N-terminal Caspase Recruitment Domain Research Articles

Understanding a point mutation signature D54K in the caspase activation recruitment domain of NOD1 capitulating concerted immunity via atomistic simulation

Point mutation D54K in the human N-terminal caspase recruitment domain (CARD) of nucleotide-binding oligomerization domain −1 (NOD1) abrogates an imperative downstream interaction with receptor-interacting protein kinase (RIPK2) that entails combating bacterial infections and inflammatory dysfunction. Here, we addressed the molecular details concerning conformational changes and interaction patterns (monomeric-dimeric states) of D54K by signature-based molecular dynamics simulation. Initially, the sequence analysis prioritized D54K as a pathogenic mutation, among other variants, based on a sequence signature. Since the mutation is highly conserved, we derived the distant ortholog to predict the sequence and structural similarity between native and mutant. This analysis showed the utility of 33 communal core residues associated with structural-functional preservation and variations, concurrently served to infer the cryptic hotspots Cys39, Glu53, Asp54, Glu56, Ile57, Leu74, and Lys78 determining the inter helical fold forming homodimers for putative receptor interaction. Subsequently, the atomistic simulations with free energy (MM/PB(GB)SA) calculations predicted structural alteration that takes place in the N-terminal mutant CARD where coils changed to helices (45 α3- L4-α4-L6- α683) in contrast to native (45T2-L4-α4-L6-T483). Likewise, the C-terminal helices 93T1-α7105 connected to the loops distorted compared to native 93α6-L7105 may result in conformational misfolding that promotes functional regulation and activation. These structural perturbations of D54K possibly destabilize the flexible adaptation of critical homotypic NOD1CARD-CARDRIPK2 interactions (α4Asp42-Arg488α5 and α6Phe86-Lys471α4) is consistent with earlier experimental reports. Altogether, our findings unveil the conformational plasticity of mutation-dependent immunomodulatory response and may aid in functional validation exploring clinical investigation on CARD-regulated immunotherapies to prevent systemic infection and inflammation. Communicated by Ramaswamy H. Sarma

ZNF205 positively regulates RLR antiviral signaling by targeting RIG-I.

Retinoic acid-inducible gene I (RIG-I) is a cytosolic viral RNA receptor. Upon viral infection, the protein recognizes and then recruits adapter protein mitochondrial antiviral signaling (MAVS) protein, initiating the production of interferons and proinflammatory cytokines to establish an antiviral state. In the present study, we identify zinc finger protein 205 (ZNF205) which associates with RIG-I and promotes the Sendai virus (SeV)-induced antiviral innate immune response. Overexpression of ZNF205 facilitates interferon-beta (IFN-β) introduction, whereas ZNF205 deficiency restricts its introduction. Mechanistically, the C-terminal zinc finger domain of ZNF205 interacts with the N-terminal tandem caspase recruitment domains (CARDs) of RIG-I; this interaction markedly promotes K63 ubiquitin-linked polyubiquitination of RIG-I, which is crucial for RIG-I activation. Thus, our results demonstrate that ZNF205 is a positive regulator of the RIG-I-mediated innate antiviral immune signaling pathway.

Pigeon MDA5 inhibits viral replication by triggering antiviral innate immunity

Pigeons are considered less susceptible, and display few or no clinical signs to infection with avian influenza virus (AIV). Melanoma differentiation-associated gene 5 (MDA5), an important mediator in innate immunity, has been linked to the virus resistance. In this study, the pigeon MDA5 (piMDA5) was cloned. The bioinformatics analysis showed that the C-terminal domain (CTD) of MDA5 is highly conserved among species while the N-terminal caspase recruitment domain (CARD) is variable. Upon infection with Newcastle diseases virus (NDV) and AIV, piMDA5 was upregulated in both pigeons and pigeon embryonic fibroblasts (PEFs). Further study found that overexpression of piMDA5 mediated the activation of interferons (IFNs) and IFN-stimulated genes (ISGs) while inhibiting NDV replication. Conversely, the knockdown of piMDA5 promoted NDV replication. Additionally, CARD was found to be essential for the activation of IFN-β by piMDA5. Furthermore, pigeon MDA5, chicken MDA5, and human MDA5 differ in inhibiting viral replication and inducing ISGs expression. These findings suggest that MDA5 contributes to suppressing viral replication by activating the IFN signal pathway in pigeons. This study provides valuable insight into the role of MDA5 in pigeons and a better understanding of the conserved role of MDA5 in innate immunity during evolution.

