大肠埃希氏菌蛋白质复合物组的鉴定分析及重要复合物的功能研究
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摘要
细胞中的蛋白质通常与其他蛋白质相互作用来共同完成生命活动,而蛋白质相互作用几乎参与了所有的生物进程,因此只有对细胞内所有蛋白质的相互作用组或蛋白质复合物组进行全面、深入的研究,并进而建立蛋白质间相互作用关系网络,才能真正阐明一个蛋白质的功能,才有可能研究细胞中某一生理活动中所有相关蛋白质的变化及作用机制,同时还有助于探索生命进程、了解疑难病症的产生机制和开发有效药物等各个方面的研究。因此对蛋白质复合物组的构成及功能的研究具有深远的理论和现实意义。
论文采用基于2-D native/SDS-PAGE的蛋白质组学方法首次对接近生理条件的E coli K-12胞浆蛋白和膜蛋白复合物组进行了较为系统的分析,分别成功地鉴定到24和18个由不同蛋白质组成的异聚体复合物和一些同聚体复合物,其中多数复合物为首次发现。利用多种蛋白质相互作用的研究技术对部分蛋白质复合物进行了证实,为构建蛋白质相互作用网络提供了可靠的相互作用信息。
对新发现的由外膜蛋白、内膜蛋白和胞浆蛋白组成的OmpW复合物进行了较为深入研究。初步确定了该复合物中各蛋白质成分间的连接方式,证明了复合物受铁离子限制调节,利用体外实验证实该复合物在抵抗人血浆补体杀菌过程中起关键作用,从而首次发现并初步阐明了游离铁介导的细菌感染性疾病的产生机制。该机制的发现,可为设计、开发治疗全身性细菌感染的药物提供全新线索。
鉴于铁离子在细菌毒力等方面具有重要作用,分析了E coli K-12胞浆蛋白在铁限制下的情况。成功的鉴定了16个差异蛋白,除EntF和GltA已知分别为两种铁转运系统中的关键蛋白外,其余蛋白均为首次发现在维持铁平衡中起重要作用,这些蛋白涉及氨基酸、蛋白质合成代谢,能量代谢和核酸代谢等多种代谢途径。细菌不仅通过提高表达铁转运蛋白来加强对铁的摄取,而且还抑制并非生长所必须的含铁蛋白的表达,最大程度的适应铁限制环境。
论文还对E coli K-12胞浆蛋白在头孢曲松和巴洛沙星两种新一代抗生素的耐药性中的作用进行了详细分析,分别获得8个和10个耐药相关蛋白。头孢曲松耐药相关蛋白主要与能量代谢相关,从而阐明了细菌对抑制细胞壁合成的抗生素的耐药新机制:细菌可通过调整能量代谢途径来而达到耐药的目的。而巴洛沙星耐药相关蛋白除AtpD与头孢曲松耐药蛋白相同且变化一致外,其他蛋白均不相同,这不仅表明细菌对不同种类抗生素耐药性的产生机制不同,而且也提示这2种抗生素可能交叉耐药较少。细菌对巴洛沙星耐药性的获得可能更主要的是与蛋白质的生物合成的改变有关。这些发现不仅有助于理解细菌对抗生素产生耐药性的机制,而且在抗生素分子的设计和筛选方面具有重要参考价值。
Life activities of a cell are performed by protein-protein interactions, and the interactions are involved in almost all of biological processes. Therefore, only when we understand exactly the networks of protein interactions which based on the systematic analysis of protein interactome or complexome of a cell, can we clarify clearly a protein's function or mechanism of a physiological activity. It can also help us to explore biological processes, uncover mechanisms of diseases occurring both in human and animals and develop efficacious drugs. Thus, the study on protein complexome on a large-scale wide is very important and it's become increasingly urgent.
Proteomics approach based on 2-D native/SDS-PAGE is firstly used for systematic analysis the complexome of cytoplasm and membrane proteins of E. coli K-12 under physiological conditions. Twenty-four and eighteen heteromeric protein complexes, as well as some hemomeric proteins, are characterized successfully. Parts of complexes are further confirmed by Far-western blotting, co-immunoprecipitation or His-pull down analysis. These results provide reliable interaction information for construction the network of protein interactions.
Moreover, the new identified OmpW complex which consists of outer membrane protein, inner membrane protein and cytoplasm protein is made an intensive investigated. The protein-protein interactions of the components of the complex are preliminary determined. The integrity of the complex, which is found regulated by iron-deprivation, is required for counteract the anti-bacterial activity of complement system of human plasma. Therefore, the mechanism of iron-mediated infection disease is revealed for the fist time in the thesis, and it may be a distinct clue for development novel drugs to cure bacterial disease.
In view of free iron functioned in virulence of pathogenic bacteria, the cytoplasmic proteins mediated by iron-starvation are investigated on proteome wide. Sixteen proteins are found responsible for iron homeostasis. Besides EntF and GltA are known key proteins relate to iron transporter, others are new identified in the study which involved in several metabolic pathway such as biosynthesis of amino acids and proteins, metabolism of energy and nuclear acids. Bacteria survive in iron-limitation conditions not only by up-regulated expression of proteins to enhance iron uptake, but also inhibited expression of iron-containing proteins which are not necessary for growth.
The cytoplasmic proteins of E. coli K-12 respond to Ceftriaxone- and Balofloxacin-resistance is also analyzed using the approach. Eight altered proteins are identified in Ceftriaxone-resistant strains and six of those proteins are involved in energy metabolism. Thus, the modification the energy metabolism pathway is uncovered as a novel mechanism of bacteria resistance to antibiotics which aim at inhibition of biosynthesis of cell wall. In Balofloxacin-resistant strains, ten altered proteins are identified and only AtpD is the same to Ceftriaxone-resistant. It shows bacteria exhibit different mechanisms to resistance to these two antibiotics and they share no cross-resistance. The acquired resistance to Balofloxacin may mainly relate to the shift of biosynthesis of amino acids and proteins. These novel identified mechanisms provide new insight for us to understanding bacteria resistance and it can also help us to develop novel antibiotics to antibacteria efficaciously.
