Identification Of Protein Complexome And Functional Characterization Of Important Complexes In Eschrichia Coli

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.