dsRNA-dependent Protein Kinase Research Articles

Salmonid Double-stranded RNA-Dependent Protein Kinase Activates Apoptosis and Inhibits Protein Synthesis.

dsRNA-dependent protein kinase R (PKR) is a key factor of innate immunity. It is involved in translation inhibition, apoptosis, and enhancement of the proinflammatory and IFN responses. However, how these antiviral functions are conserved during evolution remains largely unknown. Overexpression and knockout studies in a Chinook salmon (Oncorhynchus tshawytscha) cell line were conducted to assess the role of salmonid PKR in the antiviral response. Three distinct mRNA isoforms from a unique pkr gene, named pkr-fl (full length), pkr-ml (medium length) and pkr-sl (short length), were cloned and a pkr-/- clonal fish cell line was developed using CRISPR/Cas9 genome editing. PKR-FL includes an N-terminal dsRNA-binding domain and a C-terminal kinase domain, whereas PKR-ML and PKR-SL display a truncated or absent kinase domain, respectively. PKR-FL is induced during IFNA2 stimulation but not during viral hemorrhagic septicemia virus (VHSV) infection. Overexpression experiments showed that only PKR-FL possesses antiviral functions, including activation of apoptosis and inhibition of de novo protein synthesis. Knockout experiments confirmed that PKR is involved in apoptosis activation during the late stage of VHSV infection. Endogenous PKR also plays a critical role in translation inhibition upon poly(I:C) transfection after IFNA2 treatment. It is, however, not involved in translational arrest during VHSV infection. Extra- and intracellular titrations showed that endogenous PKR does not directly inhibit viral replication but apparently favors virion release into the supernatant, likely by triggering late apoptosis. Altogether, our data confirm that salmonid PKR has conserved molecular functions that VHSV appears to bypass with subversion strategies.

E3 ligase HECTD3 promotes RNA virus replication and virus-induced inflammation via K33-linked polyubiquitination of PKR

Uncontrolled viral replication and excessive inflammation are the main causes of death in the host infected with virus. Hence inhibition of intracellular viral replication and production of innate cytokines, which are the key strategies of hosts to fight virus infections, need to be finely tuned to eliminate viruses while avoid harmful inflammation. The E3 ligases in regulating virus replication and subsequent innate cytokines production remain to be fully characterized. Here we report that the deficiency of the E3 ubiquitin-protein ligase HECTD3 results in accelerated RNA virus clearance and reduced inflammatory response both in vitro and in vivo. Mechanistically, HECTD3 interacts with dsRNA-dependent protein kinase R (PKR) and mediates Lys33-linkage of PKR, which is the first non-proteolytic ubiquitin modification for PKR. This process disrupts the dimerization and phosphorylation of PKR and subsequent EIF2α activation, which results in the acceleration of virus replication, but promotes the formation of PKR-IKK complex and subsequent inflammatory response. The finding suggests HECTD3 is the potential therapeutic target for simultaneously restraining RNA virus replication and virus-induced inflammation once pharmacologically inhibited.

Structural study of novel vaccinia virus E3L and dsRNA-dependent protein kinase complex

E3L (RNA-binding protein E3) is one of the key IFN resistance genes encoded by VV and consists of 190 amino acids with a highly conserved carboxy-terminal double-stranded RNA-binding domain (dsRBD). PKR (dsRNA-dependent protein kinase) is an IFN-induced protein involved in anti-cell and antiviral activity. PKR inhibits the initiation of translation through alpha subunit of the initiation factor eIF2 (eIF2α) and mediates several transcription factors such as NF-κB, p53 or STATs. Activated PKR also induces apoptosis in vaccinia virus infection. E3L is required for viral IFN resistance and directly binds to PKR to block activation of PKR. In this work, we determined the three-dimensional complex structure of E3L and PKR using cryo-EM and determined the important residues involved in the interaction. In addition, PKR peptide binds to E3L and can increase protein levels of phosphorus-PKR and phosphorus-eIF2α-induced cell apoptosis through upregulation of phosphorus-PKR in HEK293 cells. Taken together, structural insights into E3L and PKR will provide a new optimization and development of vaccinia virus drugs.

