类泛素修饰蛋白bISG15抗血清在体外修饰系统中的应用

ISG15由干扰素刺激基因15编码,是最早被发现的类泛素修饰分子.病毒感染以及干扰素刺激可以强烈诱导其表达.与泛素类似,ISG15可以共价连接到其他蛋白分子上进行修饰,但ISG15及其连接修饰的功能作用还有很多尚未知.最近的研究表明,ISG15及其修饰作用在先天免疫中起着重要的作用.将牛类ISG15基因克隆进入pET28a(+)原核表达载体,并且表达了可溶的融合有His-tag标签的bISG15融合蛋白.使用Ni-NTA葡聚糖进行纯化浓缩.纯化蛋白免疫Balb/c小鼠并获得抗血清.Western印迹实验显示,抗血清可以特异地识别在真核细胞中表达的bISG15.浓缩的bISG15以及制备的抗血清用于建立bISG15的体外修饰系统.实验证明,使用该系统bISG15可以连接到细胞蛋白上进行修饰.     

ISG15 modulates development of the erythroid lineage

Activation of erythropoietin receptor allows erythroblasts to generate erythrocytes. In a search for genes that are up-regulated during this differentiation process, we have identified ISG15 as being induced during late erythroid differentiation. ISG15 belongs to the ubiquitin-like protein family and is covalently linked to target proteins by the enzymes of the ISGylation machinery. Using both in vivo and in vitro differentiating erythroblasts, we show that expression of ISG15 as well as the ISGylation process related enzymes Ube1L, UbcM8 and Herc6 are induced during erythroid differentiation. Loss of ISG15 in mice results in decreased number of BFU-E/CFU-E in bone marrow, concomitant with an increased number of these cells in the spleen of these animals. ISG15(-/-) bone marrow and spleen-derived erythroblasts show a less differentiated phenotype both in vivo and in vitro, and over-expression of ISG15 in erythroblasts is found to facilitate erythroid differentiation. Furthermore, we have shown that important players of erythroid development, such as STAT5, Globin, PLC γ and ERK2 are ISGylated in erythroid cells. This establishes a new role for ISG15, besides its well-characterized anti-viral functions, during erythroid differentiation.     

Interferon-inducible ubiquitin E2, Ubc8, is a conjugating enzyme for protein ISGylation

Protein ISGylation is unique among ubiquitin-like conjugation systems in that the expression and conjugation processes are induced by specific stimuli, mainly via the alpha/beta interferon signaling pathway. It has been suggested that protein ISGylation plays a special role in the immune response, because of its interferon-signal dependency and its appearance only in higher eukaryotic organisms. Here, we report the identification of an ISG15-conjugating enzyme, Ubc8. Like other components of the protein ISGylation system (ISG15, UBE1L, and UBP43), Ubc8 is an interferon-inducible protein. Ubc8 clearly mediates protein ISGylation in transfection assays. The reduction of Ubc8 expression by small interfering RNA causes a decrease in protein ISGylation in HeLa cells upon interferon treatment. Neither UbcH7/UbcM4, the closest homologue of Ubc8 among known ubiquitin E2s, nor the small ubiquitin-like modifier E2 Ubc9 supports protein ISGylation. These findings strongly suggest that Ubc8 is a major ISG15-conjugating enzyme responsible for protein ISGylation upon interferon stimulation. Furthermore, we established an assay system to detect ISGylated target proteins by cotransfection of ISG15, UBE1L, and Ubc8 together with a target protein to be analyzed. This method provides an easy and effective way to identify new targets for the ISGylation system and will facilitate related studies.     

