蛋白酶体对抗原蛋白酶切特异性的研究

在MHCI类分子结合的抗原表位加工呈递途径中,泛素-蛋白酶体系统对抗原蛋白的降解发挥着重要作用。为了进一步研究蛋白酶体的酶切特异性,采用偏最小二乘方法（partial least squares method,PLS）建立了蛋白酶体的酶切位点预测模型,预测准确度为82.8％;由样本数据相应氨基酸对酶切位点形成的权重系数,得出蛋白酶体酶切位点及其两侧区域氨基酸的裂解特异性,它反映了蛋白酶体对抗原蛋白酶切的相互作用信息,也表明蛋白酶体对抗原蛋白酶切处理不是随机的,而是有一定模式和选择性的。     

基于VHSE结构表征的蛋白酶体酶切位点预测及酶切特异性研究

泛素-蛋白酶体在真核生物的抗原呈递、细胞周期调控和转录因子激活等生理过程中发挥着极为重要的作用,其核心就是蛋白酶体对底物的选择性酶切作用,因此对选择性酶切位点的预测一直是计算生物学的一个重点研究内容.针对现有酶切位点预测方法的非线性和物理意义不明确等问题,借鉴定量构效关系研究方法,采用基于氨基酸物理化学性质的描述子——VHSE(Principal component score vector of hydrophobic,steric,and electronic properties)对收集的2650个MHC-I配体的源蛋白序列进行了结构表征,在此基础上利用支持向量机建立了蛋白酶体酶切位点的预测模型,其最优线性模型的灵敏度(Sensitivity)、特异性(Specificity)、接受者操作特征曲线下面积(area under receiver operatingcharacteristics curve,AUC)和马休斯相关系数(Matthews coefficient of correlation,MCC)分别为90.18%,69.63%,0.8797和0.6131.模型分析结果表明:影响酶切位点选择性的氨基酸性质由大到小依次为:疏水性、电性和立体特征;P9,P8,P4,P1,P3’,P4’和P5’位氨基酸对酶切位点的选择有重要影响,研究亦显示酶切位点上游P1位和下游P1’～P5’的"疏水势差"有利于蛋白酶体的切割作用.     

抗原肽与MHC分子相互作用QM/MM分子动力学模拟研究

在MHC I类(major histocompatibility complex class I)分子抗原加工提呈过程中抗原蛋白在抗原提呈细胞(antigen-presenting cells, APC)的胞浆中被蛋白酶体(proteasome)裂解成短肽peptide, 由转运相关蛋白(transporter associated with antigen processing, TAP)将蛋白酶体裂解产生的短肽片段从胞浆转运至内质网腔. 短肽peptide在内质网中与新生成的MHC I类分子结合, 形成peptide-MHC复合体被提呈到APC细胞表面, 与T细胞表面抗原受体(T cell receptor, TCR)特异性识别结合, 使得CTL细胞开始活化、增殖、分化, 进而对肿瘤细胞进行特异性杀伤. 目前对CTL细胞如何识别抗原肽-MHC复合物分子及抗原短肽peptide如何与主要组织相容性复合体MHC分子的相互作用识别结合的机理还不是很清楚. 传统的预测CTL细胞表位的方法没有考虑受体与配体结合过程中电子结构的变化, 电子结构的变化需要用量子力学方法来处理. 本文采用QM/MM多尺度生物大分子的分子动力学模拟方法, 以天然抗原肽TAX (LLFGYPVYVYU)与HLA-A*0201分子结合的晶体结构为模板, 替换抗原肽“锚点”氨基酸, 将口袋氨基酸残基的原子极化电荷在空间形成的静电势用电多极矩分量表示. 用箱线图分析每个口袋氨基酸分子静电势变化和功能, 确定Pocket B的Glu63和Lys66的功能是精细识别氨基酸和一级结合氨基酸, Pocket F的Asp77, Tyr84的功能是精细识别氨基酸, 而Asp77, Lys146是一级结合氨基酸, 表明QM/MM方法在提取抗原肽与MHC I类分子识别结合特异性信息是可行的, 这对了解免疫识别机理和指导肿瘤疫苗的开发都具有指导意义.     

