基于iTRAQ技术分析五倍子作用白念珠菌后的差异蛋白表达

为了探讨五倍子抗白念珠菌的作用机制,提取4 μg/mL五倍子提取液作用后的白念珠菌（SC5314）总蛋白,采用同位素标记相对和绝对定量（isobaric tags for relative and absolute quantitation,iTRAQ）蛋白质组学技术、液相色谱串联质谱（LC-MS/MS）技术分析和鉴定差异表达的蛋白质,并对差异表达蛋白进行生物信息学分析。经LC-MS/MS鉴定出3 721种蛋白质,其中,差异表达蛋白104种,包括57种表达上调蛋白和47种表达下调蛋白。通过生物信息学分析发现,上述差异蛋白参与了氧化还原反应、过氧化氢分解代谢以及能量代谢等生物学过程。研究表明,五倍子抗白念珠菌的作用机制可能是通过抑制氧化磷酸化,进而影响菌体能量代谢和物质生成,最终导致细胞结构与功能的改变。     

白念珠菌侵袭宿主毒力因子研究进展

白念珠菌是一种寄生于人类黏膜表面的条件致病菌,是导致免疫功能低下人群侵袭性真菌感染的主要病原菌。白念珠菌形成侵袭性感染的过程主要分为黏附、侵袭、播散、形成感染灶等步骤,其中黏附和侵袭过程最为关键。黏附是白念珠菌入侵宿主的前提,该过程主要依赖于细胞壁表面的黏附素。侵袭阶段主要与菌丝形成、细胞壁表面毒力蛋白表达和蛋白水解酶分泌增加有关。形成菌丝是白念珠菌侵袭宿主的关键因素,主要由细胞内cAMP/PKA和MAPK等信号通路调控;侵袭素主要位于白念珠菌细胞壁表面,可以协助其穿刺宿主上皮细胞、诱导内吞作用;白念珠菌还可分泌多种蛋白水解酶,它能够破坏宿主组织细胞,协助白念珠菌形成感染灶。该文主要对白念珠菌黏附和侵袭宿主过程中关键的毒力因子进行综述,为理解白念珠菌致病机制以及选择潜在的药物靶点提供参考。     

炭样小单孢菌JXNU-1产抗生素JX对嗜根考克氏菌肽聚糖合成的影响及其机理

为了揭示一株具有广谱抗菌活性炭样小单孢菌JXNU-1产的核苷类抗生素JX对嗜根考克氏菌肽聚糖合成的影响,本研究采用超声破壁、称重法研究抗生素对嗜根考克氏菌细胞壁含量变化,采用iTRAQ技术对抗生素处理前、后嗜根考克氏菌的蛋白质组进行比较分析。结果显示,在抗生素JX作用下,嗜根考克氏菌细胞分裂被抑制,细胞壁含量升高;iTRAQ技术鉴定了抗生素胁迫下的嗜根考克氏菌细胞中的1 780个蛋白,其中差异表达蛋白149个,包括表达上调蛋白106个,表达下调蛋白43个,上调表达蛋白中包括有一与肽聚糖合成的相关酶MurG。本研究为揭示炭样小单孢菌JXNU-1产抗生素JX的抗菌作用机制提供了基础数据。     

白念珠菌细胞壁相关基因和蛋白的研究进展

随着真菌深部感染率的上升,细胞壁作为真菌中特有和必须的细胞结构,又因其不存在于哺乳动物细胞中,已逐步成为重要的药物作用靶点。以白念珠菌为例,参与其细胞壁构成、分泌、表达等相关的结构功能基因和蛋白已逐步发现和证实,这为发现抗真菌药物新靶标、设计新型抗真菌药物奠定理论基础。     

油菜叶片蛋白质组对机械损伤应答的初步分析

 植物在对抗昆虫的长期进化过程中形成了自我防御机制,能够产生特异的抗性蛋白来应对昆虫的取食。该文用机械损伤模拟害虫取食,研究和 对比了油菜(Brassica napus cv. Westar)在机械损伤前后可溶性总蛋白的含量变化并试图通过蛋白质组学技术来检测可能发生变化的蛋白质。 蛋白质定量检测发现,同一植株同一叶片损伤前后可溶性总蛋白含量差异显著,损伤后蛋白表达量增高。 蛋白质双向凝胶电泳及其差异显示分 析损伤前后的蛋白质组,表明有8个蛋白质点发生明显的上调或下调。选择其中2个差异蛋白点经过MALDI-TOF质谱鉴定,它们分别是Rubisco小 亚基前体、果糖-1,6-二磷酸醛缩酶和粪卟啉-3-氧化酶,这些蛋白质可能在油菜叶片应答机械损伤过程中对维持植物的生理功能起到重要作用 。     

