Expression of interferon-stimulated gene 20 in vascular endothelial cells

ISG20 is an ribonuclease specific for single-stranded RNA and considered to play a role in innate immunity against virus infections. We herein show that both poly IC, an authentic double-stranded RNA, and IFN-gamma induced ISG20 expression in cultured HUVEC. Poly IC-induced ISG20 expression was inhibited by LY294002, an inhibitor of PI3K, or by RNA interference against IFN regulatory factor three. ISG20 expression was not induced by IFN-beta, loxoribine or CpG oligonucleotide. These results suggest that ISG20 induction by poly IC may not be dependent on the IRF-3-mediated type I IFN induction pathway in HUVEC. ISG20 may be involved in innate immunity against viral infection in vascular endothelial cells.     

The exonuclease ISG20 mainly localizes in the nucleolus and the Cajal (Coiled) bodies and is associated with nuclear SMN protein-containing complexes

We have previously shown that ISG20, an interferon (IFN)-induced gene, encodes a 3' to 5' exoribonuclease member of the DEDD superfamily of exonucleases. ISG20 specifically degrades single-stranded RNA. In this report, using immunofluorescence analysis, we demonstrate that in addition to a diffuse cytoplasmic and nucleoplasmic localization, the endogenous ISG20 protein was present in the nucleus both in the nucleolus and in the Cajal bodies (CBs). In addition, we show that the ectopic expression of the CBs signature protein, coilin, fused to the red fluorescent protein (coilin-dsRed) increased the number of nuclear dots containing both ISG20 and coilin-dsRed. Using electron microcopy analysis, ISG20 appeared principally concentrated in the dense fibrillar component of the nucleolus, the major site for rRNA processing. We also present evidences that ISG20 was associated with survival of motor neuron (SMN)-containing macromolecular nuclear complexes required for the biogenesis of various small nuclear ribonucleoproteins. Finally, we demonstrate that ISG20 was associated with U1 and U2 snRNAs, and U3 snoRNA. The accumulation of ISG20 in the CBs after IFN treatment strongly suggests its involvement in a new route for IFN-mediated inhibition of protein synthesis by modulating snRNA and rRNA maturation.     

ISG20, a new interferon-induced RNase specific for single-stranded RNA,defines an alternative antiviral pathway against RNA genomic viruses

Interferons (IFNs) encode a family of secreted proteins that provide the front-line defense against viral infections. Their diverse biological actions are thought to be mediated by the products of specific but usually overlapping sets of cellular genes induced in the target cells. We have recently isolated a new human IFN-induced gene that we have termed ISG20, which codes for a 3' to 5' exonuclease with specificity for single-stranded RNA and, to a lesser extent, for DNA. In this report, we demonstrate that ISG20 is involved in the antiviral functions of IFN. In the absence of IFN treatment, ISG20-overexpressing HeLa cells showed resistance to infections by vesicular stomatitis virus (VSV), influenza virus, and encephalomyocarditis virus (three RNA genomic viruses) but not to the DNA genomic adenovirus. ISG20 specifically interfered with VSV mRNA synthesis and protein production while leaving the expression of cellular control genes unaffected. No antiviral effect was observed in cells overexpressing a mutated ISG20 protein defective in exonuclease activity, demonstrating that the antiviral effects were due to the exonuclease activity of ISG20. In addition, the inactive mutant ISG20 protein, which is able to inhibit ISG20 exonuclease activity in vitro, significantly reduced the ability of IFN to block VSV development. Taken together, these data suggested that the antiviral activity of IFN against VSV is partly mediated by ISG20. We thus show that, besides RNase L, ISG20 has an antiviral activity, supporting the idea that it might represent a novel antiviral pathway in the mechanism of IFN action.     

