Investigating the essential roles of Ubiquitin-like molecules and the Ubiquitin Proteasome System

Research Project 11.
The role and regulation of the proteasome regulator PA28γ


To get better insight into PA28γ regulation, the primary goal of the project will be to explore the role(s) and the structure/function relationship of a poorly characterized protein recently discovered by S. Boulon (now in our team at the CRBM) and A. Lamond, that strongly interacts with PA28γ and was identified by mass spectrometry after PA28γ pull-down assays. This protein, that we renamed Pip30 (for PA28γ interacting protein), is of completely unknown function. Based on its interaction with PA28γ and on preliminary data showing that PIP30 and PA28γ have opposite effects on Cajal Bodies dynamics, our working hypotheses are that Pip30 could either be a negative regulator of PA28γ binding/activation of the 20S proteasome, or a protein involved in the control of the precise intranuclear localization of PA28γ (either free, or bound to the 20S proteasome). Our primary goal is to characterize the structure of both Pip30 and PIP30/PA28γ complex, as this information could greatly help understand the function and mechanism of action of Pip30. The structural analyses will be performed in close collaboration with Dr. Echalier (CBS Montpellier), associated partner within this project.

As part of this project, we will also develop cell lines stably expressing fluorescently labeled 20S proteasome subunits, PA28γ and/or Pip30, to visualize 20S / PA28γ or PA28γ / Pip30 interactions in cells by FRET/FLIM. This approach will allow us to analyze in details the role of Pip30 in the control of the nuclear localization of PA28γ and/or 20S/PA28γcomplexes. Furthermore, to better understand the role of PA28γ in nuclear dynamics, and knowing that the ubiquitin-like molecules Sumo and Nedd8 are often used to control highly dynamic processes, we will also investigate, in close collaboration with D. Xirodimas, the potential role of PA28γ on these post-translational modifications.