The interferon-inducible ubiquitin-protein isopeptide ligase (E3) EFP also functions as an ISG15 E3 ligase

The expression of the ubiquitin-like protein ISG15 and protein modification by ISG15 (ISGylation) are strongly activated by interferons. Accordingly, ISG15 expression and protein ISGylation are strongly activated upon viral and bacterial infections and during other stress conditions, suggesting important roles for the ISG15 system in innate immune responses. Here, we report the identification of the ubiquitin-protein isopeptide ligase (E3) EFP (estrogen-responsive finger protein) as the ISG15 E3 ligase for 14-3-3sigma protein. Like other known components of the protein ISGylation system (ISG15, UBE1L, UBP43, and UBC8), EFP is also an interferon-inducible protein. Expression of EFP small interfering RNA decreased the ISGylation of 14-3-3sigma in the 293T cell ISGylation system as well as in MCF-7 cells upon interferon treatment. Furthermore, the ISGylation enzyme activity of EFP was RING domain-dependent. These findings indicate that EFP is an ISG15 E3 ligase for 14-3-3sigma in vivo. The fact that both UBC8 and EFP are common components in the ubiquitin and ISG15 conjugation pathways suggests a mechanism whereby a limited set of enzymes accomplishes diverse post-translational modifications of their substrates in response to changes in environmental stimulations.     

A Theileria annulata DNA binding protein localized to the host cell nucleus alters the phenotype of a bovine macrophage cell line

The apicomplexan parasite Theileria annulata is the only intracellular eukaryote that is known to induce the proliferation of mammalian cells. However, as the parasite undergoes stage differentiation, host cell proliferation is inhibited, and the leukocyte is eventually destroyed. We have isolated a parasite gene (SuAT1) encoding an AT hook DNA binding polypeptide that has a predicted signal peptide, PEST motifs, nuclear localization signals, and domains which indicate interaction with regulatory components of the higher eukaryotic cell cycle. The polypeptide is localized to the nuclei of macroschizont-infected cells and was detected at significant levels in cells that were undergoing parasite stage differentiation. Transfection of an uninfected transformed bovine macrophage cell line, BoMac, demonstrated that SuAT1 can modulate cellular morphology and alter the expression pattern of a cytoskeletal polypeptide in a manner similar to that found during the infection of leukocytes by the parasite. Our findings indicate that Theileria parasite molecules that are transported to the leukocyte nucleus have the potential to modulate the phenotype of infected cells.     

The ISG15/USP18 ubiquitin-like pathway (ISGylation system) in hepatitis C virus infection and resistance to interferon therapy

The ISG15/USP18 pathway modulates cellular functions and is important for the host innate immune response to chronic viral infections such as Hepatitis C Virus (HCV). Interferon stimulated gene 15 (ISG15) was the first ubiquitin-like protein modifier identified. As in ubiquitination, ISG15 conjugates to target proteins (ISGylation) through the sequential enzymatic action of activating E1, conjugating E2, and ligating E3 enzymes. ISGylation modulates signal transduction pathways and host anti-viral response. The ISGylation process is reversible through the action of an ISG15 protease, USP18. Ubiquitin-like specific protease 18 (USP18) has functions that are both ISG15-dependent and ISG15-independent; the importance of the ISG15/USP18 pathway to chronic HCV infection is illustrated by the consistent finding of increased levels of ISG15 and USP18 in the liver tissue of patients who do not respond to interferon-based treatments. Mechanistically, HCV seems to exploit the ISG15/USP18 pathway to promote viral replication and evade innate anti-viral immune responses.     

A Bovine Lymphosarcoma Cell Line Infected with Theileria annulata Exhibits an Irreversible Reconfiguration of Host Cell Gene Expression

Theileria annulata, an intracellular parasite of bovine lymphoid cells, induces substantial phenotypic alterations to its host cell including continuous proliferation, cytoskeletal changes and resistance to apoptosis. While parasite induced modulation of host cell signal transduction pathways and NFκB activation are established, there remains considerable speculation on the complexities of the parasite directed control mechanisms that govern these radical changes to the host cell. Our objectives in this study were to provide a comprehensive analysis of the global changes to host cell gene expression with emphasis on those that result from direct intervention by the parasite. By using comparative microarray analysis of an uninfected bovine cell line and its Theileria infected counterpart, in conjunction with use of the specific parasitacidal agent, buparvaquone, we have identified a large number of host cell gene expression changes that result from parasite infection. Our results indicate that the viable parasite can irreversibly modify the transformed phenotype of a bovine cell line. Fifty percent of genes with altered expression failed to show a reversible response to parasite death, a possible contributing factor to initiation of host cell apoptosis. The genes that did show an early predicted response to loss of parasite viability highlighted a sub-group of genes that are likely to be under direct control by parasite infection. Network and pathway analysis demonstrated that this sub-group is significantly enriched for genes involved in regulation of chromatin modification and gene expression. The results provide evidence that the Theileria parasite has the regulatory capacity to generate widespread change to host cell gene expression in a complex and largely irreversible manner.     