Activation of Double-stranded RNA-activated Protein Kinase (PKR) by Interferon-stimulated Gene 15 (ISG15) Modification Down-regulates Protein Translation

The ubiquitin-like molecule ISG15 (UCRP) and protein modification by ISG15 (ISGylation) are strongly induced by interferon, genotoxic stress, and pathogen infection, suggesting that ISG15 plays an important role in innate immune responses. However, how ISGylation contributes to innate immune responses is not clear. The dsRNA-dependent protein kinase (PKR) inhibits translation by phosphorylating eIF2α to exert its anti-viral effect. ISG15 and PKR are induced by interferon, suggesting that a relationship exists between ISGylation and translational regulation. Here, we report that PKR is ISGylated at lysines 69 and 159. ISG15-modified PKR is active in the absence of virus infection and phosphorylates eIF2α to down-regulate protein translation. The present study describes a novel pathway for the activation of PKR and the regulation of protein translation.     

含半胱氨酸的天冬氨酸蛋白水解酶(caspase-1)对猪伪狂犬病毒复制的影响

猪伪狂犬病是伪狂犬病毒(Pseudorabies virus,PRV)感染引起的一种烈性接触性传染病,其感染宿主会触发机体先天免疫应答,引起I型干扰素(Type I interferon,IFN-1)和炎性细胞因子等细胞因子的产生,为研究可诱导产生炎性细胞因子的含半胱氨酸的天冬氨酸蛋白水解酶(Cysteinyl aspartate specific proteinase 1,caspase-1)的基因敲除对PRV复制的影响,本试验利用近年来发展迅速的一项规律性短重复回文序列簇/Cas9核酸酶(Clustered regulatory interspaced short palindromic repeat/CRISPR associated system 9,CRISPR/Cas9)基因定点修饰技术构建猪肾上皮细胞(Porcine kidney epithelial cells,PK15)caspase-1基因稳定敲除细胞系,并通过T7核酸酶检测敲除效率;细胞毒性(Cell counting kit-8,CCK-8)试剂盒检测PK15敲除caspase-1增殖影响;采用流式细胞术检测PRV-GFP感染PK15以及PK15-caspase-1^-/-的增殖差异;实时荧光定量PCR(Real-time quantitative PCR,RT-PCR)检测PRV-gB、TK及白细胞介素1β(Interleukin-1β,IL-1β)、IFN-β、干扰素刺激基因(Interferon-stimulated genes 20,ISG20)mRNA的表达;Western Blot检测PRV-gB蛋白表达;滴度测定检测子代病毒滴度。结果表明,2对特异性单链引导RNA(Single guide RNA,sgRNA)均能对caspase-1进行基因编辑,但经T7核酸酶酶切进行基因编辑效率分析结果表明sgRNA2的基因编辑效率较高;CCK-8试剂盒检测细胞活力结果表明caspase-1基因敲除对PK15以及PK15-caspase-1^-/-细胞活力无影响(P>0.05);流式细胞仪检测结果表明PRV-GFP在PK15-caspase-1^-/-中的增殖显著低于PK15细胞(P<0.05);定量RT-PCR结果表明PRV-gB、TK基因在PK15-caspase-1^-/-的mRNA表达显著低于PK15细胞(gB:P<0.05,TK:P<0.05),而IFN-β、ISG20基因在PK15-caspase-1^-/-的mRNA表达显著高于PK15细胞(gB:P<0.05,TK:P<0.05);Western Blot结果表明,PRV的gB蛋白在PK15-caspase-1^-/-的表达显著低于PK15细胞(P<0.05);滴度测定结果表明,敲除caspase-1能够抑制PRV子代病毒的增殖。以上结果均表明caspase-1基因敲除可抑制PRV在PK15细胞中复制。     

SARS 冠状病毒非结构蛋白质NSP3突变体构建及其对类泛素分子DUB活性

SARS冠状病毒(SARS-CoV) 非结构蛋白NSP3编码的木瓜蛋白酶样蛋白酶(PLpro)对泛素样分子(Ubl) 具有去泛素化酶(DUB)活性,但目前有关NSP3 DUB活性研究的报道甚少. 本研究构建包含Nsp3基因 N末端不同结构域的突变体,并检测NSP3及其一系列突变体对类泛素分子ISG15和SUMO所修饰蛋白质分子的作用特性. 实验结果表明,NSP3及其突变体NSP3AD,NSP3AE,NSP3AF具有一定的去ISG15活性,而其突变体NSP3AC则没有去ISG15 (DeISGylation) 活性. 研究结果提示,SARS NSP3具有一定的体内去ISG15活性,并且这种活性主要依赖于Nsp3基因编码的PLpro. 但SARS NSP3及其突变体NSP3AC,NSP3AD,NSP3AE和NSP3AF并不具有去SUMO (DeSUMOylation) 活性. SARS冠状病毒NSP3对类泛素样分子作用特性的研究为后续NSP3的生物学特性及其对干扰素通路的调控研究奠定了基础.     

