TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity

Retinoic-acid-inducible gene-I (RIG-I; also called DDX58) is a cytosolic viral RNA receptor that interacts with MAVS (also called VISA, IPS-1 or Cardif) to induce type I interferon-mediated host protective innate immunity against viral infection. Furthermore, members of the tripartite motif (TRIM) protein family, which contain a cluster of a RING-finger domain, a B box/coiled-coil domain and a SPRY domain, are involved in various cellular processes, including cell proliferation and antiviral activity. Here we report that the amino-terminal caspase recruitment domains (CARDs) of RIG-I undergo robust ubiquitination induced by TRIM25 in mammalian cells. The carboxy-terminal SPRY domain of TRIM25 interacts with the N-terminal CARDs of RIG-I; this interaction effectively delivers the Lys 63-linked ubiquitin moiety to the N-terminal CARDs of RIG-I, resulting in a marked increase in RIG-I downstream signalling activity. The Lys 172 residue of RIG-I is critical for efficient TRIM25-mediated ubiquitination and for MAVS binding, as well as the ability of RIG-I to induce antiviral signal transduction. Furthermore, gene targeting demonstrates that TRIM25 is essential not only for RIG-I ubiquitination but also for RIG-I-mediated interferon- production and antiviral activity in response to RNA virus infection. Thus, we demonstrate that TRIM25 E3 ubiquitin ligase induces the Lys 63-linked ubiquitination of RIG-I, which is crucial for the cytosolic RIG-I signalling pathway to elicit host antiviral innate immunity.     

拟南芥AtHHR3响应热胁迫应答的初步分析

拟南芥基因AtHHR3编码一个RING结构域的E3连接酶,通过生物信息学分析发现其可能参与植物热胁迫相关的应答.为了探索其具体的功能,构建了AtHHR3互补株系,并在DNA水平和转录水平分别鉴定了AtHHR3互补株系,用RT-PCR技术分析了AtHHR3在热处理条件下基因表达的变化情况.在热胁迫下分析了野生型、突变体athhr3、回复株系幼苗存活以及种子萌发的表型变化情况,发现突变体athhr3表现出对热胁迫的耐受性,并检测了热胁迫下不同株系的HSF、HSP等热相关基因的转录水平的变化,初步的研究表明拟南芥基因AtHHR3负调控植物对热胁迫的耐受性.     

拟南芥 BAH1 与大肠杆菌热胁迫耐受的关系分析

拟南芥BAH1含有保守的C3H4型RING结构域,与DnaJ锌指结构类似.利用原核表达纯化的BAH1进行体外泛素化实验证明了BAH1具有E3连接酶活性.然后通过表型回复实验发现BAH1融合J-domain结构域后(JdBAH1)和DnaJ一样能明显弥补danJ突变株MF634的热敏表型,在43℃存活;而转入突变锌指结构的JdBAH1C231S,C234S,C276S,C279S(JdBAH1△Zn1/2)菌株在43℃高温条件下不能存活,说明BAH1在大肠杆菌内具有类似DnaJ锌指结构的功能.因此,BAH1在E.coli中的功能有可能与DnaJ相似,通过锌指结构参与了DnaK/DnaJ伴侣系统发挥功能.     

拟南芥RING finger结构域ABRv1基因的功能初步研究

拟南芥ABscisic acid responsive RING-v protein 1基因（ABRv1）编码一个含有RING finger结构域的E3连接酶。为了分析ABRv1的基因功能,我们首先构建了高表达载体pBI121- ABRv1,经农杆菌转化,花序浸染后得到T0代转基因种子,并经卡那霉素板筛选得到转基因苗,再经过两代的筛选获取了纯合的高表达株系ABRv1。通过DNA及RNA水平鉴定了该基因的T –DNA插入突变体abrv1。对突变体、高表达转基因株系及野生型进行ABA诱导处理,检测其萌发率、气孔关闭情况进行,发现ABA处理后突变体的萌发率降低,过表达株系萌发率升高;突变体株系气孔几乎未关闭,过表达株系气孔关闭非常明显。以上结果表明ABRv1基因在拟南芥的ABA信号应答响应中可能起负调控作用,为深入分析该基因的功能打下基础。     

A Xanthomonas uridine 5'-monophosphate transferase inhibits plant immune kinases

Plant innate immunity is activated on the detection of pathogen-associated molecular patterns (PAMPs) at the cell surface, or of pathogen effector proteins inside the plant cell. Together, PAMP-triggered immunity and effector-triggered immunity constitute powerful defences against various phytopathogens. Pathogenic bacteria inject a variety of effector proteins into the host cell to assist infection or propagation. A number of effector proteins have been shown to inhibit plant immunity, but the biochemical basis remains unknown for the vast majority of these effectors. Here we show that the Xanthomonas campestris pathovar campestris type III effector AvrAC enhances virulence and inhibits plant immunity by specifically targeting Arabidopsis BIK1 and RIPK, two receptor-like cytoplasmic kinases known to mediate immune signalling. AvrAC is a uridylyl transferase that adds uridine 5'-monophosphate to and conceals conserved phosphorylation sites in the activation loop of BIK1 and RIPK, reducing their kinase activity and consequently inhibiting downstream signalling.     

