Identification of Novel ER-Resident Quality Control Players by “Interactomics”
Research area: Protein Folding and Quality Control
Group leaders: Maurizio Molinari
Status: In progress
The aim of the project is to identify new players that intervene in protein folding, quality control and ERAD in the mammalian ER lumen. We will prepare a series of stable human cell lines expressing model, epitope-tagged, folding-competent and folding-defective proteins. The model proteins are used as baits to capture interacting partners in the same immuno-complexes. Preliminary experiments (Figure) have been performed to compare the interacting partners of BACE501, a secretory protein, with the interacting partners of BACE476, a folding-defective deletion mutant of BACE501. The proteins co-immuno-isolated with the individual baits have been subjected to tryptic digestion and fragments have been separated by nano-HPLC followed by tandem MS. Fragmentation spectra of the samples have been matched to a human protein database sequence with the Mascot software. These analytic steps are performed in collaboration with Manfredo Quadroni, Coordinator of the Center for Integrative Genomics, University of Lausanne. Involvement of the model protein’s interacting partners in protein quality control will be validated upon their downregulation by specific RNA interference.
We will also determine how variations in bio-physical features of the model proteins (e.g. protein topology, glycosylation state, oxidation state, conformation of peptidyl-prolyl bonds) affect the molecular composition of the model-protein’s interactome.
Figure: Isolation of immuno-complexes containing the folding-competent BACE501 (green arrowheads) or the folding-defective BACE476 (red arrowheads). M is the immuno-isolate of Mock-transfected HEK293 cells, 501 and 476 are the immuno-isolates of HEK293 cells stably expressing BACE501 and BACE476, respectively. Ig is the BACE immunoglobulin chain used for the immunoprecipitation. Co-precipitating polypeptides are identified as described in the text.