Induced phase separation of mutant NF2 imprisons the cGAS-STING machinery to abrogate antitumor immunity.

Missense mutations of the tumor suppressor Neurofibromin 2 (NF2/Merlin/schwannomin) result in sporadic to frequent occurrences of tumorigenesis in multiple organs. However, the underlying pathogenicity of NF2-related tumorigenesis remains mostly unknown. Here we found that NF2 facilitated innate immunity by regulating YAP/TAZ-mediated TBK1 inhibition. Unexpectedly, patient-derived individual mutations in the FERM domain of NF2 (NF2m) converted NF2 into a potent suppressor of cGAS-STING signaling. Mechanistically, NF2m gained extreme associations with IRF3 and TBK1 and, upon innate nucleic acid sensing, was directly induced by the activated IRF3 to form cellular condensates, which contained the PP2A complex, to eliminate TBK1 activation. Accordingly, NF2m robustly suppressed STING-initiated antitumor immunity in cancer cell-autonomous and -nonautonomous murine models, and NF2m-IRF3 condensates were evident in human vestibular schwannomas. Our study reports phase separation-mediated quiescence of cGAS-STING signaling by a mutant tumor suppressor and reveals gain-of-function pathogenesis for NF2-related tumors by regulating antitumor immunity.

TBK1-Mediated DRP1 Targeting Confers Nucleic Acid Sensing to Reprogram Mitochondrial Dynamics and Physiology.

Mitochondrial morphology shifts rapidly to manage cellular metabolism, organelle integrity, and cell fate. It remains unknown whether innate nucleic acid sensing, the central and general mechanisms of monitoring both microbial invasion and cellular damage, can reprogram and govern mitochondrial dynamics and function. Here, we unexpectedly observed that upon activation of RIG-I-like receptor (RLR)-MAVS signaling, TBK1 directly phosphorylated DRP1/DNM1L, which disabled DRP1, preventing its high-order oligomerization and mitochondrial fragmentation function. The TBK1-DRP1 axis was essential for assembly of large MAVS aggregates and healthy antiviral immunity and underlay nutrient-triggered mitochondrial dynamics and cell fate determination. Knockin (KI) strategies mimicking TBK1-DRP1 signaling produced dominant-negative phenotypes reminiscent of human DRP1 inborn mutations, while interrupting the TBK1-DRP1 connection compromised antiviral responses. Thus, our findings establish an unrecognized function of innate immunity governing both morphology and physiology of a major organelle, identify a lacking loop during innate RNA sensing, and report an elegant mechanism of shaping mitochondrial dynamics.

CRISPR-repressed toxin-antitoxin provides herd immunity against anti-CRISPR elements.

Prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are highly vulnerable to phage-encoded anti-CRISPR (Acr) factors. How CRISPR-Cas systems protect themselves remains unclear. Here we uncovered a broad-spectrum anti-anti-CRISPR strategy involving a phage-derived toxic protein. Transcription of this toxin is normally repressed by the CRISPR-Cas effector but is activated to halt cell division when the effector is inhibited by any anti-CRISPR proteins or RNAs. We showed that this abortive infection-like effect efficiently expels Acr elements from bacterial population. Furthermore, we exploited this anti-anti-CRISPR mechanism to develop a screening method for specific Acr candidates for a CRISPR-Cas system and successfully identified two distinct Acr proteins that enhance the binding of CRISPR effector to nontarget DNA. Our data highlight the broad-spectrum role of CRISPR-repressed toxins in counteracting various types of Acr factors. We propose that the regulatory function of CRISPR-Cas confers host cells herd immunity against Acr-encoding genetic invaders whether they are CRISPR targeted or not.

Structural and Functional Characterization of a Fish Type I Subgroup d IFN Reveals Its Binding to Receptors.