Selective inhibition of PKR by C16 accelerates diabetic wound healing by inhibiting NALP3 expression in mice.

To understand the expression of dsRNA-dependent protein kinase R (PKR) in impaired diabetic wounds, hyperglycemia was induced in C57/BL6 mice with streptozotocin. Murine macrophage cell line, Raw 264.7, stimulated with high glucose and LPS was used to mimic diabetic wound environment in in-vitro. Macrophages stimulated with HG + LPS, in presence and absence of PKR inhibitor (C16) and wound tissue samples from topically treated mice with C16, were analyzed for the expression of PKR, NALP3, active caspase-1, mature IL-1β and phosphorylation of PKR and eIF2α. Wounds tissues were also analyzed for inflammatory cell infiltration by immunohistochemistry, angiogenesis by CD31 staining, collagen expression by western blotting, expression of CD206+ macrophages by flow cytometry and wound strength by texture analyzer. PKR and NALP3 were found to be upregulated in macrophages stimulated with HG + LPS as well as in impaired diabetic wounds. PKR inhibition using C16 ameliorated expression of NALP3, caspase-1, IL-1β and phosphorylation of PKR and eIF2α, in macrophages and also in diabetic wounds. Treatment with C16 promoted the wound healing in diabetic mice by increasing collagen synthesis, reducing infiltration of F4/80+ macrophages and MPO+ neutrophil cells, increased angiogenesis, and increased number of M2 macrophages. PKR inhibition using C16 accelerates the wound healing process in diabetic mice by decreasing NALP3-mediated IL-1β maturation.

PKR deficiency alleviates pulmonary hypertension via inducing inflammasome adaptor ASC inactivation

Pulmonary hypertension is a progressive fatal disease that currently has no specific therapeutic approaches. In this study, dsRNA-dependent protein kinase (PKR) was considered a candidate molecule in pulmonary hypertension. We demonstrated that PKR is activated in the endothelium of experimental pulmonary hypertension models. Deletion of PKR or treatment with the PKR activation inhibitor C16 inhibited the development of pulmonary hypertension. To explore the mechanism of PKR in pulmonary hypertension, we detected its downstream signaling and found that PKR knockout represses apoptosis-associated speck-like protein containing CARD (ASC) activation to inhibit high mobility group box 1 (HMGB1) and interleukin-1 beta release. To further explore whether ASC mediates the pro-pulmonary hypertension role of PKR, we used ASC deletion mice and found that ASC deletion inhibits the development of pulmonary hypertension and the release of HMGB1 and interleukin-1 beta. Furthermore, we co-cultured pulmonary arterial endothelial cells (PAECs) and pulmonary arterial smooth muscle cells (PASMCs) and found that endothelial PKR promotes PASMCs proliferation through the release of HMGB1 and interleukin-1 beta. In conclusion, these data indicate that endothelial PKR promotes the excessive proliferation of PASMCs by inducing ASC activation to release HMGB1 and interleukin-1 beta, which lead to the development of pulmonary hypertension. Our study will provide a novel insight that PKR is a potential target in the future treatment of pulmonary hypertension.

Association of Mutations in the NS5A-PKRBD Region and IFNL4 Genotypes with Hepatitis C Interferon Responsiveness and its Functional and Structural Analysis