A double-edged sword: role of apoptosis repressor with caspase recruitment domain (ARC) in tumorigenesis and ischaemia/reperfusion (I/R) injury.

Apoptosis repressor with caspase recruitment domain (ARC) acts as a potent and multifunctional inhibitor of apoptosis, which is mainly expressed in postmitotic cells, including cardiomyocytes. ARC is special for its N-terminal caspase recruitment domain and caspase recruitment domain. Due to the powerful inhibition of apoptosis, ARC is mainly reported to act as a cardioprotective factor during ischaemia‒reperfusion (I/R) injury, preventing cardiomyocytes from being devastated by various catastrophes, including oxidative stress, calcium overload, and mitochondrial dysfunction in the circulatory system. However, recent studies have found that ARC also plays a potential regulatory role in tumorigenesis especially in colorectal cancer and renal cell carcinomas, through multiple apoptosis-associated pathways, which remains to be explored in further studies. Therefore, ARC regulates the body and maintains the balance of physiological activities with its interesting duplex. This review summarizes the current research progress of ARC in the field of tumorigenesis and ischaemia/reperfusion injury, to provide overall research status and new possibilities for researchers.

Function conservation and disparities of zebrafish and human LGP2 genes in fish and mammalian cells responsive to poly(I:C).

Retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs) are viral RNA sensors that regulate host interferon (IFN)-mediated antiviral signaling. LGP2 (laboratory genetics and physiology 2) lacks the N-terminal caspase activation and recruitment domains (CARDs) responsible for signaling transduction in the other two RLR proteins, RIG-I and melanoma differentiation associated gene-5 (MDA5). How LGP2 regulates IFN signaling is controversial, and inconsistent results have often been obtained in overexpression assays when performed in fish cells and mammalian cells. Here we report that the differential sensitivity of fish cells and mammalian cells to poly(I:C) transfection conceals the function conservation of zebrafish and human LGP2. In fish cells, overexpression of zebrafish or human LGP2 initially activates IFN signaling in a dose-dependent manner, followed by inhibition at a critical threshold of LGP2 expression. A similar trend exists for LGP2-dependent IFN induction in response to stimulation by low and high concentrations of poly(I:C). In contrast, overexpression of zebrafish or human LGP2 alone in mammalian cells does not activate IFN signaling, but co-stimulation with very low or very high concentrations of poly(I:C) shows LGP2-dependent enhancement or inhibition of IFN signaling, respectively. Titration assays show that LGP2 promotes MDA5 signaling in mammalian cells mainly under low concentration of poly(I:C) and inhibits RIG-I/MDA5 signaling mainly under high concentration of poly(I:C). Our results suggest that fish and human LGP2s switch regulatory roles from a positive one to a negative one in increasing concentrations of poly(I:C)-triggered IFN response.

A Fluorescence-Polarization-Based Lipopolysaccharide-Caspase-4 Interaction Assay for the Development of Inhibitors.

Recognition of intracellular lipopolysaccharide (LPS) by Caspase-4 (Casp-4) is critical for host defense against Gram-negative pathogens. LPS binds to the N-terminal caspase activation and recruitment domain (CARD) of procaspase-4, leading to auto-proteolytic activation followed by pro-inflammatory cytokine release and pyroptotic cell death. Aberrant hyper-activation of Casp-4 leads to amplification of the inflammatory response linked to sepsis. While the active site of a caspase has been targeted with peptide inhibitors, inhibition of LPS–Casp-4 interaction is an emerging strategy for the development of selective inhibitors with a new mode of action for treating infectious diseases and sepsis induced by LPS. In this study, a high-throughput screening (HTS) system based on fluorescence polarization (FP) was devised to identify inhibitors of the LPS and Casp-4 interaction. Using HTS and IC50 determination and subsequently showing inhibited Casp-4 activity, we demonstrated that the LPS–Casp-4 interaction is a druggable target for Casp-4 inhibition and possibly a non-canonical inflammatory pathway.