Teleost fish type I IFNs and the associated receptors from the cytokine receptor family B (CRFB) are characterized by remarkable diversity and complexity. How the fish type I IFNs bind to their receptors is still not fully understood. In this study, we demonstrate that CRFB1 and CRFB5 constitute the receptor pair through which type I subgroup d IFN (IFNd) from large yellow croaker, Larimichthys crocea, activates the conserved JAK-STAT signaling pathway as a part of the antiviral response. Our data suggest that L. crocea IFNd (LcIFNd) has a higher binding affinity with L. crocea CRFB5 (LcCRFB5) than with LcCRFB1. Furthermore, we report the crystal structure of LcIFNd at a 1.49-Å resolution and construct structural models of LcIFNd in binary complexes with predicted structures of extracellular regions of LcCRFB1 and LcCRFB5, respectively. Despite striking similarities in overall architectures of LcIFNd and its ortholog human IFN-ω, the receptor binding patterns between LcIFNd and its receptors show that teleost and mammalian type I IFNs may have differentially selected helices that bind to their homologous receptors. Correspondingly, key residues mediating binding of LcIFNd to LcCRFB1 and LcCRFB5 are largely distinct from the receptor-interacting residues in other fish and mammalian type I IFNs. Our findings reveal a ligand/receptor complex binding mechanism of IFNd in teleost fish, thus providing new insights into the function and evolution of type I IFNs.

Crystal Structures of DNA-bound Fish IRF10 and IRF11 Reveal the Determinants of IFN Regulation.

IFN regulatory factors (IRFs) are transcription factors that mediate homeostatic mechanisms of host defense against pathogens. In addition to IRF1-9, which are conserved across vertebrates, teleost fishes have two other IRFs, IRF10 and IRF11. In zebrafish (Danio rerio), IRF10 represses the expression of IFNφ1 and IFNφ3, whereas IRF11 exerts the opposite effect. In this study, we found IRF10 could significantly inhibit the expression of IFNφ1 and IFNφ3 induced by IFN11 to synergistically regulate type I IFN expression. To clarify the synergistically regulatory mechanism of IRF10 and IRF11 in type I IFN expression, we determined and analyzed the crystal structures of the DNA-binding domains (DBDs) of zebrafish IRF10 and IRF11 bound to DNA, as well as IRF11 DBD in apo form. The interactions of IRF10-DBD and IRF11-DBD with DNA backbone were elaborated in detail. Further analysis showed that IRF10 and IRF11 have the same binding patterns and comparable affinities with the IFN-sensitive response elements of IFNφ1 and IFNφ3 promoters. Therefore, IRF10 could function as a controlling factor for IRF11 by competitive binding of the IFN-sensitive response elements to coregulate the host IFN response. Accordingly, similar to IRF1 and IRF2 in mammals, IRF10 and IRF11 act as another pair of negative and positive regulators to balance the antiviral responses in fish.

Structural transitions upon guide RNA binding and their importance in Cas12g-mediated RNA cleavage.

Cas12g is an endonuclease belonging to the type V RNA-guided CRISPR-Cas family. It is known for its ability to cleave RNA substrates using a conserved endonuclease active site located in the RuvC domain. In this study, we determined the crystal structure of apo-Cas12g, the cryo-EM structure of the Cas12g-sgRNA binary complex and investigated conformational changes that occur during the transition from the apo state to the Cas12g-sgRNA binary complex. The conserved zinc finger motifs in Cas12g undergo an ordered-to-disordered transition from the apo to the sgRNA-bound state and their mutations negatively impact on target RNA cleavage. Moreover, we identified a lid motif in the RuvC domain that undergoes transformation from a helix to loop to regulate the access to the RuvC active site and subsequent cleavage of the RNA substrate. Overall, our study provides valuable insights into the mechanisms by which Cas12g recognizes sgRNA and the conformational changes it undergoes from sgRNA binding to the activation of the RNase active site, thereby laying a foundation for the potential repurposing of Cas12g as a tool for RNA-editing.

Regulation of phosphoribosyl ubiquitination by a calmodulin-dependent glutamylase.