Background: The cellular antiviral responses induced by interferons require some cellular protein kinase for its activation. Evidence indicated that a number of Hepatitis C Virus (HCV) proteins can repress double-stranded (ds) RNA-dependent Protein Kinase (PKR) function and help HCV to escape. However, the reports are controversial, some researchers have suggested that a region in Nonstructural 5A (NS5A) gene called Protein Kinase R-Binding Domain (PKR-BD) is associated with HCV sensitivity to the antiviral effects of Interferon (IFN). In addition, the other factor that might be associated with response to PEGylated-IFNα (Peg-IFNα) and Ribavirin (RBV) combination therapy, is IFNL4 genotypes. Objective: The aim of this study was to investigate the association between amino acid (aa) substitutions in the NS5A region and the IFNL4 genotypes in two Single Nucleotide Polymorphism (SNP) (rs8099917. rs12979860) in patients with HCV genotypes 1a and 3a. We also examined their response to combination therapy and the effect of these mutations on the function and structure of PKR-BD. Methods: Eighty-six patients with hepatitis C were recruited and follow-up for 6 months. Several tests, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), viral load, IFNL4 genotyping, and PKR-BD sequencing were performed. Using several well-known and trustworthy bioinformatics tools, sequences were analyzed to define physio-chemical properties, structural features, immune epitopes and protein-protein interaction. Results: Of the 86 patients, 65.1% had high viral load at baseline, 64% had CT genotype for rs12979860 and 57% had GT genotype for rs8099917. Several aa residues changes were found in the PKR-BD region. We could not find any link between mutations in the PKR-BD region and different genotypes of IFNL4 in response to antiviral therapy. Regardless of pI, PKR-BD 1a and 3a showed similar physio-chemical properties, and 2 phosphorylation sites and one glycosylation site were estimated for both PKR-BD 1a and 3a. Trustworthy software were employed in order to predict B-cell epitopes, 3 regions (6-17, 26-32, 34-41) were found for both proteins, indicating a huge potential of PKR-BD protein to induce humoral immune system. Docking analysis determined non-responder sequences in both 1a and 3a genotypes to have higher energy value and are more compatible with PKR. Conclusion: To sum up, our results could not determine any significant relationship between mutations of PKR-BD and genotypes of IFNL4 with other factors; ALT, AST, viral load. However, docking results showed strengthened interaction between PKR-BD and PKR in non-responders that could have a momentous impact on the illness severity.

Activation of dsRNA-Dependent Protein Kinase R by MicroRNA-378 Sustains Metabolic Inflammation in Hepatic Insulin Resistance.

MicroRNAs (miRNAs) are noncoding small RNAs that regulate various pathophysiological cellular processes. Here we reported that expression of the miR-378 family was significantly induced by metabolic inflammatory inducers, a high-fructose diet, and inflammatory cytokine TNFα. Hepatic miRNA profiling revealed that expression of miR-378a was highly upregulated which, in turn, targeted the 3'-UTR of PPARα mRNA, impaired mitochondrial fatty acid β-oxidation and induced mitochondrial and ER stress. More importantly, the upregulated miR-378a can directly bind to and activate the dsRNA-dependent protein kinase R (PKR) to sustain the metabolic stress. In vivo, genetic depletion of miR-378a prevented PKR activation, ameliorated inflammatory stress and insulin resistance. Counterbalancing the upregulated miR-378a using nanoparticles encapsulated with an anti-miR-378a oligonucleotide restored PPARα activity, inhibited PKR activation and ER stress, and improved insulin sensitivity in the fructose-fed mice. Conclusion: Our study delineated a novel mechanism of miRNA-378a in the pathogenesis of metabolic inflammation and insulin resistance through targeting metabolic signaling at both mRNA (e.g., PPARα) and protein (e.g., PKR) molecules. This novel finding of functional interaction between miRNAs (e.g., miR-378a) and cellular RNA binding protein(s) (e.g., PKR) is biologically significant as it greatly broadens the potential targets of miRNAs in cellular pathophysiological processes.

Activation of dsRNA-Dependent Protein Kinase R by miR-378 Sustains Metabolic Inflammation in Hepatic Insulin Resistance.

