Fluorescence Complementation System Research Articles

Gossypium hirsutum calmodulin-like protein (CML 11) interaction with geminivirus encoded protein using bioinformatics and molecular techniques

Plant viruses are the most abundant destructive agents that exist in every ecosystem, causing severe diseases in multiple crops worldwide. Currently, a major gap is present in computational biology determining plant viruses interaction with its host. We lay out a strategy to extract virus-host protein interactions using various protein binding and interface methods for Geminiviridae, a second largest virus family. Using this approach, transcriptional activator protein (TrAP/C2) encoded by Cotton leaf curl Kokhran virus (CLCuKoV) and Cotton leaf curl Multan virus (CLCuMV) showed strong binding affinity with calmodulin-like (CML) protein of Gossypium hirsutum (Gh-CML11). Higher negative value for the change in Gibbs free energy between TrAP and Gh-CML11 indicated strong binding affinity. Consensus from gene ontology database and in-silico nuclear localization signal (NLS) tools identified subcellular localization of TrAP in the nucleus associated with Gh-CML11 for virus infection. Data based on interaction prediction and docking methods present evidences that full length and truncated C2 strongly binds with Gh-CML11. This computational data was further validated with molecular results collected from yeast two-hybrid, bimolecular fluorescence complementation system and pull down assay. In this work, we also show the outcomes of full length and truncated TrAP on plant machinery. This is a first extensive report to delineate a role of CML protein from cotton with begomoviruses encoded transcription activator protein.

Two zinc finger proteins, VdZFP1 and VdZFP2, interact with VdCmr1 to promote melanized microsclerotia development and stress tolerance in Verticillium dahliae

BackgroundMelanin plays important roles in morphological development, survival, host–pathogen interactions and in the virulence of phytopathogenic fungi. In Verticillum dahliae, increases in melanin are recognized as markers of maturation of microsclerotia which ensures the long-term survival and stress tolerance, while decreases in melanin are correlated with increased hyphal growth in the host. The conserved upstream components of the VdCmr1-regulated pathway controlling melanin production in V. dahliae have been extensively identified, but the direct activators of this pathway are still unclear.ResultsWe identified two genes encoding conserved C2H2-type zinc finger proteins VdZFP1 and VdZFP2 adjacent to VdPKS9, a gene encoding a negative regulator of both melanin biosynthesis and microsclerotia formation in V. dahliae. Both VdZFP1 and VdZFP2 were induced during microsclerotia development and were involved in melanin deposition. Their localization changed from cytoplasmic to nuclear in response to osmotic pressure. VdZFP1 and VdZFP2 act as modulators of microsclerotia melanization in V. dahliae, as confirmed by melanin biosynthesis inhibition and supplementation with the melanin pathway intermediate scytalone in albino strains. The results indicate that VdZFP1 and VdZFP2 participate in melanin biosynthesis by positively regulating VdCmr1. Based on the results obtained with yeast one- and two-hybrid (Y1H and Y2H) and bimolecular fluorescence complementation (BiFC) systems, we determined the melanin biosynthesis relies on the direct interactions among VdZFP1, VdZFP2 and VdCmr1, and these interactions occur on the cell walls of microsclerotia. Additionally, VdZFP1 and/or VdZFP2 mutants displayed increased sensitivity to stress factors rather than alterations in pathogenicity, reflecting the importance of melanin in stress tolerance of V. dahliae.ConclusionsOur results revealed that VdZFP1 and VdZFP2 positively regulate VdCmr1 to promote melanin deposition during microsclerotia development, providing novel insight into the regulation of melanin biosynthesis in V. dahliae.

IGF2BP3 Enhances the Growth of Hepatocellular Carcinoma Tumors by Regulating the Properties of Macrophages and CD8+ T Cells in the Tumor Microenvironment.

