Browse
Submit Data
Databases
API
Help

Dataset Information

325 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

LC-MS/MS analysis of Mannose 6-phosphate containing proteins

ABSTRACT: Mucolipidosis III gamma (MLIII) is clinically characterized by onset of first symptoms at an average of 5 years such as stiffness of hands and shoulders, claw hand deformities, scoliosis and progressive destruction of hip joints. The disease is caused by mutations in GNPTG encoding the gamma-subunit of the GlcNAc-1-phosphotransferase complex. This enzyme is responsible for the generation of mannose 6-phosphate (M6P) targeting signals on 70 soluble lysosomal enzymes that are required for their efficient receptor-mediated transport to lysosomes. In fibroblasts from Gnptg-KO mice the GlcNAc-1-phosphotransferase activity is reduced leading to low amounts of M6P-containing proteins. M6P affinity chromatography-assisted and mass spectrometry-based secretome analysis of Gnptg-KO fibroblasts revealed a distinct set of lysosomal enzymes modified with M6P residues.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Fibroblast

SUBMITTER: Shiva Ahmadi

LAB HEAD: Dominic Winter

PROVIDER: PXD009621 | Pride | 2018-06-06

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	MaxQuant180mingradient.zip	Other
	MaxQuant60mingradient.zip	Other
	MaxQuantTxtFiles-180mingradient.zip	Other
	MaxQuantTxtFiles-60mingradient.zip	Other
	SA_HH_Bead1.raw	Raw

Items per page:

1 - 5 of 23

Similar Datasets

LC-MS/MS analysis of Mannose 6-phosphate containing proteins

Project description:Mucolipidosis III gamma (MLIII) is clinically characterized by onset of first symptoms at an average of 5 years such as stiffness of hands and shoulders, claw hand deformities, scoliosis and progressive destruction of hip joints. The disease is caused by mutations in GNPTG encoding the gamma-subunit of the GlcNAc-1-phosphotransferase complex. This enzyme is responsible for the generation of mannose 6-phosphate (M6P) targeting signals on 70 soluble lysosomal enzymes that are required for their efficient receptor-mediated transport to lysosomes. In fibroblasts from Gnptg-KO mice the GlcNAc-1-phosphotransferase activity is reduced leading to low amounts of M6P-containing proteins. M6P affinity chromatography-assisted and mass spectrometry-based secretome analysis of Gnptg-KO fibroblasts revealed a distinct set of lysosomal enzymes modified with M6P residues.

2018-06-06 | PXD007546 | Pride

LC-MS/MS analysis of soluble lysosomal enzymes

Project description:Mucolipidosis III gamma (MLIII) is clinically characterized by onset of first symptoms at an average of 5 years such as stiffness of hands and shoulders, claw hand deformities, scoliosis and progressive destruction of hip joints. The disease is caused by mutations in GNPTG encoding the gamma-subunit of the GlcNAc-1-phosphotransferase complex. This enzyme is responsible for the generation of mannose 6-phosphate (M6P) targeting signals on 70 soluble lysosomal enzymes that are required for their efficient receptor-mediated transport to lysosomes. Complementary SILAC-based lysosomal proteomics revealed decreased amounts of several lysosomal enzymes in Gnptg-KO fibroblasts involved in the degradation of lipids, glycans and proteins.

2018-06-07 | PXD007547 | Pride

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

Project description:Lysosomes are key degradative compartments of the cell. Transport to lysosomes ismediated by tagging soluble enzymes with mannose 6-phosphate (M6P) by GlcNAc-1-phosphotransferase whose deficiency leads to the severe lysosomal storage disorder mucolipidosisII (MLII). Several viruses require lysosomal cathepsins to cleave structural proteins and thusdepend on functional GlcNAc-1-phosphotransferase. Using genome-scale CRISPR screens, weidentify LYSET as essential for infection by cathepsin-dependent viruses including SARS-CoV-2. We show that LYSET deficiency results in global loss of M6P tagging and mislocalization ofGlcNAc-1-phosphotransferase to lysosomes. Lyset knockout mice exhibit MLII-like phenotypesand human pathogenic LYSET alleles fail to restore lysosomal sorting defects. Thus, we uncoverLYSET as an indispensable component of the M6P trafficking machinery and reveal thebiochemical basis of an inherited disease caused by mutations in LYSET.

2022-09-15 | PXD029609 | Pride

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.

2022-09-09 | PXD029746 | Pride

Gene expression in the femur hip joint articular cartilage of 12-week-old Gnptg-KO mice

Project description:Mucolipidosis type III is a rare disease caused by mutations in the GNPTG gene, encoding the gamma-subunit of GlcNAc-1-phosphotransferase. Since osteoarthritis and joint stiffness are typical symptoms of the disease, we aimed to investigate the role of the gamma-subunit on cartilage homeostasis. We used microarray analysis to compare the global gene expression in the articular cartilage of Gnptg-KO and wild-type mice.

