Periodic Reporting for period 1 - MemCHAPS (Intramembrane chaperones : their role in folding membrane proteins)
Reporting period: 2019-02-01 to 2021-01-31
Misassembled or misfolded transmembrane proteins can have detrimental effects on the functioning of the cell. Destabilizing mutations in transmembrane helices that cause misassembly have been associated with serious diseases like cystic fibrosis and diabetes mellitus, emphasizing that intramembrane quality control is indispensable. Various proteins interacting with the membrane protein during these stages could serve as chaperones to ensure folding to its functional state. Taken together, this makes it imperative to understand the open question of how multi-spanning membrane proteins are chaperoned in the ER membrane
To address the vast field of intramembrane chaperoning, we defined two overall objectives of the project:
1. Identify membrane proteins interacting with ABC transporters in the ER.
2. Determine whether these interactions play a role in folding/assembly of the ABC transporters
In this reporting period we have successfully established the work-flow of the proximity labelling approach required for mass spectrometry. Since the screen was not completed, we initiated the investigation of two prospective intramembrane chaperone candidates namely Bap31 and the endoplasmic reticulum membrane protein complex (EMC). Our results show that both Bap31 and the EMC influence the folding/assembly of CFTR. Significant changes in the folding pattern are observed in the transmembrane region of CFTR, suggesting that both Bap31 and the EMC might directly be involved in the intramembrane chaperoning of CFTR.
The cystic fibrosis transmembrane conductance regulator (CFTR) was selected as a model protein, as the Braakman lab has a lot of expertise and a vast wealth of reagents and constructs to study its folding dynamics.
The method of choice was proximity-dependent biotin identification (BioID). The principle of this technique is that CFTR is genetically fused to a prokaryotic biotin ligase molecule (BirA). When expressed in HEK293T cells, the proteins in proximity of the fusion ABC transporter are biotinylated. Streptavidin-based biotin affinity capture can then allow to specifically isolate the biotinylated proteins from the cell lysate. The biotinylated proteins purified in this way can be identified using mass-spectrometry (MS) to give a list of interactors. The MS collaboration has been set up with Dr. J. Demmers in the Erasmus Medical Centre of Rotterdam.
Main results achieved:
1. Tagging CFTR at the N-terminal with BioID led to aggregation.
2. CFTR could be tagged with BioID2 at the C-terminus and within the ECL4 loop.
3. BioID2-CFTR folds and assembles similar to untagged wild type CFTR and is stably expressed at the cell surface.
4. Successful optimization of pull down of Biotinylated proteins
5. Proof-of-principle MS test run completed
6. Generation of stable BioID2 cell lines complete
7. Bap31 and the EMC influence the folding/assembly of CFTR. Significant changes in the folding pattern were observed in the transmembrane region of CFTR, suggesting that both Bap31 and the EMC might directly be involved in the intramembrane chaperoning of CFTR.
Exploitation and dissemination of results:
The knowledge generated through this work was disseminated to the scientific community, future scientists and students using different means and channels:
1. Poster presentation at the Dutch Chaperone Day
2. Poster presentation to UU Master students at the MCLS Networking meet
3. Supervision of a Master’s thesis
4. Scientific discussions at the EMBO leadership course: Project management for Scientist
Moreover, the stable cell lines generated are a valuable resource which will continue to be used in the Braakman group.
After extensive optimizations and troubleshooting, the BioID2 screen is now ready for implementation and we expect to identify novel CFTR interactors. The preliminary results obtained with Bap31 and the EMC are promising leads and will be expanded further in the Braakman group. This research aims at answering a fundamental question in cell biology pertaining to how the folding of ~ 30 % of the proteome is assisted under physiological conditions. The findings of this research could have highly relevant implications for industry in terms of the modulating the assembly of CFTR or designing strategies to inhibit the other similar complex ABC transporters like P-gp and BCRP that cause chemotherapeutic drug resistance.