Overexpression of IGF2BP3 is associated with the prognosis of hepatocellular carcinoma (HCC). However, its role in regulating tumor immune microenvironment (TME) is not well characterized. Here, we investigated the effects of IGF2BP3 on macrophages and CD8+ T cells within the TME of HCC. The relationship between IGF2BP3 and immune cell infiltration was analyzed using online bioinformatics tools. Knockout of IGF2BP3 in mouse hepatoma cell line Hepa1-6 was established using CRISPR/Cas9 technology. In vitro cell coculture and subcutaneously implanted hepatoma mice model were used to explore the effects of IGF2BP3 on immune cells. Expression of CCL5 or transforming growth factor beta 1 (TGF-β1) was detected with quantitative real-time polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay. The binding of IGF2BP3 and its target RNA was verified by trimolecular fluorescence complementation system and RNA immunoprecipitation followed by quantitative or semiquantitative polymerase chain reaction. IGF2BP3 expression was elevated in HCC and was positively correlated with macrophage infiltration. Patients with higher IGF2BP3 expression and lower macrophage infiltration had a better survival rate. We found that IGF2BP3 could bind to the mRNA of CCL5 or TGF-β1, increasing their expression, and inducing macrophage infiltration and M2 polarization while inhibiting the activation of CD8+ T cells. Furthermore, inhibition of IGF2BP3 combined with anti-CD47 antibody treatment significantly suppressed the growth of hepatoma in Hepa1-6 xenograft tumor mice. IGF2BP3 promoted the infiltration and M2-polarization of macrophages and suppressed CD8+ T activation by enhancing CCL5 and TGF-β1 expression, which facilitated the progression of Hepa1-6 xenograft tumor.

Megalin Orchestrates FcRn Endocytosis and Trafficking.

The neonatal Fc receptor (FcRn) is highly expressed in the renal proximal tubule and is important for the reclamation of albumin by cellular transcytosis to prevent its loss in the urine. The initial event of this transcellular transport mechanism is the endocytosis of albumin by the apical scavenger receptors megalin and cubilin. An interaction of megalin and FcRn was postulated, however, evidence is still missing. Similarly, the intracellular trafficking of FcRn remains unknown and shall be identified in our study. Using a Venus-based bimolecular fluorescence complementation system, we detected an interaction between megalin and FcRn in the endosomal compartment, which significantly increased with the induction of endocytosis using albumin or lactoglobulin as a ligand. The interaction between megalin and FcRn occurred at a neutral and acidic pH between the extracellular domains of both proteins. Amnionless, another transmembrane acceptor of cubilin, revealed no interaction with FcRn. With the induction of endocytosis by albumin or lactoglobulin, super resolution microscopy demonstrated a redistribution of megalin and FcRn into clathrin vesicles and early endosomes. This trafficking into clathrin vesicles was impaired in megalin-deficient cells upon albumin-induced endocytosis, supporting the role of megalin in FcRn redistribution. Our results indicate that megalin and FcRn specifically bind and interact within their extracellular domains. The availability of megalin is necessary for the redistribution of FcRn. Megalin, therefore, orchestrates FcRn endocytosis and intracellular trafficking as an early event intranscytosis.

Ebola and Marburg virus VP35 coiled-coil validated as antiviral target by tripartite split-GFP complementation

Ebola virus (EBOV) and Marburg virus (MARV) are highly pathogenic viruses in humans, against which approved antivirals are lacking. During EBOV and MARV infection, coiled-coil mediated oligomerization is essential for the virion protein 35 (VP35) polymerase co-factor function and type I interferon antagonism, making VP35 coiled-coil an elective drug target. We established a tripartite split-green fluorescent protein (GFP) fluorescence complementation (FC) system based on recombinant GFP-tagged EBOV and MARV VP35, which probes VP35 coiled-coil assembly by monitoring fluorescence on E.coli colonies, or invitro in 96/384-multiwell. Oligomerization-defective VP35 mutants showed that correct coiled-coil knobs-into-holes pairing within VP35 oligomer is pre-requisite for GFP tags and GFP detector to reconstitute fluorescing full-length GFP. The method was validated by screening a small compound library, which identified Myricetin and 4,5,6,7-Tetrabromobenzotriazole as inhibitors of EBOV and MARV VP35 oligomerization-dependent FC with low-micromolar IC50 values. These findings substantiate the VP35 coiled-coil value as antiviral target.