Role of apoptosis repressor with caspase recruitment domain (ARC) in cell death and cardiovascular disease.

Apoptosis repressor with caspase recruitment domain (ARC) is a highly effective and multifunctional inhibitor of apoptosis that is mainly expressed in postmitotic cells such as cardiomyocytes and skeletal muscle cells. ARC contains a C-terminal region rich in proline and glutamic acid residues and an N-terminal caspase recruitment domain (CARD). The CARD is originally described as a protein-binding motif that interacts with caspase through a CARD-CARD interaction. Initially, the inhibitory effect of ARC was only found in apoptosis, however, it was later found that ARC also played a regulatory role in other types of cell death. As a powerful cardioprotective factor, ARC can protect the heart by inhibiting the death of cardiomyocytes in various ways. ARC can reduce the cardiomyocyte apoptotic response to various stresses and injuries, including extrinsic apoptosis induced by death receptor ligands, cellular Ca2+ homeostasis and the dysregulation of endoplasmic reticulum (ER) stress, oxidative stress and hypoxia. In addition, changes in ARC transcription and translation levels in the heart can cause a series of physiological and pathological changes, and ARC can also perform corresponding functions through interactions with other molecules. Although there has been much research on ARC, the functional redundancy among proteins shows that ARC still has much research value. This review summarizes the molecular characteristics of ARC, its roles in the various death modes in cardiomyocytes and the roles of ARC in cardiac pathophysiology. This article also describes the potential therapeutic effect and research prospects of ARC.

Regulation of RIG-I-like receptor-mediated signaling: interaction between host and viral factors.

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are RNA sensor molecules that play essential roles in innate antiviral immunity. Among the three RLRs encoded by the human genome, RIG-I and melanoma differentiation-associated gene 5, which contain N-terminal caspase recruitment domains, are activated upon the detection of viral RNAs in the cytoplasm of virus-infected cells. Activated RLRs induce downstream signaling via their interactions with mitochondrial antiviral signaling proteins and activate the production of type I and III interferons and inflammatory cytokines. Recent studies have shown that RLR-mediated signaling is regulated by interactions with endogenous RNAs and host proteins, such as those involved in stress responses and posttranslational modifications. Since RLR-mediated cytokine production is also involved in the regulation of acquired immunity, the deregulation of RLR-mediated signaling is associated with autoimmune and autoinflammatory disorders. Moreover, RLR-mediated signaling might be involved in the aberrant cytokine production observed in coronavirus disease 2019. Since the discovery of RLRs in 2004, significant progress has been made in understanding the mechanisms underlying the activation and regulation of RLR-mediated signaling pathways. Here, we review the recent advances in the understanding of regulated RNA recognition and signal activation by RLRs, focusing on the interactions between various host and viral factors.

Crystal structure of caspase-11 CARD provides insights into caspase-11 activation

Murine caspase-11 is the centerpiece of the non-canonical inflammasome pathway that can respond to intracellular LPS and induce pyroptosis. Caspase-11 contains two components, an N-terminal caspase recruitment domain (CARD) and a C-terminal catalytic domain. The aggregation of caspase-11 is thought to promote the auto-processing and activation of caspase-11. However, the activation mechanism of caspase-11 remains unclear. In this study, we purified the caspase-11 CARD fused to an MBP tag and found it tetramerizes in solution. Crystallographic analysis reveals an extensive hydrophobic interface formed by the H1–2 helix mediating homotypic CARD interactions. Importantly, mutations of the helix H1–2 hydrophobic residues abolished the tetramerization of MBP-tagged CARD in solution and failed to induce pyroptosis in cells. Our study provides the first evidence of the homotypic interaction mode for an inflammatory caspase by crystal model. This finding demonstrates that the tetramerization of the N-terminal CARD can promote releasing of the catalytic domain auto-inhibition, leading to the caspase-11 activation.