MicroRNAs (miRNAs) are noncoding small RNAs that regulate various pathophysiological cellular processes. Here, we report that expression of the miR-378 family was significantly induced by metabolic inflammatory inducers, a high-fructose diet, and inflammatory cytokine tumor necrosis factor-α. Hepatic miRNA profiling revealed that expression of miR-378a was highly upregulated, which, in turn, targeted the 3'-untranslated region of PPARα mRNA, impaired mitochondrial fatty acid β-oxidation, and induced mitochondrial and endoplasmic reticulum stress. More importantly, the upregulated miR-378a can directly bind to and activate the double-strand RNA (dsRNA)-dependent protein kinase R (PKR) to sustain the metabolic stress. In vivo, genetic depletion of miR-378a prevented PKR activation and ameliorated inflammatory stress and insulin resistance. Counterbalancing the upregulated miR-378a using nanoparticles encapsulated with an anti-miR-378a oligonucleotide restored PPARα activity, inhibited PKR activation and ER stress, and improved insulin sensitivity in fructose-fed mice. Our study delineated a novel mechanism of miR-378a in the pathogenesis of metabolic inflammation and insulin resistance through targeting metabolic signaling at both mRNA (e.g., PPARα) and protein (e.g., PKR) molecules. This novel finding of functional interaction between miRNAs (e.g., miR-378a) and cellular RNA binding proteins (e.g., PKR) is biologically significant because it greatly broadens the potential targets of miRNAs in cellular pathophysiological processes.

Antagonism of Type I Interferon by Severe Acute Respiratory Syndrome Coronavirus 2.

The coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), warranting urgent study of the molecular mechanisms of SARS-CoV-2 infection and host immune response. Type I interferon (IFN-I) is a key component of host innate immune system responsible for eliminating the virus at the early stage of infection. In contrast, SARS-CoV-2 has evolved multiple strategies to evade innate immune response to facilitate viral replication, transmission, and pathogenesis. This review summarizes the recent progresses on SARS-CoV-2 proteins that antagonize host IFN-I production and/or signaling. These progresses have provided knowledge for new vaccine and antiviral development to prevent and control COVID-19.

Grass carp (Ctenopharyngodon idella) PACT induces cell apoptosis and activates NF-кB via PKR

As a dsRNA-dependent and interferon-induced protein kinase, PKR is involved in antiviral immune response and apoptosis mediated by various cytokines. In mammalian cells, PKR can also be activated in the absence of dsRNA. A PKR activator, PACT (PKR activating protein), also referred to as RAX (PKR-associated protein X) plays an important role. In recent years, with the increasing recognition of fish interferon system, PKR and PACT have been gradually revealed in fish. However, the function of fish PACT is unclear. In our previous work, we suggested that grass carp (Ctenopharyngodon idella) PACT must be involved in IRF2 and ATF4-mediated stress response pathways. In the present study, we found that the expression of C. idella PACT (CiPACT) and CiPKR were significantly up-regulated under the stimulation of LPS. It indicated that CiPACT and CiPKR may play an important role in response to LPS stimulation. In addition, the response time of CiPACT to LPS is earlier than that of CiPKR. It has also shown that overexpression of CiPACT in CIK cells can significantly enhance the level of p-eIF2α, induces apoptosis and translocation of Cip65 to nucleus from cytoplasm. To further understand the mechanism, we carried out the co-immunoprecipitation assay. It proved that the interaction of CiPACT and CiPKR made the phosphorylation of CiPKR. Overexpression of CiPACT induced the down-regulation of intracellular expression of bcl-2 and up-regulation of bax. However, in CiPKR knocked-down cells the expression of bcl-2 and bax were just the opposite. Therefore, the mechanism of fish PACT induces apoptosis and activates NF-кB is dependent on PKR.

