生物信息学在蛋白质结构与功能预测中的应用

生物信息学是现代生命科学与信息科学、计算机科学、数学、统计学、物理学、化学等学科相互渗透而高度交叉形成的一门新兴前沿学科。随着人类基因组计划的完成,应用生物信息学技术预测蛋白质结构与功能将成为后基因组时代的一项重要任务。本文介绍了主要的蛋白质结构和序列数据库资源,在此基础上提出了一种高效的整合方法,并简述蛋白质结构与功能预测的基本方法、进展及其改进,展望了蛋白质预测技术的前景。     

己糖激酶Ⅱ与Warburg效应

肿瘤细胞在氧气充足的情况下通过葡萄糖的无氧氧化代谢方式为细胞提供能量的现象称为Warburg效应。己糖激酶是细胞糖酵解中的第一个限速酶,其中己糖激酶-Ⅱ与肿瘤细胞的糖酵解代谢、细胞增殖和凋亡等功能有着更为密切的联系。在肿瘤细胞中,基因水平的改变,信号通路的影响,促使己糖激酶高表达和活性增加,保证了肿瘤细胞糖酵解的快速进行。己糖激酶可通过与线粒体外膜结合促进糖酵解、抑制细胞凋亡和调节细胞自噬影响肿瘤细胞的功能。己糖激酶有望成为肿瘤治疗中的重要靶点。     

大肠杆菌脂多糖对腹膜间皮细胞己糖激酶活性的影响

目的:观察大肠杆菌脂多糖对原代培养的大鼠腹膜间皮细胞己糖激酶活性的影响;探讨其活性与葡萄糖净利用的关系,初步阐释腹膜透析相关性腹膜炎引起腹膜透析失超滤的机制。方法:利用胰蛋白酶消化法行大鼠腹膜间皮细胞的原代培养及传代，第2代细胞经鉴定及同步化后进入实验；用含不同浓度的大肠杆菌脂多糖（0、0.1、1.0、10、50、100 mg/L）刺激细胞 3 h，另用 10 mg/L 脂多糖分别刺激1、3、6、12、24 h，利用6-磷酸葡萄糖脱氢酶偶联比色法观察腹膜间皮细胞己糖激酶活性的变化；利用比色法观察细胞葡萄糖净利用量。结果:大肠杆菌脂多糖对大鼠腹膜间皮细胞己糖激酶活性的影响呈剂量和时间依赖性并伴有细胞葡萄糖净利用的增加。结论:大肠杆菌感染性腹膜炎时，其脂多糖通过影响腹膜间皮细胞己糖激酶的活性参与腹膜透析失超滤的过程。     

生物信息学分析亚洲牛带绦虫WD40基因及其蛋白质的结构与功能

目的 分析和预测亚洲牛带绦虫WD40基因及其编码蛋白的结构和特性，用于指导其生物学功能的实验研究。方法 利用美国国家生物技术信息中心（NCBI，http：／／www．ncbi．nlm．nih．gov／）、瑞士生物信息学研究所的蛋白分析专家系统（ExPASY，http：／／ca．expasy．org／）和IEDBAnalysisRecource提供的各种生物信息学在线分析程序，结合其它生物信息学分析软件包如Pcgene和VectorNTIsuite，从亚洲牛带绦虫全长cDNA质粒文库中识别WIMO基因及其编码区，分析、预测该基因编码的蛋白质的理化特性、翻译后的修饰位点、功能域、亚细胞定位、拓扑结构、二级结构、三维空间构象等。结果 该基因全长1208bp，编码区为81～1056bp，编码402个氨基酸，为全长基因；无跨膜区，具有多个磷酸化位点，蛋白的理化性质稳定，理论分子量为35942．4；没有质体、线粒体定位序列；预测该蛋白表面有三个亲水性强的线性表位和三个B细胞抗原表位。结论 应用生物信息方法从亚洲牛带绦虫成虫cDNA文库中识别出了亚洲牛带绦虫WD40基因，并对其所编码的蛋白质的结构与功能进行了预测。     

