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DNA damage signaling studied by in-cell NMR: Structural and functional regulation of Mdm2 through its phosphorylation status. – Mdm2-StructReg

Mdm2-StructReg

DNA damage signaling studied by in-cell NMR: structural and functional regulation of Mdm2 through its phosphorylation status

Charaterization of Mdm2 structure and intramolecular interactions and of their regulation by phosphorylation events

We want to characterize Mdm2 structure in its different phosphorylation status. Mdm2 is a ubiquitin-ligase, which has the tumor suppressor p53 as a main target. Hence, inhibiting Mdm2 is an attractive anticancerous strategy. Mdm2 contains both folded and disordered regions, and its activity is regulated via phosphorylation events. This protein is very flexible and structural studies have thus to be performed in complex, crowded environments to be really conclusive. We want to depict the protein structure and its intra- and intermolecular interactions, depending on phosphoryaltion status and solvent conditions. This calls for important methodological developments in the field of high field NMR spectroscopy, and its applications in the characterization of post-translational modifications and of intrinsically disordered proteins. These studies will reveal new therapeutic targets against cancer.

- NMR as tool to study post-translational modifications
We develop protocols to make NMR an efficient tool in the study of post-translational modifications, and in particular those of intrinsically disordered proteins. These proteins carry often several modification sites, which are useful to integrate activities from different signaling pathways. NMR spectroscopy allows us to identify the modifications status of every residues.

- The disordered regions of Mdm2 have secondary structure propensities. Such a structural preorganization is often observed with disordered regions that are involved in protein-protein interactions. This would be consistent with our hypothesis on the importance of these regions for Mdm2 function. We have now to find which are these inteactions and whether they are intra- or intermolecular.
- We have developped a novel method to solve protein structures in cells from 2D NMR spectra. This is an important step in the field of in-cell structural biology. This is only the 2nd in-cell structure published in the litterature, and the 1st on since 2009. The method published 7 years ago was relying on the recording and analysis of 3D NMR spectra, which are very long to acquire and require high protein concentration (~1 mM). This was difficult to achieve, and thus such method has not bee reused since that time by the community. We designed paramagnetic tags that permit to solve the structure of a model protein at 50 uM in cells. This world has been published in July 2016 in the Journal of Physical Chemistry Letters (IF 8.5). We will use this method to characterize Mdm2 structure in complex, crowded environments.

- We want to map Mdmd2 phosphorylations sites established by important kinases
- We want to characterize the influence of crowded environments on Mdmd2 structural behavior
- We want to characterize Mdm2 intra- and intermolecular interactions
- We want to validate these interactions as therapeutic targets using peptides as inhibitors in cells

Since the beginning of the project (June 2016)
- 1 scientific paper linked to the project: Müntener, Haüssinger, Selenko, Theillet (2016) In-cell protein structure from 2D NMR experiments. J Phys Chem Lett, 7(14), 2821-2825.
- 6 scientific art

We want to characterize Mdm2 structure in its various phosphorylation states, and in complex with its main protein partners. The oncoprotein Mdm2, the main regulator of the tumor suppressor p53, contains both folded and large disordered regions, whose phosphorylation controls the protein's activity. Its structure in full-length, and after post-translational modification is unknown. This project will give us the opportunity to further develop methods that will be useful for this important class of proteins containing folded and disordered regions.
Indeed, about 30% of eukaryote proteomes code for proteins combining both folded and disordered regions. These proteins control cell-signaling mechanisms to a large extent. They are very often involved in cell pluripotency, cancers, or neurodegenerative diseases. A better description of their structural behavior is thus an important task for the near future, if we want to turn these flexible proteins into therapeutic targets. Importantly, such proteins are rarely investigated as entire proteins in structural studies. Their structure and control by post-translational modifications (PTMs) have been sparsely studied. Moreover, we do not know to which degree the intracellular environment influences their structural behavior. We want to bring decisive improvements in the field, both in experimental techniques and in fundamental knowledge. To this purpose, we will apply and further develop methods that I established over the last years for the characterization of PTMs by NMR and for in-cell structural biology.
The proposed project focuses on the description of the structure of Mdm2, a major therapeutic target against cancer. A number of phosphorylation events on its disordered regions control its activity. We will map these phosphorylation sites and monitor their kinetics. We will describe secondary, tertiary and quaternary structures of Mdm2 in its different phosphorylation status. In addition, we will study the influence of the intracellular environment on Mdm2 structure. Further, we will provide improved information on intermolecular contacts between Mdm2 and p53, using full-length, phosphorylated constructs. Finally, we will check the therapeutic interest of the newly identified phosphorylation sites and of intra- or intermolecular contacts.
By developing this project, our ambition is also to establish in French research facilities and further improve novel techniques using NMR spectroscopy for structural biology and biochemistry. Moreover, we will work in an international context, carrying on close collaborations with foreign laboratories that are leaders in their fields – lanthanides cages chemistry, EPR spectroscopy, fluorescence microscopy or systems biology modeling. We anticipate that our novel technical methods have the potential to be applied to every protein containing folded and disordered regions, and post-translational modifications sites. A number of these types of proteins are already studied in the proposed host facilities, the Laboratory for Structural Biology and Radiobiology (LBSR) and the future Institute for Integrative Biology of the Cell (I2BC) of Gif-sur-Yvette, which are thus ideal environments for our research.
Thanks to the evoked technical and cognitive improvements, we will make essential steps towards the understanding of the structural behavior of these flexible proteins. This work will turn intrinsically disordered proteins from objects of academic studies into biomedical targets.

Project coordinator

Monsieur Francois-Xavier Theillet (Institut de Biologie Intégrative de la Cellule (I2BC))

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

I2BC Institut de Biologie Intégrative de la Cellule (I2BC)

Help of the ANR 499,940 euros
Beginning and duration of the scientific project: May 2015 - 48 Months

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