基于ISC-SVR方法预测Th细胞表位

外源性抗原蛋白被抗原提呈细胞(APC)摄取送入溶酶体中被降解为长度不一的肽段. 在HLA-DM分子辅助下, MHC II类分子相关恒定链多肽(class II-associated invariant chain peptide, CLIP)从MHC II类分子的肽结合槽解离, 使得外源性抗原降解的肽段进入MHC II类分子空的肽结合槽中, 形成稳定的抗原肽-MHC II类分子复合物. 之后再被提呈到APC细胞表面供CD4⁺Th细胞的TCR识别, 激活Th细胞分泌细胞因子, 促进CTL细胞特异性杀伤靶细胞或辅助B细胞产生抗体. Th细胞的活化对机体的细胞免疫和体液免疫功能都有重要的辅助作用. 本工作基于迭代自洽策略与支持向量回归机(ISC-SVR)方法建立了MHC II类分子与外源性抗原肽结合亲合力预测模型, 采用13mer扩展核心结合序列可以提高预测模型的性能, 分别按照均值法、最大值法、结合法、加权平均值法4种方法计算MHC II类分子与抗原肽的结合亲合力值. 对17种MHC II类分子配型进行了回归分析, 与其他预测模型相比较, 本工作模型都得到了最佳AUC值. 最后, 以HLA DRB1*0101分子为例, 分析讨论了MHC II类分子与抗原肽的结合特异性.     

基于VHSE结构表征的TAP亲和活性预测及选择特异性分析

应用氨基酸描述子VHSE(Principal component score vector of hydrophobic, steric, and electronic properties)对613个抗原9肽进行结构表征, 在此基础上, 采用支持向量机结合逐步回归变量筛选方法, 成功建立了抗原肽抗原处理相关转运蛋白(Transporter associated with antigen processing, TAP)亲和活性预测模型, 最优线性支持向量机模型的R2, Q2和R2ext分别为0.7386, 0.7270和0.6057. 模型结果分析表明, 影响TAP亲和活性的首要因素是电性, 其次是立体和疏水性质; 底物9肽的P1(N端)及P2, P7和P9(C端)位氨基酸物化性质对TAP亲和活性有重要影响, 而P3, P4, P5和P6位对模型贡献相对较小, P8位则与活性无关. 依据最优模型对模拟点突变9肽的TAP亲和活性的预测结果, 并结合变量载荷分析, 对TAP底物选择特异性进行了分析和总结.     

基于遗传算法的G蛋白偶联受体(GPCR)家族的分类预测研究

G蛋白偶联受体广泛参与各类生理活动的调控,目前市场上1/2的小分子药物均是以GPCR为药物靶标。由于G蛋白偶联受体晶体结构缺乏,采用理论方法对G蛋白受体耦合特异性进行分类预测在药物研发领域有着重要的学术和应用价值。因此,本文采用模式识别方法,基于GPCR序列,以伪氨基酸算法以及遗传算法为基础,用支持向量机方法建立了G蛋白偶联受体耦合特异性的预测模型,取得了可达82.5%的较高的预测准确度。     

应用独立成分分析方法预测CTL表位

基于独立成分分析方法分别采用3 z-scale和5 z-scale氨基酸结构描述符, 建立了抗原肽与MHC分子(major histocompatibility complex, MHC)相互作用结合的定量构效关系模型. 该两个模型训练集样本数是316, 预测集样本数是786. 结果表明: 3 z-scale模型的预测准确度和AUC值分别为70.3%, 0.70; 5 z-scale模型的预测准确度和AUC值分别为70.9%, 0.79. 本文建立CTL表位预测模型对进一步了解抗原肽与MHC I类分子相互作用机理具有一定的帮助.     

