泛素/26S蛋白酶体途径调节作物种子大小的研究进展

泛素/26S蛋白酶体系统（ubiquitin/26S proteasome system, UPS）是蛋白质泛素化的重要途径，作为蛋白质翻译后重要的修饰系统，在作物生长发育的各个阶段发挥重要作用。大量研究表明，UPS中泛素受体蛋白、E3泛素连接酶、去泛素化酶等能相互协调，通过在目标蛋白上连接或去除不同数量的泛素来介导目标蛋白的降解、改变亚细胞定位、蛋白活性等，从而调控种子的大小。对不同作物中泛素/26S蛋白酶体系统调节种子大小方面的研究进展进行了综述，并对其未来的发展进行了展望，为研究种子大小的调控机制及育种改良提供参考。     

鸭维甲酸诱导基因I克隆及其结构域功能分析

【目的】克隆鸭维甲酸诱导基因I（retinoic acid inducible gene I,RIG-I）,分析其不同结构域的功能。【方法】根据GenBank上公布的鸭RIG-I序列设计引物,利用RT-PCR克隆鸭RIG-I基因CDS（coding sequence）区,根据保守结构域预测结果,构建携带6*his组氨酸标签的不同结构域缺失突变体的真核表载体（RIG-I-Full、RIG-I-N和RIG-I-C）,转染鸡胚成纤维细胞DF1,经RT-PCR、间接免疫荧光方法鉴定重组质粒在细胞中转录与表达;同时,利用RT-qPCR检测RLR抗病毒信号通路中的IFN-β、Mx1和PKR等下游基因的表达变化。【结果】鸭RIG-I基因CDS区全长2 802 bp,共编码933个氨基酸;不同结构域缺失突变体的真核表载体转染DF1细胞后,重组蛋白均在DF1细胞中表达;RT-qPCR结果显示,N端能显著激活RLR通路上IFN-β、Mx1及PKR基因的表达上调。【结论】 duRIG-I及不同区段均能在DF1细胞中表达,其中N端在调节RLR抗病毒信号通路下游基因的表达过程中发挥了重要作用。     

前沿动态

<正>泛素复合物高分辨率晶体结构解析揭示识别K27-连接泛素链的分子机理泛素是一类在真核生物中普遍存在且高度保守的小分子多肽,其与底物蛋白质的结合过程被称为泛素化。蛋白质的泛素化是生物体内十分重要的翻译后修饰过程,在植物光合作用及抗逆、抗旱等多种生理过程中扮演着重要调控角色,在病毒、病菌等致病微生物的侵入及畜禽等宿主免疫防御方面也同样具有重要作用。细胞内的泛素化形式多种多样,不同泛素链的分子结构     

猪源Toll样受体家族(TLRs)及其在抗病育种中的应用

Toll样受体(toll like receptors, TLRs)作为模式识别受体,不仅能够对机体特异性配体进行识别,并通过多种信号传导通路(由髓样分化蛋白88或由β-干扰素TLR结构域衔接蛋白介导)启动信号传导继而引发特异性的免疫应答,同时还在一些由支原体、病毒、细菌等感染引起的免疫应答过程中发挥了重要的调控功能。因为其重要的免疫调控作用,Toll样受体家族已成为近些年研究的热点,对畜禽抗病育种工作也具有重要的科学意义和应用前景。文章综述了猪源TLRs的种类、功能、遗传变异以及介导的信号通路,并重点介绍了猪源TLRs在抗病育种中的应用,旨在为猪Toll样受体家族基因功能研究及有效遗传标记的筛选提供参考依据。     

