Identification and Herc5-mediated ISGylation of novel target proteins

Department of Biochemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan.
Biochemical and Biophysical Research Communications (Impact Factor: 2.3). 10/2006; 348(2):473-7. DOI: 10.1016/j.bbrc.2006.07.076
Source: PubMed


ISG15, a protein containing two ubiquitin-like domains, is an interferon-stimulated gene product that functions in antiviral response and is conjugated to various cellular proteins (ISGylation) upon interferon stimulation. ISGylation occurs via a pathway similar to the pathway for ubiquitination that requires the sequential action of E1/E2/E3: the E1 (UBE1L), E2 (UbcH8), and E3 (Efp/Herc5) enzymes for ISGylation have been hitherto identified. In this study, we identified six novel candidate target proteins for ISGylation by a proteomic approach. Four candidate target proteins were demonstrated to be ISGylated in UBE1L- and UbcH8-dependent manners, and ISGylation of the respective target proteins was stimulated by Herc5. In addition, Herc5 was capable of binding with the respective target proteins. Thus, these results suggest that Herc5 functions as a general E3 ligase for protein ISGylation.

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    • "As a result, the levels of ISG15 conjugates are tightly controlled by IFN. In several studies, hundreds of target proteins for ISGylation have been identified which are involved in diverse cellular pathways [4], [18], [20], [24]–[27]. Only some substrates were found in all studies which may be attributed to the fact that different cell lines were used [18], [20], [24]–[26]. "
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    ABSTRACT: 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.
    PLoS ONE 06/2012; 7(6):e38294. DOI:10.1371/journal.pone.0038294 · 3.23 Impact Factor
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    • "HERC5 is up-regulated in a variety of primary cells and immortalized cell lines by interferon, lipopolysaccharide, tumor necrosis factor α, and interleukin-1β [6,13,16]. Moreover, HERC5 broadly targets newly synthesized proteins for ISG15 conjugation, and many endogenous targets of HERC5 have been identified that function in a variety of cellular pathways including RNA splicing, chromatin remodeling/polII transcription, cytoskeleton organization and regulation, stress responses, translation, glycolysis, interferon signaling and antiviral responses [17-22]. Here, we show that HERC5 inhibits HIV-1 replication by targeting a unique step of HIV-1 particle assembly at the plasma membrane. "
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    ABSTRACT: The identification and characterization of several interferon (IFN)-induced cellular HIV-1 restriction factors, defined as host cellular proteins or factors that restrict or inhibit the HIV-1 life cycle, have provided insight into the IFN response towards HIV-1 infection and identified new therapeutic targets for HIV-1 infection. To further characterize the mechanism underlying restriction of the late stages of HIV-1 replication, we assessed the ability of IFNbeta-induced genes to restrict HIV-1 Gag particle production and have identified a potentially novel host factor called HECT domain and RCC1-like domain-containing protein 5 (HERC5) that blocks a unique late stage of the HIV-1 life cycle. HERC5 inhibited the replication of HIV-1 over multiple rounds of infection and was found to target a late stage of HIV-1 particle production. The E3 ligase activity of HERC5 was required for blocking HIV-1 Gag particle production and correlated with the post-translational modification of Gag with ISG15. HERC5 interacted with HIV-1 Gag and did not alter trafficking of HIV-1 Gag to the plasma membrane. Electron microscopy revealed that the assembly of HIV-1 Gag particles was arrested at the plasma membrane, at an early stage of assembly. The mechanism of HERC5-induced restriction of HIV-1 particle production is distinct from the mechanism underlying HIV-1 restriction by the expression of ISG15 alone, which acts at a later step in particle release. Moreover, HERC5 restricted murine leukemia virus (MLV) Gag particle production, showing that HERC5 is effective in restricting Gag particle production of an evolutionarily divergent retrovirus. HERC5 represents a potential new host factor that blocks an early stage of retroviral Gag particle assembly. With no apparent HIV-1 protein that directly counteracts it, HERC5 may represent a new candidate for HIV/AIDS therapy.
    Retrovirology 11/2011; 8(1):95. DOI:10.1186/1742-4690-8-95 · 4.19 Impact Factor
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    • "An additional RING-containing ubiquitin E3 ligase, called HHARI (human homolog of Drosophila ariadne), also serves as an ISG15 E3 ligase for 4EHP [96]. HERC5 (HECT domain and RCC1-like domain containing protein 5) is an ISG15 E3 ligase that contains the HECT domain [97] [98] [99]. Like EFP, HERC5 is a type I IFN-inducible protein. "
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    ABSTRACT: ISG15, the product of interferon (IFN)-stimulated gene 15, is the first identified ubiquitin-like protein, consisting of two ubiquitin-like domains. ISG15 is synthesized as a precursor in certain mammals and, therefore, needs to be processed to expose the C-terminal glycine residue before conjugation to target proteins. A set of three-step cascade enzymes, an E1 enzyme (UBE1L), an E2 enzyme (UbcH8), and one of several E3 ligases (e.g., EFP and HERC5), catalyzes ISG15 conjugation (ISGylation) of a specific protein. These enzymes are unique among the cascade enzymes for ubiquitin and other ubiquitin-like proteins in that all of them are induced by type I IFNs or other stimuli, such as exposure to viruses and lipopolysaccharide. Mass spectrometric analysis has led to the identification of several hundreds of candidate proteins that can be conjugated by ISG15. Some of them are type I IFN-induced proteins, such as PKR and RIG-I, and some are the key regulators that are involved in IFN signaling, such as JAK1 and STAT1, implicating the role of ISG15 and its conjugates in type I IFN-mediated innate immune responses. However, relatively little is known about the functional significance of ISG15 induction due to the lack of information on the consequences of its conjugation to target proteins. Here, we describe the recent progress made in exploring the biological function of ISG15 and its reversible modification of target proteins and thus in their implication in immune diseases.
    Biochimica et Biophysica Acta 02/2010; 1802(5):485-96. DOI:10.1016/j.bbadis.2010.02.006 · 4.66 Impact Factor
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