Mechanisms of mono- and poly-ubiquitination : ubiquitination specificity depends on compatibility between the E2 catalytic core and amino acid residues proximal to the lysine

Ubiquitination involves the attachment of ubiquitin to lysine residues on substrate proteins or itself, which can result in protein monoubiquitination or polyubiquitination. Ubiquitin attachment to different lysine residues can generate diverse substrate-ubiquitin structures, targeting proteins to different fates. The mechanisms of lysine selection are not well understood. Ubiquitination by the largest group of E3 ligases, the RING-family E3 s, is catalyzed through co-operation between the non-catalytic ubiquitin-ligase (E3) and the ubiquitin-conjugating enzyme (E2), where the RING E3 binds the substrate and the E2 catalyzes ubiquitin transfer. Previous studies suggest that ubiquitination sites are selected by E3-mediated positioning of the lysine toward the E2 active site. Ultimately, at a catalytic level, ubiquitination of lysine residues within the substrate or ubiquitin occurs by nucleophilic attack of the lysine residue on the thioester bond linking the E2 catalytic cysteine to ubiquitin. One of the best studied RING E3/ E2 complexes is the Skp1/Cul1/F box protein complex, SCFCdc4, and its cognate E2, Cdc34, which target the CDK inhibitor Sic1 for K48-linked polyubiquitination, leading to its proteasomal degradation. Our recent studies of this model system demonstrated that residues surrounding Sic1 lysines or lysine 48 in ubiquitin are critical for ubiquitination. This sequence-dependence is linked to evolutionarily conserved key residues in the catalytic region of Cdc34 and can determine if Sic1 is mono- or poly-ubiquitinated. Our studies indicate that amino acid determinants in the Cdc34 catalytic region and their compatibility to those surrounding acceptor lysine residues play important roles in lysine selection. This may represent a general mechanism in directing the mode of ubiquitination in E2 s.

Characterization of epitopes for virus-neutralizing monoclonal antibodies to Ross River virus E2 using phage-displayed random peptide libraries

Ross River virus (RRV) is the predominant cause of epidemic polyarthritis in Australia, yet the antigenic determinants are not well defined. We aimed to characterize epitope(s) on RRV-E2 for a panel of monoclonal antibodies (MAbs) that recognize overlapping conformational epitopes on the E2 envelope protein of RRV and that neutralize virus infection of cells in vitro. Phage-displayed random peptide libraries were probed with the MAbs T1E7, NB3C4, and T10C9 using solution-phase and solid-phase biopanning methods. The peptides VSIFPPA and KTAISPT were selected 15 and 6 times, respectively, by all three of the MAbs using solution-phase biopanning. The peptide LRLPPAP was selected 8 times by NB3C4 using solid-phase biopanning; this peptide shares a trio of amino acids with the peptide VSIFPPA. Phage that expressed the peptides VSIFPPA and LRLPPAP were reactive with T1E7 and/or NB3C4, and phage that expressed the peptides VSIFPPA, LRLPPAP, and KTAISPT partially inhibited the reactivity of T1E7 with RRV. The selected peptides resemble regions of RRV-E2 adjacent to sites mutated in neutralization escape variants of RRV derived by culture in the presence of these MAbs (E2 210-219 and 238-245) and an additional region of E2 172-182. Together these sites represent a conformational epitope of E2 that is informative of cellular contact sites on RRV.

In vivo delivery of bovine viral diahorrea virus, E2 protein using hollow mesoporous silica nanoparticles

Our work focuses on the application of mesoporous silica nanoparticles as a combined delivery vehicle and adjuvant for vaccine applications. Here we present results using the viral protein, E2, from bovine viral diarrhoea virus (BVDV). BVDV infection occurs in the target species of cattle and sheep herds worldwide and is therefore of economic importance. E2 is a major immunogenic determinant of BVDV and is an ideal candidate for the development of a subunit based nanovaccine using mesoporous silica nanoparticles. Hollow type mesoporous silica nanoparticles with surface amino functionalisation (termed HMSA) were characterised and assessed for adsorption and desorption of E2. A codon-optimised version of the E2 protein (termed Opti-E2) was produced in Escherichia coli. HMSA (120 nm) had an adsorption capacity of 80 [small mu ]g Opti-E2 per mg HMSA and once bound E2 did not dissociate from the HMSA. Immunisation studies in mice with a 20 [small mu ]g dose of E2 adsorbed to 250 [small mu ]g HMSA was compared to immunisation with Opti-E2 (50 [small mu ]g) together with the traditional adjuvant Quillaja saponaria Molina tree saponins (QuilA, 10 [small mu ]g). The humoral responses with the Opti-E2/HMSA nanovaccine although slightly lower than those obtained for the Opti-E2 + QuilA group demonstrated that HMSA particles are an effective adjuvant that stimulated E2-specific antibody responses. Importantly the cell-mediated immune responses were consistently high in all mice immunised with Opti-E2/HMSA nanovaccine formulation. Therefore we have shown the Opti-E2/HMSA nanoformulation acts as an excellent adjuvant that gives both T-helper 1 and T-helper 2 mediated responses in a small animal model. This study has provided proof-of-concept towards the development of an E2 subunit nanoparticle based vaccine.