人泛素结合酶UbcH5c活性突变体的构建·纯化以及催化活性研究

[目的]构建人泛素结合酶UbcH5c的活性位点突变体S22R和F62A,同时分析这2种突变体蛋白与野生型蛋白在泛素化反应中的活性差异。[方法]通过点突变(Site-Directed Mutagenesis)的方法构建2种突变体质粒,利用0.5 mmol/L IPTG分别诱导2种突变体蛋白在大肠杆菌中进行表达;将菌体超声破碎后,依次利用阳离子交换层析法对UbcH5c突变蛋白进行分离纯化,最后利用体外泛素化酶促反应结合蛋白免疫印迹杂交的方法分析野生型UbcH5c与其活性突变体UbcH5c S22R和UbcH5c F62A在泛素化修饰上的差异。[结果]人泛素结合酶UbcH5c的2个突变体蛋白S22R、F62A的泛素化修饰程度明显弱于野生型蛋白。[结论]突变体S22R和F62A突变均不同程度的改变了人泛素结合酶UbcH5c与泛素分子之间的结合活性,即2个突变的位点中第62位苯丙氨酸残基及第22位丝氨酸残基均是UbcH5c结合泛素的关键位点,而且后者对于UbcH5c结合泛素活性的影响更大。     

组蛋白H2B泛素化修饰研究进展

组蛋白翻译后修饰包括乙酰化、磷酸化、甲基化、泛素化和糖基化等。其中,组蛋白泛素化可能与基因的转录调控、异染色质的基因沉默、DNA修复等有关。笔者介绍了组蛋白H2B的泛素化机制及其意义。     

植物U-box蛋白研究进展

U-box结构是真核生物体内尤其是植物中广泛存在的一种高度保守的基序,其构象与RING-finger极其相似,两者都能发挥泛素连接酶E3的作用,促进底物蛋白泛素化降解。U-box基序还是泛素链聚集酶E4最主要的功能因子。在植物体内U-box经常与ARM重复耦联形成U-box/ARM结构,影响蛋白-蛋白互作,参与信号传导级联反应。许多植物U-box蛋白(PUB)能有效提高植株对干旱、高盐、异常温度等非生物胁迫的抵抗能力。此外,在植物对许多病原物的抗病反应中也需要U-box蛋白的参与。     

SCF泛素连接酶的体外泛素化体系构建与检测

为研究蛋白质的体外泛素化过程,利用大肠杆菌表达系统异源表达和纯化APPBP1/UBA3 （E1）、UBC12 （E2）、Cullin1-Rbx1 （E3）和Nedd8 （neural precursor cell expressed developmentally down-regulated protein 8）蛋白,制备FITC-Cysteine 绿色荧光素标记的泛素Ub（Ubiquitin）,构建了SCF泛素连接酶的体外泛素化体系,同时实现快速检测SCF泛素连接酶中Cullin1蛋白的自泛素化修饰。结果表明,建立的体外SCF E3自泛素化活性反应体系具有较高的可操作性和便利性。     

STAT3蛋白真核表达载体的构建及其泛素化功能研究

为研究信号转导与转录激活因子3(STAT3)的活性调控机制,克隆人源STAT3基因,并将其构建在真核表达载体上。之后,通过细胞内共表达STAT3和泛素化质粒的方法,验证了STAT3蛋白可以发生多种类型的泛素化修饰。结果表明,K63位非降解功能的泛素化影响STAT3蛋白的磷酸化,从而影响STAT3的活性及功能的发挥;而K48位降解功能的泛素化影响STAT3蛋白的降解,使STAT3蛋白得以更新,这2种类型的泛素化对STAT3蛋白及其功能都十分重要。     