论文采用基于2-D native/SDS-PAGE的蛋白质组学方法首次对接近生理条件的E coli K-12胞浆蛋白和膜蛋白复合物组进行了较为系统的分析,分别成功地鉴定到24和18个由不同蛋白质组成的异聚体复合物和一些同聚体复合物,其中多数复合物为首次发现。利用多种蛋白质相互作用的研究技术对部分蛋白质复合物进行了证实,为构建蛋白质相互作用网络提供了可靠的相互作用信息。
对新发现的由外膜蛋白、内膜蛋白和胞浆蛋白组成的OmpW复合物进行了较为深入研究。初步确定了该复合物中各蛋白质成分间的连接方式,证明了复合物受铁离子限制调节,利用体外实验证实该复合物在抵抗人血浆补体杀菌过程中起关键作用,从而首次发现并初步阐明了游离铁介导的细菌感染性疾病的产生机制。该机制的发现,可为设计、开发治疗全身性细菌感染的药物提供全新线索。
鉴于铁离子在细菌毒力等方面具有重要作用,分析了E coli K-12胞浆蛋白在铁限制下的情况。成功的鉴定了16个差异蛋白,除EntF和GltA已知分别为两种铁转运系统中的关键蛋白外,其余蛋白均为首次发现在维持铁平衡中起重要作用,这些蛋白涉及氨基酸、蛋白质合成代谢,能量代谢和核酸代谢等多种代谢途径。细菌不仅通过提高表达铁转运蛋白来加强对铁的摄取,而且还抑制并非生长所必须的含铁蛋白的表达,最大程度的适应铁限制环境。
论文还对E coli K-12胞浆蛋白在头孢曲松和巴洛沙星两种新一代抗生素的耐药性中的作用进行了详细分析,分别获得8个和10个耐药相关蛋白。头孢曲松耐药相关蛋白主要与能量代谢相关,从而阐明了细菌对抑制细胞壁合成的抗生素的耐药新机制:细菌可通过调整能量代谢途径来而达到耐药的目的。而巴洛沙星耐药相关蛋白除AtpD与头孢曲松耐药蛋白相同且变化一致外,其他蛋白均不相同,这不仅表明细菌对不同种类抗生素耐药性的产生机制不同,而且也提示这2种抗生素可能交叉耐药较少。细菌对巴洛沙星耐药性的获得可能更主要的是与蛋白质的生物合成的改变有关。这些发现不仅有助于理解细菌对抗生素产生耐药性的机制,而且在抗生素分子的设计和筛选方面具有重要参考价值。
Life activities of a cell are performed by protein-protein interactions, and the interactions are involved in almost all of biological processes. Therefore, only when we understand exactly the networks of protein interactions which based on the systematic analysis of protein interactome or complexome of a cell, can we clarify clearly a protein's function or mechanism of a physiological activity. It can also help us to explore biological processes, uncover mechanisms of diseases occurring both in human and animals and develop efficacious drugs. Thus, the study on protein complexome on a large-scale wide is very important and it's become increasingly urgent.
Proteomics approach based on 2-D native/SDS-PAGE is firstly used for systematic analysis the complexome of cytoplasm and membrane proteins of E. coli K-12 under physiological conditions. Twenty-four and eighteen heteromeric protein complexes, as well as some hemomeric proteins, are characterized successfully. Parts of complexes are further confirmed by Far-western blotting, co-immunoprecipitation or His-pull down analysis. These results provide reliable interaction information for construction the network of protein interactions.
Moreover, the new identified OmpW complex which consists of outer membrane protein, inner membrane protein and cytoplasm protein is made an intensive investigated. The protein-protein interactions of the components of the complex are preliminary determined. The integrity of the complex, which is found regulated by iron-deprivation, is required for counteract the anti-bacterial activity of complement system of human plasma. Therefore, the mechanism of iron-mediated infection disease is revealed for the fist time in the thesis, and it may be a distinct clue for development novel drugs to cure bacterial disease.
In view of free iron functioned in virulence of pathogenic bacteria, the cytoplasmic proteins mediated by iron-starvation are investigated on proteome wide. Sixteen proteins are found responsible for iron homeostasis. Besides EntF and GltA are known key proteins relate to iron transporter, others are new identified in the study which involved in several metabolic pathway such as biosynthesis of amino acids and proteins, metabolism of energy and nuclear acids. Bacteria survive in iron-limitation conditions not only by up-regulated expression of proteins to enhance iron uptake, but also inhibited expression of iron-containing proteins which are not necessary for growth.
The cytoplasmic proteins of E. coli K-12 respond to Ceftriaxone- and Balofloxacin-resistance is also analyzed using the approach. Eight altered proteins are identified in Ceftriaxone-resistant strains and six of those proteins are involved in energy metabolism. Thus, the modification the energy metabolism pathway is uncovered as a novel mechanism of bacteria resistance to antibiotics which aim at inhibition of biosynthesis of cell wall. In Balofloxacin-resistant strains, ten altered proteins are identified and only AtpD is the same to Ceftriaxone-resistant. It shows bacteria exhibit different mechanisms to resistance to these two antibiotics and they share no cross-resistance. The acquired resistance to Balofloxacin may mainly relate to the shift of biosynthesis of amino acids and proteins. These novel identified mechanisms provide new insight for us to understanding bacteria resistance and it can also help us to develop novel antibiotics to antibacteria efficaciously.
引文
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