The bacterial pathogen Legionella pneumophila creates an intracellular niche permissive for its replication by extensively modulating host-cell functions using hundreds of effector proteins delivered by its Dot/Icm secretion system1. Among these, members of the SidE family (SidEs) regulate several cellular processes through a unique phosphoribosyl ubiquitination mechanism that bypasses the canonical ubiquitination machinery2-4. The activity of SidEs is regulated by another Dot/Icm effector known as SidJ5; however, the mechanism of this regulation is not completely understood6,7. Here we demonstrate that SidJ inhibits the activity of SidEs by inducing the covalent attachment of glutamate moieties to SdeA-a member of the SidE family-at E860, one of the catalytic residues that is required for the mono-ADP-ribosyltransferase activity involved in ubiquitin activation2. This inhibition by SidJ is spatially restricted in host cells because its activity requires the eukaryote-specific protein calmodulin (CaM). We solved a structure of SidJ-CaM in complex with AMP and found that the ATP used in this reaction is cleaved at the α-phosphate position by SidJ, which-in the absence of glutamate or modifiable SdeA-undergoes self-AMPylation. Our results reveal a mechanism of regulation in bacterial pathogenicity in which a glutamylation reaction that inhibits the activity of virulence factors is activated by host-factor-dependent acyl-adenylation.

Legionella pneumophila regulates the activity of UBE2N by deamidase-mediated deubiquitination.

The Legionella pneumophila effector MavC induces ubiquitination of the E2 ubiquitin-conjugating enzyme UBE2N by transglutamination, thereby abolishing its function in the synthesis of K63 -type polyubiquitin chains. The inhibition of UBE2N activity creates a conundrum because this E2 enzyme is important in multiple signaling pathways, including some that are important for intracellular L. pneumophila replication. Here, we show that prolonged inhibition of UBE2N activity by MavC restricts intracellular bacterial replication and that the activity of UBE2N is restored by MvcA, an ortholog of MavC (50% identity) with ubiquitin deamidase activity. MvcA functions to deubiquitinate UBE2N-Ub using the same catalytic triad required for its deamidase activity. Structural analysis of the MvcA-UBE2N-Ub complex reveals a crucial role of the insertion domain in MvcA in substrate recognition. Our study establishes a deubiquitination mechanism catalyzed by a deamidase, which, together with MavC, imposes temporal regulation of the activity of UBE2N during L. pneumophila infection.

Structure of the Spring Viraemia of Carp Virus Ribonucleoprotein Complex Reveals Its Assembly Mechanism and Application in Antiviral Drug Screening.

Spring viremia of carp virus (SVCV) is a highly pathogenic Vesiculovirus infecting the common carp, yet neither a vaccine nor effective therapies are available to treat spring viremia of carp (SVC). Like all negative-sense viruses, SVCV contains an RNA genome that is encapsidated by the nucleoprotein (N) in the form of a ribonucleoprotein (RNP) complex, which serves as the template for viral replication and transcription. Here, the three-dimensional (3D) structure of SVCV RNP was resolved through cryo-electron microscopy (cryo-EM) at a resolution of 3.7 Å. RNP assembly was stabilized by N and C loops; RNA was wrapped in the groove between the N and C lobes with 9 nt nucleotide per protomer. Combined with mutational analysis, our results elucidated the mechanism of RNP formation. The RNA binding groove of SVCV N was used as a target for drug virtual screening, and it was found suramin had a good antiviral effect. This study provided insights into RNP assembly, and anti-SVCV drug screening was performed on the basis of this structure, providing a theoretical basis and efficient drug screening method for the prevention and treatment of SVC. IMPORTANCE Aquaculture accounts for about 70% of global aquatic products, and viral diseases severely harm the development of aquaculture industry. Spring viremia of carp virus (SVCV) is the pathogen causing highly contagious spring viremia of carp (SVC) disease in cyprinids, especially common carp (Cyprinus carpio), yet neither a vaccine nor effective therapies are available to treat this disease. In this study, we have elucidated the mechanism of SVCV ribonucleoprotein complex (RNP) formation by resolving the 3D structure of SVCV RNP and screened antiviral drugs based on the structure. It is found that suramin could competitively bind to the RNA binding groove and has good antiviral effects both in vivo and in vitro. Our study provides a template for rational drug discovery efforts to treat and prevent SVCV infections.