iTRAQ技术在真菌研究中的应用进展

iTRAQ技术是一种新的、功能强大的、可以最多同时比较8种不同样品中蛋白质相对或绝对含量的蛋白质组学方法,结合多维液相色谱和串联质谱分析,iTRAQ技术已成为差异蛋白质组学定量研究的主要工具之一。而真菌的致病作用是多种蛋白质共同参与的真菌?宿主相互作用的复杂过程,因此整体、定量地分析真菌致病过程中的差异表达蛋白质谱,对于研究真菌的致病机制具有重要作用。该文重点就iTRAQ技术在真菌研究中的应用进展进行综述。     

分泌型蛋白介导白念珠菌与宿主相互作用分子机制的研究进展

白念珠菌是人类最常见的条件性致病真菌之一,主要定植于人体粘膜表面。在白念珠菌与宿主相互作用过程中,分泌型蛋白起着非常重要的作用。针对分泌蛋白功能及其作用机理的研究有助于阐明白念珠菌致病分子机制,并为诊断、预防和治疗真菌感染提供新的理论策略。本文综述了白念珠菌分泌型蛋白在介导病原与宿主相互作用分子机制方面的最新研究进展,概括了分泌蛋白在组织侵入损伤、营养获取、细胞壁维持以及免疫逃避等方面的功能,同时对未来值得重点关注的研究方向进行了探讨。     

白头翁汤正丁醇提取物对白念珠菌细胞壁抑制作用研究

目的探讨白头翁汤正丁醇提取物(Butyl alcohol extract of BaiTouWeng decoction,BAEB)对白念珠菌细胞壁的抑制作用。方法以spot assay检测BAEB对白念珠菌细胞活性的影响;流式细胞仪和酶标仪检测BAEB对白念珠菌细胞壁β-1,3-葡聚糖及几丁质变化;qRT-PCR检测白念珠菌细胞壁β-1,3-葡聚糖合成相关基因FKS-1及几丁质合成相关基因CHS1、CHS2、CHS3、CHS8的表达;透射电镜观察BAEB对白念珠菌细胞壁结构影响。结果 BAEB干预后白念珠菌活性降低,256、512、1 024μg/mL BAEB组白念珠菌细胞壁β-1,3-葡聚糖暴露与几丁质暴露逐渐增多(P0.05);1 024μg/mL BAEB干预组FKS1、CHS1、CHS2、CHS3、CHS8分别下调5.57、2.96、3.29、4.47、3.00倍;透射电镜观察BAEB干预后白念珠菌细胞壁结构有破损。结论 BAEB可通过增加β-1,3-葡聚糖和几丁质的暴露,抑制β-1,3-葡聚糖、几丁质及其生物合成相关基因的表达,进而破坏白念珠菌细胞壁完整性。     

应用iTRAQ标记技术结合2DLC-MS/MS分析家蝇幼虫免疫诱导后血淋巴蛋白质组

目的分析家蝇幼虫免疫诱导前后血淋巴中的免疫防御相关靶标,探索其先天性免疫机制。方法采用同位素标记及相对定量技术(isobaric tags for relative and absolute quantification,iTRAQ)结合2D LC-MS/MS对家蝇幼虫诱导前后的血淋巴蛋白质组进行研究。结果与对照组比较研究后共获得237个不同肽段,鉴定到13个具有定量信息的差异蛋白。免疫刺激后显著上调的蛋白有9个,下调蛋白4个(P0.05),对鉴定到的差异蛋白进行理化性质分析和GO(gene ontology)注释分析,发现这些蛋白分别具有抗菌、抗氧化、催化结合等功能,并参与了免疫、代谢、应激、转运等生物学过程,表明对家蝇幼虫经诱导后血淋巴中多种功能蛋白的表达发生了变化。结论 iTRAQ标记技术结合2D LC-MS/MS可以有效地分离鉴定昆虫血淋巴蛋白质组,为深入研究差异蛋白的功能奠定了基础。     

