泛素-蛋白酶体途径在病毒感染中的作用

泛素-蛋白酶体途径——降解溶酶体外蛋白的主要细胞内系统,在许多细胞功能中发挥重要作用。为自身利益如病毒出芽、凋亡抑制和免疫逃避,许多病毒已经进化出了利用泛素-蛋白酶体途径的不同策略。深入理解泛素-蛋白酶体途径在病毒感染中的作用有助于揭示一些病毒病的致病机理和发现新的分子靶标以开发抗病毒药物。因此,将泛素-蛋白酶体途径在病毒感染中的作用方面的最新进展作一综述。     

病毒利用UPP逃避抗病毒反应的研究进展

泛素-蛋白酶体途径是溶酶体外蛋白降解的主要系统,在许多细胞功能中发挥重要作用。越来越多的证据表明病毒参与泛素-蛋白酶体途径,干扰IFN信号通路和免疫受体表达、凋亡抑制及介导病毒潜伏。深入理解病毒利用泛素-蛋白酶体途径逃避宿主抗病毒反应的策略,有助于揭示病毒的致病机理和鉴定抗病毒药物新靶标。     

病毒攻击泛素蛋白酶体逃逸先天免疫的策略

泛素蛋白酶体水解途径是蛋白质的选择性降解中一种非常重要的机制,它通过选择性地降解蛋白质控制着体内许多重要的生物学过程。病毒侵染宿主细胞后,细胞的泛素蛋白酶体途径与一些重要的病毒蛋白相互作用,参与调节病毒的生命周期。同时,病毒的某些蛋白也会影响泛素蛋白酶体途径,以逃避宿主的防御机制。     

降解的智慧

蛋白质的"泛素—蛋白酶体"降解途径是生物体最基本的生命过程之一,它保证了细胞内各种蛋白的稳态和氨基酸的循环利用。Ci蛋白作为Hedgehog信号通路中的关键转录因子,能通过蛋白酶体发生完全降解和部分降解,而其中的分子机制一直未知。基于此,中国科学院上海生命科学研究院生物化学与细胞生物学研究所赵允课题组利用多种生物化学和果蝇遗传学手段,最终发现Ter94复合物和K11连接形式泛素化修饰共同参与了蛋白酶体的选择性降解。该发现一方面揭示了Hedgehog信号通路中Ci蛋白的调控机制,为未来相关癌症的治疗提供了参考;另一方面揭示了Ter94和K11泛素化修饰的新功能,为蛋白酶体降解领域的研究提供了新的思路。     

自噬和泛素-蛋白酶体系统之间的交联

自噬和泛素-蛋白酶体系统作为细胞内最重要的两大降解途径,对细胞稳态及细胞正常生理功能的维持都具有十分重要的作用。目前,越来越多的证据显示,这两大降解途径之间存在多种交联方式。首先,自噬和泛素-蛋白酶体系统都能以泛素作为共同标签,从而将泛素化底物降解;其次,泛素化的蛋白酶体可以通过自噬被清除,自噬相关蛋白质也可以通过蛋白酶体系统被降解;再次,这两条途径在细胞内能协同降解同一种底物;最后,它们之间可以相互调节活性,任一条途径被干扰都将影响另一条途径的活性。自噬和泛素-蛋白酶体系统之间的交联对细胞稳态的维持至关重要。交联失调不仅导致细胞功能异常,还可引起多种疾病的发生。本文主要对自噬和泛素-蛋白酶体系统之间的交联方式及其分子机制进行阐述,有助于深入了解细胞的分解代谢过程,进一步理解细胞稳态的维持机制,继而加深对相关疾病病理机制的认识。     

泛素化连接酶HUWE1在相关疾病中的研究进展

泛素化连接酶是泛素蛋白酶体系统中的关键酶之一,主要负责靶蛋白特异性识别以及泛素化系统活性的调控。HUWE1是一类具有HETC(homologous to E6AP C terminus)功能域的泛素化连接酶。研究发现,HUWE1参与细胞凋亡、基因组DNA损伤、肿瘤抑制因子调控和神经细胞分化增殖等重要生理过程。现主要针对泛素化连接酶HUWE1在肿瘤发生、男性生殖系统损伤、胚胎发育异常与神经发育疾病中的研究进展以及调节机制进行综述。     

泛素/26S蛋白酶体途径与植物的生长发育

泛素/26S蛋白酶体途径在植物蛋白降解系统中起重要作用,泛素分子主要通过泛素活化酶(E1)、泛素结合酶(E2)和泛素连接酶(E3)将靶蛋白泛素化,泛素化的蛋白最后被26S蛋白酶体识别和降解。泛素蛋白酶体途径参与植物体内的多种生理过程,如花和胚的发育、光形态建成、植物生长物质等几乎所有的生长发育过程,本文主要对泛素/26S蛋白酶体途径及其在植物生长发育过程中的精确调控作用进行综述。     