A Bright Monomeric Near-Infrared Fluorescent Protein with an Excitation Peak at 633 nm for Labeling Cellular Protein and Reporting Protein-Protein Interaction.

Bright monomeric near-infrared fluorescent proteins (NIR-FPs) are useful as markers for labeling proteins and cells and as sensors for reporting molecular activities in living cells and organisms. However, current monomeric NIR-FPs are dim under excitation with common 633/635/640 nm lasers, limiting their broad use in cellular/subcellular level imaging. Here, we report a bright monomeric NIR-FP with maximum excitation at 633 nm, named mIFP663, engineered from Xanthomonas campestris pv Campestris phytochrome (XccBphP). mIFP663 has high molecular brightness with a large extinction coefficient (86,600 M-1 cm-1) and a decent quantum yield (19.4%), and high cellular brightness that is 3-6 times greater than those of spectrally similar NIR-FPs in HEK293T cells in the presence of exogenous BV. Moreover, we demonstrate that mIFP663 is able to label critical cellular and viral proteins without perturbing subcellular localization and virus replication, respectively. Finally, with mIFP663, we engineer improved bimolecular fluorescence complementation (BiFC) and new bioluminescent resonance energy transfer (BRET) systems to detect protein-protein interactions in living cells.

The smallest near-infrared fluorescence complementation system for imaging protein-protein and RNA-protein interactions.

Bimolecular fluorescence complementation (BiFC) and its derivative molecular biosensor systems provide effective tools for visualizing biomolecular interactions. The introduction of red and near-infrared fluorescence emission proteins has expanded the spectrum of signal generating modules, enabling BiFC for in vivo imaging. However, the large size of the signal module of BiFC can hinder the interaction between proteins under investigation. In this study, we constructed the near-infrared BiFC and TriFC systems by splitting miRFP670nano, the smallest cyanobacteriochrome-evolved phytochrome available. The miRFP670nano-BiFC sensor system identified and enabled visualization of protein–protein interactions in living cells and live mice, and afforded a faster maturation rate and higher photostability and cellular stability when compared with those of reported near-infrared BiFC systems. We used the miRFP670nano-BiFC sensor system to identify interactions between the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and cellular stress granule proteins in living cells and found that the N protein downregulated the expression level of granule protein G3BP1. With the advantages of small size and long wavelength emission of the signal module, the proposed molecular biosensor system should be suitable for various applications in cell imaging studies.

Grass Carp Reovirus VP56 Allies VP4, Recruits, Blocks, and Degrades RIG-I to More Effectively Attenuate IFN Responses and Facilitate Viral Evasion.

ABSTRACTGrass carp reovirus (GCRV), the most virulent aquareovirus, causes epidemic hemorrhagic disease and tremendous economic loss in freshwater aquaculture industry. VP56, a putative fibrin inlaying the outer surface of GCRV-II and GCRV-III, is involved in cell attachment. In the present study, we found that VP56 localizes at the early endosome, lysosome, and endoplasmic reticulum, recruits the cytoplasmic viral RNA sensor retinoic acid-inducible gene I (RIG-I) and binds to it. The interaction between VP56 and RIG-I was detected by endogenous coimmunoprecipitation (co-IP), glutathione S-transferase (GST) pulldown, and subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) and was then confirmed by traditional co-IPs and a novel far-red mNeptune-based bimolecular fluorescence complementation system. VP56 binds to the helicase domain of RIG-I. VP56 enhances K48-linked ubiquitination of RIG-I to degrade it by the proteasomal pathway. Thus, VP56 impedes the initial immune function of RIG-I by dual mechanisms (blockade and degradation) and attenuates signaling from RIG-I recognizing viral RNA, subsequently weakening downstream signaling transduction and interferon (IFN) responses. Accordingly, host antiviral effectors are reduced, and cytopathic effects are increased. These findings were corroborated by RNA sequencing (RNA-seq) and VP56 knockdown. Finally, we found that VP56 and the major outer capsid protein VP4 bind together in the cytosol to enhance the degradation of RIG-I and more efficiently facilitate viral replication. Collectively, the results indicated that VP56 allies VP4, recruits, blocks, and degrades RIG-I, thereby attenuating IFNs and antiviral effectors to facilitate viral evasion more effectively. This study reveals a virus attacking target and an escaping strategy from host antiviral immunity for GCRV and will help understand mechanisms of infection of reoviruses.IMPORTANCE Grass carp reovirus (GCRV) fibrin VP56 and major outer capsid protein VP4 inlay and locate on the outer surface of GCRV-II and GCRV-III, which causes tremendous loss in grass carp and black carp industries. Fibrin is involved in cell attachment and plays an important role in reovirus infection. The present study identified the interaction proteins of VP56 and found that VP56 and VP4 bind to the different domains of the viral RNA sensor retinoic acid-inducible gene I (RIG-I) in grass carp to block RIG-I sensing of viral RNA and induce RIG-I degradation by the proteasomal pathway to attenuate signaling transduction, thereby suppressing interferons (IFNs) and antiviral effectors, facilitating viral replication. VP56 and VP4 bind together in the cytosol to more efficiently facilitate viral evasion. This study reveals a virus attacking a target and an escaping strategy from host antiviral immunity for GCRV and will be helpful in understanding the mechanisms of infection of reoviruses.

