Ordered assembly of the cytosolic RNA-sensing MDA5-MAVS signaling complex via binding to unanchored K63-linked poly-ubiquitin chains

Bin, Song; Chen, Yun; Liu, Xin; Yuan, Fei; Tan, Eddie Yong Jun; Lei, Yixuan; Song, Ning; Han, Yinqi; Pascal, Bruce D.; Griffin, Patrick R.; Luo, Cheng; Wu, Bin; Luo, Dahai; Zheng, Jie

doi:10.1016/j.immuni.2021.09.008

Cited by 38 publications

(25 citation statements)

References 59 publications

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“…However, there is no reported HEL2i mutation associated with MDA5 that could lead to these autoimmune diseases. This could be due to the fact that MDA5 lacks HEL2i-CARD2 interface and adopts an open CARDs conformation in solutions ( 21 ). As a result, the HEL2i domain of MDA5 plays a distinctive role in recognizing self RNA and placing CARDs activation as compared with RIG-I.…”

Section: Discussionmentioning

confidence: 99%

“…Lys63-linked ubiquitin chains (K63-polyUb n ) were generated from a reaction containing 1 mM ubiquitin, 1 μM mE1, 10 μM UbcH13 and 10 μM Uev1A in buffer (10 mM ATP, 20 mM PC, 1.2 U/ml IPP, 1.2 U/ml CPK, 50 mM Tris pH 7.5, 10 mM MgCl 2 , 2 mM DTT). The K63-polyUb n synthesis reaction was performed at 37°C for 3–6 h. Synthesized polyUb n chains were purified as described previously ( 21 ). Briefly, ubiquitin chains were diluted 5-fold into 50 mM ammonium acetate, pH 4.5, and 0.1 M NaCl and separated over a 0.1–1.0 M NaCl gradient in 50 mM ammonium acetate, pH 4.5, using a HiTrap TM Heparin HP column (GE Healthcare).…”

Section: Methodsmentioning

confidence: 99%

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A loosened gating mechanism of RIG-I leads to autoimmune disorders

Luo

Zheng

2022

Nucleic Acids Research

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DDX58 encodes RIG-I, a cytosolic RNA sensor that ensures immune surveillance of nonself RNAs. Individuals with RIG-IE510V and RIG-IQ517H mutations have increased susceptibility to Singleton-Merten syndrome (SMS) defects, resulting in tissue-specific (mild) and classic (severe) phenotypes. The coupling between RNA recognition and conformational changes is central to RIG-I RNA proofreading, but the molecular determinants leading to dissociated disease phenotypes remain unknown. Herein, we employed hydrogen/deuterium exchange mass spectrometry (HDX-MS) and single molecule magnetic tweezers (MT) to precisely examine how subtle conformational changes in the helicase insertion domain (HEL2i) promote impaired ATPase and erroneous RNA proofreading activities. We showed that the mutations cause a loosened latch-gate engagement in apo RIG-I, which in turn gradually dampens its self RNA (Cap2 moiety:m7G cap and N1-2-2′-O-methylation RNA) proofreading ability, leading to increased immunopathy. These results reveal HEL2i as a unique checkpoint directing two specialized functions, i.e. stabilizing the CARD2-HEL2i interface and gating the helicase from incoming self RNAs; thus, these findings add new insights into the role of HEL2i in the control of antiviral innate immunity and autoimmunity diseases.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A loosened gating mechanism of RIG-I leads to autoimmune disorders

Luo

Zheng

2022

Nucleic Acids Research

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“…To determine Rubpro activity on Ubiquitin (Ub) and the Ub-like (Ubl) modifier interferon stimulated gene 15 (ISG15), we purified K63-triubiquitin, K48-triubiquitin and M1-triubiquitin(linear) chains and ISG15-HIS according to our previous work [65]. Starting with ubiquitin, robust RubPro activity and high specificity was observed towards Lys48-linked polyubiquitin (Fig 5B), where triubiquitin was cleaved to monoubiquitin products, but not Lys63-linked triubiquitin (Fig 5A) and linear-linked triubiquitin (Fig 5D).…”

Section: Resultsmentioning

confidence: 99%

Section: Rubpro Is a K48-linkage-specific Deubiquitinasementioning

confidence: 99%

Crystal structure of the Rubella virus protease reveals a unique papain-like protease fold

Cheong

Quek

Liu

et al. 2022

Preprint

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Rubella is well-controlled due to an effective vaccine, but outbreaks are still occurring without any available antiviral treatments. There is still much to learn about the rubella virus (RUBV) papain-like protease (RubPro) that could be a potential drug target. This protease is crucial to RUBV replication, cleaving the non-structural polyprotein p200 into 2 multi-functional proteins, p150 and p90. Here we report a novel crystal structure of RubPro at 1.64 Angstrom resolution. It has a similar catalytic core structure to that of SARS-CoV-2 and foot-mouth-disease virus (FMDV) proteases. RubPro has well-conserved sequence motifs that are also found in its newly discovered Rubivirus relatives. The RubPro construct was shown to have protease activity in trans against a construct of RUBV protease-helicase and fluorogenic peptide. A protease-helicase construct was also cleaved in E. coli expression. RubPro was demonstrated to possess deubiquitylation activity, suggesting a potential role of RubPro in modulating the host's innate immune responses. The structural and functional insights of the RubPro will advance our current understanding of its function and point to more structure-based research into the RUBV replication machinery, in hopes of developing antiviral therapeutics in the future.

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“…Particularly, ubiquitin ligase TRIM65 specifically recognizes RNA-bound MDA5 filaments to catalyze K63-linked ubiquitination and activation of MDA5 ( Kato et al 2021 ). Similar to RIG-I, an unanchored but longer K63-linked polyubiquitin chain is thought to trigger oligomerisation and activation of MDA5 ( Song et al 2021 ). Besides, different MDA5 interacting partners are also involved in MDA5 regulation.…”

Section: Introductionmentioning

confidence: 99%

Cytoplasmic RNA sensors and their interplay with RNA-binding partners in innate antiviral response: theme and variations

Chan

Jin

2022

RNA

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Sensing of pathogen-associated molecular patterns including viral RNA by innate immunity represents the first line of defense against viral infection. In addition to RIG-I-like receptors and NOD-like receptors, several other RNA sensors are known to mediate innate antiviral response in the cytoplasm. Double-stranded RNA-binding protein PACT interacts with prototypic RNA sensor RIG-I to facilitate its recognition of viral RNA and induction of host interferon response, but variations of this theme are seen when the functions of RNA sensors are modulated by other RNA-binding proteins to impinge on antiviral defense, proinflammatory cytokine production and cell death programs. Their discrete and coordinated actions are crucial to protect the host from infection. In this review, we will focus on cytoplasmic RNA sensors with an emphasis on their interplay with RNA-binding partners. Classical sensors such as RIG-I will be briefly reviewed. More attention will be brought to the new insights on how RNA-binding partners of RNA sensors modulate innate RNA sensing and how viruses perturb the functions of RNA-binding partners.

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Ordered assembly of the cytosolic RNA-sensing MDA5-MAVS signaling complex via binding to unanchored K63-linked poly-ubiquitin chains

Cited by 38 publications

References 59 publications

A loosened gating mechanism of RIG-I leads to autoimmune disorders

A loosened gating mechanism of RIG-I leads to autoimmune disorders

Crystal structure of the Rubella virus protease reveals a unique papain-like protease fold

Cytoplasmic RNA sensors and their interplay with RNA-binding partners in innate antiviral response: theme and variations

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