甲状腺功能异常患者与血清糖、脂肪、蛋白质水平的相关性分析

本文通过对甲亢与甲减患者血清甲状腺激素（ＴＨ）及血清糖、脂肪、蛋白质水平变化的分析 ,旨在观察甲状腺功能异常时 ,ＴＨ水平对人血清糖、脂肪、蛋白质变化的影响。对象和方法一、对象 :甲状腺功能异常患者共６５例。其中甲亢组４０例（男１６,女２４） ,年龄１８～２５岁。甲减组２５例（男１０ ,女１５） ,年龄１８～５９岁。所有受试对象均未经甲状腺药物治疗。健康对照组２５例（男１２ ,女１３） ,年龄２０～５２岁。甲亢组、甲减组及健康对照组所有受试对象 ,均无家族性高血脂症或高血压病、糖尿病、肝脏病或肾脏病病史 ,近一个月…     

己糖激酶2通过抑制线粒体凋亡通路减少LPS引起的人肺上皮BEAS-2B细胞凋亡

目的:探讨脂多糖(LPS)诱导人正常肺上皮BEAS-2B细胞凋亡的分子机制,并对己糖激酶2(HK2)在该效应中的作用进行分析。方法:采用不同浓度的LPS作用于BEAS-2B细胞建立损伤模型,CCK-8实验检测细胞存活率; Hoechst 33342染色及Annexin V/PI双染法分析细胞凋亡水平;通过使用线粒体凋亡通路抑制剂或者外在凋亡通路抑制剂鉴定细胞凋亡通路;在BEAS-2B细胞中转染HK2过表达质粒以验证HK2对上述效应的影响。Western blot法确认HK2过表达效果;免疫荧光实验检测HK2亚细胞定位。结果:CCK-8实验结果显示,LPS以时间和剂量依赖性方式降低BEAS-2B细胞活力; Hoechst 33342染色结果表明,给予LPS处理后的BEAS-2B细胞核出现固缩和碎裂;同时Annexin V/PI双染实验结果表明,处于凋亡状态的细胞由2. 89%增加至42. 4%,细胞凋亡率明显升高(P 0. 05)。线粒体凋亡通路执行蛋白caspase-9特异性抑制剂可显著抑制细胞凋亡,而caspase-8抑制剂却无此效应。在BEAS-2B细胞的凋亡过程中伴随着HK2的表达下调,而HK2过表达可以有效阻止以上事件的发生。结论:己糖激酶2可通过抑制线粒体凋亡通路减少LPS引起的人肺上皮细胞凋亡。     

己糖胺途径与糖基化修饰关系的研究进展

蛋白质糖基化 (protein glycosylation) 是一种广泛的翻译后修饰, 包括合成不同的多糖核心, 并将其添加到蛋白质一致序列内的特定氨基酸中。 大多数糖蛋白从涉及内质网和高尔基体的分泌系统获得糖基, 并分泌到与质膜相关的细胞壁或细胞外空间。 糖基化修饰可以通过影响糖蛋白的折叠、 分类、 定位、丰度和活性来调节糖蛋白的功能。 近年来多项研究报道葡萄糖代谢影响糖基化修饰。 蛋白质糖基化修饰涉及多种代谢疾病和肿瘤的发生, 己糖胺生物合成途径 ( hexosaminebiosynthesis pathway, HBP) 与机体代谢和蛋白质糖基化修饰关系密切。     

蛋白质序列信息的提取与蛋白质结构预测

蛋白质结构预测是生物信息学的一项重要任务.本文介绍了目前在蛋白质结构预测中,基于人工神经网络和小波分析蛋白质序列信息的提取方法.从蛋白质序列提取更多的生物学特征和机器学习相结合的方法将有效提高蛋白质结构预测的精度.     

