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Validation of a novel shotgun proteomic workflow for the discovery of protein-protein interactions: focus on ZNF521

ABSTRACT: The study of protein-protein interactions is increasingly relying on mass spectrometry (MS). The classical approach of separating immunoprecipitated proteins by SDS-PAGE followed by in-gel digestion is long and labour-intensive. Besides, it is difficult to integrate it with most quantitative MS-based workflows, except for stable isotopic labelling of amino acids in cell culture (SILAC). This work describes a fast, flexible and quantitative workflow for the discovery of novel proteinprotein interactions. A cleavable cross-linker, dithiobis[succinimidyl propionate] (DSP), is utilized to stabilize protein complexes before immunoprecipitation. Protein complex detachment from the antibody is achieved by limited proteolysis. Finally, protein quantitation is performed via 18O labelling. The workflow has been optimized concerning: (i) DSP concentration and (ii) incubation times for limited proteolysis, using the stem cell-associated transcription co-factor ZNF521 as a model target. The interaction of ZNF521 with the core components of the nuclear remodelling and histone deacetylase (NuRD) complex, already reported in the literature, was confirmed. Additionally, interactions with newly discovered molecular partners of potentially relevant functional role, such as ZNF423, Spt16, Spt5, were discovered and validated by Western blotting.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Professor Giovanni Cuda

PROVIDER: MSV000080723 | MassIVE | Wed Mar 29 01:09:00 BST 2017

SECONDARY ACCESSION(S): PXD001609

REPOSITORIES: MassIVE

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Validation of a novel shotgun proteomic workflow for the discovery of protein-protein interactions: focus on ZNF521.

Bernaudo Francesca F Monteleone Francesca F Mesuraca Maria M Krishnan Shibu S Chiarella Emanuela E Scicchitano Stefania S Cuda Giovanni G Morrone Giovanni G Bond Heather M HM Gaspari Marco M

Journal of proteome research 20150324 4

The study of protein-protein interactions is increasingly relying on mass spectrometry (MS). The classical approach of separating immunoprecipitated proteins by SDS-PAGE followed by in-gel digestion is long and labor-intensive. Besides, it is difficult to integrate it with most quantitative MS-based workflows, except for stable isotopic labeling of amino acids in cell culture (SILAC). This work describes a fast, flexible and quantitative workflow for the discovery of novel protein-protein intera ...[more]

PMID: 25774781

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Project description:Purpose: The exosome plays major roles in RNA processing and surveillance but the in vivo target range and substrate acquisition mechanisms remain unclear. We applied an in vivo cross-linking technique coupled with deep sequencing (CRAC) that captures transcriptome-wide interactions between individual yeast exosome subunits and their targets in a living cell. Methods: We apply CRAC to HTP-tagged proteins (HTP: His6 - TEV cleavage site - two copies of the z-domain of Protein A): Two nucleases (Rrp44, Rrp6) and two structural subunits (Rrp41, Csl4) of the yeast exosome. At least two independent experiments were performed in each case and analyzed separately. We performed CRAC on wild-type (WT) Rrp44 and two catalytic mutants, rrp44-endo (D91N, E120Q, D171N, D198N) and rrp44-exo (D551N). We further developed CRAC using cleavable proteins (split-CRAC) to compare endonuclease and exonuclease targets of Rrp44. Plasmids designed for split-CRAC contain a PreScission protease cleavage site (PP) inserted between aa 241 and 242 in the RRP44 ORF to allow in vitro cleavage of purified protein, and a His6 tag to select the respective cleaved fragment. Results: Analysis of wild-type Rrp44 and catalytic mutants showed that both the CUT and SUT classes of noncoding RNA, snoRNAs and, most prominently, pre-tRNAs and other Pol III transcripts are targeted for oligoadenylation and exosome degradation. Unspliced pre-mRNAs were also identified as targets for Rrp44 and Rrp6. CRAC performed using cleavable proteins (split-CRAC) revealed that Rrp44 endonuclease and exonuclease activities cooperate on most substrates. Mapping oligoadenylated reads suggests that the endonuclease activity may release stalled exosome substrates. Rrp6 was preferentially associated with structured targets, which frequently did not associate with the core exosome. This indicates that substrates can follow multiple pathways to the nucleases. Conclusion: Our study represents the first transcriptome-wide map of substrates for the yeast exosome nuclease complex. Identification of targets for individual exosome subunits in wild-type and mutant yeast cells.

Dataset Information

Validation of a novel shotgun proteomic workflow for the discovery of protein-protein interactions: focus on ZNF521

Publications

Validation of a novel shotgun proteomic workflow for the discovery of protein-protein interactions: focus on ZNF521.

Similar Datasets

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