2018 Fiscal Year Annual Research Report
Establishing a framework for EGFR attenuation through structure-guided targeting of GGA2
|Allocation Type||Single-year Grants |
|Research Institution||Fukushima Medical University |
BOKHOVE MARCEL 福島県立医科大学, 医学部, 博士研究員 (30825526)
|Project Period (FY)
2018-08-24 – 2020-03-31
|Keywords||GGA2 / EGFR / Crystallography / Signal-disruption / Protein complex / FLIM / Confocal microscopy|
|Outline of Annual Research Achievements
The goal of this project is to elucidate the structure of the GGA2 cargo domain in complex with the jxt region of EGFR to design GGA2 inhibitors to interfere with cancer development in EGFR-dependent tumors.
I have managed to construct all the necessary vectors to evaluate the interaction between GGA2 and jxt for structural studies. These constructs include GST-fusions of GGA2 VHS/VHS-GAT and several fused/unfused jxt constructs including wild-type, truncations and point mutants. These jxt constructs, used in pull downs, are essential for crystallographic studies because the length of jxt needs to be reduced from the current 80 amino acids to the minimally required binding site since large flexible regions inhibit crystal growth. Furthermore, a GGA2 hinge region construct was developed to produce peptides for competition experiments and the development of a GGA2-specific monoclonal antibody for histology. Using these constructs I managed to produce large quantities of pure and stable VHS/VHS-GAT and jxt.
GGA2-hinge was sent to a company who are currently generating monoclonal antibodies. The GGA2-, and jxt-domain constructs are being used in pull-downs to pinpoint the region of jxt recognized by GGA2. However, within the confines of the in vitro conditions needed for crystallography I am having issues replicating the pull-down experiments. Therefore, I have developed constructs to produce binding-protein/ligand-complexes as FRET (Forster resonance energy transfer) pairs to to evaluate protein-protein interactions in vivo using FLIM (fluorescence lifetime imaging microscopy).
|Current Status of Research Progress
Current Status of Research Progress
4: Progress in research has been delayed.
Currently the project concerning structural investigations into the complex between the GGA2 cargo-binding domain and jxt is halted. Within the confines of a crystallographic project, wherein the ultimate goal is to obtain the structure of a protein complex, I cannot corroborate that these two entities bind.
Therefore, I have been studying the binding between jxt and GGA2 using FLIM inside living cells. A disadvantage of this technique is that it cannot be used directly in my endeavors towards structural studies of the GGA2-jxt complex. However, this technique can be used to study the interaction between these two proteins in an environment closely resembling the in vivo situation including molecular crowding, oxidative pressure and the presence of protein and non-protein cofactors. Results of such experiments, including mutants and truncations, could be extrapolated to the constructs employed in crystallography. As a positive control I will utilize the original GGA2 ligand, which is the C-terminal acid dileucine motif of the cation independent mannose-6-phosphate receptor (CIMPR).
Surprisingly, while the CIMPR experiments performed perfectly, I could not observe binding between GGA2 and jxt neither in C-, nor N-terminal fluorescence protein fusions of jxt. These data do not dismiss that GGA2 and jxt interact, but could rather imply that the interaction between GGA2 and jxt is weak or transient. Such interactions are hard to study using some techniques and may not be the ideal target for structure-based drug design without additional knowledge on interacting partners.
|Strategy for Future Research Activity
Given these caveats I am compelled to shift my focus towards another protein involved in EGFR trafficking and recycling. This project, when successful, will have an impact comparable to the originally intended project and it is still achievable within the time limit.
I would like to focus on the cargo recognition region of one of the adaptor protein complexes. This protein is of interest because there is evidence that supports its involvement in EGFR trafficking and recycling. Furthermore, sequence alignments have revealed that this region has a ligand-binding region unique among its homologues, indicating that it has a markedly different ligand specificity. The different binding site would allow us, after elucidation of its crystal structure in complex with its ligand, to design small molecule inhibitors that are highly specific. I am currently in the process of developing expression constructs that are compatible with structural studies.