Long noncoding RNA HOTAIR interacts with Y-Box Protein-1 (YBX1) to regulate cell proliferation.

HOTAIR is a long noncoding RNA (lncRNA) which serves as an important factor regulating diverse processes linked with cancer development. Here, we used comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) to explore the HOTAIR-protein interactome. We were able to identify 348 proteins interacting with HOTAIR, allowing us to establish a heavily interconnected HOTAIR-protein interaction network. We further developed a novel near-infrared fluorescent protein (iRFP)-trimolecular fluorescence complementation (TriFC) system to assess the interaction between HOTAIR and its interacting proteins. Then, we determined that HOTAIR specifically binds to YBX1, promotes YBX1 nuclear translocation, and stimulates the PI3K/Akt and ERK/RSK signaling pathways. We further demonstrated that HOTAIR exerts its effects on cell proliferation, at least in part, through the regulation of two YBX1 downstream targets phosphoenolpyruvate carboxykinase 2 (PCK2) and platelet derived growth factor receptor β. Our findings revealed a novel mechanism, whereby an lncRNA is able to regulate cell proliferation via altering intracellular protein localization. Moreover, the imaging tools developed herein have excellent potential for future in vivo imaging of lncRNA-protein interaction.

Nanoscale Imaging of RNA-Protein Interactions with a Photoactivatable Trimolecular Fluorescence Complementation System.

Imaging RNA-protein interaction in the cellular space with single molecule sensitivity is attractive for studying gene expression and regulation, but remains a challenge. In this study, we reported a photoactivatable trimolecular fluorescence complementation (TriFC) system based on fluorescent protein, mIrisFP, to identify and visualize RNA-protein interactions in living mammalian cells. We also combined this TriFC system with photoactivated localization microscopy (PALM), named the TriFC-PALM technique, which allowed us to image the RNA-protein interactions with single molecule sensitivity. Using this TriFC-PALM technique, we identified the actin-bundling protein, FSCN1, specifically interacting with the HOX Transcript Antisense RNA (HOTAIR). The TriFC-PALM imaging acquired a higher resolution compared with the traditional method of total internal reflection (TIRF) imaging. The TriFC-PALM thus provides a useful tool for imaging and identifying the RNA-protein interactions inside cells at the nanometer scale.