泛素-蛋白酶体系统对烟粉虱体内番茄黄曲叶病毒的影响

【目的】泛素化修饰广泛参与细胞周期、信号传导、转录调控、免疫应答等多个方面,是细胞内一种非常重要的蛋白水平可逆修饰。但目前关于泛素-蛋白酶体系统是否参与介体昆虫传播病毒还鲜有报道。本研究旨在探索泛素-蛋白酶体系统在烟粉虱Bemisia tabaci传播番茄黄曲叶病毒(Tomato yellow leaf curl virus,TYLCV)过程中所起的作用。【方法】通过RT-qPCR分析取食感染TYLCV和不感染TYLCV番茄植物的烟粉虱成虫体内泛素和蛋白酶体亚基基因的表达,采用Western blot检测TYLCV侵染后烟粉虱成虫体内泛素蛋白含量的变化;饲喂抑制剂和注射双链RNA的方法抑制烟粉虱成虫体内蛋白酶体活性后,通过RT-qPCR测定其体内TYLCV含量的变化。【结果】烟粉虱成虫携带TYLCV后,泛素以及26S蛋白酶体亚基4、6B和β基因的表达量没有发生显著变化,体内游离泛素和缀合泛素的含量以及两者的比例也都没有显著改变,表明TYLCV侵染不会影响烟粉虱体内泛素-蛋白酶体的活性。但饲喂抑制剂(Bortezomib或MG132)或沉默Rpn11抑制蛋白酶体活性后,烟粉虱成虫体内的TYLCV含量显著升高。【结论】泛素-蛋白酶体系统对烟粉虱体内的TYLCV起负调控作用,该系统可能通过直接降解病毒或激活免疫反应等方式抑制病毒含量,进而帮助烟粉虱应对TYLCV带来的不利影响。     

自噬与泛素化蛋白降解途径的分子机制及其功能

细胞内所有的蛋白质和大多数的细胞外蛋白都在不断的进行更新,即它们在不断地被降解,并被新合成的蛋白质取代。细胞内蛋白的降解主要通过两个途径,即自噬和泛素蛋白酶体系统。自噬是一种由溶酶体介导的细胞内过多或异常蛋白质的降解机制。在细胞内主要有3种类型的自噬,即分子伴侣介导的自噬、微自噬和巨自噬。泛素蛋白酶体系统是由泛素介导的一种高度复杂的蛋白降解机制,它参与降解细胞内许多蛋白质并且这个过程具有高度特异性。细胞内蛋白质的降解参与调节许多细胞过程,包括细胞周期、DNA修复、细胞生长和分化、细胞质量的控制、病原生物的感染反应和细胞凋亡等。许多严重的人类疾病被认为是由于蛋白质降解系统的紊乱而引起的。文章综述了自噬和泛素化途径及其分子机制,以及蛋白质降解系统紊乱的病理学意义。     

去泛素化酶在乳腺癌中的研究新进展

泛素-蛋白酶体系统(ubiquitin-proteasome-system,UPS)是控制蛋白质降解的主要系统,也是细胞基本活动的关键调节器。去泛素化酶(deubiquitinating enzymes,DUBs)是泛素-蛋白酶体系统的组成部分,主要参与调节蛋白质泛素化和去泛素化的动态平衡,对细胞增殖、信号转导、神经病变或肿瘤发生意义重大。不同的DUBs在乳腺癌中的作用不同,最新发现去泛素化酶BAP1、OTUD3、ATXN3L主要调节乳腺癌细胞增殖,某些DUBs小分子抑制剂可以间接诱导三阴性乳腺癌细胞凋亡。本文主要综述这三个DUBs及去泛素化酶抑制剂在乳腺癌中的研究新进展,为寻找新型的乳腺癌分子靶向药物提供理论依据。     

Ubiquitin enzymes in the regulation of immune responses

Ubiquitination plays a central role in the regulation of various biological functions including immune responses. Ubiquitination is induced by a cascade of enzymatic reactions by E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme, and E3 ubiquitin ligase, and reversed by deubiquitinases. Depending on the enzymes, specific linkage types of ubiquitin chains are generated or hydrolyzed. Because different linkage types of ubiquitin chains control the fate of the substrate, understanding the regulatory mechanisms of ubiquitin enzymes is central. In this review, we highlight the most recent knowledge of ubiquitination in the immune signaling cascades including the T cell and B cell signaling cascades as well as the TNF signaling cascade regulated by various ubiquitin enzymes. Furthermore, we highlight the TRIM ubiquitin ligase family as one of the examples of critical E3 ubiquitin ligases in the regulation of immune responses.     