铝纳米粉末活化树突状细胞的作用

采用电爆炸法制备金属铝纳米粉末(ALEX),验证了其免疫增强效应及肿瘤预防效果.首先,利用铝金属丝电爆炸法,并应用羧基端基聚合物配体反应的方法合成了分散性良好的ALEX.利用静电物理吸附将抗原卵清蛋白(OVA)、抗原多肽(Peptide)接枝于ALEX表面,傅里叶变换红外光谱检测证明抗原与ALEX成功覆合.其次,验证了ALEX增强特异性免疫反应的效果.通过体外实验证明, ALEX可提高树突状细胞(DCs)的抗原提呈效果;同时,对免疫后小鼠血清抗体进行了定量检测,结果表明ALEX可显著提高小鼠体内抗原特异性抗体的水平.最后,利用小鼠黑色素瘤模型,验证了含有ALEX的肿瘤疫苗能够有效预防肿瘤的发生.     

hBAF53 多克隆抗体的制备与活性分析

用PCR方法扩增了人的BAF53全长eDNA序列，用酶切后的△BAF53 eDNA片段构建了表达质粒．转化大肠杆菌后，经IPTG诱导表达，再经包涵体裂解、纯化，得到hBAF53抗原多肽．从免疫动物身上获得实验动物的抗血清．通过斑点印迹方法测得hBAF53抗血清的特异性和效价，又用已提取的HeLa细胞核蛋白(含有天然BAF53蛋白)进行免疫印迹分析，证明天然。BAF53蛋白是该抗血清的抗原，说明获得的抗血清具有比较高的特异性，可用于染色体改构复合物各亚基问相互作用分析及调控基因转录机理方面的研究．     

氨基酸广义信息因子分析标度组合支持向量机模型用于β-turns预测及特征分析

提出一种新的组合方法用于β-turns预测和特征分析.该方法包括两步:如何表征β-turns特征和如何构建其预测模型.第一步应用氨基酸广义信息因子分析标度表征蛋白质中β-turns的结构特征,该标度涉及氨基酸的疏水性、α-螺旋与转角倾向、体积性质、构成特征、局部柔性及静电性.第二步以426个蛋白质为训练集样本,通过留1/7法交互验证,基于支持向量机建立β-turns预测模型.该模型分别成功地预测547和823个蛋白的β-turns.所得结果与所对比方法结果相当,更重要的是,SVM模型提供了一些关于β-turns特征的重要结构信息.该组合方法可以进一步尝试用于蛋白质结构预测及特征分析.     

Specificity of the proteasome cleavage to the antigen protein

In the MHC classⅠmolecule binding antigenic peptides processing and presentation pathway,the ubiquitin-proteasome system plays a key role in degrading the protein substrate.For the purpose of studying the specificities of proteasomal cleavage sites,partial least squares method is used to predict the proteasomal cleavage sites,and the predictive accuracy of the model is 82.8%.The specificities of the cleavage sites and the adjacent positions come from the contribution of the amino acids of the samples to the...     

Proteasome function in antigen presentation: immunoproteasome complexes, Peptide production, and interactions with viral proteins

Proteasomes are the major nonlysosomal protein degradation machinery in eukaryotic cells and they are largely responsible for the processing of antigens for presentation by the MHC class I pathway. This review concentrates on recent developments in the area of antigen processing. Specialized proteasomes called immunoproteasomes and an 11S regulator of proteasomes (PA28) are induced by interferon-gamma, but it is not entirely clear why changes in proteasome structure are beneficial for antigen presentation. Different proteasome complexes have distinct subcellular distributions and subtle differences in cleavage specificity. Thus it is likely that the efficiency of production of MHC class I binding peptides varies in different locations. Immunoproteasome subunits are enriched at the ER where TAP transports peptides for association with newly synthesized MHC class I molecules. There is recent evidence to suggest that antigen presentation from viral expression vectors, or from peptides that are either delivered by bacterial toxins or derived from signal peptides, require proteasome activity for generation of the correct C-terminus of the epitope. The correct N-terminus may be generated by recently identified ER associated aminopeptidases. A number of viral protein interactions with proteasome subunits have been reported and such interactions may interfere with host anti-viral defenses and also contribute to mechanisms of cell transformation.     