The helicase DDX41 recognizes the bacterial secondary messengers cyclic di-GMP and cyclic di-AMP to activate a type I interferon immune response.

The induction of type I interferons by the bacterial secondary messengers cyclic di-GMP (c-di-GMP) or cyclic di-AMP (c-di-AMP) is dependent on a signaling axis that involves the adaptor STING, the kinase TBK1 and the transcription factor IRF3. Here we identified the heliase DDX41 as a pattern-recognition receptor (PRR) that sensed both c-di-GMP and c-di-AMP. DDX41 specifically and directly interacted with c-di-GMP. Knockdown of DDX41 via short hairpin RNA in mouse or human cells inhibited the induction of genes encoding molecules involved in the innate immune response and resulted in defective activation of STING, TBK1 and IRF3 in response to c-di-GMP or c-di-AMP. Our results suggest a mechanism whereby c-di-GMP and c-di-AMP are detected by DDX41, which forms a complex with STING to signal to TBK1-IRF3 and activate the interferon response.

Molecular and Structural Basis of Receptor Binding and Signaling of a Fish Type I IFN with Three Disulfide Bonds.

In mammals, type I IFNs, which commonly contain one or two disulfide bonds, activate the JAK-STAT signaling pathway through binding to the common cell surface receptor formed by IFN-α/ß receptor (IFNAR)1 and IFNAR2 subunits. Although type I IFNs are also known to be essential for antiviral defense in teleost fish, very little is known about mechanisms underlying the recognition of fish type I IFNs by associated receptors. In this study, we demonstrate that a type I IFN of large yellow croaker Larimichthys crocea (LcIFNi), belonging to a new subgroup of fish type I IFNs, triggers antiviral response via the conserved JAK-STAT pathway through stable binding with a heterodimeric receptor comprising subunits LcCRFB5 and LcCRFB2. LcIFNi binds to LcCRFB5 with a much higher affinity than to LcCRFB2. Furthermore, we determined the crystal structure of LcIFNi at a 1.39 Å resolution. The high-resolution structure is, to our knowledge, the first reported structure of a type I IFN with three disulfide bonds, all of which were found to be indispensable for folding and stability of LcIFNi. Using structural analysis, mutagenesis, and biochemical assays, we identified key LcIFNi residues involved in receptor interaction and proposed a structural model of LcIFNi bound to the LcCRFB2-LcCRFB5 receptor. The results show that LcIFNi-LcCRFB2 exhibits a similar binding pattern to human IFN-ω-IFNAR2, whereas the binding pattern of LcIFNi-LcCRFB5 is quite different from that of IFN-ω-IFNAR1. Altogether, our findings reveal the structural basis for receptor interaction and signaling of a type I IFN with three disulfide bonds and provide new insights into the mechanisms underlying type I IFN recognition in teleosts.

Structural insights into target DNA recognition and cleavage by the CRISPR-Cas12c1 system.

Cas12c is the recently characterized dual RNA-guided DNase effector of type V-C CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein) systems. Due to minimal requirements for a protospacer adjacent motif (PAM), Cas12c is an attractive candidate for genome editing. Here we report the crystal structure of Cas12c1 in complex with single guide RNA (sgRNA) and target double-stranded DNA (dsDNA) containing the 5'-TG-3' PAM. Supported by biochemical and mutation assays, this study reveals distinct structural features of Cas12c1 and the associated sgRNA, as well as the molecular basis for PAM recognition, target dsDNA unwinding, heteroduplex formation and recognition, and cleavage of non-target and target DNA strands. Cas12c1 recognizes the PAM through a mechanism that is interdependent on sequence identity and Cas12c1-induced conformational distortion of the PAM region. Another special feature of Cas12c1 is the cleavage of both non-target and target DNA strands at a single, uniform site with indistinguishable cleavage capacity and order. Location of the sgRNA seed region and minimal length of target DNA required for triggering Cas12c1 DNase activity were also determined. Our findings provide valuable information for developing the CRISPR-Cas12c1 system into an efficient, high-fidelity genome editing tool.