The ‘ubiquitous’ reality of vector immunology

Ubiquitination (ubiquitylation) is a common protein modification that regulates a multitude of processes within the cell. This modification is typically accomplished through the covalent binding of ubiquitin to a lysine residue onto a target protein and is catalysed by the presence of three enzymes: an activating enzyme (E1), ubiquitin‐conjugating enzyme (E2) and ubiquitin‐protein ligase (E3). In recent years, ubiquitination has risen as a major signalling regulator of immunity and microbial pathogenesis in the mammalian system. Still, little is known about how ubiquitin relates specifically to vector immunology. Here, we provide a brief overview of ubiquitin biochemistry and describe how ubiquitination regulates immune responses in arthropods of medical relevance. We also discuss scientific gaps in the literature and suggest that, similar to mammals, ubiquitin is a major regulator of immunity in medically important arthropods.     

A spectrophotometric assay for conjugation of ubiquitin and ubiquitin-like proteins

Ubiquitination is a widely studied regulatory modification involved in protein degradation, DNA damage repair, and the immune response. Ubiquitin is conjugated to a substrate lysine in an enzymatic cascade involving an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin ligase. Assays for ubiquitin conjugation include electrophoretic mobility shift assays and detection of epitope-tagged or radiolabeled ubiquitin, which are difficult to quantitate accurately and are not amenable to high-throughput screening. We have developed a colorimetric assay that quantifies ubiquitin conjugation by monitoring pyrophosphate released in the first enzymatic step in ubiquitin transfer, the ATP-dependent charging of the E1 enzyme. The assay is rapid, does not rely on radioactive labeling, and requires only a spectrophotometer for detection of pyrophosphate formation. We show that pyrophosphate production by E1 is dependent on ubiquitin transfer and describe how to optimize assay conditions to measure E1, E2, and E3 activity. The kinetics of polyubiquitin chain formation by Ubc13–Mms2 measured by this assay are similar to those determined by gel-based assays, indicating that the data produced by this method are comparable to methods that measure ubiquitin transfer directly. This assay is adaptable to high-throughput screening of ubiquitin and ubiquitin-like conjugating enzymes.     

Structural modeling of protein ensembles between E3 RING ligases and SARS-CoV-2: The role of zinc binding domains

BackgroundThe ubiquitin system is a modification process with many different cellular functions including immune signaling and antiviral functions. E3 ubiquitin ligases are enzymes that recruit an E2 ubiquitin-conjugating enzyme bound to ubiquitin in order to catalyze the transfer of ubiquitin from the E2 to a protein substrate. The RING E3s, the most abundant type of ubiquitin ligases, are characterized by a zinc (II)-binding domain called RING (Really Interesting New Gene). Viral replication requires modifying and hijacking key cellular pathways within host cells such as cellular ubiquitination. There are well-established examples where a viral proteins bind to RING E3s, redirecting them to degrade otherwise long-lived host proteins or inhibiting E3’s ubiquitination activity. Recently, three binary interactions between SARS-CoV-2 proteins and innate human immune signaling Ε3 RING ligases: NSP15-RNF41, ORF3a-TRIM59 and NSP9-MIB1 have been experimentally established.MethodsIn this work, we have investigated the mode of the previous experimentally supported NSP15-RNF41, ORF3a,-TRIM59 and NSP9-MIB1 binary interactions by in silico methodologies intending to provide structural insights of E3-virus interplay that can help identify potential inhibitors that could block SARS-CoV-2 infection of immune cells.ConclusionIn silico methodologies have shown that the above human E3 ligases interact with viral partners through their Zn(II) binding domains. This RING mediated formation of stable SARS-CoV-2-E3 complexes indicates a critical structural role of RING domains in immune system disruption by SARS-CoV-2-infection.Data AvailabilityThe data used to support the findings of this research are included within the article and are labeled with references.     

