocarcinoma, caused by activating mutation in K-Ras, is an aggressive malignancy due to its early invasion and matastasis. Ral GTPases, negatively regulated by RalGAP, are activated downstream of Ras and play a crucial role in development and progression of pancreatic ductal adenocarcinoma. However, the underlying mechanisms remain unclear. We used microarrays to detail the global programme of gene expression underlying the human pancreatic ductal adenocarcinoma cell line, MIA PaCa-2 with RalGAPβ deficiency or not, and identified distinct classes of Ral activation-related mRNA.

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The human disease gene TMEM251 is essential for lysosomal transport and viral infection

Project description:After gaining access to the endo-lysosomal pathway, several viruses depend on lysosomal cathepsin proteases to cleave their structural proteins triggering productive entry. Targeting of cathepsins and other luminal lysosomal proteins to lysosomes requires their modification with mannose 6-phosphate (M6P) signals. Key to M6P tagging is N-acetylglucosamine (GlcNAc)-1-phosphotransferase whose deficiency leads to the severe lysosomal storage disorder mucolipidosis II (MLII). Here, using genome-scale CRISPR screens, we identify the transmembrane protein TMEM251 as critically important for viral infection by cathepsin-dependent viruses including reovirus, Ebola virus, and SARS-CoV-2. We demonstrate that Golgi-resident TMEM251 is essential for lysosomal sorting and activity of cathepsins. Mechanistically, we show that TMEM251 deficiency in human cells results in global loss of M6P on luminal lysosomal proteins by destabilizing GlcNAc-1-phosphotransferase. Tmem251 knockout mice reveal characteristics typical of MLII including hypersecretion of lysosomal enzymes and accumulation of lysosomal storage material in isolated fibroblasts. Finally, we demonstrate that human pathogenic TMEM251 alleles fail to rescue lysosomal sorting defects in knockout cells. Our results uncover a crucial role of TMEM251 in M6P-dependent lysosomal transport and viral infection. Overall, work here reveals the biochemical basis of an inherited MLII-like disease caused by mutations in TMEM251 and provides insights into infection mechanisms of a broad class of medically important viruses.

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The Human Disease Gene LYSET is Essential for Lysosomal Enzyme Transport and Viral Infection

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Project description:Among solid tumors, pancreatic cancer (PC) remains a leading cause of death. In PC, the protein ANXA1 has been identified as an oncogenic factor acting in an autocrine/paracrine way, and also as a component of tumor-deriving extracellular vesicles. Here, we proposed the experimental protocol to obtain spheroids from the two cell lines, wild-type (WT) and Annexin A1 (ANXA1) knock-out (KO) MIA PaCa-2, this last previously obtained through CRISPR/Cas9 genome editing system. The use of three-dimensional (3D) models, like spheroids, can be useful to mimic tumor characteristics and for preclinical chemo-sensitivity studies. By using PC spheroids, we have assessed the activity of intracellular and extracellular ANXA1. Indeed, we have proved that the intracellular protein influences in vitro tumor development and growth by spheroids analysis, in addition to defining the modification about cell protein pattern in ANXA1 KO model compared to the WT one. Moreover, we have tested the response to FOLFIRINOX chemotherapy regimen whose cytostatic effect appeared notably increased in ANXA1 KO spheroids. Additionally, this study has highlighted that the extracellular ANXA1 action is strengthened through the EVs supporting spheroids growth and resistance to drug treatment, mainly affecting tumor progression. Thus, our data interestingly suggest the relevance of ANXA1 as a potential therapeutic PC marker.

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