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.     

Dynamic regulation of innate immunity by ubiquitin and ubiquitin-like proteins

Protein post-translational modifications (PTMs) are central to the host innate immune regulations. Dynamically, PTMs fine-tune the spatial and temporary responses of immune- and non-immune-cells, in accordance with extracellular and intracellular stresses. Ubiquitin and ubiquitin-like proteins (Ubls) are emerging as the important multi-functional signals, controlling the activation, stability, affinity and location of many signaling proteins. Recent investigations, at the molecular-cellular-animal models, have shed new light on the versatility of the ubiquitin, SUMO and ISG15, for shaping the strength and duration of the innate immune responses. This review summarizes our current knowledge on the functions and regulatory mechanisms of the ubiquitin and Ubls in the innate immunity, the first line of host defense against microbial infection.     

The implication of SUMO in intrinsic and innate immunity

Since its discovery, SUMOylation has emerged as a key post-translational modification involved in the regulation of host-virus interactions. SUMOylation has been associated with the replication of a large number of viruses, either through the direct modification of viral proteins or through the modulation of cellular proteins implicated in antiviral defense. SUMO can affect protein function via covalent or non-covalent binding. There is growing evidence that SUMO regulates several host proteins involved in intrinsic and innate immunity, thereby contributing to the process governing interferon production during viral infection; as well as the interferon-activated Jak/STAT pathway. Unlike the interferon-mediated innate immune response, intrinsic antiviral resistance is mediated by constitutively expressed antiviral proteins (defined as restriction factors), which confer direct viral resistance through a variety of mechanisms. The aim of this review is to evaluate the role of SUMO in intrinsic and innate immunity; highlighting the involvement of the TRIM family proteins, with a specific focus on the mechanism through which SUMO affects i- interferon production upon viral infection, ii-interferon Jak/STAT signaling and biological responses, iii-the relationship between restriction factors and RNA viruses.     

Control of nuclear HIPK2 localization and function by a SUMO interaction motif

The serine/threonine kinase HIPK2 regulates gene expression programs controlling differentiation and cell death. HIPK2 localizes in subnuclear speckles, but the structural components allowing this localization are not understood. A point mutation analysis allowed mapping two nuclear localization signals and a SUMO interaction motif (SIM) that also occurs in HIPK1 and HIPK3. The SIM binds all three major isoforms of SUMO (SUMO-1-3), while only SUMO-1 is capable of covalent conjugation to HIPK2. Deletion or mutation of the SIM prevented SUMO binding and precluded localization of HIPK2 in nuclear speckles, thus causing localization of HIPK2 to the entire cell. Functional inactivation of the SIM prohibited recruitment of HIPK2 to PML nuclear bodies and disrupted colocalization with other proteins such as the polycomb protein Pc2 in nuclear speckles. Interaction of HIPK2 with Pc2 or PML in intact cells was largely dependent on a functional SIM in HIPK2, highlighting the relevance of SUMO/SIM interactions as a molecular glue that serves to enhance protein/protein interaction networks. HIPK2 mutants with an inactive SIM showed changed activities, thus revealing that non-covalent binding of SUMO to the kinase is important for the regulation of its function.     

类泛素蛋白--SUMO

SUMO(small ubiquitin-related modifier)是泛素(ubiquitin)类蛋白家族的重要成员之一。尽管SUMO的生化反应途径与泛素相似,但不像泛素那样诱导底物蛋白降解。SUMO化能够使蛋白质更加稳定,进而调节许多关键的细胞活动。现从分类、结构、生化途径和生物学功能等方面介绍SUMO及SUMO化过程。     

Identification and Herc5-mediated ISGylation of novel target proteins

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.     

