Dataset Information

Analysis of intact mannose-6-phosphate glycopeptides

ABSTRACT: We have repurposed standard phosphoproteomics workflow based on Fe3+IMAC for enrichment ofmannose-6-phosphate modified glycopeptides. This worklfow was used to profile lysosomal acid hydrolases in HeLa and CHO cell lines. We have combined this approach with CRISPR/Cas9 KO of acid phosphatases 2 and 5 responsible for dephosphorylation of mannose-6-phosphate glycopeptides in the lysosome. This combined approach enabled significantly deeper coverage of CHO mannose-6-phosphate glycoproteome.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human) Cricetulus Griseus (chinese Hamster) (cricetulus Barabensis Griseus)

TISSUE(S): Cell Suspension Culture, Hela Cell

SUBMITTER: TOMISLAV CAVAL

LAB HEAD: Albert J R Heck

PROVIDER: PXD010333 | Pride | 2018-12-18

REPOSITORIES: Pride

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Publications

Targeted Analysis of Lysosomal Directed Proteins and Their Sites of Mannose-6-phosphate Modification.

Čaval Tomislav T Zhu Jing J Tian Weihua W Remmelzwaal Sanne S Yang Zhang Z Clausen Henrik H Heck Albert J R AJR

Molecular & cellular proteomics : MCP 20180920 1

Mannose-6-phosphate (M6P) is a distinctive post-translational modification critical for trafficking of lysosomal acid hydrolases into the lysosome. Improper trafficking into the lysosome, and/or lack of certain hydrolases, results in a toxic accumulation of their substrates within the lysosomes. To gain insight into the enzymes destined to the lysosome these glycoproteins can be distinctively enriched and studied using their unique M6P tag. Here we demonstrate, by adapting a protocol optimized f ...[more]

PMID: 30237200

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Project description:The mitogen-activated protein kinase (MAPK) pathway is one of the most altered pathways in cancer. It is involved in the control of cell proliferation, invasion, metabolism, and resistance to therapy. A number of aggressive malignancies, including melanoma, colon cancer and glioma, are driven by an activating missense mutation (V600E) in one component of the pathway, BRAF. BRAF V600E mutated cancers may respond initially to MEK inhibition, but may develop resistance mediated by increased reliance on mTOR signaling. We have previously demonstrated that the combination of the MEK inhibitor trametinib with the dual mTORC1/2 inhibitor TAK228 improved survival and decreased vascularization in a BRAFV600E mutant glioma model. To elucidate the mechanism of action of, and the changes in response to, MEK and mTOR inhibition, we performed comprehensive unbiased proteomic and phosphoproteomic characterization of BRAFV600E mutant glioma xenografts after short-course treatment with trametinib and TAK228, alone and in combination. We identified distinct response signatures for each monotherapy and combination therapy and validated that combination treatment inhibited activation of the MAPK and mTOR pathways, increased apoptotic signaling and suppressed angiogenesis signaling. Furthermore, we found that trametinib and TAK228 combination treatment broadly suppressed the activity of the cyclin-dependent kinases and increased the levels of proteins (and their activating phosphorylations) involved in glycolysis, the TCA cycle, nucleotide biosynthesis and DNA replication. We also demonstrated activation of both receptor tyrosine kinase and histone deacetylase proteins. This study reports a detailed (phospho)proteomic analysis of the response of BRAFV600E mutant glioma to combined MEK and mTOR pathway inhibition and identifies a number of targetable upregulated proteins and pathways, providing new avenues for the development of additional rational combination therapies for aggressive BRAF-driven tumors.

Dataset Information

Analysis of intact mannose-6-phosphate glycopeptides

Publications

Targeted Analysis of Lysosomal Directed Proteins and Their Sites of Mannose-6-phosphate Modification.

Similar Datasets

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