The conserved protein DCN-1/Dcn1p is required for cullin neddylation in C. elegans and S. cerevisiae

SCF-type E3 ubiquitin ligases are multi-protein complexes required for polyubiquitination and subsequent degradation of target proteins by the 26S proteasome. Cullins, together with the RING-finger protein Rbx1, form the catalytic core of the ligase, and recruit the substrate-recognition module. Cycles of covalent modification of cullins by the ubiquitin-like molecule Nedd8 (neddylation) and removal of Nedd8 by the COP9 signalosome (deneddylation) positively regulate E3 ligase activity. Here we report the identification and analysis of a widely conserved protein that is required for cullin neddylation in the nematode Caenorhabditis elegans and the yeast Saccharomyces cerevisiae. C. elegans DCN-1 and S. cerevisiae Dcn1p (defective in cullin neddylation) are characterized by a novel UBA-like ubiquitin-binding domain and a DUF298 domain of unknown function. Consistent with their requirements for neddylation, DCN-1 and Dcn1p directly bind Nedd8 and physically associate with cullins in both species. Moreover, overexpression of Dcn1p in yeast results in the accumulation of Nedd8-modified cullin Cdc53p. Both in vivo and in vitro experiments indicate that Dcn1p does not inhibit deneddylation of Cdc53p by the COP9 signalosome, but greatly increases the kinetics of the neddylation reaction.     

拟南芥 AtTR1 在盐胁迫应答中的功能初探

本研究主要探索油菜中E3泛素连接酶BnTR1在拟南芥中的同源基因AtTR1(At3g47550)的功能.通过体外泛素化实验证明AtTR1具有E3连接酶活性.基因表达分析显示该基因受200mmol/L NaCl显著诱导,说明该基因可能在响应盐胁迫中发挥一定的功能.为了更深入的探究该基因在植物耐盐中的作用,构建了植物表达载体pZH01-AtTR1转化突变体.在含有潮霉素的培养基上筛选阳性苗,并利用荧光定量PCR检测表明AtTR1基因已经成功转入突变体中.     

Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein

Macrophages and dendritic cells have key roles in viral infections, providing virus reservoirs that frequently resist antiviral therapies and linking innate virus detection to antiviral adaptive immune responses. Human immunodeficiency virus 1 (HIV-1) fails to transduce dendritic cells and has a reduced ability to transduce macrophages, due to an as yet uncharacterized mechanism that inhibits infection by interfering with efficient synthesis of viral complementary DNA. In contrast, HIV-2 and related simian immunodeficiency viruses (SIVsm/mac) transduce myeloid cells efficiently owing to their virion-associated Vpx accessory proteins, which counteract the restrictive mechanism. Here we show that the inhibition of HIV-1 infection in macrophages involves the cellular SAM domain HD domain-containing protein 1 (SAMHD1). Vpx relieves the inhibition of lentivirus infection in macrophages by loading SAMHD1 onto the CRL4(DCAF1) E3 ubiquitin ligase, leading to highly efficient proteasome-dependent degradation of the protein. Mutations in SAMHD1 cause Aicardi-Goutières syndrome, a disease that produces a phenotype that mimics the effects of a congenital viral infection. Failure to dispose of endogenous nucleic acid debris in Aicardi-Goutières syndrome results in inappropriate triggering of innate immune responses via cytosolic nucleic acids sensors. Thus, our findings show that macrophages are defended from HIV-1 infection by a mechanism that prevents an unwanted interferon response triggered by self nucleic acids, and uncover an intricate relationship between innate immune mechanisms that control response to self and to retroviral pathogens.     

TNF-RII and c-IAP1 mediate ubiquitination and degradation of TRAF2

Tumour necrosis factor-alpha (TNF-alpha) is a proinflammatory mediator that exerts its biological functions by binding two TNF receptors (TNF-RI and TNF-RII), which initiate biological responses by interacting with adaptor and signalling proteins. Among the signalling components that associate with TNF receptors are members of the TNF-R-associated factor (TRAF) family. TRAF2 is required for TNF-alpha-mediated activation of c-Jun N-terminal kinase (JNK), contributes to activation of NF-kappaB, and mediates anti-apoptotic signals,. TNF-RI and TNF-RII signalling complexes also contain the anti-apoptotic ('inhibitor of apoptosis') molecules c-IAP1 and c-IAP2 (refs 5, 6), which also have RING domain-dependent ubiquitin protein ligase (E3) activity. The function of IAPs in TNF-R signalling is unknown. Here we show that binding of TNF-alpha to TNF-RII induces ubiquitination and proteasomal degradation of TRAF2. Although c-IAP1 bound TRAF2 and TRAF1 in vitro, it ubiquitinated only TRAF2. Expression of wild-type c-IAP1, but not an E3-defective mutant, resulted in TRAF2 ubiquitination and degradation. Moreover, E3-defective c-IAP1 prevented TNF-alpha-induced TRAF2 degradation and inhibited apoptosis. These findings identify a physiologic role for c-IAP1 and define a mechanism by which TNF-RII-regulated ubiquitin protein ligase activity can potentiate TNF-induced apoptosis.     