Loss of mitochondrial ClpP, Lonp1, and Tfam triggers transcriptional induction of Rnf213, a susceptibility factor for moyamoya disease

Human RNF213, which encodes the protein mysterin, is a known susceptibility gene for moyamoya disease (MMD), a cerebrovascular condition with occlusive lesions and compensatory angiogenesis. Mysterin mutations, together with exposure to environmental trigger factors, lead to an elevated stroke risk since childhood. Mysterin is induced during cell stress, to function as cytosolic AAA+ ATPase and ubiquitylation enzyme. Little knowledge exists, in which context mysterin is needed. Here, we found that genetic ablation of several mitochondrial matrix factors, such as the peptidase ClpP, the transcription factor Tfam, as well as the peptidase and AAA+ ATPase Lonp1, potently induces Rnf213 transcript expression in various organs, in parallel with other components of the innate immune system. Mostly in mouse fibroblasts and human endothelial cells, the Rnf213 levels showed prominent upregulation upon Poly(I:C)-triggered TLR3-mediated responses to dsRNA toxicity, as well as upon interferon gamma treatment. Only partial suppression of Rnf213 induction was achieved by C16 as an antagonist of PKR (dsRNA-dependent protein kinase). Since dysfunctional mitochondria were recently reported to release immune-stimulatory dsRNA into the cytosol, our results suggest that mysterin becomes relevant when mitochondrial dysfunction or infections have triggered RNA-dependent inflammation. Thus, MMD has similarities with vasculopathies that involve altered nucleotide processing, such as Aicardi-Goutières syndrome or systemic lupus erythematosus. Furthermore, in MMD, the low penetrance of RNF213 mutations might be modified by dysfunctions in mitochondria or the TLR3 pathway.

Apoptosis Induction by dsRNA-Dependent Protein Kinase R (PKR) in EPC Cells via Caspase 8 and 9 Pathways.

dsRNA-dependent protein kinase R (PKR) is an interferon-inducible protein that mediates antiviral effects and induces apoptosis. We studied PKR-related apoptosis mechanisms by transfecting wild type pcDNA-carp-wtPKR, a catalytically inactive mutant pcDNA-mut-carpPKR, and empty plasmid in Epithelioma papulosum cyprini (EPC) cells, designated wtPKR, mutPKR, and pcDNA3.1, respectively. PKR was inefficiently expressed from wtPKR unlike mutPKR that produced high PKR levels detected by western blot. eIF2α phosphorylation increased in wtPKR-transfected cells, while for mutPKR, phosphorylation was not different from non-transfected controls. Flow-cytometry revealed high level of apoptosis in wtPKR transfected cells, corresponding with high cytopathic effect. mutPKR and pcDNA3.1 transfection gave significantly less apoptosis and were not different from each other. Caspase-8 and -9 were activated for wtPKR, suggesting death receptor-caspase-8 and mitochondrion-dependent caspase-9 activated pathways, similar to mammalian cells. These findings suggest that the induction of apoptosis via the caspase-8 and -9 pathways are conserved in vertebrate taxa and likely play a role in viral infections of lower vertebrates.

Visualization of Double-Stranded RNA Colocalizing With Pattern Recognition Receptors in Arenavirus Infected Cells.

An important step in the initiation of the innate immune response to virus infection is the recognition of non-self, viral RNA, including double-stranded RNA (dsRNA), by cytoplasmic pattern recognition receptors (PRRs). For many positive-sense RNA viruses and DNA viruses, the production of viral dsRNA, and the interaction of viral dsRNA and PRRs are well characterized. However, for negative-sense RNA viruses, viral dsRNA was thought to be produced at low to undetectable levels and PRR recognition of viral dsRNA is still largely unclear. In the case of arenaviruses, the nucleocaspid protein (NP) has been identified to contain an exoribonuclease activity that preferentially degrades dsRNA in biochemical studies. Nevertheless, pathogenic New World (NW) arenavirus infections readily induce an interferon (IFN) response in a RIG-I dependent manner, and also activate the dsRNA-dependent Protein Kinase R (PKR). To better understand the innate immune response to pathogenic arenavirus infection, we used a newly identified dsRNA-specific antibody that efficiently detects viral dsRNA in negative-sense RNA virus infected cells. dsRNA was detected in NW arenavirus infected cells colocalizing with virus NP in immunofluorescence assay. Importantly, the dsRNA signals also colocalized with cytoplasmic PRRs, namely, PKR, RIG-I and MDA-5, as well as with the phosphorylated, activated form of PKR in infected cells. Our data clearly demonstrate the PRR recognition of dsRNA and their activation in NW arenavirus infected cells. These findings provide new insights into the interaction between NW arenaviruses and the host innate immune response.