Explorations of the Apoptotic Protease Activating Factor 1 (Apaf‐1) in its Formation of the Seven‐Spoked Apoptosome Responsible for Programmed Cell Death

Multicellular life forms first appeared in the fossil record in a burst of evolution termed during the Cambrian period. With multicellularity came the development and specialization of cells, alongside their coordination in the formation of tissues and organs. An adult human, composed of 30–40 trillion cells, progresses through repeated choreographed rounds of growth and cell division from its microscopic begins as a single celled zygote. Some have argued that this is the most critical stage of one’s life. Equally important for development, relatively overlooked, is the intrinsic process of cell death. For example, the webbing between our fingers and toes disappears around week six of gestation. This macroscopic observation during fetal development is the result of a complex series of molecular events initiated by the apoptotic protease activating factor 1 (Apaf‐1) as it forms the holo‐enzyme the apoptosome. The Blue Valley CAPS 2020 SMART Team (Students Modeling a Research Topic), with support from the CBM at MSOE, modeled the Apaf‐1 using JMol and 3D printing technologies in order to better understand how this molecular machine carries out programmed cell death. Structurally, Apaf‐1 is composed of 1249 amino acid residues that form a C‐terminal regulatory domain, a nucleotide binding and oligomerization domain (NOD), and an N‐terminal caspase recruitment domain (CARD). The regulatory domain is composed of two WD40 b‐propellers that are responsible for binding cytochrome c, which itself is released from the inner membrane of the mitochrondria under conditions of stress. Cytochrome c bonding causes a disruption in a salt bridge between Lys192 and Asp616 leading to changes in the orientation of the propellers. The nucleotide binding and oligomerization domain is composed of a number of helical subdomains. Conformational changes in the regulatory domain stimulates nucleotide exchange, where an ADP molecule bond in Apaf‐1’s inactive and closed monomeric state is exchanged for an ATP molecule leading to a more open monomeric state and the subsequent formation of the heptameric apoptosome. The Arg265 trigger which stabilizes an aspartate triad in the inactive monomeric state becomes compromised after ATP binding leading to conformational changes that open up Apaf‐1. Once the polymeric apoptosome forms, the CARD binds and activates procaspase‐9, which itself cleaves loops in procaspases 3 and 7 which become active and responsible of the apoptosomes proteolytic function. Proteolysis ultimately leads to cell death.

The influenza NS1 protein modulates RIG-I activation via a strain-specific direct interaction with the second CARD of RIG-I

The influenza NS1 protein modulates RIG-I activation via a strain-specific direct interaction with the second CARD of RIG-I

Characterization and immune function of the interferon-β promoter stimulator-1 in the barbel chub, Squaliobarbus curriculus

To elucidate the immunity-protecting role of the interferon-β promoter stimulator-1 (ScIPS-1) in barbel chub Squaliobarbus curriculus, the full-length cDNA of ScIPS-1 was cloned and expression levels in response to stimulation were investigated. In addition, the function of ScIPS-1 and its domains were analyzed. The full-length cDNA of ScIPS-1 is 2524 bp and encodes 601 aa. The N-terminal caspase activation and recruitment domain, central proline-rich domain, C-terminal transmembrane domain, C2HC-zinc finger, and Cwf21 domains were identified. The mRNA level of ScIPS-1 was the highest in the kidney, whereas the highest protein level was observed in the liver. The ScIPS-1 expressions were significantly up-regulated after lipopolysaccharide and poly I:C treatment. The ScIPS-1 protein level was up-regulated at 12 h in the head kidney and was up-regulated at 12 h and then down-regulated from 12 to 48 h in the liver after grass carp reovirus (GCRV) infection. The CiIFN and CiMx transcription levels were significantly enhanced in pEGFP-C1-IPS-1 and pcDNA3.1-ΔCwf21 overexpressing cells after GCRV infection. The results indicate that ScIPS-1 may function in the immune response against pathogens and provide a basis for achieving resistance to diseases in fish breeding.

Oligomerization of RIG-I and MDA5 2CARD domains.