Effects of exogenous soluble dextrans on insoluble glucan synthesis by Streptococcus mutans glucosyltransferase

Production of water-insoluble glucan (ISG) from sucrose by cell-free Streptococcus mutans AHT glucosyltransferase (GTF) first rapidly increased, and then sharply declined, as the amounts of water-soluble Dextrans T20 approximately T500 present, were increased. The decline of ISG synthesis was accompanied by an increased synthesis of the water-soluble fraction (SG). Prolonged incubation, however, induced enhanced synthesis of ISG even at higher dextran concentrations. The concentration of dextran required to stimulate or suppress ISG synthesis depended on the amounts of GTF used, but the extent of the stimulation was almost identical for the same GTF/dextran ratio. Thus, ISG synthesis is stimulated by the presence of dextrans at relatively low concentrations, but retarded at higher concentrations by being shifted to SG synthesis. ISG produced in the presence of dextrans contained higher proportions of alpha-1,6 glucosidic linkage and lower molecular size fractions, and possessed lower viscosity. These ISG products did not exhibit the coalescence of two component fibrils as observed with control ISG. These changes combined may contribute to the reduction of ISG-dependent adherence to glass of S. mutans cells by the presence of soluble dextrans, irrespective of their molecular size and concentration.     

Negative regulation of ISG15 E3 ligase EFP through its autoISGylation

The function of ubiquitin-like protein ISG15 and protein modification by ISG15 (ISGylation) has been an enigma for many years. Recently, the research of ISGylation has been accelerated by the identification of the enzymes involved in the ISG15 conjugation process. Our previous study identified the interferon inducible protein EFP as an ISG15 isopeptide ligase (E3) for 14-3-3σ. In this study, we show that ISG15 E3 ligase EFP can be modified by ISG15. Two ubiquitin E2 conjugating enzymes, UbcH6 and UbcH8, can support ISGylation of EFP. The Ring-finger domain of EFP is important for its ISGylation. Full-length EFP can enhance the ISGylation of Ring domain deleted EFP, indicating EFP can function as an ISG15 E3 ligase for itself. We also determined the ISGylation site of EFP and created its ISGylation resistant mutant EFP-K117R. Compared to the wild-type EFP, this mutant further increases the ISGylation of 14-3-3σ. Thus we propose that autoISGylation of EFP negatively regulates its ISG15 E3 ligase activity for 14-3-3σ.     

Crystal structure of human ISG20, an interferon-induced antiviral ribonuclease

ISG20 is an interferon-induced antiviral exoribonuclease that acts on single-stranded RNA and also has minor activity towards single-stranded DNA. It belongs to the DEDDh group of RNases of the DEDD exonuclease superfamily. We have solved the crystal structure of human ISG20 complexed with two Mn2+ ions and uridine 5'-monophosphate (UMP) at 1.9 A resolution. Its structure, including that of the active site, is very similar to those of the corresponding domains of two DEDDh-group DNases, the epsilon subunit of Escherichia coli DNA polymerase III and E. coli exonuclease I, strongly suggesting that its catalytic mechanism is identical to that of the two DNases. However, ISG20 also has distinctive residues, Met14 and Arg53, to accommodate hydrogen bonds with the 2'-OH group of the UMP ribose, and these residues may be responsible for the preference of ISG20 for RNA substrates.     