家蚕bHLH转录因子Bmdimm与Bmchip的相互作用

【目的】bHLH转录因子Bmdimm是家蚕（Bombyx mori）5龄后期调控蚕丝蛋白丝素重链基因表达的重要转录因子。分析发现Bmdimm蛋白中包含有可能发生泛素化修饰的赖氨酸残基K43,因此推测Bmdimm可能发生泛素化修饰而被降解。Bmchip属于家蚕泛素连接酶E3家族中的U-box亚家族,在家蚕5龄期后部丝腺中有表达。论文旨在明确Bmdimm与Bmchip之间的相互作用,以便于更好地理解Bmdimm在家蚕体内的泛素化修饰及其对丝素重链基因转录调控的生物学意义。【方法】利用家蚕各组织基因芯片表达谱分析Bmdimm和Bmchip在后部丝腺中的表达情况;分别通过PCR和基因合成获得Bmdimm和Bmchip的CDS序列,将其构建到不同的原核表达载体并转化大肠杆菌表达目的蛋白,筛选最优的表达条件;利用镍柱亲和层析纯化融合蛋白,加入蛋白酶酶切,并再次通过镍柱亲和层析去除融合标签,纯化Bmdimm和Bmchip蛋白;利用凝胶过滤层析分析Bmdimm和Bmchip在溶液中的聚集状态;利用圆二色光谱研究Bmdimm和Bmchip蛋白的二级结构;利用Far-western blotting和Pull-down研究Bmdimm和Bmchip在体外的相互作用;利用微量热泳动研究Bmdimm和Bmchip结合的亲和力。【结果】基因芯片表达谱揭示Bmdimm和Bmchip在家蚕5龄期后部丝腺中都有表达。构建了Bmdimm和Bmchip多种表达载体,发现ppSUMO-Bmdimm和pET-32M·3C-Bmchip表达载体在16℃,0.3 mmol·L^-1 IPTG诱导下表达最好,并以此为基础表达纯化了Bmdimm和Bmchip蛋白;凝胶过滤分析表明Bmdimm和Bmchip蛋白在溶液中主要以二聚体的形式存在;圆二色光谱显示Bmdimm和Bmchip蛋白都含有α-螺旋结构; Far-western blotting和Pull-down结果表明Bmdimm和Bmchip可以在体外结合;利用微量热泳动实验计算出Bmdimm和Bmchip结合的平衡解离常数为(750±28.6)μmol·L^-1。【结论】家蚕bHLH转录因子Bmdimm和泛素连接酶Bmchip在家蚕5龄期后部丝腺中均有表达,二者可以在体外发生瞬时相互作用,暗示了Bmdimm可能通过Bmchip介导发生泛素化修饰从而下调丝素重链基因的转录。     

DUBA: a deubiquitinase that regulates type I interferon production

Production of type I interferon (IFN-I) is a critical host defense triggered by pattern-recognition receptors (PRRs) of the innate immune system. Deubiquitinating enzyme A (DUBA), an ovarian tumor domain-containing deubiquitinating enzyme, was discovered in a small interfering RNA-based screen as a regulator of IFN-I production. Reduction of DUBA augmented the PRR-induced IFN-I response, whereas ectopic expression of DUBA had the converse effect. DUBA bound tumor necrosis factor receptor-associated factor 3 (TRAF3), an adaptor protein essential for the IFN-I response. TRAF3 is an E3 ubiquitin ligase that preferentially assembled lysine-63-linked polyubiquitin chains. DUBA selectively cleaved the lysine-63-linked polyubiquitin chains on TRAF3, resulting in its dissociation from the downstream signaling complex containing TANK-binding kinase 1. A discrete ubiquitin interaction motif within DUBA was required for efficient deubiquitination of TRAF3 and optimal suppression of IFN-I. Our data identify DUBA as a negative regulator of innate immune responses.     

Proteasome-independent functions of ubiquitin in endocytosis and signaling

Ubiquitination is a reversible posttranslational modification of cellular proteins, in which a 76-amino acid polypeptide, ubiquitin, is primarily attached to the epsilon-amino group of lysines in target proteins. Ubiquitination is a major player in regulating a broad host of cellular processes, including cell division, differentiation, signal transduction, protein trafficking, and quality control. Aberrations in the ubiquitination system are implicated in pathogenesis of some diseases, certain malignancies, neurodegenerative disorders, and pathologies of the inflammatory immune response. Here, we discuss the proteasome-independent roles of ubiquitination in signaling and endocytosis.     