结合蛋白质互作与功能类的可分性预测蛋白质功能

当前蛋白质功能注释体系的欠完备性限制了生物学及医药研究的进一步发展和应用,有必要将基因本体功能知识体系 (GO)的功能注释信息进一步深化,把蛋白质注释到GO中更具体的功能节点.为此,提出一种结合互作信息的新的预测策略,将酵母蛋白质准确地预测到更具体的功能类中.针对GO中的每个候选预测空间来构建分类器,并选用功能类分离性指标对候选预测空间进行评价,选出该指标大于一定阈值的候选预测空间,再将父节点中的蛋白质预测到子节点中.通过扩展深化预测的范围,可将预测空间一直上溯到根节点,对蛋白质功能进行深层预测,得到很好的预测结果.以上溯两层的预测空间为例,平均真阳性率和覆盖率分别达到94.02%和95.82%.     

Human basophils release interleukin-4 after stimulation with Schistosoma mansoni egg antigen

The elevated interleukin (IL)-4 and IgE production in Schistosoma mansoni infection seems to be induced essentially by the egg stage of the parasite. The underlying mechanism, however, is not known. Since basophils from human peripheral blood can produce IL-4, we asked, whether soluble S. mansoni egg antigens (SEA) would trigger basophils to release IL-4. Basophils from healthy human donors (n = 32) without prior history of schistosomiasis were incubated with SEA in the presence of IL-3. In all donors, IL-4 was produced at different concentrations. The IL-4 production was dependent on the dose of SEA, was correlated with the purity of the basophil preparation, and the IL-4 concentration in the culture supernatant was maximal 5 h after stimulation with SEA. In addition to its IL-4-stimulatory effect, SEA triggered basophils to degranulate, thereby releasing histamine and sulfidoleukotrienes. Stripping of receptor-bound IgE from basophils inhibited both SEA- and anti-IgE-induced, but not ionomycin-induced IL-4 production. Moreover, resensitization of stripped basophils with stripping supernatants or human serum restored SEA-induced IL-4 production. This suggests that IgE is involved in the mechanism of IL-4 induction by SEA. Since IL-4 is induced in basophils from nonexposed donors, basophils may play a role as an early source of IL-4 in S. mansoni infection.     

Primary in vitro sensitization to heterogeneous soluble antigens of Schistosoma mansoni adult worms

Primary in vitro sensitization of normal mouse spleen cells was accomplished using a saline soluble heterogeneous antigenic preparation from the whole adult worms (SWAP) of Schistosoma mansoni. A SWAP-specific rosette-forming cell (RFC) assay, using antigen on sheep erythrocytes, was used to detect sensitization due to S. mansoni infection and primary in vitro SWAP exposure. The latter exposure was facilitated by SWAP adsorption to the culture vessel via poly-L-lysine. RFC expression was maximal on day 3 of culture. It was antigen-specific in regard to unexposed controls, SWAP and human gamma globulin, and as controlled by RFC detected by parallel treated (control) erythrocytes. The RFC assay was best read between 90 and 180 min after initiation of rosette formation. Assay erythrocytes gave reproducible results for at least 3 weeks after preparation.     

Molecular cloning of Schistosoma mansoni calcineurin subunits and immunolocalization to the excretory system

In order to explain the schistosomicidal effect of cyclosporin A, the hypothesis was advanced that the drug, complexed with cyclophilin, inhibits the phosphatase activity of parasite calcineurin (CN), with mechanisms similar to those operating in its immunosuppressive action. As a preparatory step to the testing of this hypothesis, we report the molecular cloning of both CN subunits in Schistosoma mansoni. The catalytic (A) subunit has a predicted sequence of 607 amino acids and shows substantial similarity to other cloned CNs, except for the carboxy-terminal end that is highly divergent. The regulatory (B) subunit consists of 169 amino acids that are 86% identical to those of the human counterpart and, from its anomalous electrophoretic mobility, it appears to be myristoylated. The results of Southern blotting experiments are compatible with the existence of multiple genes for CNA and a single gene for CNB. Western blots showed that both subunits are present at all stages of the parasite life cycle and can be detected both in the soluble and in the membrane fraction. Immunofluorescence confocal microscopy revealed a striking concentration of the anti-CNA reactivity in 6–8 discrete spots in the schistosomula and in distinct spots along the body of the adult parasite, corresponding to the expected localization of flame cells. Both patterns were confirmed by a perfect co-localization of the anti-CNA signal with that of a previously characterized anti-flame cell monoclonal antibody. The preferential confinement of schistosome CN to the protonephridial system suggests that the enzyme in the parasite may fulfil similar functions to those performed in mammalian kidneys.     