A tandem near-infrared fluorescence complementation system with enhanced fluorescence for imaging protein–protein interactions in vivo

Bimolecular fluorescence complementation (BiFC) is an effective tool for visualizing protein–protein interactions (PPIs). However, a BiFC system with long wavelength and high fluorescence intensity is yet to be developed for in vivo imaging. In this study, we constructed a tandem near-infrared BiFC (tBiFC) system by splitting a near-infrared phytochrome, IFP2.0. This system allows the identification and visualization of PPIs in live cells and living mice. The photophysical properties of the complementary fluorescence of the tBiFC system were similar to those of its parent protein IFP2.0, but the intensity was twice that of a single-copy IFP2.0-based BiFC system. Compared with previously reported near infrared BiFC systems—iRFP-BiFC and IFP1.4-BiFC—the signal intensity of the tBiFC system increased by ~1.48- and ~400-fold for weak PPIs in living cells, and ~1.51- and ~8-fold for strong PPIs, respectively. When applied to imaging PPIs in live mice, the complementary fluorescence intensity of the tBiFC system was also significantly higher than that of the other near-infrared BiFC systems. The use of this bright phytochrome in a tandem arrangement constitutes a powerful tool for imaging PPIs in the near infrared region.

Grass Carp Reovirus Major Outer Capsid Protein VP4 Interacts with RNA Sensor RIG-I to Suppress Interferon Response.

Diseases caused by viruses threaten the production industry and food safety of aquaculture which is a great animal protein source. Grass carp reovirus (GCRV) has caused tremendous loss, and the molecular function of viral proteins during infection needs further research, as for most aquatic viruses. In this study, interaction between GCRV major outer capsid protein VP4 and RIG-I, a critical viral RNA sensor, was screened out by GST pull-down, endogenous immunoprecipitation and subsequent LC-MS/MS, and then verified by co-IP and an advanced far-red fluorescence complementation system. VP4 was proved to bind to the CARD and RD domains of RIG-I and promoted K48-linked ubiquitination of RIG-I to degrade RIG-I. VP4 reduced mRNA and promoter activities of key genes of RLR pathway and sequential IFN production. As a consequence, antiviral effectors were suppressed and GCRV replication increased, resulting in intensified cytopathic effect. Furthermore, results of transcriptome sequencing of VP4 stably expressed CIK (C. idella kidney) cells indicated that VP4 activated the MyD88-dependent TLR pathway. Knockdown of VP4 obtained opposite effects. These results collectively revealed that VP4 interacts with RIG-I to restrain interferon response and assist GCRV invasion. This study lays the foundation for anti-dsRNA virus molecular function research in teleost and provides a novel insight into the strategy of immune evasion for aquatic virus.

βC1, pathogenicity determinant encoded by Cotton leaf curl Multan betasatellite, interacts with calmodulin-like protein 11 (Gh-CML11) inGossypium hirsutum.

Begomoviruses interfere with host plant machinery to evade host defense mechanism by interacting with plant proteins. In the old world, this group of viruses are usually associated with betasatellite that induces severe disease symptoms by encoding a protein, βC1, which is a pathogenicity determinant. Here, we show that βC1 encoded by Cotton leaf curl Multan betasatellite (CLCuMB) requires Gossypium hirsutum calmodulin-like protein 11 (Gh-CML11) to infect cotton. First, we used the in silico approach to predict the interaction of CLCuMB-βC1 with Gh-CML11. A number of sequence- and structure-based in-silico interaction prediction techniques suggested a strong putative binding of CLCuMB-βC1 with Gh-CML11 in a Ca+2-dependent manner. In-silico interaction prediction was then confirmed by three different experimental approaches: The Gh-CML11 interaction was confirmed using CLCuMB-βC1 in a yeast two hybrid system and pull down assay. These results were further validated using bimolecular fluorescence complementation system showing the interaction in cytoplasmic veins of Nicotiana benthamiana. Bioinformatics and molecular studies suggested that CLCuMB-βC1 induces the overexpression of Gh-CML11 protein and ultimately provides calcium as a nutrient source for virus movement and transmission. This is the first comprehensive study on the interaction between CLCuMB-βC1 and Gh-CML11 proteins which provided insights into our understating of the role of βC1 in cotton leaf curl disease.

