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Analysis of the GFP-labelled β-dystroglycan interactome in Hek-293 transfected cells reveals novel intracellular networks

ABSTRACT: So far very little is known about the fine regulation of some of these intracellular interactions of β-DG and how they are perturbed in diseases. To start filling this gap, HEK-293 cells were transiently transfected with a plasmid carrying the β-DG subunit with GFP fused at its C-terminus. Immunoprecipitation by anti-GFP antibodies followed by shotgun proteomic analysis to investigate the proteins exclusively matching for β-DG binding, applying the following filters to MS identification data: high confidence, peptide rank 1, peptide length minimum 9 amino acid residues and selecting 2 peptides per protein. A series of already known β-DG interactors have been found, whilst significant new matches, which include potential novel β-DG interactors and their related networks, were identified in diverse subcellular compartments.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Claudia Desiderio

LAB HEAD: Claudia Desiderio, Andrea Brancaccio, Francesca Sciandra

PROVIDER: PXD043909 | Pride | 2024-02-12

REPOSITORIES: Pride

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			Action	DRS
	BDG_GFPIP_IIIrun1_04042022.mgf	Mgf
	BDG_GFPIP_IIIrun1_04042022.msf	Msf
	BDG_GFPIP_IIIrun1_04042022.raw	Raw
	BDG_GFPIP_IIIrun2_04042022.mgf	Mgf
	BDG_GFPIP_IIIrun2_04042022.msf	Msf

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Project description:Although therapy responsiveness to therapy in Burkitt lymphoma (BL) is high, relapsed disease and and chemoresistance remain a clinical challenge, and complete mechanisms underlying BL chemoresistance and how it can be circumvented is yet to be fully elucidated. In this study we present data showing that chymotrypsin-like serine proteases inhibitor Nα-tosyl-L-phenylalanine chloromethylketone (TPCK) and specific NF-κB inhibitor Bay-11 7082 can induce caspase-independent apoptosis in chemoresistant BL cells. We also demonstrate that both TPCK and Bay-11 7082-treatment leads to decreased NF-κB nuclear activity and that this is associated with sensitization of chemoresistant Burkitt lymphoma cells. Furthermore we investigated global transcriptional changes induced by Bay-11 7082 and TPCK in the DG-75 and Raji cell lines, respectively, by microarray analysis using Illumina BeadChips. TPCK-treatment of Raji and DG-75 cells resulted in 59 and 21 differently expressed genes, respectively, while Bay-11-treated Raji and DG-75 cells displayed 1403 and 8 differently expressed genes, respectively. Gene Ontology (GO) categorization confirmed enrichment of multiple GOs in Bay 11-treated Raji and DG-75 cells. Fifty percent of the 61 categories in Raji cells were categories sorting under Biological Processes and represented mostly increased gene expression. In DG-75 cells Bay-11 7082 induced significant gene ontology enrichment in only two categories, where the increased/decreased ratio was 1:1. Further unsupervised and supervised bioinformatics processing by Ingenuity Pathway Analysis indicated significant networks in response to TPCK and Bay 11 respectively, including association to NF-κB. Bay-11 7082 demonstrated deregulated NF-κB related members of receptor mediated cell death signaling, i.e TRAF2 and TRADD, as well as deregulated members of the NF-κB signaling pathway from the cytoplasmic compartment, i.e RELB, in Raji cells. Comparably NF-κB network analysis of Raji- and DG-75 cells treated with Bay-11 7082 and Raji cells treated with TPCK demonstrated deregulation of NF-κB target genes CD69 and IL8. These data indicates that NF-kB may play a role in overcoming chemoresistance in BL cells with defective classical apoptosis signaling. NF-κB network analysis of Raji- and DG-75 cells treated with Bay-11 7082 and Raji cells treated with TPCK

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Analysis of the GFP-labelled β-dystroglycan interactome in Hek-293 transfected cells reveals novel intracellular networks

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