Structural basis for the dual catalytic activity of the Legionella pneumophila ovarian tumor (OTU) domain deubiquitinase LotA.

Legionella pneumophila, a bacterial pathogen that causes a severe pneumonia known as Legionnaires' disease, extensively exploits the ubiquitin (Ub) pathway in the infected host cells through certain virulence effectors excreted by the Dot/Icm system. To date, several Dot/Icm effectors have been found to act as Ub ligases, and four effectors, including LotA, LotB, LotC, and Ceg7, have been identified as deubiquitinases (DUBs) from the ovarian tumor (OTU) domain family. LotA is unique among other OTU DUBs because it possesses two distinct DUB domains and exclusively exhibits catalytic activity against K6-linked diUb and polyUb chains. However, the structure of LotA and the molecular mechanism for the dual DUB activity remains elusive. In this study, we solved the structure of LotA in complex with proximally bound Ub and distal covalently bound Ub. Both Ub molecules are bound to the DUB1 domain and mimic a K6-linked diUb. Structural analysis reveals that the DUB1 domain utilizes a distinct mechanism for recognition of the K6-linked diUb within a large S1' binding site that is uncommon to OTU DUBs. Structural fold of the LotA DUB2 domain closely resembles LotB and LotC, similarly containing an extra α-helix lobe that has been demonstrated to play an important role in Ub binding. Collectively, our study uncovers the structural basis for the dual catalytic activity of the unique OTU family DUB LotA.

NLRC3, a member of the NLR family of proteins, is a negative regulator of innate immune signaling induced by the DNA sensor STING.

Stimulator of interferon genes (STING, also named MITA, MYPS, or ERIS) is an intracellular DNA sensor that induces type I interferon through its interaction with TANK-binding kinase 1 (TBK1). Here we found that the nucleotide-binding, leucine-rich-repeat-containing protein, NLRC3, reduced STING-dependent innate immune activation in response to cytosolic DNA, cyclic di-GMP (c-di-GMP), and DNA viruses. NLRC3 associated with both STING and TBK1 and impeded STING-TBK1 interaction and downstream type I interferon production. By using purified recombinant proteins, we found NLRC3 to interact directly with STING. Furthermore, NLRC3 prevented proper trafficking of STING to perinuclear and punctated region, known to be important for its activation. In animals, herpes simplex virus 1 (HSV-1)-infected Nlrc3(-/-) mice exhibited enhanced innate immunity and reduced morbidity and viral load. This demonstrates the intersection of two key pathways of innate immune regulation, NLR and STING, to fine tune host response to intracellular DNA, DNA virus, and c-di-GMP.

Cryo-EM structures of the human PA200 and PA200-20S complex reveal regulation of proteasome gate opening and two PA200 apertures.

Proteasomes are highly abundant and conserved protease complexes that eliminate unwanted proteins in the cells. As a single-chain ATP-independent nuclear proteasome activator, proteasome activator 200 (PA200) associates with 20S core particle to form proteasome complex that catalyzes polyubiquitin-independent degradation of acetylated histones, thus playing a pivotal role in DNA repair and spermatogenesis. Here, we present cryo-electron microscopy (cryo-EM) structures of the human PA200-20S complex and PA200 at 2.72 Å and 3.75 Å, respectively. PA200 exhibits a dome-like architecture that caps 20S and uses its C-terminal YYA (Tyr-Tyr-Ala) to induce the α-ring rearrangements and partial opening of the 20S gate. Our structural data also indicate that PA200 has two openings formed by numerous positively charged residues that respectively bind (5,6)-bisdiphosphoinositol tetrakisphosphate (5,6[PP]2-InsP4) and inositol hexakisphosphate (InsP6) and are likely to be the gates that lead unfolded proteins through PA200 and into the 20S. Besides, our structural analysis of PA200 found that the bromodomain (BRD)-like (BRDL) domain of PA200 shows considerable sequence variation in comparison to other human BRDs, as it contains only 82 residues because of a short ZA loop, and cannot be classified into any of the eight typical human BRD families. Taken together, the results obtained from this study provide important insights into human PA200-induced 20S gate opening for substrate degradation and the opportunities to explore the mechanism for its recognition of H4 histone in acetylation-mediated proteasomal degradation.