Novel role of PKR in palmitate-induced Sirt1 inactivation and endothelial cell senescence.

Endothelial cell senescence is regarded as a vital characteristic of cardiovascular diseases. Elevated palmitate (PA) is an independent risk factor of cardiovascular diseases, but its role in endothelial cell senescence is currently unknown. During the course of studying the prosenescent role of PA, we discovered a key role of dsRNA-dependent protein kinase [protein kinase R (PKR)] in endothelial senescence. Exposure of human umbilical vein endothelial cells (HUVECs) to PA-induced cell senescence is characterized by increased levels of senescence-associated β-galactose glucosidase activity, excessive production of reactive oxygen species production, impaired cellular proliferation, and G1 phase arrest. This phenomenon is associated with an increase of PKR autophosphorylation and decreased activity of sirtuin 1 (Sirt1), a pivotal antisenescent factor. PKR inactivation by PKR siRNA or its phosphorylation inhibitor 2-aminopurine significantly attenuated PA-induced HUVEC senescence by reversing Sirt1 activity and its downstream signaling. Moreover, to study the regulatory mechanism between PKR and Sirt1, we found that PKR promotes JNK activation to inhibit Sirt1 activity and that this effect could be reversed by the JNK inhibitor SP600125. These findings provide evidence that PKR mediates PA-induced HUVEC senescence by inhibiting Sirt1 signaling. Our study provides novel insights into the actions and mechanisms of PKR in endothelial senescence. NEW & NOTEWORTHY This study first provides a novel observation that dsRNA-dependent protein kinase (PKR) mediates palmitate-induced sirtuin 1 inactivation and subsequent human umbilical vein endothelial cell senescence. Most importantly, these new findings will provide a potential therapeutic strategy to improve free fatty acid-induced endothelial senescence by targeting PKR in cardiovascular diseases.

Impact of chemical modifications in the structure of isRNA on its antiproliferative and immunostimulatory properties

Short double-stranded RNAs, depending on their structure, sequence, and the method of delivery to cells, can activate the system of innate and adaptive immunity. The immunostimulatory activity of nucleic acids can be used in antitumor and antiviral therapy. We have previously identified a biologically active immunostimulatory 19-bp dsRNA (isRNA) with 3′-nucleotide overhangs, which inhibits the proliferation of tumor cells, stimulates interferon synthesis, and suppresses the growth and metastasis of tumors. Here, we have studied the impact of chemical modifications in the isRNA structure and the type of the transfection agent on the antiproliferative and immunostimulatory properties of isRNA. It has been shown that the attachment of an aminohexyl group or a cholesterol residue to the 5′-terminus of the first strand of the isRNA duplex does not impair its antiproliferative and immunostimulatory properties in vitro and in vivo when it is used in complex with a transfection agent, whereas the modification at the 5′-end of the second strand has an adverse effect on the biological activity of isRNA. It has been found that, when used without the transfection agent, isRNA conjugated with a cholesterol residue does not display antiproliferative and immunostimulatory properties. The use of isRNA in complex with low-toxicity liposomes 2Х3-DOPE enhances its activity: the intravenous injection of the isRNA/2Х3-DOPE complex induces a 55-fold increase in the level of interferon α (INF-α) in the murine blood 6 h after the injection, which is significantly higher than the INF-α level after the injection of the isRNA/Lipofectamine 2000 complex. The cytoplasmic localization of isRNA is crucially important for the manifestation of its antiproliferative activity since the selective inhibition of the dsRNA cytoplasmic sensor PKR (dsRNA-dependent protein kinase R) by 2-aminopurine completely blocks the antiproliferative effect of isRNA.