The innate immune system is the first line of defense against invading pathogens. The retinoic acid-inducible gene I (RIG-I) like receptors (RLRs), RIG-I and melanoma differentiation-associated protein 5 (MDA5), are critical for host recognition of viral RNAs. These receptors contain a pair of N-terminal tandem caspase activation and recruitment domains (2CARD), an SF2 helicase core domain, and a C-terminal regulatory domain. Upon RLR activation, 2CARD associates with the CARD domain of MAVS, leading to the oligomerization of MAVS, downstream signaling and interferon induction. Unanchored K63-linked polyubiquitin chains (polyUb) interacts with the 2CARD domain, and in the case of RIG-I, induce tetramer formation. However, the nature of the MDA5 2CARD signaling complex is not known. We have used sedimentation velocity analytical ultracentrifugation to compare MDA5 2CARD and RIG-I 2CARD binding to polyUb and to characterize the assembly of MDA5 2CARD oligomers in the absence of polyUb. Multi-signal sedimentation velocity analysis indicates that Ub4 binds to RIG-I 2CARD with a 3:4 stoichiometry and cooperatively induces formation of an RIG-I 2CARD tetramer. In contrast, Ub4 and Ub7 interact with MDA5 2CARD weakly and form complexes with 1:1 and 2:1 stoichiometries but do not induce 2CARD oligomerization. In the absence of polyUb, MDA5 2CARD self-associates to forms large oligomers in a concentration-dependent manner. Thus, RIG-I and MDA5 2CARD assembly processes are distinct. MDA5 2CARD concentration-dependent self-association, rather than polyUb binding, drives oligomerization and MDA5 2CARD forms oligomers larger than tetramer. We propose a mechanism where MDA5 2CARD oligomers, rather than a stable tetramer, function to nucleate MAVS polymerization.

Identification of a novel RIG-I isoform and its truncating variant in Japanese eel, Anguilla japonica

Retinoic acid-inducible gene-I (RIG-I) is a cytoplasmic viral RNA sensor that triggers the production of type I interferons (IFNs) and proinflammatory cytokines during viral infection. RIG-I gene has been identified previously in Japanese eel, Anguilla japonica. In the present study, we have characterized a novel isoform of RIG-I (designated as AjRIG-Ib) and its truncated variant (AjRIG-Ibv). The AjRIG-Ib encodes 940 amino acids (aa) consisting of two N-terminal caspase activation and recruitment domains (CARDs), a DEX(D/H) box RNA helicase domain, and a C-terminal regulatory domain (CTD). The AjRIG-Ibv encodes a protein of 843 aa, that shares similar structural organization with AjRIG-Ib, but lacking CTD. The gene expression analyses showed that AjRIG-Ib and AjRIG-Ibv were detectable in all tissues/organs examined, and AjRIG-Ib was the predominant form. The mRNA level of AjRIG-Ibv was upregulated rapidly at 8 h after the Poly I:C injection, and the significant increase of AjRIG-Ib was observed at 16 and 24 h post-injection (hpi). Laser confocal microscopy showed that AjRIG-Ib and AjRIG-Ibv were both located in cytoplasm. In addition, the overexpression of AjRIG-Ib or AjRIG-Ibv led to the increased activity of IFN promoter in transient transfection assay. Taken together, our results indicated that AjRIG-Ib and AjRIG-Ibv may play cooperative or somewhat complementary roles in coordinating the antiviral response in fish.

Structural bioinformatics insights into the CARD-CARD interaction mediated by the mitochondrial antiviral-signaling protein of black carp.