Silica vesicles as nanocarriers and adjuvants for generating both antibody and T-cell mediated immune resposes to Bovine Viral Diarrhoea Virus E2 protein

Bovine Viral Diarrhoea Virus (BVDV) is widely distributed in cattle industries and causes significant economic losses worldwide annually. A limiting factor in the development of subunit vaccines for BVDV is the need to elicit both antibody and T-cell-mediated immunity as well as addressing the toxicity of adjuvants. In this study, we have prepared novel silica vesicles (SV) as the new generation antigen carriers and adjuvants. With small particle size of 50 nm, thin wall (similar to 6 nm), large cavity (similar to 40 nm) and large entrance size (5.9 nm for SV-100 and 16 nm for SV-140), the SV showed high loading capacity (similar to 250 mu g/mg) and controlled release of codon-optimised E2 (oE2) protein, a major immunogenic determinant of BVDV. The in vivo functionality of the system was validated in mice immunisation trials comparing oE2 plus Quil A (50 mu g of oE2 plus 10 mu g of Quil A, a conventional adjuvant) to the oE2/SV-140 (50 mu g of oE2 adsorbed to 250 mu g of SV-140) or oE2/SV-140 together with 10 mu g of Quil A. Compared to the oE2 plus Quil A, which generated BVDV specific antibody responses at a titre of 10(4), the oE2/SV-140 group induced a 10 times higher antibody response. In addition, the cell-mediated response, which is essential to recognise and eliminate the invading pathogens, was also found to be higher [1954-2628 spot forming units (SFU)/million cells] in mice immunised with oE2/SV-140 in comparison to oE2 plus Quil A (512-1369 SFU/million cells). Our study has demonstrated that SV can be used as the next-generation nanocarriers and adjuvants for enhanced veterinary vaccine delivery. (C) 2014 Elsevier Ltd. All rights reserved.

Silica Vesicle Nanovaccine Formulations Stimulate Long-Term Immune Responses to the Bovine Viral Diarrhoea Virus E2 Protein

Bovine Viral Diarrhoea Virus (BVDV) is one of the most serious pathogen, which causes tremendous economic loss to the cattle industry worldwide, meriting the development of improved subunit vaccines. Structural glycoprotein E2 is reported to be a major immunogenic determinant of BVDV virion. We have developed a novel hollow silica vesicles (SV) based platform to administer BVDV-1 Escherichia coli-expressed optimised E2 (oE2) antigen as a nanovaccine formulation. The SV-140 vesicles (diameter 50 nm, wall thickness 6 nm, perforated by pores of entrance size 16 nm and total pore volume of 0.934 cm(3)g(-1)) have proven to be ideal candidates to load oE2 antigen and generate immune response. The current study for the first time demonstrates the ability of freeze-dried (FD) as well as non-FD oE2/SV140 nanovaccine formulation to induce long-term balanced antibody and cell mediated memory responses for at least 6 months with a shortened dosing regimen of two doses in small animal model. The in vivo ability of oE2 (100 mu g)/SV-140 (500 mu g) and FD oE2 (100 mu g)/SV-140 (500 mu g) to induce long-term immunity was compared to immunisation with oE2 (100 mu g) together with the conventional adjuvant Quil-A from the Quillaja saponira (10 mu g) in mice. The oE2/SV-140 as well as the FD oE2/SV-140 nanovaccine generated oE2-specific antibody and cell mediated responses for up to six months post the final second immunisation. Significantly, the cell-mediated responses were consistently high in mice immunised with oE2/SV-140 (1,500 SFU/million cells) at the six-month time point. Histopathology studies showed no morphological changes at the site of injection or in the different organs harvested from the mice immunised with 500 mu g SV-140 nanovaccine compared to the unimmunised control. The platform has the potential for developing single dose vaccines without the requirement of cold chain storage for veterinary and human applications.