基于Hog-MAPK信号通路探讨白念珠菌氟康唑耐药机制的实验研究

目的探讨Hog-MAPK信号通路在白念珠菌氟康唑耐药机制中的作用。方法通过Real-Time PCR、Western blot等方法比较白念珠菌氟康唑敏感株与耐药株Hog-MAPK信号通路相关的HOG1等基因的mRNA表达,以及磷酸化p38 MAPK蛋白表达的差异,并应用微量液基稀释法检测白念珠菌HOG1基因缺陷株及其原始亲代菌株/标准株对氟康唑MIC值的差异。结果白念珠菌氟康唑敏感株与耐药株之间Hog-MAPK信号通路相关基因mRNA表达和蛋白表达存在一定差异性,敏感株的表达在一定程度上低于耐药株,HOG1基因缺陷株对氟康唑更为敏感。结论白念珠菌氟康唑耐药性可能与Hog-MAPK信号通路中部分基因和蛋白表达有关,这些基因和蛋白表达的降低可能使耐药性发生改变。     

A proteomic-based approach for the identification of Candida albicans protein components present in a subunit vaccine that protects against disseminated candidiasis

Candidiasis has become a prevalent infection in different types of immunocompromised patients. The cell wall of Candida albicans plays important functions during the host-fungus interactions. Cell wall (surface) proteins of C. albicans are major elicitors of host immune responses during candidiasis, and represent candidates for vaccine development. Groups of mice were vaccinated subcutaneously with a beta-mercaptoethanol (beta-ME) extract from C. albicans containing cell wall proteins. Vaccinated mice were then infected with a lethal dose of C. albicans. Increased survival and decreased fungal burden were observed in vaccinated mice as compared to a control group, and 75% of vaccinated mice with the beta-ME extract survived this otherwise lethal infection. We used a proteomic approach (2-DE followed by immunoblotting) to demonstrate a complex polypeptidic pattern associated with the beta-ME extract used in the vaccine formulation and to detect immunogenic components recognized by antibodies in immune sera from vaccinated animals. Reactive protein spots were identified by MALDI-TOF-MS and searches in genomic databases. As a conclusion, vaccination strategies using C. albicans cell wall proteins induce protective responses. These antigens can be identified by proteomic approaches and may be used as components of subcellular vaccines against candidiasis.     

Proteomic characterization of the Pseudomonas putida KT2440 global response to a monocyclic aromatic compound by iTRAQ analysis and 1DE-MudPIT

Pseudomonas putida KT2440 is a metabolically versatile soil bacterium. To examine the effects of an aromatic compound on the proteome of this bacterium, cytosolic proteins induced by the presence of benzoate and succinate were analyzed using two liquid chromatography (LC)-based proteomic approaches: an isobaric tag for relative and absolute quantitation (iTRAQ) for quantitative analysis and one-dimensional gel electrophoresis/multidimensional protein identification technology (1-DE MudPIT) for protein identification. In total, 1286 proteins were identified by 1-DE MudPIT; this represents around 23.3% of the total proteome. In contrast, 570 proteins were identified and quantified by iTRAQ analysis. Of these, 55 and 52 proteins were up- and down-regulated, respectively, in the presence of benzoate. The proteins up-regulated included benzoate degradation enzymes, chemotaxis-related proteins, and ABC transporters. Enzymes related to nitrogen metabolism and pyruvate metabolism were down-regulated. These data suggest that a combination of 1-DE MudPIT and iTRAQ is an appropriate method for comprehensive proteomic analysis of biodegradative bacteria.     

Use of green fluorescent protein fusions to analyse the N- and C-terminal signal peptides of GPI-anchored cell wall proteins in Candida albicans

Glycophosphatidylinositol (GPI)-anchored proteins account for 26-35% of the Candida albicans cell wall. To understand the signals that regulate these proteins' cell surface localization, green fluorescent protein (GFP) was fused to the N- and C-termini of the C. albicans cell wall proteins (CWPs) Hwp1p, Als3p and Rbt5p. C. albicans expressing all three fusion proteins were fluorescent at the cell surface. GFP was released from membrane fractions by PI-PLC and from cell walls by beta-glucanase, which implied that GFP was GPI-anchored to the plasma membrane and then covalently attached to cell wall glucans. Twenty and 25 amino acids, respectively, from the N- and C-termini of Hwp1p were sufficient to target GFP to the cell surface. C-terminal substitutions that are permitted by the omega rules (G613D, G613N, G613S, G613A, G615S) did not interfere with GFP localization, whereas some non-permitted substitutions (G613E, G613Q, G613R, G613T and G615Q) caused GFP to accumulate in intracellular ER-like structures and others (G615C, G613N/G615C and G613D/G615C) did not. These results imply that (i) GFP fusions can be used to analyse the N- and C-terminal signal peptides of GPI-anchored CWPs, (ii) the omega amino acid in Hwp1p is G613, and (iii) C can function at the omega+2 position in C. albicans GPI-anchored proteins.     