The innate immune system offers the first line of defense against invading microbial pathogens through the recognition of conserved pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). The host innate immune system through PRRs, the sensors for PAMPs, induces the production of cytokines. Among different families of PRRs, the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), and its mitochondrial adaptor ie, the mitochondrial antiviral-signaling (MAVS) protein, are crucial for RLR-triggered interferon (IFN) antiviral immunity. Recent studies have shownthat the N-terminal caspase recruitment domain (CARD) and transmembrane domain playa pivotal role in oligomerization of black carp MAVS (BcMAVS), crucial for the host innate immune response against viral invasion. In this study, we have used molecular modeling, docking, and molecular dynamics (MD) simulation approaches to shed molecular insights into the oligomerization mechanism of BcMAVSCARD . MD simulation and interaction analysis portrayed that the type-I surface patches of BcMAVS CARD makethe major contributionto the interaction. Moreover, the evidence from surface patches and critical residues involved in the said interaction is found to be similar tothat of the human counterpart and requires further investigation for legitimacy. Altogether, our study provided crucial information on oligomerization of BcMAVS CARDs and might be helpful for clarifying the innate immune response against pathogens and downstream signaling in fishes.

Cloning, expression and bioinformatics analysis of a putative pigeon melanoma differentiation-associated gene 5

ABSTRACT1. Melanoma differentiation-associated gene 5 (MDA5) is a critical member of cytosolic pattern recognition receptors (PRRs) that recognise viral RNA and mediate type I interferon secretion in host cells.2. The objective of the present study was to identify and characterise the structure and expression of pigeon MDA5.3. The full-length MDA5 cDNA was cloned from pigeon spleen using RT-PCR and RACE. The distribution and expression level of pigeon MDA5 in different tissues were determined by QRT-PCR.4. The results showed that the full-length pigeon MDA5 cDNA had 3858 nucleotides (containing a 210-bp 5ʹ-UTR, a 3030-bp open reading frame and a 618-bp 3ʹ-UTR) encoding a polypeptide of 1009 amino acids. The deduced amino acid sequence contained six conserved structural domains typical of RIG-I-like receptor (RLR), including two tandem arranged N-terminal caspase activation and recruitment domains (CARDs), a DEAH/DEAD box helicase domain (DExDc), a helicase superfamily c-terminal domain (HELICc), a type III restriction enzyme (ResIII) and a C-terminal regulatory domain (RD).5. The pigeon MDA5 showed 84.8%, 87.3%, 87.9% and 87.2% amino acid sequence identities with previously described homologues from chicken, duck, goose and Muscovy ducks, respectively, and phylogenetic analysis revealed a close relationship among these MDA5.6. Pigeon MDA5 transcript was ubiquitously expressed in all seven tissues tested in healthy pigeons and showed a high level in the thymus gland and kidney.7. These findings lay the foundation for further research on the function and mechanism of MDA5 in innate immune responses related to vaccinations and infectious diseases in the pigeon.

Cloning, expression, and bioinformatics analysis of a putative pigeon retinoid acid-inducible gene-I

Retinoid acid-inducible gene-I (RIG-I) is a major cytoplasmic RNA sensor, playing an essential role in detecting viral RNA and triggering antiviral innate immune responses. The objective of the present study was to characterize the structure and expression of the RIG-I gene in pigeons. The pigeon RIG-I (piRIG-I) was cloned from splenic lymphocytes of pigeons using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. The cDNA of piRIG-I contains a 147 bp 5′-untranslated regions (UTRs), a 2787 bp open reading frame, and 2962 bp 3′-UTRs. Based on this sequence, the encoded piRIG-I protein is predicted to consist of 928 amino acids, and it has conserved domains typical of RIG-I-like receptors (RLRs) including two tandem arranged N-terminal caspase recruitment domains, a domain with the signature of DExD/H box helicase (helicase domain), and a C-terminal repression domain similar to finch RIG-I, duck RIG-I, goose RIG-I, human RIG-I, and mouse RIG-I. The piRIG-I shows 82.1%, 78.6%, and 78.2% amino acid sequence identity with previously described finch RIG-I, duck RIG-I, and goose RIG-I, respectively, and 49.7%–53.8% sequence identity with mammalian homologs. Quantitative RT-PCR (qRT-PCR) analysis indicated that the piRIG-I mRNA is scarcely detected in healthy tissues, and it is strongly expressed in the thymus gland, kidney, spleen, and bursa of Fabricius. These findings lay the foundation for further research on the function and mechanism of avian RIG-I in innate immune response related to vaccinations and infectious diseases in the pigeon.