Mathematical Model of Viral Kinetics In Vitro Estimates the Number of E2-CD81 Complexes Necessary for Hepatitis C Virus Entry

Interaction between the hepatitis C virus (HCV) envelope protein E2 and the host receptor CD81 is essential for HCV entry into target cells. The number of E2-CD81 complexes necessary for HCV entry has remained difficult to estimate experimentally. Using the recently developed cell culture systems that allow persistent HCV infection in vitro, the dependence of HCV entry and kinetics on CD81 expression has been measured. We reasoned that analysis of the latter experiments using a mathematical model of viral kinetics may yield estimates of the number of E2-CD81 complexes necessary for HCV entry. Here, we constructed a mathematical model of HCV viral kinetics in vitro, in which we accounted explicitly for the dependence of HCV entry on CD81 expression. Model predictions of viral kinetics are in quantitative agreement with experimental observations. Specifically, our model predicts triphasic viral kinetics in vitro, where the first phase is characterized by cell proliferation, the second by the infection of susceptible cells and the third by the growth of cells refractory to infection. By fitting model predictions to the above data, we were able to estimate the threshold number of E2-CD81 complexes necessary for HCV entry into human hepatoma-derived cells. We found that depending on the E2-CD81 binding affinity, between 1 and 13 E2-CD81 complexes are necessary for HCV entry. With this estimate, our model captured data from independent experiments that employed different HCV clones and cells with distinct CD81 expression levels, indicating that the estimate is robust. Our study thus quantifies the molecular requirements of HCV entry and suggests guidelines for intervention strategies that target the E2-CD81 interaction. Further, our model presents a framework for quantitative analyses of cell culture studies now extensively employed to investigate HCV infection.

Intravenous immunoglobulin expands regulatory T cells via induction of cyclooxygenase-2-dependent prostaglandin E2 in human dendritic cells

CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) play a critical role in the maintenance of immune tolerance. Intravenous immunoglobulin (IVIg), a therapeutic preparation of normal pooled human IgG, expands Tregs in various experimental models and in patients. However, the cellular and molecular mechanisms by which IVIg expands Tregs are relatively unknown. As Treg expansion in the periphery requires signaling by antigen-presenting cells such as dendritic cells (DCs) and IVIg has been demonstrated to modulate DC functions, we hypothesized that IVIg induces distinct signaling events in DCs that subsequently mediate Treg expansion. We demonstrate that IVIg expands Tregs via induction of cyclooxygenase (COX)-2-dependent prostaglandin E2 (PGE(2)) in human DCs. However, costimulatory molecules of DCs such as programmed death ligands, OX40 ligand, and inducible T-cell costimulator ligands were not implicated. Inhibition of PGE(2) synthesis by COX-2 inhibitors prevented IVIg-mediated Treg expansion in vitro and significantly diminished IVIg-mediated Treg expansion in vivo and protection from disease in experimental autoimmune encephalomyelitis model. IVIg-mediated COX-2 expression, PGE(2) production, and Treg expansion were mediated in part via interaction of IVIg and F(ab('))(2) fragments of IVIg with DC-specific intercellular adhesion molecule-3-grabbing nonintegrin. Our results thus uncover novel cellular and molecular mechanism by which IVIg expands Tregs.

人和猴T淋巴细胞表面TRBC受体及其配体不同于E2分子和CD2的配体

T淋巴细胞表面的TRBC受体不同介导E花结形成的E受体(CO_(2))和E2分子 。CD_(2)的配体, 人红细胞表面的CD58(LFA-3)和绵羊红细胞表面的T11TS, S42, S14及S110-220, 与TRBC受体的配体无关,TRBC玫瑰花结的形成是通过不同E 花结和人自身玫瑰花结的受体—配体相互作用来实现的, 进一步表明, 人和猴T 淋巴细胞表面和TRBC表面,可能都有独特的蛋白质分子介导TRBC玫瑰花结的形成 。表4参16