A split fluorescent reporter with rapid and reversible complementation

Interactions between proteins play an essential role in metabolic and signaling pathways, cellular processes and organismal systems. We report the development of splitFAST, a fluorescence complementation system for the visualization of transient protein-protein interactions in living cells. Engineered from the fluorogenic reporter FAST (Fluorescence-Activating and absorption-Shifting Tag), which specifically and reversibly binds fluorogenic hydroxybenzylidene rhodanine (HBR) analogs, splitFAST displays rapid and reversible complementation, allowing the real-time visualization of both the formation and the dissociation of a protein assembly.

Investigating the Role of BATF3 in Grass Carp (Ctenopharyngodon idella) Immune Modulation: A Fundamental Functional Analysis.

Basic leucine zipper transcription factor ATF-like (BATF)-3, belonging to activator protein 1 (AP-1) superfamily transcription factors, is essential for homeostatic development of CD8α+ classical dendritic cells activating CD8 T-cell responses to intracellular pathogens. In this study, the characteristics and cDNA cloning of the CiBATF3 molecule were described in grass carp (Ctenopharyngodon idella). CiBATF3 had abundant expression in immune-related organizations, including liver, spleen and gill, and grass carp reovirus (GCRV) infection had significantly changed its expression level. After Ctenopharyngodon idella kidney (CIK) cells were challenged with pathogen-associated molecular patterns (PAMPs), polyinosinic:polycytidylic acid (poly(I:C)) stimulation induced higher mRNA levels of CiBATF3 than that of lipopolysaccharide (LPS). Subcellular localization showed that CiBATF3-GFP was entirely distributed throughout cells and nuclear translocation of CiBATF3 was found after poly(I:C) treatment. Additionally, the interaction between CiBATF3 and interleukin 10 (IL-10) was proven by bimolecular fluorescence complementation (BiFC) system. The small interfering RNA (siRNA)-mediated CiBATF3 silencing showed that the mRNA of CiBATF3 and its downstream genes were down-regulated in vitro and in vivo. CiBATF3 played a negative regulatory role in the transcriptional activities of AP-1 and NF-κB reporter gene. In summary, the results may provide valuable information on fundamental functional mechanisms of CiBATF3.

Characterisation and function of TRIM23 in grass carp (Ctenopharyngodon idella)

Tripartite motif (TRIM) proteins are key components of the innate immune system, functioning as antiviral restriction factors or modulating signaling cascades that lead to proinflammatory cytokine induction. In the present study, the TRIM family gene TRIM23 from grass carp (Ctenopharyngodon idella) was cloned and characterised. TRIM23 was moderately expressed in the examined tissues, and the significantly altered expression was observed after grass carp reovirus (GCRV) and poly(I:C) infection. Dual-luciferase activity assay showed that TRIM23, especially its C-terminal domain ARF, depressed the promoter activity of IRF3 and IRF7. The subcellular localisation showed that TRIM23 protein was located in the cytoplasm and could be recruited by both TRAF6 and MyD88. Furthermore, TRIM23 was confirmed to interact with either TRAF6 or MyD88 by the bimolecular fluorescence complementation (BiFC) system in CIK cells. Additionally, autophagy was enhanced by over-expressed TRIM23 in 293T cells. Taken together, our results demonstrate that TRIM23 gene plays an important role in innate immune regulation and provide new insights into understanding the functional characteristics of the TRIM23 in teleosts.

Trimolecular Fluorescence Complementation (TriFC) Assay for Visualization of RNA-Protein Interaction in Plants.

RNA-protein interactions play important roles in various eukaryotic biological processes. Molecular imaging of subcellular localization of RNA-protein complexes in plants is critical for understanding these interactions. However, methods to image RNA-protein interactions in living plants have not yet been developed until now. Recently, we have developed a trimolecular fluorescence complementation (TriFC) system for in vivo visualization of RNA-protein interaction by transient expression in tobacco leaves. In this method, we combined conventional bimolecular fluorescence complementation (BiFC) system with the MS2 system (phage MS2 coat protein [MCP] and its binding RNA sequence [MS2 sequence]) to tag lncRNA. Target RNA is tagged with 6xMS2, and MCP and RNA-binding protein are fused with YFP fragments. DNA constructs encoding such fusion RNA and proteins are infiltrated into tobacco leaves with Agrobacterium suspensions. RNA-protein interaction in vivo is observed by confocal microscopy.