Identification of paramyosin T cell epitopes associated with human resistance to Schistosoma mansoni reinfection

Paramyosin, a Schistosoma mansoni myoprotein associated with human resistance to infection and reinfection, is a candidate antigen to compose a subunit vaccine against schistosomiasis. In this study, 11 paramyosin peptides selected by TEPITOPE algorithm as promiscuous epitopes were produced synthetically and tested in proliferation and in vitro human leucocyte antigen (HLA)-DR binding assays. A differential proliferative response was observed in individuals resistant to reinfection compared to individuals susceptible to reinfection in response to Para (210-226) peptide stimulation. In addition, this peptide was able to bind to all HLA-DR molecules tested in HLA-DR binding assays, confirming its promiscuity. Para (6-22) and Para (355-371) were also shown to be promiscuous peptides, because they were able to bind to the six and eight most prevalent HLA-DR alleles used in HLA-DR binding assays, respectively, and were also recognized by T cells of the individuals studied. These results suggest that these paramyosin peptides are promising antigens to compose an anti-schistosomiasis vaccine.     

Sequence and level of endogenous expression of calcium channel beta subunits in Schistosoma mansoni displaying different susceptibilities to praziquantel

Kohn et al. [J. Biol. Chem. 276 (2001) 36873] demonstrated that cells expressing the structurally unusual schistosome β subunit SmCa_vβ1 in their voltage-operated calcium channels, exhibit an increased current amplitude in the presence of praziquantel (PZQ). This suggests that the beta subunit is involved in PZQ activity and is consistent with the known pharmacological effects of the drug. If this is so, the low susceptibility to PZQ noted in some Schistosoma mansoni strains could be due to some mutation(s) in the gene coding for this protein. We have sequenced the cDNAs coding for the SmCa_vβ1 and SmCa_vβ2 subunits of different sensitive and resistant strains and we have not been able to detect any meaningful differences. As an alternative hypothesis, the different sensitivity of schistosomes to PZQ action could be due to the expression of different β subunits in the parasite. This interpretation could also explain the low PZQ susceptibility of immature worms (28 days). We analyzed Northern blots of various strains and various developmental stages, but we were unable to demonstrate major quantitative differences in the expression of the β subunits.     

LIM‐only protein FHL2 regulates experimental pulmonary Schistosoma mansoni egg granuloma formation

LIM‐only protein FHL2 is associated with several immune and inflammatory diseases such as arthritis, influenza A virus infection, and lung inflammation. However, the role of FHL2 in macrophage differentiation and in the development of granuloma formation is unknown. Here, we show that expression of FHL2 is induced in mouse bone marrow derived macrophages (BMMs) following stimulation with M2 cytokines such as IL‐4 and IL‐10. FHL2‐knockout (FHL2‐KO) BMMs exhibit a proinflammatory M1 phenotype after LPS treatment and display a reduced anti‐inflammatory M2 phenotype following IL‐4 treatment. Furthermore, thioglycollate‐induced migration of macrophages and B cells is enhanced in FHL2‐KO mice. To evaluate the importance of FHL2 in the development of pulmonary granuloma formation, FHL2‐KO mice were challenged with Schistosoma mansoni eggs. FHL2‐KO mice show an enhanced number of granulomas and display decreased expression of Th2 markers and an exacerbated Th1 type of inflammation, characterized by enhanced expression of neutrophil markers and Th1 cytokines. Furthermore, the expression of barrier proteins is reduced in FHL2‐KO lung compared to WT. Collectively, these data identify a previously unrecognized role for FHL2 in the pathogenesis of pulmonary granulomatous inflammation, partly through its effect on macrophage polarization, modulation of the Th1/Th2 balance and regulation of permeability in lung.