2019 Fiscal Year Annual Research Report
Establishing a framework for EGFR attenuation through structure-guided targeting of GGA2
|Allocation Type||Multi-year Fund |
|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)
2019-04-01 – 2020-03-31
|Keywords||GGA2 / EGFR / Crystallography / Signal-disruption / Protein complex / FLIM / Confocal microscopy / Adaptor protein complex|
|Outline of Annual Research Achievements
The project goal is to elucidate the structure of GGA2 in complex with the jxt region of EGFR to design GGA2 inhibitors that interfere with cancer development in EGFR-dependent tumours.
I produced numerous constructs to evaluate the interaction between GGA2 and jxt for structural studies. I could not corroborate the established pull-down experiments. Therefore,I generated fluorescent protein fusions to produce complexes as Forster resonance energy transfer pairs to evaluate protein-protein interactions in vivo using fluorescence lifetime imaging microscopy. In spite of the fact that I generated almost 30 constructs to screen for binding interactions using many techniques I have been unable to obtain a GGA2-jxt complex. Therefore I decided to shift my project to the m-protein of the adaptor protein complex (APm) involved in EGFR trafficking and recycling.
For the structural investigations of APm involved in EGFR trafficking and recycling over 20 constructs were generated for expression in bacteria and mammalian cells. However, I was unable to obtain material for structural studies. Further experiments are needed, however this falls outside the project’s time limit.
A side project I contributed to, is the generation of a monoclonal antibody against GGA2. This antibody will be an invaluable tool in histological detection of GGA2 in tumours of patients with EGFR-dependent cancers. I overexpressed pure GGA2 peptide used to generate a GGA2-specific monoclonal antibody. I purified the antibody from hybridoma culture medium, which is currently being used to analyze patient material.
Research Products (1 results)