Identification, characterisation and preliminary functional analysis of IRAK-M in grass carp (Ctenopharyngodon idella)

Interleukin-1 receptor-associated kinase (IRAK) family members play important roles in myeloid differentiation primary response 88 (MyD88)-dependent toll-like receptor (TLR) signaling, the crucial innate immune pathway in vertebrates. In the present study, the IRAK family gene IRAK-M (also called IRAK3) from grass carp (Ctenopharyngodon idella) was cloned and characterised. IRAK-M was mainly enriched in the spleen, and the significantly altered expression was observed after grass carp reovirus (GCRV) infection. Subcellular localisation showed that IRAK-M protein distributed uniformly in the entire cell and co-localised with MyD88 in the cytoplasm of transfected cells. Additionally, the interaction between IRAK-M and MyD88 was confirmed by bimolecular fluorescence complementation (BiFC) system. Moreover, deficient of IRAK-M in C. idella kidney cell line (CIK) with small interference RNA (siRNA) upregulated polyinosinic:polycytidylic acid (poly(I:C))-induced inflammatory cytokines production, including interleukin 8 (IL-8), IL-6, and tumour necrosis factor α (TNF-α), which reveals that IRAK-M functions as a negative regulator of inflammatory cytokines. Taken together, our results demonstrate that IRAK-M gene plays an important role in innate immune regulation and provide new insights into understanding the functional characteristics of the IRAK-M in teleosts.

Molecular cloning and functional characterisation of NLRX1 in grass carp (Ctenopharyngodon idella)

The nucleotide-binding domain and leucine-rich-repeat-containing (NLR) proteins regulate innate immunity. Although the positive regulatory impact of NLRs is clear, their inhibitory roles are not well defined. In the present study, the NLR family gene NLRX1 from grass carp (Ctenopharyngodon idella) was cloned and characterised. NLRX1 was widely expressed in all tissues examined, albeit at varying levels. After exposure to the grass carp reovirus (GCRV), NLRX1 mRNA expression levels were altered in immune organs, and dramatically altered in liver. Subcellular localisation indicated that NLRX1 protein co-localised with the mitochondria in the transfected cells. Additionally, the bimolecular fluorescence complementation (BiFC) system was introduced to detect the interaction between tumour necrosis factor (TNF) receptor associated factor 6 (TRAF6) and NLRX1. Moreover, deficient of NLRX1 in CIK cells with small interference RNA (siRNA) promoted polyinosinic:polycytidylic acid (poly (I:C))-induced IFN-related genes production, including IRF3, IRF7, and IFN-I, which reveals that NLRX1 is a negative regulator of IFN. Taken together, our results demonstrate that NLRX1 gene plays an important role in innate immune regulation and provide new insights into understanding the functional characteristics of the NLRX1 in teleosts.

Dimer-specific immunoprecipitation of active caspase-2 identifies TRAF proteins as novel activators.

Caspase‐2 has been shown to initiate apoptotic cell death in response to specific intracellular stressors such as DNA damage. However, the molecular mechanisms immediately upstream of its activation are still poorly understood. We combined a caspase‐2 bimolecular fluorescence complementation (BiFC) system with fluorophore‐specific immunoprecipitation to isolate and study the active caspase‐2 dimer and its interactome. Using this technique, we found that tumor necrosis factor receptor‐associated factor 2 (TRAF2), as well as TRAF1 and 3, directly binds to the active caspase‐2 dimer. TRAF2 in particular is necessary for caspase‐2 activation in response to apoptotic cell death stimuli. Furthermore, we found that dimerized caspase‐2 is ubiquitylated in a TRAF2‐dependent manner at K15, K152, and K153, which in turn stabilizes the active caspase‐2 dimer complex, promotes its association with an insoluble cellular fraction, and enhances its activity to fully commit the cell to apoptosis. Together, these data indicate that TRAF2 positively regulates caspase‐2 activation and consequent cell death by driving its activation through dimer‐stabilizing ubiquitylation.