title: An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client.
abstract: Tandem affinity purification-mass spectrometry (TAP-MS) is a popular strategy for the identification of protein-protein interactions, characterization of protein complexes, and entire networks. Its employment in cellular settings best fitting the relevant physiology is limited by convenient expression vector systems. We developed an easy-to-handle, inducible, dually selectable retroviral expression vector allowing dose- and time-dependent control of bait proteins bearing the efficient streptavidin-hemagglutinin (SH)-tag at their N- or C termini. Concomitant expression of a reporter fluorophore allows to monitor bait-expressing cells by flow cytometry or microscopy and enables high-throughput phenotypic assays. We used the system to successfully characterize the interactome of the neuroblastoma RAS viral oncogene homolog (NRAS) Gly12Asp (G12D) mutant and exploited the advantage of reporter fluorophore expression by tracking cytokine-independent cell growth using flow cytometry. Moreover, we tested the feasibility of studying cytotoxicity-mediating proteins with the vector system on the cell death-inducing mixed lineage kinase domain-like protein (MLKL) Ser358Asp (S358D) mutant. Interaction proteomics analysis of MLKL Ser358Asp (S358D) identified heat shock protein 90 (HSP90) as a high-confidence interacting protein. Further phenotypic characterization established MLKL as a novel HSP90 client. In summary, this novel inducible expression system enables SH-tag-based interaction studies in the cell line proficient for the respective phenotypic or signaling context and constitutes a valuable tool for experimental approaches requiring inducible or traceable protein expression.
authors.affiliation: From the ‡CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria;
authors.forename: Johannes W
authors.initials: JW
authors.lastname: Bigenzahn
authors.affiliation: From the ‡CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria;
authors.forename: Astrid
authors.initials: A
authors.lastname: Fauster
authors.affiliation: From the ‡CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria;
authors.forename: Manuele
authors.initials: M
authors.lastname: Rebsamen
authors.affiliation: From the ‡CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria;
authors.forename: Richard K
authors.initials: RK
authors.lastname: Kandasamy
authors.affiliation: From the ‡CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria;
authors.forename: Stefania
authors.initials: S
authors.lastname: Scorzoni
authors.affiliation: From the ‡CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria;
authors.forename: Gregory I
authors.initials: GI
authors.lastname: Vladimer
authors.affiliation: From the ‡CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria;
authors.forename: André C
authors.initials: AC
authors.lastname: Müller
authors.affiliation: §Department of Biology, Institute of Mol. Syst. Biol., ETH Zürich, Zürich, Switzerland;
authors.forename: Matthias
authors.initials: M
authors.lastname: Gstaiger
authors.affiliation: ¶Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), 1030 Vienna, Austria;
authors.forename: Johannes
authors.initials: J
authors.lastname: Zuber
authors.affiliation: From the ‡CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria;
authors.forename: Keiryn L
authors.initials: KL
authors.lastname: Bennett
authors.affiliation: From the ‡CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria; ‖Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria gsuperti@cemm.oeaw.ac.at.
authors.forename: Giulio
authors.initials: G
authors.lastname: Superti-Furga
mesh_terms: D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D057809:HEK293 Cells; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D019073:HT29 Cells; D006801:Humans; D020014:K562 Cells; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009154:Mutation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D012190:Retroviridae / Q000235:genetics*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D018841:HSP90 Heat-Shock Proteins; D008565:Membrane Proteins; C570693:MLKL protein, human; D011494:Protein Kinases; D020558:GTP Phosphohydrolases; C579846:NRAS protein, human