Determinants of small ubiquitin-like modifier 1 (SUMO1) protein specificity, E3 ligase, and SUMO-RanGAP1 binding activities of nucleoporin RanBP2

The RanBP2 nucleoporin contains an internal repeat domain (IR1-M-IR2) that catalyzes E3 ligase activity and forms a stable complex with SUMO-modified RanGAP1 and UBC9 at the nuclear pore complex. RanBP2 exhibits specificity for SUMO1 as RanGAP1-SUMO1/UBC9 forms a more stable complex with RanBP2 compared with RanGAP1-SUMO2 that results in greater protection of RanGAP-SUMO1 from proteases. The IR1-M-IR2 SUMO E3 ligase activity also shows a similar preference for SUMO1. We utilized deletions and domain swap constructs in protease protection assays and automodification assays to define RanBP2 domains responsible for RanGAP1-SUMO1 protection and SUMO1-specific E3 ligase activity. Our data suggest that elements in both IR1 and IR2 exhibit specificity for SUMO1. IR1 protects RanGAP1-SUMO1/UBC9 and functions as the primary E3 ligase of RanBP2, whereas IR2 retains the ability to interact with SUMO1 to promote SUMO1-specific E3 ligase activity. To determine the structural basis for SUMO1 specificity, a hybrid IR1 construct and IR1 were used to determine three new structures for complexes containing UBC9 with RanGAP1-SUMO1/2. These structures show more extensive contacts among SUMO, UBC9, and RanBP2 in complexes containing SUMO1 compared with SUMO2 and suggest that differences in SUMO specificity may be achieved through these subtle conformational differences.     

Acetylation of SUMO1 Alters Interactions with the SIMs of PML and Daxx in a Protein-Specific Manner

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The SUMO2/3 specific E3 ligase ZNF451-1 regulates PML stability

The small ubiquitin related modifier SUMO regulates protein functions to maintain cell homeostasis. SUMO attachment is executed by the hierarchical action of E1, E2 and E3 enzymes of which E3 ligases ensure substrate specificity. We recently identified the ZNF451 family as novel class of SUMO2/3 specific E3 ligases and characterized their function in SUMO chain formation. The founding member, ZNF451 isoform1 (ZNF451-1) partially resides in PML bodies, nuclear structures organized by the promyelocytic leukemia gene product PML. As PML and diverse PML components are well known SUMO substrates the question arises whether ZNF451-1 is involved in their sumoylation. Here, we show that ZNF451-1 indeed functions as SUMO2/3 specific E3 ligase for PML and selected PML components in vitro. Mutational analysis indicates that substrate sumoylation employs an identical biochemical mechanism as we described for SUMO chain formation. In vivo, ZNF451-1 RNAi depletion leads to PML stabilization and an increased number of PML bodies. By contrast, PML degradation upon arsenic trioxide treatment is not ZNF451-1 dependent. Our data suggest a regulatory role of ZNF451-1 in fine-tuning physiological PML levels in a RNF4 cooperative manner in the mouse neuroblastoma N2a cell-line.     

The interferon regulated ubiquitin-like protein, ISG15, in tumorigenesis: friend or foe?

The interferon-stimulated gene, 15 kDa (ISG15) is an interferon regulated gene that is induced as a primary response to diverse microbial and cell stress stimuli, and encodes the founding member of the ubiquitin-like protein family. ISG15 post-translationally modifies proteins via a pathway parallel to, and partially overlapping with, that of ubiquitin. In addition, ISG15 is released from cells to mediate extracellular cytokine-like activities. Although the biological activities of ISG15 have yet to be fully elucidated, it is clear that ISG15 has the capacity to modulate diverse cellular and physiologic functions. Consistent with this view, alterations in the ISG15 pathway have been identified in human tumors and in tumor cell lines. Here we review evidence of a role for ISG15 as an endogenous tumor suppressor that, when dysregulated in malignant cells, can be subverted to promote oncogenesis.     

