iTRAQ-Based Proteomics Analysis of Response to Solanum tuberosum Leaves Treated with the Plant Phytotoxin Thaxtomin A

Thaxtomin A (TA) is a phytotoxin secreted by Streptomyces scabies that causes common scab in potatoes. However, the mechanism of potato proteomic changes in response to TA is barely known. In this study, the proteomic changes in potato leaves treated with TA were determined using the Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) technique. A total of 693 proteins were considered as differentially expressed proteins (DEPs) following a comparison of leaves treated with TA and sterile water (as a control). Among the identified DEPs, 460 and 233 were upregulated and downregulated, respectively. Based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, many DEPs were found to be involved in defense and stress responses. Most DEPs were grouped in carbohydrate metabolism, amino acid metabolism, energy metabolism, and secondary metabolism including oxidation–reduction process, response to stress, plant–pathogen interaction, and plant hormone signal transduction. In this study, we analyzed the changes in proteins to elucidate the mechanism of potato response to TA, and we provided a molecular basis to further study the interaction between plant and TA. These results also offer the option for potato breeding through analysis of the resistant common scab.     

不同产地太子参的iTRAQ定量蛋白质组学研究

采用同位素标记相对和绝对定量（iTRAQ）技术对不同产地的太子参进行定量蛋白质组学研究,探讨蛋白质组表达水平的差异。所提取的太子参蛋白质样品经FASP酶解、iTRAQ试剂标记、高pH-RPLC分离、RPLC-MS分离分析,获取的串联质谱数据通过Protein Pilot 5.0软件搜库进行蛋白质鉴定,通过蛋白质相对定量的比较寻找差异表达蛋白;再对差异蛋白质进行GO（gene ontology）、KEGG代谢通路和STRING网络通路分析。实验共鉴定出3 775个蛋白质,其中3676个蛋白质具有定量信息,与传统产区相比,种植基地太子参上调差异蛋白质54个,下调差异蛋白质86个;通过生物信息学分析得到44个目标差异蛋白,主要分为9类：热休克蛋白、氧化还原酶、转移酶、水解酶、裂解酶、异构酶、Rubisco大亚基结合蛋白、伴侣蛋白质、胞腔结合蛋白。结果显示,传统产区太子参中氧化还原酶和转移酶的分解代谢、碳水化合物代谢以及抗应激能力比种植基地太子参强,但热休克蛋白、异构酶、Rubisco大亚基结合蛋白、伴侣蛋白质和胞腔结合蛋白中的蛋白质折叠和抗应激能力比种植基地太子参弱,水解酶和裂解酶的分解代谢能力与种植基地太子参相差不大;ADG1和TKTA可能是调节不同产地太子参蔗糖变化的2个关键蛋白;MFP2是导致不同产地太子参脂肪酸差异的关键蛋白。本研究可为解析不同产地太子参次生代谢物差异的成因及其药材品质形成的蛋白质机制提供参考。     

~(137)Csγ射线累积照射SD大鼠后肝脏蛋白质组的分析

采用相对和绝对定量的同位素标签技术(Isobaric tags for relative and absolute quantitation,i TRAQ)分析累积受137Csγ射线照射大鼠肝脏组织的蛋白质表达,筛选出差异蛋白质,对差异表达的蛋白质进行GO注释分析及相互作用网络分析,探讨137Csγ射线累积照射对SD大鼠肝脏蛋白质的影响。结果显示,质谱数据经蛋白质数据库搜索,在3组样品中有定量信息的蛋白质有2 220个;0.2 Gy照射组与对照组相比,差异蛋白有149种,52种蛋白上调,97种蛋白下调;2 Gy照射组有243种差异蛋白,123种上调,120种下调;两组共同表达差异的蛋白有122种,其中,52种蛋白表达上调,70种蛋白表达下调;利用GO注释对122个共同差异蛋白质功能进行聚类分析,结果表明:根据分子功能注释,差异蛋白以酶催化、蛋白质结合、核酸结合等功能为主,另外少数蛋白还有翻译调控活性;根据细胞组成注释,多数蛋白参与细胞胞浆、细胞核、细胞膜的组成;根据生物学过程注释,多数蛋白参与代谢调节和生物过程调节。利用PAJEK软件对两个剂量组共同差异表达的蛋白进行相互作用网络分析。结果显示SF3B1、ATP6V1C1是蛋白相互作用网络的重要节点,这两个蛋白直接或间接地与其它差异蛋白存在相互作用。上述研究结果表明,137Csγ射线累积照射可诱导SD大鼠肝脏组织蛋白质组的改变,SF3B1、ATP6V1C1蛋白可能在累积照射的肝脏损伤机制中发挥作用。     

