CryoEM studies and functional characterization of the regulation mechanism of the disease-related human flippases ATP8B1 and ATP8B4

  Flippases (or P4-ATPases) are eukaryotic membrane proteins capable of transporting phospholipids from the exoplasmic leaflet to the cytosolic leaflet of membranes using energy from ATP hydrolysis. This lipid transfer is crucial for maintaining membrane lipid asymmetry, which is essential for cellular processes such as membrane trafficking, signal transduction, and resistance to mechanical and chemical stress. The human genome encodes 14 P4-ATPases, which form heterodimers with a chaperone protein, CDC50A. Dysfunction of these flippases is linked to genetic diseases, including progressive familial intrahepatic cholestasis (ATP8B1 mutations) and Alzheimer’s disease (ATP8B4 mutations).

   Despite their importance, the precise molecular mechanisms regulating flippase activation and their roles in disease remain poorly understood. Previous work has revealed key insights into ATP8B1 regulation, including its auto-inhibition mechanism mediated by the N- and C-terminal regions and the role of post-translational modifications in releasing this inhibition. ATP8B4 shares significant structural similarities with ATP8B1, suggesting analogous regulatory mechanisms.

  This PhD project aims to identify the post-translational modifications and protein partners regulating ATP8B1 and ATP8B4 activity and to structurally and functionally characterize these interactions, providing insights into their roles in health and disease contexts. The PhD student will work at the interface of cell biology, biochemistry, and structural biology and will be involved in:

-  Identifying post-translational modifications and protein partners involved in ATP8B1 and ATP8B4 activation using proteomics and phosphoproteomics approaches.

 - Structurally characterizing the ATP8B1/CDC50A and ATP8B4/CDC50A complexes and their interactions with partners using cryo-electron microscopy (cryo-EM).

-  Functionally investigating the enzymatic activity and substrate specificity of these flippases in relation to their activation mechanisms.

The PhD project will be conducted in the Laboratory of Membrane Proteins and Membrane Systems (LPSM) under the supervision of Thibaud Dieudonné. The LPSM is part of the Biochemistry, Biophysics, and Structural Biology Department (B3S) at the Institute for Integrative Biology of the Cell (I2BC). The I2BC is located in Gif-sur-Yvette, in the southern Paris area.

The laboratory is currently equipped with all the necessary tools for carrying out the proposed thesis, and they will be made available to the student. The project will benefit from the team's access to the institute's mass spectrometry and cryo-EM (200 kV Glacios) platforms. For structural studies, the student will also be able to take advantage of the proximity of the 300 kV Titan Krios microscope at the SOLEIL synchrotron.

Candidates with experience in protein biochemistry and/or structural biology is expected, previous experience with membrane proteins would be a plus, although not mandatory. Good English skills are expected.