MHC-I biogenesis and degradation at the endoplasmic reticulum membrane

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

Reporting period: 2019-02-01 to 2021-01-31

Summary of the context and overall objectives of the project

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.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

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.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

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