Vaccinia virus K1 ankyrin repeat protein inhibits NF-κB activation by preventing RelA acetylation.

The vaccinia virus (VACV) K1 protein inhibits dsRNA-dependent protein kinase (PKR) activation. A consequence of this function is that K1 inhibits PKR-induced NF-κB activation during VACV infection. However, transient expression of K1 also inhibits Toll-like receptor (TLR)-induced NF-κB activation. This suggests that K1 has a second NF-κB inhibitory mechanism that is PKR-independent. This possibility was explored by expressing K1 independently of infection and stimulating NF-κB under conditions that minimized or excluded PKR activation. K1 inhibited both TNF- and phorbol 12-myristate 13-acetate (PMA)-induced NF-κB activation, as detected by transcription of synthetic (e.g. luciferase) and natural (e.g. CXCL8) genes controlled by NF-κB. K1 also inhibited NF-κB activity in PKRkd cells, cells that have greatly decreased amounts of PKR. K1 no longer prevented IκBα degradation or NF-κB nuclear translocation in the absence of PKR, suggesting that K1 acted on a nuclear event. Indeed, K1 was present in the nucleus and cytoplasm of stimulated and unstimulated cells. K1 inhibited acetylation of the RelA (p65) subunit of NF-κB, a nuclear event known to be required for NF-κB activation. Moreover, p65-CBP (CREB-binding protein) interactions were blocked in the presence of K1. However, K1 did not preclude NF-κB binding to oligonucleotides containing κB-binding sites. The current interpretation of these data is that NF-κB-promoter interactions still occur in the presence of K1, but NF-κB cannot properly trigger transcriptional activation because K1 antagonizes acetylation of RelA. Thus, in comparison to all known VACV NF-κB inhibitory proteins, K1 acts at one of the most downstream events of NF-κB activation.

The Receptor for Advanced Glycation End Products Activates the AIM2 Inflammasome in Acute Pancreatitis.

Severe acute pancreatitis (AP) is responsible for significant human morbidity and mortality worldwide. Currently, no specific treatments for AP exist, primarily due to the lack of a mechanistic understanding of sterile inflammation and the resultant multisystem organ dysfunction, the pathologic response of AP linked to early death. In this study, we demonstrate that the class III major histocompatibility region III receptor for advanced glycation end products (RAGE) contributes to AP by modulating inflammasome activation in macrophages. RAGE mediated nucleosome-induced absent in melanoma 2 (but not NLRP3) inflammasome activation by modulating dsRNA-dependent protein kinase phosphorylation in macrophages. Pharmacological and genetic inhibition of the RAGE-dsRNA-dependent protein kinase pathway attenuated the release of inflammasome-dependent exosomal leaderless cytokines (e.g., IL-1β and high-mobility group box 1) in vitro. RAGE or absent in melanoma 2 depletion in mice limited tissue injury, reduced systemic inflammation, and protected against AP induced by l-arginine or cerulein in experimental animal models. These findings define a novel role for RAGE in the propagation of the innate immune response with activation of the nucleosome-mediated inflammasome and will help guide future development of therapeutic strategies to treat AP.

Betaine Inhibits Hepatitis B Virus with an Advantage of Decreasing Resistance to Lamivudine and Interferon α.