SUMO与乳腺癌

小泛素修饰物（Small ubiquitin-like modifier,SUMO）是结构上与泛素类似的一种修饰蛋白,能与一些特定的靶蛋白共价连接。与泛素介导蛋白质的降解不同,SUMO化修饰调控主要对靶蛋白的功能,如在蛋白质的稳定性、细胞定位、信号转导、基因转录调控等方面均发挥着重要的作用。最近的研究表明：SUMO与乳腺癌的发生发展密切相关,它是通过SUMO化修饰参与并影响雌激素受体信号通路来实现的,本文将就此做一综述。     

The dynamics and mechanism of SUMO chain deconjugation by SUMO-specific proteases

SUMOylation of proteins is a cyclic process that requires both conjugation and deconjugation of SUMO moieties. Besides modification by a single SUMO, SUMO chains have also been observed, yet the dynamics of SUMO conjugation/deconjugation remain poorly understood. Using a non-deconjugatable form of SUMO we demonstrate the underappreciated existence of SUMO chains in vivo, we highlight the importance of SUMO deconjugation, and we demonstrate the highly dynamic nature of the SUMO system. We show that SUMO-specific proteases (SENPs) play a crucial role in the dynamics of SUMO chains in vivo by constant deconjugation. Preventing deSUMOylation in Schizosaccharomyces pombe results in slow growth and a sensitivity to replication stress, highlighting the biological requirement for deSUMOylation dynamics. Furthermore, we present the mechanism of SUMO chain deconjugation by SENPs, which occurs via a stochastic mechanism, resulting in cleavage anywhere within a chain. Our results offer mechanistic insights into the workings of deSUMOylating proteases and highlight their importance in the homeostasis of (poly)SUMO-modified substrates.     

ISG15 modification of Ubc13 suppresses its ubiquitin-conjugating activity

ISG15 is one of the interferon-stimulated genes and is classified as a ubiquitin-like protein. Upon interferon stimuli, ISG15 is upregulated and becomes conjugated to various cellular proteins (ISGylation). Several target proteins for ISGylation have recently been identified, but the biological consequence of protein ISGylation remains unclear. In the course of our study to identify components of the ISGylation system, we found that Ubc13, an E2 enzyme for ubiquitin conjugation, is covalently modified with ISG15. To determine the meaning of ISGylation of Ubc13, we isolated ISG15-modified Ubc13 protein and compared its ubiquitin-conjugating activity with that of an unmodified one. We found that ISGylation of Ubc13 suppresses its ability to form a thioester intermediate with ubiquitin.     

Tripartite Motif Ligases Catalyze Polyubiquitin Chain Formation through a Cooperative Allosteric Mechanism

Ligation of polyubiquitin chains to proteins is a fundamental post-translational modification, often resulting in targeted degradation of conjugated proteins. Attachment of polyubiquitin chains requires the activities of an E1 activating enzyme, an E2 carrier protein, and an E3 ligase. The mechanism by which polyubiquitin chains are formed remains largely speculative, especially for RING-based ligases. The tripartite motif (TRIM) superfamily of ligases functions in many cellular processes including innate immunity, cellular localization, development and differentiation, signaling, and cancer progression. The present results show that TRIM ligases catalyze polyubiquitin chain formation in the absence of substrate, the rates of which can be used as a functional readout of enzyme function. Initial rate studies under biochemically defined conditions show that TRIM32 and TRIM25 are specific for the Ubc5 family of E2-conjugating proteins and, along with TRIM5α, exhibit cooperative kinetics with respect to Ubc5 concentration, with submicromolar [S]_0.5 and Hill coefficients of 3–5, suggesting they possess multiple binding sites for their cognate E2-ubiquitin thioester. Mutation studies reveal a second, non-canonical binding site encompassing the C-terminal Ubc5α-helix. Polyubiquitin chain formation requires TRIM subunit oligomerization through the conserved coiled-coil domain, but can be partially replaced by fusing the catalytic domain to GST to promote dimerization. Other results suggest that TRIM32 assembles polyubiquitin chains as a Ubc5-linked thioester intermediate. These results represent the first detailed mechanistic study of TRIM ligase activity and provide a functional context for oligomerization observed in the superfamily.     