~(129)Xe~(30+)轰击Ni表面激发靶原子偶极跃迁和禁戒(M1和E2)跃迁的特征光谱线

报道了用高电荷态离子1 2 9Xe30 + (15 0keV)轰击金属Ni表面 ,激发的 2 0 0— 10 0 0nmNiⅠ和NiⅡ的特征光谱线的实验结果 .实验结果表明 :用电荷态足够高的离子作光谱激发源 ,无需很强的束流强度 (nA量级 ) ,便可有效地产生原子和离子的复杂组态间跃迁所形成的可见光波段的特征谱线 ,特别是NiⅠ和NiⅡ偶极禁戒的电四极跃迁E2和磁偶极跃迁M1的特征光谱线 .通过分析发现 ,在禁戒跃迁的谱线中 ,有些是电子组态相同而原子态不同的偶极禁戒跃迁光谱线而且NiⅡ的 6 84 84nm谱线较强

Two-electron-one-photon M1 and E2 transitions between the states of the 2p(3) and 2s(2)2p odd configurations for B-like ions with 18 <= Z <= 92

Two-electron-one-photon (TEOP) M1 and E2 transition energies, line strengths and transition probabilities between the states of the 2p(3) and 2s(2)2p odd configurations for B-like ions with 18 <= Z <= 92 have been calculated using the GRASP2K package based on the multiconfiguration Dirac-Hartree-Fock (MCDHF) method. Employing active-space techniques to expand the configuration list, we have systematically considered the valence, core-valence and core-core electron correlation effects. Breit interaction and quantum electrodynamical (QED) effects were also included to correct atomic state wavefunctions and the corresponding energies. Influences of electron correlation, Breit interaction and QED effects on transition energies and line strengths of the TEOP M1 and E2 transitions were analysed in detail. The present results were also compared with other theoretical and experimental values.

牙鲆性腺分化发育及类固醇激素T和E2对性腺分化的影响

本文采用组织学手段研究了牙鲆性腺在分化、发育和成熟过程中的变化。然后，通过放射性免疫方法（RIA）测定了牙鲆仔稚幼鱼全组织匀浆液中的性类固醇激素—睾酮（T）和雌二醇（E2）的含量，并结合牙鲆血清中T和E2含量的年周期测定，从内分泌学水平探讨了T和E2在其性腺分化、发育和成熟过程中水平的变化规律。同时，采用高温和雌性激素对性腺未分化的普通和雌核发育牙鲆仔稚鱼进行诱导处理，获得了较高比例的雄性鱼/假雄鱼或100%雌性鱼；并研究了这些外界环境因子对牙鲆性腺分化、性别比率及体内T和E2水平的影响，藉此探讨了牙鲆性别决定与性腺分化的细胞学和内分泌学机制。 对牙鲆仔稚幼鱼性腺的组织切片观察发现，培育水温18~20℃下，孵化后第45天、平均全长<22.0±2.8 mm的牙鲆，其性腺分化尚未开始，属于原始性腺；在孵化后70日龄、平均全长为38.0±1.7 mm左右，部分个体中观察到卵巢的雏形，其余个体的性腺在此阶段以及之后的一段时间内变化并不明显；到了第110天、平均全长达到86.5±5.9 mm时，雌性个体卵巢出现了卵原细胞向卵母细胞的转变，标志着卵巢分化的结束。在90日龄、平均全长为63.5±3.4 mm的雄性牙鲆中，精原细胞快速增殖，并观察到了输精管结构；进一步的细胞学分化则出现在100日龄、平均全长为76.0±8.6 mm的个体中，此时可以看到精小叶的形成；在平均全长为140.0±15.2 mm时，精巢中出现初级精母细胞，标志着性腺分化的基本完成。 对牙鲆仔稚幼鱼全组织匀浆和成鱼血清中的T和E2水平的比较发现，在全长为6 mm左右的仔鱼中T和E2含量均较高。随后，在性腺分化过程中T含量大大降低，E2的含量急剧增高，而性腺分化后期E2含量又降到较低的水平。在雄性牙鲆成鱼中， 从精巢第Ⅲ期开始，T含量随着精巢的发育而增加，到了精巢第Ⅴ期性腺发育成熟并排精后，又降低到较低的水平；E2含量在从精巢第Ⅲ期发育至精巢第Ⅴ期过程中略呈降低的趋势，但是总体上来说没有明显的差异。在雌性牙鲆成鱼中，卵巢从第Ⅱ期到第Ⅳ期的过程中，T水平逐渐升高，在第Ⅴ期时则明显降低；而E2含量在卵巢第Ⅱ期时保持较低的水平，随着卵巢的发育，E2含量逐渐增高，在卵巢第Ⅳ期时达到最高水平，在第Ⅴ期产卵后又有所降低。在雌雄个体中T和E2均呈现周期性的变化。5月份随着水温的升高，雄性个体T和E2含量显著上升；到了9月份又逐渐下降至最低值。雌性个体E2含量自3月份开始增高，在5月份急剧升高，并在6月份达到最高值；在7月份的时候，E2突然降低，而到了8月份又有所回升；9月份之后E2逐渐降低并在1月份左右降到最低；而T的含量分别在2月份和6月份出现两次高峰。 温度诱导牙鲆幼鱼性腺分化的结果表明，牙鲆中存在明显的TSD机制，即其性腺分化因饲育水温的不同而变化：在一定温度范围内，随着饲育温度的增加，牙鲆的雄性比例逐渐增高，常温对照组和21℃组中的雄性比例分别为51.62%、60.00%，而在24℃和28℃高温组中，雄性比例显著高于对照组，分别达到73.33%和87.27%。T和E2含量测定显示，在性腺分化时期，高温和对照组中T含量没有明显的变化，而温度处理组中的E2水平则低于对照组，特别是在28℃高温组，其E2水平显著低于对照组（P<0.05）。外源E2处理性腺未分化的牙鲆幼鱼的结果也表明，牙鲆的死亡率与雌性化比率均为雌性激素剂量依赖型的。随着外源E2剂量的增加，雌性比率增加，但同时死亡率也增高。此期间T和E2水平比较发现，在性腺分化时期，对照组中的T含量稍高于雌激素处理组；而对照组中的E2含量高于0.2 ppm和2 ppm两个低剂量组，却低于20 ppm和100 ppm两个高剂量组。 同时，还对人工诱导培育的雌核发育牙鲆和性反转牙鲆进行了性腺发育观察，在所观察的雌核发育牙鲆个体中，其雌性比例为83.33%，而高温28℃饲育群体中的雄性比例（即假雄鱼比例）为91.67%；在雌性个体中，也有一定比例的个体性腺发育不正常，有的性腺发育较小，有的则缺少部分性腺。进一步对雌核发育成体的血清中T和E2含量进行测量，发现在普通牙鲆个体中T含量显著低于雌核发育个体，而E2含量则高于雌核发育牙鲆；在雌核发育牙鲆中，性腺发育不正常的个体比性腺发育正常的个体中的T含量稍高，而E2含量则显著低于正常雌核发育牙鲆个体和普通牙鲆个体（P< 0.05）。