Iron Restriction Alleviates Atherosclerosis in ApoE KO Mice: An iTRAQ Proteomic Analysis

The “iron hypothesis” of atherosclerosis has long been controversial. Several studies have shown that dietary iron restriction or low-iron diets can effectively alleviate atherosclerosis in rabbits and mice. However, the underlying molecular mechanisms of these phenomena remain to be elucidated. In this study, we further evaluated possible correlations between a low-iron diet and atherosclerosis alleviation by using a quantitative proteomic approach. For this purpose, apolipoprotein E knockout (ApoE KO) mice were divided into three groups and fed a normal diet (ND), a high-fat diet (HFD), or a high-fat +low-iron diet (HFD + LI). Our results showed that the HFD-LI improved atherosclerosis by decreasing en face lesions of the aorta and reducing the accumulation of macrophages and disordered smooth muscle cells. HFD-LI also decreased iron levels, serum hepcidin levels and the serum concentration of low-density lipoprotein cholesterol (LDL-C). The use of the isobaric tag for absolute quantification (iTRAQ) proteomic method and subsequent multi-technique molecular validation indicated that many of the proteins involved in atherosclerotic inflammation, vascular remodeling, and focal adhesion had significant changes in their expression among the diet groups. Importantly, the proteins Gal-3 and VCAM1, which are key participants of atherosclerosis pathogenesis, revealed lower expression after a low-iron diet. The present findings widely support the “iron hypothesis” of atherosclerosis. Further studies are suggested to fully understand the implications of these results.     

Incomptine A Induces Apoptosis,ROS Production and a Differential Protein Expression on Non-Hodgkin’s Lymphoma Cells

Sesquiterpene lactones are of pharmaceutical interest due their cytotoxic and antitumor properties, which are commonly found within plants of several genera from the Asteraceae family such as the Decachaeta genus. From Decachaeta incompta four heliangolide, namely incomptines A-D have been isolated. In this study, cytotoxic properties of incomptine A (IA) were evaluated on four lymphoma cancer cell lines: U-937, Farage, SU-DHL-2, and REC-1. The type of cell death induced by IA and its effects on U-937 cells were analyzed based on its capability to induce apoptosis and produce reactive oxygen species (ROS) through flow cytometry with 4′,6-diamidino-2-phenylindole staining, dual annexin V/DAPI staining, and dichlorofluorescein 2′,7′-diacetate, respectively. A differential protein expression analysis study was carried out by isobaric tags for relative and absolute quantitation (iTRAQ) through UPLC-MS/MS. Results reveal that IA exhibited cytotoxic activity against the cell line U-937 (CC₅₀ of 0.12 ± 0.02 μM) and the incubation of these cells in presence of IA significantly increased apoptotic population and intracellular ROS levels. In the proteomic approach 1548 proteins were differentially expressed, out of which 587 exhibited a fold-change ≥ 1.5 and 961 a fold-change ≤ 0.67. Most of these differentially regulated proteins are involved in apoptosis, oxidative stress, glycolytic metabolism, or cytoskeleton structuration.     

Comparative Proteomic Analysis of Gossypium thurberi in Response to Verticillium dahliae Inoculation

Verticillium wilt is threatening cotton productivity globally. This disease is caused by soil-borne Verticillium dahliae which directly infects cotton roots, and exclusively colonizes and occludes xylem vessels, finally resulting in necrosis, defoliation, and most severely, plant death. For the first time, iTRAQ (isobaric tags for relative and absolute quantification) was applied to screen the differentially expressed proteins of Gossypium thurberi inoculated with V. dahliae. A total of 6533 proteins were identified from the roots of G. thurberi after inoculation with V. dahliae, and 396 showed up- and 279 down-regulated in comparison to a mock-inoculated roots. Of these identified proteins, the main functional groups were those involved in cell wall organization and reinforcement, disease-resistant chemicals of secondary metabolism, phytohormone signaling, pathogenesis-related proteins, and disease-resistant proteins. Physiological and biochemical analysis showed that peroxidase activity, which promotes the biosynthesis and accumulation of lignin, was induced early in the hypocotyl after inoculation with V. dahliae. Similarly, salicylic acid also accumulated significantly in hypocotyl of the seedlings after inoculation. These findings provide an important knowledge of the molecular events and regulatory networks occurring during G. thurberi-V. dahliae interaction, which may provide a foundation for breeding disease-resistance in cotton.