Rap80 protein recruitment to DNA double-strand breaks requires binding to both small ubiquitin-like modifier (SUMO) and ubiquitin conjugates

Ubiquitin (Ub) modifications at sites of DNA double-strand breaks (DSBs) play critical roles in the assembly of signaling and repair proteins. The Ub-interacting motif (UIM) domain of Rap80, which is a component of the BRCA1-A complex, interacts with Ub Lys-63 linkage conjugates and mediates the recruitment of BRCA1 to DSBs. Small ubiquitin-like modifier (SUMO) conjugation also occurs at DSBs and promotes Ub-dependent recruitment of BRCA1, but its molecular basis is not clear. In this study, we identified that Rap80 possesses a SUMO-interacting motif (SIM), capable of binding specifically to SUMO2/3 conjugates, and forms a tandem SIM-UIM-UIM motif at its N terminus. The SIM-UIM-UIM motif binds to both Ub Lys-63 linkage and SUMO2 conjugates. Both the SIM and UIM domains are required for efficient recruitment of Rap80 to DSBs immediately after damage and confer cellular resistance to ionizing radiation. These findings propose a model in which SUMO and Ub modification is coordinated to recruit Rap80 and BRCA1 to DNA damage sites.     

SUMO conjugation in plants

Covalent attachment of small proteins to substrates can regulate protein activity in eukaryotes. SUMO, the small ubiquitin-related modifier, can be covalently linked to a broad spectrum of substrates. An understanding of SUMOs role in plant biology is still in its infancy. In this review, we briefly summarize the enzymology of SUMO conjugation (sumoylation), and the current knowledge of SUMO modification in Arabidopsis thaliana (L.) Heynh. and other plants, in comparison to animals and fungi. Furthermore, we assemble a list of potential pathway components in the genome of A. thaliana that have either been functionally defined, or are suggested by similarity to pathway components from other organisms.     

类泛素化修饰蛋白SUMO1的表达纯化及抗体制备

SUMO是近年发现的类泛素化修饰蛋白,可通过异肽键共价连接到靶蛋白上,影响靶蛋白的细胞内定位、稳定性及与其它生物大分子的相互作用. 为研究蛋白质的SUMO化修饰,本文表达并利用亲和层析的方法纯化了重组的人SUMO1,制备了兔抗hSUMO1的多克隆抗体. 经ELISA和免疫印迹检测,获得了灵敏度高、特异性好的抗体,可用于SUMO化修饰靶蛋白的鉴定及SUMO化修饰的生物学功能研究.     

SUMO and SUMOylation in plants

The traditional focus on the central dogma of molecular biology, from gene through RNA to protein, has now been replaced by the recognition of an additional mechanism. The new regulatory mechanism, post-translational modifications to proteins, can actively alter protein function or activity introducing additional levels of functional complexity by altering cellular and sub-cellular location, protein interactions and the outcome of biochemical reaction chains. Modifications by ubiquitin (Ub) and ubiquitin-like modifiers systems are conserved in all eukaryotic organisms. One of them, small ubiquitin-like modifier (SUMO) is present in plants. The SUMO mechanism includes several isoforms of proteins that are involved in reactions of sumoylation and de-sumoylation. Sumoylation affects several important processes in plants. Outstanding among those are responses to environmental stresses. These may be abiotic stresses, such as phosphate deficiency, heat, low temperature, and drought, or biotic stressses, as well including defense reactions to pathogen infection. Also, the regulations of flowering time, cell growth and development, and nitrogen assimilation have recently been added to this list. Identification of SUMO targets is material to characterize the function of sumoylation or desumoylation. Affinity purification and mass spectrometric identification have been done lately in plants. Further SUMO noncovalent binding appears to have function in other model organisms and SUMO interacting proteins in plants will be of interest to plant biologists who dissect the dynamic function of SUMO. This review will discuss results of recent insights into the role of sumoylation in plants.