Functional Role Of Molecular Disorder In Androgen Receptor
Reference : PhD Bruno KIEFFER
Offer publication : April 6, 2016
The androgen receptor (AR) plays an important role in the progression of prostate cancer. It is so far the only clearly identified therapeutic target for advanced forms of this cancer. All the molecules used or developed to fight against prostate cancer are targeted at the hormone binding domain (LBD) of the receptor, the 3D structure of which beeing well characterized by X-ray crystallography. It is believed that long-term
effectiveness of these molecules will face the same treatment failure problems because the molecular target (the LBD) is identical.
Our project is to explore the possibility of designing new classes of androgen receptor inhibitors able to interfere with the AF-1 activation function. Given the location of this function in a disordered region of the AR receptor at its N-terminal side (the NTD domain), there is currently no information on the molecular
mechanisms of this AF-1 function, nor on the interactions between the ligands and the AR NTD. We will use an experimental approach that integrates protein NMR methods with those of synthetic biology for the development of mutant receptors and their characterization in vitro and in vivo. NMR is the only method currently capable of describing disordered protein domains with atomic resolution, offering the opportunity to study the interactions between ligands specific domain NTD. The similarity between the molecular structures of endocrine disruptors (such as bisphenol A) and NTD ligands suggests a common mechanism of interaction that we propose to explore using several biophysical methods (NMR, mass spectrometry, fluorescence, thermophérèse ...).
Preliminary results obtained by our team identified a NTD region involved in the formation of amyloid fibers suggestive of
supramolecular organization of the receiver. The functional role of these peculiar forms of the receptor will also be studied in connection with their ligand-binding properties.
This project involves the development and the use of innovative methods to address such issues, such as Fluorine-19 NMR
or synthetic biology approaches. The knowledge gained from this work will be particularly useful to guide the development of new classes of molecules for the treatment of prostate cancer.
The student will focus on the following questions:
- Are there any parts of the AR NTD domain involved in the modulation of the formation of amyloid fibers? In particular, it will focus on poly-glutamine sequences whose length has been shown to be an important factor in the formation of fibers.
- Does the oligomerization of AR occur within the cell? Is it possible to observe this phenomenon using in-cell NMR methods ?
The project will start with the cloning of different fragments of the AR receptor, containing different parts of the NTD domain (528 aa). The proteins will be expressed and their ability to form fibers and the oligomerization rate will be measured by light scattering and NMR. These measurements will then be adapted to be performed in 96-well plate in-order to allow the high througput screening for interactions with molecules capable of interacting with AR oligomerization. Finally, synthetic receptors will be designed from simpler NTD domains that retain their ability to self-assemble in a controlled manner. The properties of these receptors will be analyzed using luciferase gene expression assays, a technique mastered by our collaborator:
Jocelyn Céraline. We will then design methods enabling the observation of receptor oligomerization in a cellular context using fluorine NMR 19. Fluorinated amino acids such as fluoro-tryptophan-fluoro will be introduced in the NTD sequence. This labelilng will allow us to follow the receptor oligomerization in complex mixtures such as cell extracts.
- WISHED SKILLS :
- Molecular Biology
- Molecular Biophysics
- EXPERTISES WHICH WILL BE ACQUIRED DURING THE TRAINING :
- Biomolecular NMR
- Expression of isotopically labeled proteins
- Data analysis using modern tools
Application Deadline : Dec. 31, 2016