ER/PR阳性和阴性乳腺癌的定量蛋白质组学和生物信息学比较研究

目的:建立雌/孕激素受体(ER/PR)阴性和阳性乳腺癌的蛋白质表达谱,寻找ER/PR阴性和阳性乳腺癌中差异表达蛋白,为乳腺癌患者提供新的预后预测指标和治疗新靶点。方法:应用蛋白质组学i TRAQ技术建立ER/PR阳性和阴性乳腺癌的蛋白质差异表达谱,鉴定两组乳腺癌的差异表达蛋白,对部分差异表达蛋白进行生物信息学分析,包括蛋白功能注释和分类GO分析和KEGG通路分析。结果:应用i TRAQ蛋白质组学技术对乳腺癌组织进行了蛋白组学分析,鉴定出ER/PR阳性和阴性组间有差异表达的蛋白4999种,以ER/PR阳性:ER/PR阴性≥3为上调标准,确定ER/PR阳性组上调蛋白101种。以ER/PR阳性:ER/PR阴性≤0.5为下调标准,ER/PR阳性组下调蛋白122种。GO分析结果显示ER/PR受体阴性和阳性乳腺癌的差异表达蛋白的分子功能、生物过程、细胞定位较为复杂,并且在上调蛋白和下调蛋白上存在分布差异。KEGG通路分析发现部分差异表达蛋白涉及201条信号通路。结论:ER/PR阳性和阴性乳腺癌间存在差异表达蛋白,这些蛋白涉及复杂的分子功能、生物过程和信号通路。     

Influence of histatin 5 on Candida albicans mitochondrial protein expression assessed by quantitative mass spectrometry

Individual aspects of the mode of action of histatin 5, a human salivary antifungal protein, have been partially elucidated, but the mechanism likely involves a complex set of events that have not been characterized. Previous evidence points toward histatin-induced alterations in mitochondrial function. The purpose of the present study was to verify and quantify changes in the mitochondrial proteome of Candida albicans treated with histatin 5. Cell killing was determined by plating and differential protein expression levels in the mitochondrial samples were determined by quantitative proteomics approaches employing mTRAQ and ICAT labeling and Western blotting. Relative quantitation ratios were established for 144 different proteins. Up-regulated mitochondrial proteins were predominantly involved in genome maintenance and gene expression, whereas proteins that constitute the respiratory enzyme complexes were mostly down-regulated. The differential expression of ATP synthase gamma chain and elongation factor 1-alpha were confirmed by Western blotting by comparison to levels of cytochrome c which were unchanged upon histatin treatment. The mTRAQ and ICAT proteomics results suggest that key steps in the histatin 5 antifungal mechanism involve a bioenergetic collapse of C. albicans, caused essentially by a decrease in mitochondrial ATP synthesis.     

iTRAQ-facilitated proteomic analysis of human prostate cancer cells identifies proteins associated with progression

The unpredictable behavior of prostate cancer presents a major clinical challenge during patient management. In order to gain an insight into the molecular mechanisms associated with prostate cancer progression, we employed the shot-gun proteomic approach of isobaric tags for relative and absolute quantitation (iTRAQ), followed by 2D-LC-MS/MS, using the poorly metastatic LNCaP cell line and its highly metastatic variant LNCaP-LN3 cell line as a model. A total number of 280 unique proteins were identified (> or =95% confidence), and relative expression data was obtained for 176 of these. Ten proteins were found to be significantly up-regulated (> or =1.50 fold), while 4 proteins were significantly down-regulated (> or = -1.50 fold), in LNCaP-LN3 cells. Differential expression of brain creatine kinase (CKBB), soluble catechol-O-methyltransferase (S-COMT), tumor rejection antigen (gp96), and glucose regulated protein, 78 kDa (grp78), was confirmed by Western blotting or independent 2D-PAGE analysis. Additionally, iTRAQ analysis identified absence of the lactate dehydrogenase-B (LDH-B) subunit in LNCaP-LN3 cells, confirming our published data. The clinical relevance of gp96 was assessed by immunohistochemistry using prostate tissues from benign ( n = 95), malignant ( n = 66), and metastatic cases ( n = 3). Benign epithelium showed absent/weak gp96 expression in the basal cells, in contrast to the moderate/strong expression seen in malignant epithelium. Furthermore, there was a statistically significant difference in the intensity of gp96 expression between benign and malignant cases ( p < 0.0005, Mann-Whitney U). Our study is the first to report the application of iTRAQ technology and its potential for the global proteomic profiling of prostate cancer cells, including the identification of absent protein expression.