HIV protease-mediated activation of sterically capped proteasome inhibitors and substrates

Strategies for selectively killing HIV-infected cells present an appealing alternative to traditional antiretroviral drugs. We show here the first example of an inactive “Trojan horse” molecule that releases a cytotoxic, small-molecule proteasome inhibitor upon cleavage by HIV-1 protease. As a proof-of-concept strategy, the protein avidin was used to block entry of the compound into the proteasome in the absence of HIV-1 protease. We demonstrate that this strategy is also feasible without requiring an exogenous protein; a polylysine dendrimer-containing molecule is unable to enter the proteasome until cleaved by HIV-1 protease. These results demonstrate that conditional proteasome inhibitors could prove useful in the development of new tools for chemical biology and future therapeutics.     

Extended peptide-based inhibitors efficiently target the proteasome and reveal overlapping specificities of the catalytic beta-subunits

BACKGROUND: The 26S proteasome is responsible for most cytosolic proteolysis, and is an important protease in major histocompatibility complex class I-mediated antigen presentation. Constitutively expressed proteasomes from mammalian sources possess three distinct catalytically active species, beta1, beta2 and beta5, which are replaced in the gamma-interferon-inducible immunoproteasome by a different set of catalytic subunits, beta1i, beta2i and beta5i, respectively. Based on preferred cleavage of short fluorogenic peptide substrates, activities of the proteasome have been assigned to individual subunits and classified as 'chymotryptic-like' (beta5), 'tryptic-like' (beta2) and 'peptidyl-glutamyl peptide hydrolyzing' (beta1). Studies with protein substrates indicate a far more complicated, less strict cleavage preference. We reasoned that inhibitors of extended size would give insight into the extent of overlapping substrate specificity of the individual activities and subunits. RESULTS: A new class of proteasome inhibitors, considerably extended in comparison with the commonly used fluorescent substrates and peptide-based inhibitors, has been prepared. Application of the safety catch resin allowed the generation of the target compounds using a solid phase protocol. Evaluation of the new compounds revealed a set of highly potent proteasome inhibitors that target all individual active subunits with comparable affinity, unlike the other inhibitors described to date. Modification of the most active compound, adamantane-acetyl-(6-aminohexanoyl)(3)-(leucinyl)(3)-vinyl-(methyl)-sulfone (AdaAhx(3)L(3)VS), itself capable of proteasome inhibition in living cells, afforded a new set of radio- and affinity labels. CONCLUSIONS: N-terminal extension of peptide vinyl sulfones has a profound influence on both their efficiency and selectivity as proteasome inhibitors. Such extensions greatly enhance inhibition and largely obliterate selectivity towards the individual catalytic activities. We conclude that for the interaction with larger substrates, there appears to be less discrimination of different substrate sequences for the catalytic activities than is normally assumed based on the use of small peptide-based substrates and inhibitors. The compounds described here are readily accessible synthetically, and are more potent inhibitors in living cells than their shorter peptide vinyl sulfone counterparts.     

Locating apoptosis proteins by incorporating the signal peptide cleavage sites into the general form of Chou's Pseudo amino acid composition

Apoptosis proteins play an essential role in the development and homeostasis of an organism. The accurate prediction of subcellular location for apoptosis proteins is helpful for understanding the mechanism of programmed cell death and their biological functions. In this article, a new apoptosis proteins localization algorithm, named PSSP, is proposed based on the predicted cleavage sites of primary protein sequences. First, protein chains are divided into N‐terminal signal parts and mature protein parts according to their predicted cleavage sites by SignalP. Then, amino acid composition (ACC) of the individual subsequence together with pseudo‐ACC and stereochemical properties of whole chain were extracted to represent a given protein sequence. Jackknife test by support vector machine on three broadly used datasets (ZD98, ZW225, and CL317 datasets) of apoptosis proteins demonstrated that the total accuracies by this approach are 93.9, 87.6, and 91.5%, respectively. In addition, an independent nonapoptosis benchmark dataset (NNPSL) was also used to evaluate the performance of this method, and predictive accuracies for eukaryotic and prokaryotic proteins are also comparable to existing methods. © 2013 Wiley Periodicals, Inc.     