Advances in Research on Type 2 Diabetes Mellitus Targets and Therapeutic Agents.

Diabetes mellitus is a chronic multifaceted disease with multiple potential complications, the treatment of which can only delay and prolong the terminal stage of the disease, i.e., type 2 diabetes mellitus (T2DM). The World Health Organization predicts that diabetes will be the seventh leading cause of death by 2030. Although many antidiabetic medicines have been successfully developed in recent years, such as GLP-1 receptor agonists and SGLT-2 inhibitors, single-target drugs are gradually failing to meet the therapeutic requirements owing to the individual variability, diversity of pathogenesis, and organismal resistance. Therefore, there remains a need to investigate the pathogenesis of T2DM in more depth, identify multiple therapeutic targets, and provide improved glycemic control solutions. This review presents an overview of the mechanisms of action and the development of the latest therapeutic agents targeting T2DM in recent years. It also discusses emerging target-based therapies and new potential therapeutic targets that have emerged within the last three years. The aim of our review is to provide a theoretical basis for further advancement in targeted therapies for T2DM.

Structural and functional insights into a novel two-component endolysin encoded by a single gene in Enterococcus faecalis phage.

Using bacteriophage-derived endolysins as an alternative strategy for fighting drug-resistant bacteria has recently been garnering renewed interest. However, their application is still hindered by their narrow spectra of activity. In our previous work, we demonstrated that the endolysin LysIME-EF1 possesses efficient bactericidal activity against multiple strains of Enterococcus faecalis (E. faecalis). Herein, we observed an 8 kDa fragment and hypothesized that it contributes to LysIME-EF1 lytic activity. To examine our hypothesis, we determined the structure of LysIME-EF1 at 1.75 Å resolution. LysIME-EF1 exhibits a unique architecture in which one full-length LysIME-EF1 forms a tetramer with three additional C-terminal cell-wall binding domains (CBDs) that correspond to the abovementioned 8 kDa fragment. Furthermore, we identified an internal ribosomal binding site (RBS) and alternative start codon within LysIME-EF1 gene, which are demonstrated to be responsible for the translation of the truncated CBD. To elucidate the molecular mechanism for the lytic activity of LysIME-EF1, we combined mutagenesis, lytic activity assays and in vivo animal infection experiments. The results confirmed that the additional LysIME-EF1 CBDs are important for LysIME-EF1 architecture and its lytic activity. To our knowledge, this is the first determined structure of multimeric endolysin encoded by a single gene in E. faecalis phages. As such, it may provide valuable insights into designing potent endolysins against the opportunistic pathogen E. faecalis.

Functional and structural characteristics of HLA-B*13:01-mediated specific T cells reaction in dapsone-induced drug hypersensitivity.

