MHC-I biogenesis and degradation at the endoplasmic reticulum membrane

Informations projet

MHCIbiopic

N° de convention de subvention: 792575

DOI

10.3030/792575

Projet clôturé

Date de signature de la CE 15 Mars 2018

Date de début 1 Février 2019

Date de fin 31 Janvier 2021

Financé au titre de

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Coût total

€ 165 598,80

Contribution de l’UE

€ 165 598,80

Coordonné par

UNIVERSITEIT UTRECHT
Netherlands

Periodic Reporting for period 1 - MHCIbiopic (MHC-I biogenesis and degradation at the endoplasmic reticulum membrane)

Période du rapport: 2019-02-01 au 2021-01-31

Résumé du contexte et des objectifs généraux du projet

One third of the proteins of a human cell are synthetized and degraded at the membrane of a cellular compartment called the Endoplamsic Reticulum (ER). Dysfunction of these processes generate a stress on this compartment which is prevalent in diseases like Parkinson and Alzheimer diseases, diabetes, liver diseases and some cancers. However, because these processes occur at a membrane, they are particularly tedious to reconstitute and study in vitro and we currently lack detailed structural and mechanistic understanding of these crucial events.
The objective of this project was to use a microscopy technique called Cryo Electron Tomography (CET) on isolated ER to image some of these ER processes and provide new molecular details on their mechanism.

Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent

In this work we developped methods to isolate functional ER from different cell sources. We then established protocols to recapitulate in vitro the synthesis and translocation of cellular proteins into these isolated ER. We collected CET data on those and the processing revealed a low efficiency of the system and a high heterogeneity of the complexes to study, thus precluding a high resolution CET study. In a second part of the project we used specialized viral molecules to trigger the ER degradation of an immune molecule called MHC-I. We worked on the purification of the degradation complexes formed and their biochemical characterization for later high resolution single particle Cryo-EM studies.

Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent)

The cryoEM skills acquired during this work led us to contribute to a COVID19 project in collaboration with the virology group at Utrecht University. We used single particle cryoEM to elucidate the structure of the complex formed by a SARS-CoV and SARS-CoV-2 cross-neutralizing antibody called 47D11 and the corresponding spike proteins. We determined the binding mode of this antibody, highlighting a vulnerability patch on the spike Receptor Binding Domain and providing a structural roadmap for the development of this antibody as a prophylactic or post-exposure therapy for COVID-19.

slice through a tomogram of ER derived microsomes and corresponding subtomogram average