2020-10-14 | GSE157180 | GEO

Targeting of non-phosphorylated lysosomal enzymes involves secretion and Lrp1-mediated recapture

Project description:Most lysosomal enzymes require mannose 6-phosphate (M6P) residues for efficient receptor-mediated lysosomal targeting. Although the lack of M6P results in missorting and hypersecretion, selected lysosomal enzymes reach normal levels in lysosomes of various cell types suggesting the existence of M6P-independent transport routes. SILAC-based analysis of purified lysosomes from M6P-deficient mouse fibroblasts (PTki) revealed unchanged amounts of 11 lysosomal enzymes, including cathepsin D and B (CtsD, CtsB), while demonstrating mossiorting of a variety of soluble lysosomal enzymes

2015-05-01 | PXD001221 | Pride

Multi-omic Analysis of an Mucolipidosis II Neuronal Cell Model Uncovers Involvement of Pathways Related to Neurodegeneration and Drug Metabolism

Project description:Defining the molecular consequences of lysosomal dysfunction in neuronal cell types remains an area of investigation that is needed to understand many underappreciated phenotypes associated with lysosomal disorders. Here we characterize GNPTAB-knockout DAOY medulloblastoma cells using different genetic and proteomic approaches, with a focus on how altered gene expression and cell surface abundance of glycoproteins may explain emerging neurological issues in individual with mucolipidosis (ML). The two knockout clones that were characterized demonstrated all the biochemical hallmarks of this disease, including loss of intracellular glycosidase activity due to impaired mannose 6-phosphate-dependent lysosomal sorting, lysosomal cholesterol accumulation, and increased markers of autophagic dysfunction. RNA sequencing identified altered transcript abundance of several neuronal markers and genes involved in drug metabolism and transport, and neurodegeneration-related pathways. Using selective exo-enzymatic labeling (SEEL) coupled with proteomics to profile cell surface glycoproteins, we demonstrated altered abundance of several glycoproteins in the knockout cells. Most striking was increased abundance of the amyloid precursor protein and apolipoprotein B, indicating that loss of GlcNAc-1-phosphotransferase function in these cells corresponds with elevation in proteins associated with neurodegeneration. The implication of these findings on lysosomal disease pathogenesis and the emerging neurological manifestations of GNPTAB-related disorders is discussed

2024-10-28 | GSE273794 | GEO

Targeted Quantification of the Lysosomal Proteome in Complex Samples

Project description:The lysosome, as the main degradative organelle of eukaryotic cells, is involved in numerous cellular processes. A defect in one of its proteins often results in lysosomal storage diseases (LSDs). For the study of lysosomal proteins, mass spectrometry (MS) has emerged as the method of choice. Lysosomal proteins are, however, low-abundant, restricting the analysis of lysosomal proteins in unbiased approaches to the investigation of lysosome-enriched fractions. The use of targeted MS provides an attractive alternative to analyze lysosomal proteins in a complex background. In this study, the two targeted MS approaches data-independent acquisition (DIA) and parallel reaction monitoring (PRM) were compared with regard to their ability to analyse lysosomal proteins. These experiments were conducted with samples of different complexity: low-complex lysosome-enriched fractions, medium-complex mouse embryonic fibroblasts (MEF), and high-complex liver whole tissue lysate. While both MS approaches were able to identify and quantify lysosomal proteins, PRM outperformed DIA, especially in high-complex samples.

2021-03-08 | PXD023278 | Pride

Presenilin deficiency results in cellular cholesterol accumulation by impairment of protein glycosylation and NPC1 function.

Project description:Presenilin (PS) proteins represent the catalytic subunits of γ-secretase, and play a critical role in the generation of the Amyloid β (Aβ) peptide and the pathogenesis of Alzheimer’s disease (AD). Indeed, mutations in presenilin 1 (PS1) or PS2 are a major cause of early onset familial Alzheimer’s disease (FAD). However, PS proteins also exert multiple functions beyond Aβ generation. In this study, we examine the individual roles of PS1 and PS2 in cellular cholesterol metabolism. Deletion of PS1 or PS2 in mouse models led to cholesterol accumulation in cerebral neurons. Cholesterol accumulation was also observed in lysosomes of embryonic fibroblasts from PS1-KO and PS2-KO mice, and associated with decreased expression of the Niemann-Pick type C1 (NPC1) protein involved in intracellular cholesterol transport at late endosome/lysosome. Mass spectrometry and complementary biochemical methods also revealed abnormal N-glycosylation of NPC1 and several other membrane proteins in PS1-KO and PS2-KO cells. Interestingly, pharmacological inhibition of N-glycosylation resulted in intracellular cholesterol accumulation prominently in lysosomes as well as decrease of NPC1 expression in wild-type MEFs, thereby resembling the changes in PS1-KO and PS2-KO MEFs. In turn, treatment of PS1-KO and PS2-KO MEFs with a chaperone inducer, Arimoclomol, attenuated decrease of NPC1 and rescued the lysosomal cholesterol accumulation. Additionally, the intracellular cholesterol accumulation in PS1-KO and PS2-KO MEFs was prevented by NPC1 overexpression. Collectively, these data indicate that PS/γ-secretase dysfunction results in an impairment of protein N-glycosylation, which eventually causes NPC1 decrease and intracellular cholesterol accumulation. 3'RNASeq of mouse embryonic fibroblasts with PS1- or PS2-deficiency compared with controls. Biological replicates

2024-05-26 | GSE223572 | GEO

Anp32e is a histone chaperone that removes H2A.Z from chromatin

Project description:We report a genome wide enrichment, redistribution and accumulation of H2A.Z at specific chromatin control regions, in particular at enhancers and insulators, in mouse embryonic fibroblasts depleted for Anp32e (MEFs Anp32e-/-). H2A.Z ChIP-seq in MEFs WT (+/+) or KO (-/-) for Anp32e.

2014-01-22 | E-GEOD-51579 | biostudies-arrayexpress