Zika Virus Non-structural Protein 4A Blocks the RLR-MAVS Signaling.

Flaviviruses have evolved complex mechanisms to evade the mammalian host immune systems including the RIG-I (retinoic acid-inducible gene I) like receptor (RLR) signaling. Zika virus (ZIKV) is a re-emerging flavivirus that is associated with severe neonatal microcephaly and adult Guillain-Barre syndrome. However, the molecular mechanisms underlying ZIKV pathogenesis remain poorly defined. Here we report that ZIKV non-structural protein 4A (NS4A) impairs the RLR-mitochondrial antiviral-signaling protein (MAVS) interaction and subsequent induction of antiviral immune responses. In human trophoblasts, both RIG-I and melanoma differentiation-associated protein 5 (MDA5) contribute to type I interferon (IFN) induction and control ZIKV replication. Type I IFN induction by ZIKV is almost completely abolished in MAVS-/- cells. NS4A represses RLR-, but not Toll-like receptor-mediated immune responses. NS4A specifically binds the N-terminal caspase activation and recruitment domain (CARD) of MAVS and thus blocks its accessibility by RLRs. Our study provides in-depth understanding of the molecular mechanisms of immune evasion by ZIKV and its pathogenesis.

Characterization of Rabbit Nucleotide-Binding Oligomerization Domain 1 (NOD1) and the Role of NOD1 Signaling Pathway during Bacterial Infection.

Nucleotide-binding oligomerization domain 1 (NOD1) is the most prominent of all NOD-like receptors, which in the mammalian innate immune system, serve as intracellular receptors for pathogens and endogenous molecules during tissue injury. From rabbit kidney cells, we cloned rabbit NOD1 (rNOD1) and identified an N-terminal caspase activation and recruitment domain, a central NACHT domain, and C-terminal leucine-rich repeat domains. rNOD1 was expressed in all tested tissues; infection with Escherichia coli induced significantly higher expression in the spleen, liver, and kidney compared to other tissues. The overexpression of rNOD1 induced the expression of proinflammatory cytokines Il1b, Il6, Il8, Ifn-γ, and Tnf and defensins, including Defb124, Defb125, Defb128, Defb135, and Np5 via activation of the nuclear factor (NF)-κB pathway. Overexpression of rNOD1 inhibited the growth of E. coli, whereas knockdown of rNOD1 or inhibition of the NF-κB pathway promoted the growth of E. coli. rNOD1 colocalized with LC3, upregulated autophagy pathway protein LC3-II, and increased autolysosome formation in RK-13 cells infected with E. coli. In summary, our results explain the primary signaling pathway and antibacterial ability of rNOD1, as well as the induction of autophagy that it mediates. Such findings suggest that NOD1 could contribute to therapeutic strategies such as targets of new vaccine adjuvants or drugs.

Zyxin stabilizes RIG-I and MAVS interactions and promotes type I interferon response

RIG-I and MDA5 are cytoplasmic viral RNA sensors that belong to the RIG-I-like receptors (RLRs), which induce antiviral innate immune responses, including the production of type I interferon and other pro-inflammatory cytokines. After recognition of viral RNA, the N-terminal caspase activation and recruitment domains (CARDs) of RIG-I and MDA5 bind to a CARD in the MAVS adaptor molecule, resulting in MAVS oligomerization and downstream signaling. To reveal the molecular mechanism of MAVS-dependent signaling, we performed a yeast two-hybrid screening and identified zyxin as a protein that binds to MAVS. Zyxin co-immunoprecipitated with MAVS in human cells. A proximity ligation assay showed that zyxin and MAVS partly co-localized on mitochondria. Ectopic expression of zyxin augmented MAVS-mediated IFN-β promoter activation, and knockdown of zyxin (ZYX) attenuated the IFN-β promoter activation. Moreover, ZYX knockdown reduced the expression of type I IFN and an interferon-inducible gene after stimulation with polyI:C or influenza A virus RNA. Interestingly, physical interactions between RLRs and MAVS were abrogated by ZYX knockdown. These observations indicate that zyxin serves as a scaffold for the interactions between RLRs and MAVS.