Betaine (BET) is a native compound known for its ability to protect the liver from toxicants. However, few studies have examined the effects of BET on the most common cause of liver disease, hepatitis B virus (HBV). In this study, the anti-HBV activity of BET was assessed in vitro and in vivo using enzyme-linked immunosorbent assay, quantitative polymerase chain reaction, and Southern blotting. The resistance of HBV to lamivudine and interferon α is challenging in the clinical treatment of HBV. The effect of BET on resistance was also investigated. The results showed that the secretion of HBsAg (HBV surface antigen), HbeAg (HBV e antigen), and HBV DNA in HepG2.2.15 cells was significantly decreased by BET via suppression of GRP78 expression. In duck HBV (DHBV)-infected ducklings, 1.0 or 2.0 g/kg BET significantly reduced serum DHBV DNA, and DHBV DNA did not rebound after the 5 day withdrawal period. BET suppressed HBV DNA rebound produced by the resistance of HBV to lamivudine and decreased the resistance mutation (rtM204V/I) of HBV DNA. Supplementation of BET may improve the anti-HBV effect of interferon α by increasing the expression of antiviral dsRNA-dependent protein kinase induced by the JAK-STAT (JAK = Janus kinase; STAT = signal transducer and activator of transcription) signaling pathway. These results may provide useful information for the clinical application of BET and solution of HBV drug resistance in anti-HBV therapy.

Autism-associated R451C mutation in neuroligin3 leads to activation of the unfolded protein response in a PC12 Tet-On inducible system.

Several forms of monogenic heritable autism spectrum disorders are associated with mutations in the neuroligin genes. The autism-linked substitution R451C in neuroligin3 induces local misfolding of its extracellular domain, causing partial retention in the ER (endoplasmic reticulum) of expressing cells. We have generated a PC12 Tet-On cell model system with inducible expression of wild-type or R451C neuroligin3 to investigate whether there is activation of the UPR (unfolded protein response) as a result of misfolded protein retention. As a positive control for protein misfolding, we also expressed the mutant G221R neuroligin3, which is known to be completely retained within the ER. Our data show that overexpression of either R451C or G221R mutant proteins leads to the activation of all three signalling branches of the UPR downstream of the stress sensors ATF6 (activating transcription factor 6), IRE1 (inositol-requiring enzyme 1) and PERK [PKR (dsRNA-dependent protein kinase)-like endoplasmic reticulum kinase]. Each branch displayed different activation profiles that partially correlated with the degree of misfolding caused by each mutation. We also show that up-regulation of BiP (immunoglobulin heavy-chain-binding protein) and CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein] was induced by both mutant proteins but not by wild-type neuroligin3, both in proliferative cells and cells differentiated to a neuron-like phenotype. Collectively, our data show that mutant R451C neuroligin3 activates the UPR in a novel cell model system, suggesting that this cellular response may have a role in monogenic forms of autism characterized by misfolding mutations.

Effects of interferons and double-stranded RNA on human prostate cancer cell apoptosis.

Prostate cancer is the second most commonly diagnosed cancer among men in the United States. Prostate cancer therapy is severely hampered by lack of response and development of resistance to conventional chemotherapeutic drugs in patients. Therefore, the development and discovery of new drugs have become an urgent clinical need. Interferons (IFNs), a family of pleiotropic cytokines, exert antitumor activities due to their anti-proliferative, immunomodulatory and proapoptotic functions. Here, we report that pretreatment of prostate cancer PC-3 cells with IFNs sensitized these cells to double-stranded RNAs (dsRNAs)-induced apoptosis. The enhancement effect of IFN treatment was dependent on IFN subtypes, in particular, IFN γ. In comparison with IFN α or β, IFN γ treatment remarkably augmented apoptosis in PC-3 cells induced with polyinosinic:polycytidylic acid (poly I:C), a synthesized form of dsRNA. We demonstrated that IFN-signaling was necessary for these effects by using mutant cell lines. Transfection of 2-5A, the activator of RNase L, or silencing of dsRNA-dependent protein kinase R (PKR) by siRNA did not have any significant impact on this event, suggesting that neither RNase L nor PKR was involved in poly I:C/IFN γ-induced apoptosis in the cells. Further investigation of the apoptotic pathway revealed that Bak, a pro-apoptotic member of the Bcl-2family, was synergistically up-regulated by IFN γ and poly I:C, whereas other members of the family were not affected. Knocking down of Bak demonstrated its contribution to poly I:C/IFN γ-induced apoptosis in the cells. We believeour findings will precipitate the design of novel therapeutic strategies for prostate cancer.