Proteomic Analysis On Viral Hepatitis

The main contributions of this dissertation are: the differential proteomic analysis is carried out for human liver carcinoma cell lines Huh7 and Huh7 cells harboring Hepatitis C virus subgenomic replicon (Huh7-HCV) by combining sub-proteomics and comparative proteomic technologies; proteomic analysis about membrane and lipid raft in Huh7-HCV is performed by using 1D SDS-PAGE/LC/MS and 2DE/MALDI MS; differential expression profiles about hepatitis B surface antigen- positive and hepatitis B surface antigen- negative mouse livers are studied; in addition, improvement of the signal-noise ratio of MS measurement by using additives to the MALDI matrix is achieved and its applications in mouse liver proteomics are carried out.Proteomics — the analysis of genomic complements of proteins — has burst onto the scientific scene with stunning rapidity over the past few years. By studying global patterns of protein content and activity and how these change during development or in response to disease, proteomics research has boosted our understanding of systems-level cellular behaviour and mechanism of disease. In addition, proteomics benefits the identification of new drug targets and the development of new diagnostic markers in clinical research.Viral Hepatitis is now caused at least by hepatitis A, B, C, delta or E virus, etc., while Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are the most common causes of liver disease worldwide and continues as an important public health concern. However, many questions such as the mechanism and machinery of hepatitis viral replication are still poorly understood.Based on the pioneer work of Institutes of Biomedical Sciences of Fudan University, and Key Laboratory of Medical Molecular Virology, Medical College of Fudan University, proteomic studies on viral hepatitis including HBV and HCV were carried out in detail in this work. On the basis of two-dimensional electrophoresis (2DE) technique for the comparative proteomic studies on kinds of biosystems, it has been verified that 2DE is an efficient means to identify those high or medium abundant proteins, and gives an object profile of differentially expressed proteins. Such different protein-expression maps are valuable to the discovery of targetmolecules and the study of cell signaling pathways, etc., although there are still some intrinsic shortcomings in the quantitative proteomic analysis based on 2DE techniques.This dissertation consists of 5 parts and the contents are summarized as follows:In the first chapter, the status quo of proteomics and the newest technical development in proteomics such as 2DE, bio-mass spectrometry, chromatography, protein chip. Yeast two-hybrid system, tandem affinity purification, and so on, were summarized. A review of liver proteomics study was presented.The research works described in the second charpter centers on the proteome alterations between human liver carcinoma cell lines Huh7 and Huh7 cells harboring Hepatitis C virus subgenomic replicon (Huh7-HCV). In this thesis, the differential expression profile about Huh7 and Huh7-HCV were studied by combining sub-proteomics and comparative proteomic technologies, and the 2DE profile and protein database of Huh7-HCV were established for the first time. Three sub-cellular fractions (nuclei, membrane and cytosol) were purified by differential centrifugation. After 2DE, 289 differentially expressed protein spots in three fractions were in-gel digested manually and identified by tandem mass spectrometry. A total of 179 proteins were identified definitely, including proteins associated with host cytoskeleton, intracellular traffic, oxidative and ER stress, proteasome degradation, translation, apoptosis and proliferation etc.. Some of the proteome results were verified by western blot. Host proteins known to interact with HCV proteins, such as HSP27, alpha actinin, nucleolin, eukaryotic initiation factor 4A-I, were elevated in Huh7-HCV cells. Our study provided the global information of proteomic alteration of Huh7 cells in the presence of HCV replication and also provided the clues for further understanding of the mechanism of HCV replication and pathogenesis.In the works mentioned in the third chapter, membrane and lipid raft proteome in Huh7-HCV were studied by using ID SDS-PAGE/LC/MS and 2DE/MALDI MS. Membrane protein, as the main executor of membrane functions, has a series of crucial functions such as energy transfer, material transport, signal transduction, etc. Lipid rafts are microdomains that are enriched in cholesterol and sphingolipid and play critical roles in many biological processes such as regulators and organizing centers of signal transduction and membrane traffic pathways. Recent studies have also demeonstrated that many infectious agents, including viruses, bacteria, andparasites, utilize rafts to populate in the host cells. In this study, we established the database of membrane proteome and lipid raft proteome in human liver carcinoma Huh7 cells harboring HCV subgenomic replicon for the first time. A total of 462 proteins was identified by MS, which included binding proteins, enzymes, transporters, signal transducers, etc. and involved the physiological process, metabolism, development, cell organization, and so on, which was an substantial complement to the human liver proteome database. At the same time, we analyzed synthetically the results and found that 82 lipid raft associated proteins were the differentially expressed proteins identified in chapter 2. What's more, some biological experiments were carried out to verify the functions of these lipid raft associated proteins. hVAP33, Signal molecular Ras-GTPase-activating protein binding protein 1 (G3BP-1), prohibitin, Nucleophosmin (NPM), snapy, PA28J3 were colocalized with HCV NS5A, while RhoA, syntaxinl7, Ubiquitin fusion degradation protein 1 homolog (UFD1L) were partially colocalized with NS5A. All these data suggested that HCV RNA replication was associated with lipid raft associated proteins nearly. The membrane and lipid raft proteome provided major bases for the elucidation of mechanisms of HCV replication and pathogenesis and for the identification of new diagnostic markers and therapeutic targets. In addition, the superiority of ID SDS-PAGE/LC/MS for analyzing the hydrophobic proteins and the complement of 2DE/MALDI MS method were demonstrated.In the fourth chapter, a comparative proteomics was described to study the given model that was associated with HBV infection. The 126 differentially expressed protein spots were selected via 2DE separation and PDQuest software analysis to make further in-gel digestion and MS/MS identification, in which 77 protein spots were up-regulated in the disease group and 49 protein spots were highly expressed in the control group. A total of 95 proteins were identified unambiguously of which 61 proteins were elevated and 34 proteins were down-regulated in the disease group. These proteins mainly involved in signal transduction, fat and lipoid metabolism, energy and electron transport, carbohydrate metabolism, amino acid metabolism, and so on. The proteomic results offered some candidate proteins for further study of this kind of disease and provided important reference to interpret the mechanism of HBV chronic infection.In a study relative to the fifth part, improving the signal-noise ratio of MS measurement by using additives to the MALDI matrix was investigated and its...