Interferon-stimulated gene 15 enters posttranslational modifications of p53

The tumor suppressor protein p53 is a central governor of various cellular signals. It is well accepted that ubiquitination as well as ubiquitin-like (UBL) modifications of p53 protein is critical in the control of its activity. Interferon-stimulated gene 15 (ISG15) is a well-known UBL protein with pleiotropic functions, serving both as a free intracellular molecule and as a modifier by conjugating to target proteins. Initially, attentions have historically focused on the antiviral effects of ISG15 pathway. Remarkably, a significant role in the processes of autophagy, DNA repair, and protein translation provided considerable insight into the new functions of ISG15 pathway. Despite the deterministic revelation of the relation between ISG15 and p53, the functional consequence of p53 ISGylation appears somewhat confused. More important, more recent studies have hinted p53 ubiquitination or other UBL modifications that might interconnect with its ISGylation. Here, we aim to summarize the current knowledge of p53 ISGylation and the differences in other significant modifications, which would be beneficial for the development of p53-based cancer therapy.     

泛素样蛋白ISG15抗病毒机制研究进展

ISG15(Interferon stimulated gene 15,ISG15)蛋白是由干扰素诱导产生的一种泛素样蛋白分子,分子量大小约为15kD。ISG15同泛素分子相类似可以被共价结合于其他蛋白分子上,这种现象称为ISG化(ISGylation)现象。ISG化系统包括ISG15、UBE1L、UBCH8和HERC5四类蛋白分子,协同完成ISG化过程。ISG15及ISG化系统在抗病毒反应中具有重要作用。近几年对于ISG15的抗病毒作用和机制的研究已经有了很大的突破,ISG15的抗病毒作用也越来越受到人们重视,了解清楚ISG15抗病毒机制对于研制新的抗病毒药物及提出新的抗病毒策略具有重要意义。本文对ISG15在不同种病毒中的抗病毒机制研究进展进行了简要综述。     

Viral defense, carcinogenesis and ISG15: novel roles for an old ISG

Recent studies have established that type I interferon modulates expression of large number of cellular genes. While the proteins encoded by some of these genes have a direct antiviral activity, the functions of the majority of the others have not yet been determined. One of the first identified IFN stimulated gene, encodes ubiquitin like protein ISG15 that is also expressed in response to different stress stimuli. Although it was shown that ISG15 functions as protein modifier, it has been only recently that the targets of ISG15 conjugation were identified. Recent studies have also revealed mechanism of ISG15 conjugation and its interaction with the ubiquitin conjugation pathway. This review is focused on the possible role of ISG15 in the antiviral response, regulation of cell growth and carcinogenesis.     

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。     

Interferon-mediated ISG15 conjugation restricts dengue virus 2 replication

ISGylation, an ubiquitin-like post-translational modification by ISG15, has been reported to participate in the interferon (IFN)-mediated antiviral response. In this study, we analyzed the functional role of ISGylation in dengue virus 2 (DENV-2) replication. Overexpression of ISG15 was found to significantly suppress the amount of extracellular infectious virus released, while intracellular viral RNA was unaffected. This effect was not observed with a conjugation-defective ISG15 mutant. In addition, extracellular virus infectivity was decreased by ISG15 overexpression. To further clarify the role of ISGylation in the anti-DENV-2 response, we depleted endogenous ISG15 by RNA interference and analyzed the virus production in the absence or presence of type-I IFN. Results showed a significant reduction in extracellular DENV-2 RNA levels for cells treated with IFN, and that these DENV-2 RNA levels could be partially restored by the ISG15 knockdown. Among various DENV-2 proteins, NS3 and NS5 were subjected to the ISGylation. These results demonstrate that IFN-inducible ISGylation suppresses DENV-2 particle release, and that ISG15 is one of the mediators of IFN-induced inhibition of DENV-2 replication. ISG15 therefore functions as a host antiviral factor against DENV-2 infection.     

Post-Translational Modifications of PCNA in Control of DNA Synthesis and DNA Damage Tolerance-the Implications in Carcinogenesis

The faithful DNA replication is a critical event for cell survival and inheritance. However, exogenous or endogenous sources of damage challenge the accurate synthesis of DNA, which causes DNA lesions. The DNA lesions are obstacles for replication fork progression. However, the prolonged replication fork stalling leads to replication fork collapse, which may cause DNA double-strand breaks (DSB). In order to maintain genomic stability, eukaryotic cells evolve translesion synthesis (TLS) and template switching (TS) to resolve the replication stalling. Proliferating cell nuclear antigen (PCNA) trimer acts as a slide clamp and encircles DNA to orchestrate DNA synthesis and DNA damage tolerance (DDT). The post-translational modifications (PTMs) of PCNA regulate these functions to ensure the appropriate initiation and termination of replication and DDT. The aberrant regulation of PCNA PTMs will result in DSB, which causes mutagenesis and poor response to chemotherapy. Here, we review the roles of the PCNA PTMs in DNA duplication and DDT. We propose that clarifying the regulation of PCNA PTMs may provide insights into understanding the development of cancers.     

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.     

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.     

Large-Scale Arrayed Analysis of Protein Degradation Reveals Cellular Targets for HIV-1 Vpu

1. Download high-res image (299KB)
2. Download full-size image

     

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.