Proteomic identification of proteins conjugated to ISG15 in mouse and human cells

Though the interferon-inducible protein ISG15 was one of the first ubiquitin-like modifiers to be discovered, much remains unknown about the identity of proteins conjugated to ISG15 or the biologic consequences of modification. To gain a better understanding of the cellular pathways affected by ISG15, we identified proteins targeted for ISGylation using a proteomic approach. Mass spectrometric analysis identified 76 candidate ISGylation targets in anti-ISG15 immunoprecipitates from interferon-treated mouse or human cells. Twenty-one proteins were found in both mouse and human samples, including STAT1, a known target of ISGylation. Candidates identified in both species were tested for ISGylation in a transfection system: 18 of 19 proteins tested were ISGylated in this system. Two candidates, EF-2 and VCP, were also shown to be ISGylated in an interferon-dependent manner in the absence of exogenous over-expression. Seven proteins identified from a single species, but functionally related to candidates found in both species, were also ISGylated in the over-expression system. Proteins that can be ISGylated play important roles in translation, glycolysis, stress responses, and cell motility. These data indicate that ISGylation targets proteins found in several fundamentally important cellular pathways and will contribute to understanding the physiologic role of interferon-induced ISG15 and ISG15 conjugation.     

Interplay between interferon-stimulated gene 15/ISGylation and interferon gamma signaling in breast cancer cells

Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like protein that conjugates to its target proteins to modify them through ISGylation, but the relevance of ISG15 expression and its effects have been not completely defined. Herein, we examined the interplay between ISG15/ISGylation and the interferon-gamma (IFN-γ) signaling pathway in mammary tumors and compared it with that in normal mammary tissues. Our results indicated that mammary tumors had higher levels of ISG15 mRNA and ISG15 protein than the adjacent normal mammary tissue. Furthermore, the expression of IFN-γ signaling components was altered in breast cancer. Interestingly, IFN-γ treatment induced morphological changes in MCF-7 and MDA-MB-231 breast cancer cell lines due to cytoskeletal reorganization. This cellular process seems to be related to the increase in ISGylation of cytoplasmic IQ Motif Containing GTPase Activating Protein 1 (IQGAP1). Interactome analysis also indicated that IFN-γ signaling and the ISGylation system are associated with several proteins implicated in cytoskeletal remodeling, including IQGAP1. Thus, ISG15 may present a potential biomarker for breast cancer, and IFN-γ signaling and protein ISGylation may participate in the regulation of the cytoskeleton in breast cancer cells.     

干扰素刺激基因15在人类免疫缺陷病毒感染中作用的研究进展

随着有效的联合抗反转录病毒疗法(combination antiretroviral therapy,cART)的普及,人类免疫缺陷病毒(human immunodeficiency virus,HIV)感染者的生存期逐步延长。这一过程中,HIV感染者自身免疫反应对免疫系统功能的恢复也发挥了至关重要的作用。HIV感染激活干扰素信号通路,诱导干扰素刺激基因(interferon-stimulated gene,ISG)上调表达,从而发挥抗病毒作用。其中,类泛素蛋白ISG15在HIV感染者中显著上调,通过ISG化抑制HIV颗粒的出芽和释放;而HIV的非结构蛋白则通过干扰ISG化过程或结合干扰素信号通路关键分子,逆转ISG15对病毒的抑制作用。本文从ISG15的生物学特性、在不同细胞亚群中的表达、抗病毒功能及病毒逃逸机制等方面进行综述,为进一步解析ISG15在HIV感染中扮演的角色、探索如何获得以抗HIV感染宿主因子为契机的治疗策略提供了思路。     