The structural basis for activation of plant immunity by bacterial effector protein AvrPto

Pathogenic microbes use effectors to enhance susceptibility in host plants. However, plants have evolved a sophisticated immune system to detect these effectors using cognate disease resistance proteins, a recognition that is highly specific, often elicits rapid and localized cell death, known as a hypersensitive response, and thus potentially limits pathogen growth. Despite numerous genetic and biochemical studies on the interactions between pathogen effector proteins and plant resistance proteins, the structural bases for such interactions remain elusive. The direct interaction between the tomato protein kinase Pto and the Pseudomonas syringae effector protein AvrPto is known to trigger disease resistance and programmed cell death through the nucleotide-binding site/leucine-rich repeat (NBS-LRR) class of disease resistance protein Prf. Here we present the crystal structure of an AvrPto-Pto complex. Contrary to the widely held hypothesis that AvrPto activates Pto kinase activity, our structural and biochemical analyses demonstrated that AvrPto is an inhibitor of Pto kinase in vitro. The AvrPto-Pto interaction is mediated by the phosphorylation-stabilized P+1 loop and a second loop in Pto, both of which negatively regulate the Prf-mediated defences in the absence of AvrPto in tomato plants. Together, our results show that AvrPto derepresses host defences by interacting with the two defence-inhibition loops of Pto.     

BICP0 and its RING finger domain act as ubiquitin E3 ligases <Emphasis Type="Italic">in vitro</Emphasis>

Bovine infected-cell protein 0 (BICP0) encoded by bovine herpes virus 1 (BHV-1) immediate early gene is necessary for efficient productive infection, in a large part,because it activates all 3 classes of BHV-1 genes. It also has the ability to efficiently transactivate promoters that are not derived from BHV-1. To investigate the mechanism by which BICP0 achieves these effects, we expressed and purified BICP0 and its different mutants in E. coil In vitro assays showed that both full-length BICP0 and its isolated RING finger domain induce the accumulation of polyubiquitin chains. Mutations within the RING finger region that abolish the in vitro ubiquitination activity also cause severe reductions in BICP0 activity in other assays. Based on these, we conclude that BICP0 has the potential to act as an E3 ubiquitin ligase during viral infection and its RING finger domain is necessary for this function. These strongly support the hypothesis that BICPO might influence virus infection through its ability to interact with the ubiquitin-proteasome pathway.     

A saponin-detoxifying enzyme mediates suppression of plant defences

Plant disease resistance can be conferred by constitutive features such as structural barriers or preformed antimicrobial secondary metabolites. Additional defence mechanisms are activated in response to pathogen attack and include localized cell death (the hypersensitive response). Pathogens use different strategies to counter constitutive and induced plant defences, including degradation of preformed antimicrobial compounds and the production of molecules that suppress induced plant defences. Here we present evidence for a two-component process in which a fungal pathogen subverts the preformed antimicrobial compounds of its host and uses them to interfere with induced defence responses. Antimicrobial saponins are first hydrolysed by a fungal saponin-detoxifying enzyme. The degradation product of this hydrolysis then suppresses induced defence responses by interfering with fundamental signal transduction processes leading to disease resistance.     

A ubiquitin ligase transfers preformed polyubiquitin chains from a conjugating enzyme to a substrate

In eukaryotic cells, many short-lived proteins are conjugated with Lys 48-linked ubiquitin chains and degraded by the proteasome. Ubiquitination requires an activating enzyme (E1), a conjugating enzyme (E2) and a ligase (E3). Most ubiquitin ligases use either a HECT (homologous to E6-associated protein C terminus) or a RING (really interesting new gene) domain to catalyse polyubiquitination, but the mechanism of E3 catalysis is poorly defined. Here we dissect this process using mouse Ube2g2 (E2; identical at the amino acid level to human Ube2g2) and human gp78 (E3), an endoplasmic reticulum (ER)-associated conjugating system essential for the degradation of misfolded ER proteins. We demonstrate by expressing recombinant proteins in Escherichia coli that Ube2g2/gp78-mediated polyubiquitination involves preassembly of Lys 48-linked ubiquitin chains at the catalytic cysteine of Ube2g2. The growth of Ube2g2-anchored ubiquitin chains seems to be mediated by an aminolysis-based transfer reaction between two Ube2g2 molecules that each carries a ubiquitin moiety in its active site. Intriguingly, polyubiquitination of a substrate can be achieved by transferring preassembled ubiquitin chains from Ube2g2 to a lysine residue in a substrate.