Fas ligand expression and tumour immune evasion; the role of prostaglandin E2 and the EP1 receptor

Background and Aim: During carcinogenesis, tumours develop multiple mechanisms to evade the immune system and suppress the anti-tumour immune response. Upregulation of Fas Ligand (FasL/CD95L) expression may represent one such mechanism. FasL is a member of the tumour necrosis factor superfamily that triggers apoptotic cell death following ligation to its receptor Fas. Numerous studies have demonstrated upregulated FasL expression in tumor cells, with FasL expression associated with numerous pro-tumorigenic effects. However, little is known about the mechanisms that regulate FasL expression in tumours. The cyclooxgenase (COX) signalling pathway may play an important role in colon carcinogenesis, via the production of prostaglandins, in particular PGE2. PGE2 signals through four different receptor subtypes, EP1 – EP4. Thus, the aim of this study was to investigate the effect of targeting the PGE2-FasL signaling pathway. Results: (i) PGE2 induces FasL expression via the EP1 receptor in colon cancer cells. (ii) Suppression of FasL expression in colon tumour cells in vivo significantly delays and reduces tumour growth. (iii) Blocking EP1 receptor signaling, or suppression of the EP1 receptor in colon tumour cells, reduces tumour growth in vivo. Suppression of tumour growth correlates in part with suppression of FasL expression. (iv) The reduction in tumour growth is associated with an improved anti-tumour immune response. Tumour infiltration by Treg cells and macrophages was reduced, and the cytotoxic activity of CTL generated from splenocytes isolated from these mice increased. Conclusion: 1) Targeting FasL expression by blocking PGE2-EP1 receptor signalling reduces tumour development in vivo. 2) The mechanism is indirect but is associated with an increased anti-tumour immune response. Thus, unraveling the mechanisms regulating FasL expression and the pro-tumorigenic effects of the EP1 receptor may aid in the search for new therapeutic targets against colon cancer.