Direct ubiquitination of pattern recognition receptor FLS2 attenuates plant innate immunity

Innate immune responses are triggered by the activation of pattern-recognition receptors (PRRs). The Arabidopsis PRR FLAGELLIN-SENSING 2 (FLS2) senses bacterial flagellin and initiates immune signaling through association with BAK1. The molecular mechanisms underlying the attenuation of FLS2 activation are largely unknown. We report that flagellin induces recruitment of two closely related U-box E3 ubiquitin ligases, PUB12 and PUB13, to FLS2 receptor complex in Arabidopsis. BAK1 phosphorylates PUB12 and PUB13 and is required for FLS2-PUB12/13 association. PUB12 and PUB13 polyubiquitinate FLS2 and promote flagellin-induced FLS2 degradation, and the pub12 and pub13 mutants displayed elevated immune responses to flagellin treatment. Our study has revealed a unique regulatory circuit of direct ubiquitination and turnover of FLS2 by BAK1-mediated phosphorylation and recruitment of specific E3 ligases for attenuation of immune signaling.     

Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway

Stimulation of Toll-like receptors (TLRs) triggers activation of a common MyD88-dependent signaling pathway as well as a MyD88-independent pathway that is unique to TLR3 and TLR4 signaling pathways leading to interferon (IFN)-beta production. Here we disrupted the gene encoding a Toll/IL-1 receptor (TIR) domain-containing adaptor, TRIF. TRIF-deficient mice were defective in both TLR3- and TLR4-mediated expression of IFN-beta and activation of IRF-3. Furthermore, inflammatory cytokine production in response to the TLR4 ligand, but not to other TLR ligands, was severely impaired in TRIF-deficient macrophages. Mice deficient in both MyD88 and TRIF showed complete loss of nuclear factor kappa B activation in response to TLR4 stimulation. These findings demonstrate that TRIF is essential for TLR3- and TLR4-mediated signaling pathways facilitating mammalian antiviral host defense.     

番茄USP1响应干旱和高温胁迫的研究

DDB1作为CUL4泛素连接酶的核心组件参与细胞内许多重要的生命活动。前期研究发现番茄CUL4-DDB1复合物（CRL4）调节植物细胞的非生物胁迫应答。为进一步阐明DDB1在植物抗逆中的调控机制和生理功能,通过酵母双杂交筛选,发现普遍应激蛋白USP1与DDB1存在相互作用, USPs家族蛋白参与提高生物体对逆境胁迫的耐受;进一步研究发现USP1定位于细胞质中,且其基因表达受到多种胁迫条件诱导。此外泛素化实验揭示USP1可以被泛素化修饰降解,上调USP1表达导致植株对干旱和高温抗性的增强,表明USP1可能正调控番茄植株对逆境下胁迫的应答,且它的作用受CUL4-DDB1泛素连接酶的靶向降解调控。结果不仅揭示新的植物抗逆机理,而且为植物抗逆分子育种提供新的基因资源和技术途径。     

A cell's sense of direction

In eukaryotic cells directional sensing is mediated by heterotrimeric guanine nucleotide-binding protein (G protein)-linked signaling pathways. In Dictyostelium discoideum amoebae and mammalian leukocytes, the receptors and G-protein subunits are uniformly distributed around the cell perimeter. Chemoattractants induce the transient appearance of binding sites for several pleckstrin homology domain-containing proteins on the inner face of the membrane. In gradients of attractant these sites are persistently present on the side of the cell facing the higher concentration, even in the absence of a functional actin cytoskeleton or cell movement. Thus, the cell senses direction by spatially regulating the activity of the signal transduction pathway.     

Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells

Toll-like receptors (TLRs) control activation of adaptive immune responses by antigen-presenting cells (APCs). However, initiation of adaptive immune responses is also controlled by regulatory T cells (TR cells), which act to prevent activation of autoreactive T cells. Here we describe a second mechanism of immune induction by TLRs, which is independent of effects on costimulation. Microbial induction of the Toll pathway blocked the suppressive effect of CD4+CD25+ TR cells, allowing activation of pathogen-specific adaptive immune responses. This block of suppressor activity was dependent in part on interleukin-6, which was induced by TLRs upon recognition of microbial products.