BACKGROUND: Severe cutaneous adverse drug reactions (SCARs) are a group of serious clinical conditions caused by immune reaction to certain drugs. The allelic variance of human leukocyte antigens of HLA-B*13:01 has been strongly associated with hypersensitivities induced by dapsone (DDS). T-cell receptor mediated activation of cytotoxic T lymphocytes (CTLs) has also been suggested to play an essential role in pathogenesis of SCARs. However, HLA-B*13:01-DDS-TCR immune synapse that plays role in drug-induced hypersensitivity syndrome (DIHS) associated T cells activation remains uncharacterized. METHODS: To investigate the molecular mechanisms for HLA-B*13:01 in the pathogenesis of Dapsone-induced drug hypersensitivity (DDS-DIHS), we performed crystallization and expanded drug-specific CTLs to analyze the pathological role of DDS-DIHS. RESULTS: Results showed the crystal structure of HLA-B*13:01-beta-2-microglobulin (ß2M) complex at 1.5 Å resolution and performed mutation assays demonstrating that I118 or I119, and R121 of HLA-B*13:01 were the key residues that mediate the binding of DDS. Subsequent single-cell TCR and RNA sequencing indicated that TCRs composed of paired TRAV12-3/TRBV28 clonotype with shared CDR3 region specifically recognize HLA-B*13:01-DDS complex to trigger inflammatory cytokines associated with DDS-DIHS. CONCLUSION: Our study identified the novel p-i-HLA/TCR as the model of interaction between HLA-B*13:01, DDS and the clonotype-specific TCR in DDS-DIHS.

NOD1 Promotes Antiviral Signaling by Binding Viral RNA and Regulating the Interaction of MDA5 and MAVS.

Nucleotide oligomerization domain-like receptors (NLRs) and RIG-I-like receptors (RLRs) detect diverse pathogen-associated molecular patterns to activate the innate immune response. The role of mammalian NLR NOD1 in sensing bacteria is well established. Although several studies suggest NOD1 also plays a role in sensing viruses, the mechanisms behind this are still largely unknown. In this study, we report on the synergism and antagonism between NOD1 and MDA5 isoforms in teleost. In zebrafish, the overexpression of NOD1 enhances the antiviral response and mRNA abundances of key antiviral genes involved in RLR-mediated signaling, whereas the loss of NOD1 has the opposite effect. Notably, spring viremia of carp virus-infected NOD1-/- zebrafish exhibit reduced survival compared with wild-type counterparts. Mechanistically, NOD1 targets MDA5 isoforms and TRAF3 to modulate the formation of MDA5-MAVS and TRAF3-MAVS complexes. The cumulative effects of NOD1 and MDA5a (MDA5 normal form) were observed for the binding with poly(I:C) and the formation of the MDA5a-MAVS complex, which led to increased transcription of type I IFNs and ISGs. However, the antagonism between NOD1 and MDA5b (MDA5 truncated form) was clearly observed during proteasomal degradation of NOD1 by MDA5b. In humans, the interactions between NOD1-MDA5 and NOD1-TRAF3 were confirmed. Furthermore, the roles that NOD1 plays in enhancing the binding of MDA5 to MAVS and poly(I:C) are also evolutionarily conserved across species. Taken together, our findings suggest that mutual regulation between NOD1 and MDA5 isoforms may play a crucial role in the innate immune response and that NOD1 acts as a positive regulator of MDA5/MAVS normal form-mediated immune signaling in vertebrates.

Novel polyadenylylation-dependent neutralization mechanism of the HEPN/MNT toxin/antitoxin system.

The two-gene module HEPN/MNT is predicted to be the most abundant toxin/antitoxin (TA) system in prokaryotes. However, its physiological function and neutralization mechanism remains obscure. Here, we discovered that the MntA antitoxin (MNT-domain protein) acts as an adenylyltransferase and chemically modifies the HepT toxin (HEPN-domain protein) to block its toxicity as an RNase. Biochemical and structural studies revealed that MntA mediates the transfer of three AMPs to a tyrosine residue next to the RNase domain of HepT in Shewanella oneidensis. Furthermore, in vitro enzymatic assays showed that the three AMPs are transferred to HepT by MntA consecutively with ATP serving as the substrate, and this polyadenylylation is crucial for reducing HepT toxicity. Additionally, the GSX10DXD motif, which is conserved among MntA proteins, is the key active motif for polyadenylylating and neutralizing HepT. Thus, HepT/MntA represents a new type of TA system, and the polyadenylylation-dependent TA neutralization mechanism is prevalent in bacteria and archaea.