Role of ISG15 protease UBP43 (USP18) in innate immunity to viral infection

Innate immune responses provide the host with an early protection barrier against infectious agents, including viruses, and help shape the nature and quality of the subsequent adaptive immune responses of the host. Expression of ISG15 (UCRP), a ubiquitin-like protein, and protein ISGylation are highly increased upon viral infection. We have identified UBP43 (USP18) as an ISG15 deconjugating protease. Protein ISGylation is enhanced in cells deficient in UBP43 (ref. 6). Here we have examined the role of UBP43, encoded by the gene Usp18, in innate immunity to virus infection. Usp18(-/-) mice were resistant to the fatal lymphocytic choriomeningitis and myeloencephalitis that developed in wild-type mice after intracerebral inoculation with lymphocytic choriomeningitis virus (LCMV) or vesicular stomatitis virus (VSV), respectively. Survival of Usp18(-/-) mice after intracerebral LCMV infection correlated with a severe inhibition of LCMV RNA replication and antigen expression in the brain and increased levels of protein ISGylation. Consistent with these findings, mouse embryonic fibroblasts (MEF) and bone marrow-derived macrophages from Usp18(-/-) mice showed restricted LCMV replication. Moreover, MEF from Usp18(-/-) mice showed enhanced interferon-mediated resistance to the cytopathic effect caused by VSV and Sindbis virus (SNV). This report provides the first direct evidence that the ISG15 protease UBP43 and possibly protein ISGylation have a role in innate immunity against viral infection.     

Covalent protein modification with ISG15 via a conserved cysteine in the hinge region

The ubiquitin-like protein ISG15 (interferon-stimulated gene of 15 kDa) is strongly induced by type I interferons and displays antiviral activity. As other ubiquitin-like proteins (Ubls), ISG15 is post-translationally conjugated to substrate proteins by an isopeptide bond between the C-terminal glycine of ISG15 and the side chains of lysine residues in the substrates (ISGylation). ISG15 consists of two ubiquitin-like domains that are separated by a hinge region. In many orthologs, this region contains a single highly reactive cysteine residue. Several hundred potential substrates for ISGylation have been identified but only a few of them have been rigorously verified. In order to investigate the modification of several ISG15 substrates, we have purified ISG15 conjugates from cell extracts by metal-chelate affinity purification and immunoprecipitations. We found that the levels of proteins modified by human ISG15 can be decreased by the addition of reducing agents. With the help of thiol blocking reagents, a mutational analysis and miRNA mediated knock-down of ISG15 expression, we revealed that this modification occurs in living cells via a disulphide bridge between the substrates and Cys78 in the hinge region of ISG15. While the ISG15 activating enzyme UBE1L is conjugated by ISG15 in the classical way, we show that the ubiquitin conjugating enzyme Ubc13 can either be classically conjugated by ISG15 or can form a disulphide bridge with ISG15 at the active site cysteine 87. The latter modification would interfere with its function as ubiquitin conjugating enzyme. However, we found no evidence for an ISG15 modification of the dynamin-like GTPases MxA and hGBP1. These findings indicate that the analysis of potential substrates for ISG15 conjugation must be performed with great care to distinguish between the two types of modification since many assays such as immunoprecipitation or metal-chelate affinity purification are performed with little or no reducing agent present.     

类泛素修饰蛋白质ISG15及其修饰酶系的功能

受干扰素诱导表达的干扰素刺激基因15编码蛋白质(ISG15)是第1个被鉴定的类泛素修饰蛋白质.目前已在病毒感染细胞和肿瘤细胞中发现了多种ISG15的作用靶蛋白,提示ISG15可能在免疫调节和肿瘤发生等方面发挥重要作用.本文介绍ISG15的结构与生化特点,探讨ISG15在相关酶系作用下修饰目标蛋白质的机制,总结ISG15及其修饰酶系的抗病毒和抗肿瘤作用及其相关机制.