M2 internship in proteomics at the Institute of Pharmacology and Structural Biology in Toulouse

Posted 23 Aug 2022

Institut de Pharmacologie et de Biologie Structurale (https://www.ipbs.fr/)

France (Student)

https://proteotoul.ipbs.fr/proteotoul-research/reg...


Title of the project: Exploring the interactome and composition of variants of proteasome complexes involved in NeuroDevelopmental Disorders using proteomics and MS approaches

Supervision: Marie-Pierre Bousquet (MCF UPS) / marie-pierre.bousquet@ipbs.fr & Julien Marcoux (CR CNRS) / julien.marcoux@ipbs.fr & Angelique Dafun (PhD student) / angelique.dafun@ipbs.fr Team: «Proteomics and Biological Mass Spectrometry», IPBS CNRS UMR 5089, 205 route de Narbonne, Toulouse.

Background Neurodevelopmental disorders (NDDs) are a major public health concern affecting 3-10% of children worldwide [1]. 10-15% of the genes involved in NDDs encode components of the ubiquitin-proteasome system (UPS) [2]. This pathway is essential for the proteostatic equilibrium of eukaryotic cells. It ensures the specific degradation of denatured proteins that have become non-functional or even toxic. The UPS has been shown essential to neuronal development and function. The recognition of UPS-related NDDs (UPS-NDDs) is fairly recent, and, although about 1,200 genes contribute to the UPS pathway, amongst which many are highly expressed in the brain, only a limited number of UPS-related NDDs (UPS-NDDs) have been identified to date. 150 pathogenic or likely pathogenic variants are associated with NDD in 17 UPS genes and of which 8 genes are proteasomal subunits. Biological samples from 67 families from child and unaffected parents, have been collected by our collaborators at the CHU of Nantes, resulting in a biobank of 140 individuals. Preliminary functional studies from affected individuals T cells showed impaired proteolytic activity and/or assembly of proteasome 26S caused by PSMD12, PSMC3, PSMC5, PSMB3 or PSMB5 variants.

The main objective of this project is to study the consequences of the mutations in NDD-UPS genes on proteasome complexes composition (20S core subunits as well as 20S-associated regulators), proteasome assembly, but also on the proteasome interactome, as these three levels of regulation are known to affect proteasome cellular activity both in physiological and pathological situations [3]. The experiments will involve:  The biochemical purification of endogenous 20S proteasome [4],  The functional characterization of purified proteasome from mutant T cells and their controls,  Their analysis using bottom-up proteomics and top-down proteomics using already established methods [4].

References [1] Ebstein et al. MedRxiv. (2021). [2] Alvarez-Castelao et al. (2015). [3] Coux O. et al. (2020) Adv. Exp. Med. Biol. [4] Fabre et al. (2015) Mol. Sys. Biol. ; Zivkovic D. et al (2022) PNAS.

Techniques used during the internship:

  • Protein analysis (SDS-PAGE, Western Blot), Protein complexes purification (immunopurification) and biochemical enrichment (Size Exclusion Chromatography),
  • Proteasomal enzymatic activity measurement (Fluorescence-based kinetic assay),
  • Bottom-up and top-down mass spectrometry analysis (nanoHPLC ESI-MS/MS on different types of Orbitrap mass spectrometers),
  • Mass spectrometry-based differential quantification and statistical analysis.

Perks

Join our highly dynamic group and work on various proteomic and structural MS workflows using modern MS instruments. Visit our website for more information: https://proteotoul.ipbs.fr/

How to Apply

Send a CV and a motivation letter to Marie-Pierre Bousquet (marie-pierre.bousquet@ipbs.fr) & Julien Marcoux ((julien.marcoux@ipbs.fr)