The protein substrate binding site of the ubiquitin-protein ligase system

In order to gain insight into the mechanisms that determine the selectivity of the ubiquitin proteolytic pathway, the protein substrate binding site of the ubiquitin-protein ligase system was identified and examined. Previous studies had shown that the ligase system consists of three components: a ubiquitin-activating enzyme (E1), ubiquitin-carrier protein (E2), and a third enzyme, E3, the mode of action of which has not been defined. E3 from rabbit reticulocytes was further purified by a combination of affinity chromatography, hydrophobic chromatography, and gel filtration procedures. A 180-kDa protein was identified as the subunit of E3. Two independent methods indicate that E3 has the protein binding site of the ubiquitin ligase system. These are the chemical cross-linking of 125I-labeled proteins to the E3 subunit and the functional conversion of enzyme-bound labeled proteins to ubiquitin conjugates in pulse-chase experiments. The trapping of E3-bound protein for labeled product formation was allowed by the slow dissociation of E3 X protein complex. The specificity of binding of different proteins to E3, examined by both methods, showed a direct correlation with their susceptibility to degradation by the ubiquitin system. Proteins with free alpha-NH2 groups, which are good substrates, bind better to E3 than corresponding proteins with blocked NH2 termini, which are not substrates. Oxidation of methionine residues to sulfoxide derivatives greatly increases the susceptibility of some proteins to ligation with ubiquitin, with a corresponding increase in their binding to E3. However, a protein derivative which was subjected to both amino group modification and oxidation binds strongly to the enzyme, even though it cannot be ligated to ubiquitin. It thus seems that the substrate binding site of E3 participates in determining the specificity of proteins that enter the ubiquitin pathway of protein degradation.     

植物蛋白的体外泛素化检测方法

泛素激活酶(E1)、泛素耦联酶(E2)和泛素连接酶(E3)是蛋白质泛素化修饰的关键酶。在真核基因组上有大量基因编码这些泛素化相关的酶类或蛋白。检测这些泛素化修饰酶及其底物蛋白的生化特性和特异性是分析其生物学功能的重要内容。该文提供了一种简便快速检测体外泛素化反应的方法, 不仅可通过检测对DTT敏感的硫酯键的形成来判断E2的活性、检测E3的体外泛素化活性, 而且可以检测E2-E3和E3-底物的特异性。所用蛋白主要来源于拟南芥(Arabidopsis thaliana), 包括分属于绝大多数E2亚家族的成员, 可用于不同RING类型E3的活性检测。该方法不仅可以采用多种E2进行E3活性分析, 而且可以分析不同组合的E2-RING E3、RING E3-底物的泛素化活性等, 亦可应用于真核生物蛋白质尤其是植物蛋白的体外泛素化活性分析。     

植物蛋白的体外泛素化检测方法

泛素激活酶(E1)、泛素耦联酶(E2)和泛素连接酶(E3)是蛋白质泛素化修饰的关键酶。在真核基因组上有大量基因编码这些泛素化相关的酶类或蛋白。检测这些泛素化修饰酶及其底物蛋白的生化特性和特异性是分析其生物学功能的重要内容。该文提供了一种简便快速检测体外泛素化反应的方法, 不仅可通过检测对DTT敏感的硫酯键的形成来判断E2的活性、检测E3的体外泛素化活性, 而且可以检测E2-E3和E3-底物的特异性。所用蛋白主要来源于拟南芥(Arabidopsis thaliana), 包括分属于绝大多数E2亚家族的成员, 可用于不同RING类型E3的活性检测。该方法不仅可以采用多种E2进行E3活性分析, 而且可以分析不同组合的E2-RING E3、RING E3-底物的泛素化活性等, 亦可应用于真核生物蛋白质尤其是植物蛋白的体外泛素化活性分析。     

LUBAC synthesizes linear ubiquitin chains via a thioester intermediate

The linear ubiquitin chain assembly complex (LUBAC) is a RING E3 ligase that regulates immune and inflammatory signalling pathways. Unlike classical RING E3 ligases, LUBAC determines the type of ubiquitin chain being formed, an activity normally associated with the E2 enzyme. We show that the RING-in-between-RING (RBR)-containing region of HOIP-the catalytic subunit of LUBAC-is sufficient to generate linear ubiquitin chains. However, this activity is inhibited by the N-terminal portion of the molecule, an inhibition that is released upon complex formation with HOIL-1L or SHARPIN. Furthermore, we demonstrate that HOIP transfers ubiquitin to the substrate through a thioester intermediate formed by a conserved cysteine in the RING2 domain, supporting the notion that RBR ligases act as RING/HECT hybrids.     

Degradation of misfolded protein in the cytoplasm is mediated by the ubiquitin ligase Ubr1

Protein quality control and subsequent elimination of terminally misfolded proteins occurs via the ubiquitin-proteasome system. Tagging of misfolded proteins with ubiquitin for degradation depends on a cascade of reactions involving an ubiquitin activating enzyme (E1), ubiquitin conjugating enzymes (E2) and ubiquitin ligases (E3). While ubiquitin ligases responsible for targeting misfolded secretory proteins to proteasomal degradation (ERAD) have been uncovered, no such E3 enzymes have been found for elimination of misfolded cytoplasmic proteins in yeast. Here we report on the discovery of Ubr1, the E3 ligase of the N-end rule pathway, to be responsible for targeting misfolded cytosoplasmic protein to proteasomal degradation.