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ZHENG Jie Group Revealed Assembly Program and Activation Mechanism of MDA5-MAVS Antiviral Signal Pathway

 

MDA5 is an allogeneic RNA monitoring protein in cells and an important member of RIG-I-like receptor family (RLRs). As an important barrier of natural immunity, it is mainly involved in regulating the immune response caused by RNA viruses. The RLRs family has three members: RIG-I, MDA5 and LGP2. The N-terminal of RIG-I and MDA5 have tandem CARDs domains, which can recruit MAVS through CARD-CARD homotypic interaction and finally promote the activation of type I interferon (IFN) pathway. During the activation of RLRs antiviral signal, K63-linked polyubiquitin chain (K63-polyUb) plays a key role in regulating RLRs signal pathway. However, how K63-polyUb regulates the assembly of MDA5CARDs and the molecular mechanism of recruiting and activating MAVSCARD remains a problem to be solved.

In a study published in Immunity lately, ZHENG Jie's group of Shanghai Institute of Materia Medica, Chinese Academy of Sciences revealed the molecular mechanism that long chain, unanchored K63-polyUb promotes MDA5-MAVS assembly program and signal transduction.

Firstly, the researchers established a biochemical synthesis platform for K63- , K48- linked ubiquitin chains, and prepared K63-polyUbn with different lengths. Then, through hydrogen deuterium exchange mass spectrometry (HDX-MS), researchers found that the oligomerization of MDA5CARDs and RIG-ICARDs depends on different lengths of k63 polyUbn (MDA5: n ≥ 8; RIG-I: n ≥ 3), but not K48-polyUbn (n ≥ 10).

In order to study the assembly mechanism of oligomers of MDA5CARDs mediated by K63-polyUbn, the structure of the complex of MDA5CARDs-K63 polyUb13 with a resolution of 3.3 Å was analyzed by Cryo-Electron Microscope (Cryo-EM) for the first time. The structure shows eight UB molecules surround a left-handed helical, cyclic CARDs tetramer. This is also the first Cryo-EM structure of MDA5CARDs with near atomic resolution. But how does the heterotetramer of MDA5CARDs-K63 polyUbn recruit its downstream signal protein MAVS? The researchers further obtained the "bottom-up" left-handed helical MDA5CARDs-MAVSCARD complex tethered by long chain K63-polyUb11 with a resolution of 3.2 Å by Cryo-EM analysis.

Moreover, the researchers proved for the first time that the CARDs of human MDA5 full-length protein are in an open conformation in the initial state and can bind to the long chain K63-polyUb10 through HDX-MS. However, in early studies, it was proved by HDX-MS that RIG-ICARDs has a closed conformation in the initial state. This also directly proved the great difference in the conformation of cards of RIG-I and MDA5 in solution. In addition, they found that the stability of K63-polyUb10 tethered MDA5CARDs complex in solution is regulated by the RNA dependent ATPase activity of MDA5, the effective recognition of RNA by MDA5 near mitochondrial membrane containing high concentration of ATP helps to remotely regulate the stability of K63 polyUb10-MDA5CARDs complex and help activate MAVS (Fig).


Figure. Signal transduction mechanism of full-length MDA5 under the action of its recognition ligand or substrate (dsRNA / ATP / K63-polyUb)

This study fills the gap in the research of MDA5 activation and signal transduction, reveals the immunological function of long-chain, non-anchored K63-polyUb as an endogenous agonist in cells, and provides a new clue for understanding the role of ubiquitin molecular diversity in anti-RNA virus natural immune signal transduction.

Links: https://doi.org/10.1016/j.immuni.2021.09.008