ISG20L2, a novel vertebrate nucleolar exoribonuclease involved in ribosome biogenesis

Proteomics analyses of human nucleoli provided molecular bases for an understanding of the multiple functions fulfilled by these nuclear domains. However, the biological roles of about 100 of the identified proteins are unpredictable. The present study describes the functional characterization of one of these proteins, ISG20L2. We demonstrate that ISG20L2 is a 3' to 5' exoribonuclease involved in ribosome biogenesis at the level of 5.8 S rRNA maturation, more specifically in the processing of the 12 S precursor rRNA. The use of truncated forms of ISG20L2 demonstrated that its N-terminal half promotes the nucleolar localization and suggested that its C-terminal half bears the exoribonuclease activity. Identification of the binding partners of ISG20L2 confirmed its involvement in the biogenesis of the large ribosomal subunit. These results strongly support the notion that, in human, as it was demonstrated in yeast, 5.8 S rRNA maturation requires several proteins in addition to the exosome complex. Furthermore this observation greatly sustains the idea that the extremely conserved need for correctly processed rRNAs in vertebrates and yeast is achieved by close but different mechanisms.     

Unique Expression of HRF20 (CD59) in Human Nervous Tissue

Damage to autologous tissue by complement is limited by several widely distributed membrane-associated glycoproteins which restrict the action of the complement in homologous species. These include decay accelerating factor (DAF), membrane cofactor protein (MCP) and 20 kDa homologous restriction factor (HRF20,CD59). Using immunohistochemical techniques, we examined the localization of these proteins in the centra] nervous system (CNS) and peripheral nervous system (PNS) using non-neurological human nervous tissue since some complement components have been demonstrated to be synthesized in the CNS. There was no evidence of parenchymal staining by anti-DAF or anti-MCP antibodies in either type of tissue except for the staining of the endothelium in capillaries. On the other hand, anti-HRF20 antibody clearly stained myelinated axons in the CNS as well as Schwann cells in the PNS. In addition, we detected positive staining by anti-DAF antibody in the PNS of a Paroxysmal nocturnal hemoglobinuria (PNH) patient who is genetically deficient in HRF20.     

Expression profiles of interferon‐stimulated gene 15 and prostaglandin synthases in the ovine lymph nodes during early pregnancy

Lymph nodes are distributed all over the body and are part of the lymphatic system. The interferon‐stimulated gene 15 kDa protein (ISG15) and prostaglandins (PGs) are involved in the establishment of pregnancy and are expressed in the uterus during early pregnancy in sheep. In this study, the ovine lymph nodes were obtained on Day 16 of the estrous cycle, and Days 13, 16, and 25 of pregnancy, and the expression of ISG15 and PG synthases, including cyclooxygenase 1 (COX‐1), COX‐2, prostaglandin E (PGE) synthase (PTGES), and a PGF synthase (aldo‐keto reductase family 1, member B1, AKR1B1) were detected by quantitative real‐time polymerase chain reaction, western blot analysis, and immunohistochemistry analysis. Our results showed that there were peaks in the expression of mRNAs and the proteins of ISG15, COX‐1, COX‐2, PTGES, and AKR1B1 in the lymph nodes during early pregnancy and that the COX‐2 and AKR1B1 proteins were limited to the subcapsular sinus and lymph sinuses. In conclusion, the ISG15, COX‐1, COX‐2, PTGES, and AKR1B1 were upregulated in the maternal lymph nodes, which may be beneficial for the development of conceptus, maternal systemic immunoregulation, and anti‐luteolysis during early pregnancy in sheep.     

Proadrenomedullin NH2-terminal 20 peptide has cAMP-mediated vasodilator activity in the mesenteric vascular bed of the cat

Responses to proadrenomedullin NH₂-terminal 20 peptide (hPAMP), a truncated analogue [hPAMP(12–20)], and adrenomedullin (hADM) were investigated in the mesenteric vascular bed of the cat. Under constant-flow conditions, injections of hPAMP, hPAMP(12–20), and hADM caused dose-related decreases in mesenteric perfusion pressure. hADM was 100-fold more potent than hPAMP, and 1000-fold more potent than hPAMP(12–20). Vasodilator responses to hPAMP and hADM were not altered by adrenergic-blocking agents, were similar in innervated and denervated preparations, and were similar when tone was increased by sympathetic nerve stimulation or phenylephrine infusion. Vasodilator responses to hPAMP and hADM were increased in duration by rolipram, a cAMP phosphodiesterase inhibitor. The present data suggest that vasodilator responses to the hPAMP and hADM are mediated by an increase in cAMP and that an interaction with the adrenergic nervous system is not involved.