Substrate specificity of the human proteasome

BACKGROUND: Regulated proteolysis by the proteasome is crucial for a broad array of cellular processes, from control of the cell cycle to production of antigens. RESULTS: The rules governing the N-terminal primary and extended substrate specificity of the human 20S proteasome in the presence or absence of 11S proteasome activators (REGalpha/beta and REGgamma) have been elaborated using activity-based proteomic library tools. CONCLUSIONS: The 11S proteasome activators are shown to be important for both increasing the activity of the 20S proteasome and for altering its cleavage pattern and substrate specificity. These data also establish that the extended substrate specificity is an important factor for proteasomal cleavage. The specificities observed have features in common with major histocompatibility complex (MHC) class I ligands and can be used to improve the prediction of MHC class I restricted cytotoxic T-cell responses.     

Carboxylator: incorporating solvent-accessible surface area for identifying protein carboxylation sites

In proteins, glutamate (Glu) residues are transformed into γ-carboxyglutamate (Gla) residues in a process called carboxylation. The process of protein carboxylation catalyzed by γ-glutamyl carboxylase is deemed to be important due to its involvement in biological processes such as blood clotting cascade and bone growth. There is an increasing interest within the scientific community to identify protein carboxylation sites. However, experimental identification of carboxylation sites via mass spectrometry-based methods is observed to be expensive, time-consuming, and labor-intensive. Thus, we were motivated to design a computational method for identifying protein carboxylation sites. This work aims to investigate the protein carboxylation by considering the composition of amino acids that surround modification sites. With the implication of a modified residue prefers to be accessible on the surface of a protein, the solvent-accessible surface area (ASA) around carboxylation sites is also investigated. Radial basis function network is then employed to build a predictive model using various features for identifying carboxylation sites. Based on a five-fold cross-validation evaluation, a predictive model trained using the combined features of amino acid sequence (AA20D), amino acid composition, and ASA, yields the highest accuracy at 0.874. Furthermore, an independent test done involving data not included in the cross-validation process indicates that in silico identification is a feasible means of preliminary analysis. Additionally, the predictive method presented in this work is implemented as Carboxylator (), a web-based tool for identifying carboxylated proteins with modification sites in order to help users in investigating γ-glutamyl carboxylation.     

Specific cell-permeable inhibitor of proteasome trypsin-like sites selectively sensitizes myeloma cells to bortezomib and carfilzomib

Proteasomes degrade the majority of proteins in mammalian cells, are involved in the regulation of multiple physiological functions, and are established targets of anticancer drugs. The proteasome has three types of active sites. Chymotrypsin-like sites are the most important for protein breakdown and have long been considered the only suitable targets for antineoplastic drugs; however, our recent work demonstrated that inhibitors of caspase-like sites sensitize malignant cells to inhibitors of the chymotrypsin-like sites. Here, we describe the development of specific cell-permeable inhibitors and an activity-based probe of the trypsin-like sites. These compounds selectively sensitize multiple myeloma cells to inhibitors of the chymotrypsin-like sites, including antimyeloma agents bortezomib and carfilzomib. Thus, trypsin-like sites are cotargets for anticancers drugs. Together with inhibitors of chymotrypsin- and caspase-like sites developed earlier, we provide the scientific community with a complete set of tools to separately modulate proteasome active sites in living cells.     

Aggregation Patterns of Proteasome in Injured Neurons Induced by Transient Cerebral Ischemia

Proteasome activity reduction is an important pathological phenomenon, resulting in proteins aggregation and neuronal death in the injured neurons induced by transient ischemia. Our previous report showed that the trap of proteasome in the protein aggregates was a reason to lead to the reduction of proteasome activity. However, the patterns of proteasome entered into protein aggregates are not clear. In this study, we used a global ischemia model, Hematoxylin-Eosin staining, differential centrifuge, proteas...