89 research outputs found
Das Buch der Bücher
No full textThe firms and markets of today's complex socio-economic system developed in a spontaneous process termed evolution, in just the same way as the universe, the solar system, the Earth and all that lives upon it. Darwin's theory of evolution clearly demonstrated that evolution involved increasing organization. As we began to explore the molecular basis of life and its evolution, it became equally clear that it depended on the processing and communication of information. This book develops a consistent theory of evolution in its wider sense, examining the information based laws and forces that drive it. Exploring subjects as diverse as economics and the theories of thermodynamics, the author revisits the paradox of the apparent conflict between the laws of thermodynamics and evolution to arrive at a systems theory, tracing a continuous line of evolving information sets that connect the Big-Bang to the firms and markets of our current socio-economic system
A proteomic and molecular approach to decipher the repertoire of protein modifications during influenza A virus infection
No full textInfluenza virus is an important human respiratory pathogen that continues to have a major impact on global public health. The ever-evolving nature of influenza virus has restricted the development of a universal vaccine as well as the efficacy of currently available vaccines. In the absence of a universal vaccine, antiviral drugs are serving as the first line of defense against novel influenza virus strains. However, the emergence of drug-resistant variants of influenza virus has made the available drugs almost ineffective. Therefore, there is an undeniable need for developing universal and long-lasting antiviral strategies. Exploring new approaches such as targeting critical factors for influenza virus infection might lead to the development of novel antiviral strategies. There is growing evidence that influenza exploits host protein modification machinery to proliferate and escape the host innate immune response. Therefore, a detailed profiling of the protein modification events during influenza virus infection is required.
Protein modifications occurring co- or post-translationally regulate the structure and function of a protein in various ways. Here, we used global mass spectrometry approach in association with bioinformatics analysis to identify and characterize the modifications of viral and host proteins in uninfected and IAV-infected human lung epithelial cells. To the best of our knowledge, this is the first time such a comprehensive approach has been employed in influenza virus proteomics. We identified 8 viral proteins: matrix protein (M1), nucleoprotein (NP), two non-structural proteins (NS1, NS2) and haemagglutinin (HA), modified by methylation, acetylation, allysine and/or ubiquitination. Three viral proteins, M1, NP and PA showed multiple modifications on the same amino acid residue, indicating potential crosstalk among protein modifications. The majority of modified amino acids exhibited sequence homology across IAV subtypes and strains, suggesting the fundamental importance of these modifications in IAV biology. Furthermore, the location of modified amino acids in structural context suggested the functional significance of modified sites in multiple steps of IAV life cycle. In addition, a total of 116 methylated, 103 acetylated, 29 allysine modified, 2 ubiquitinated and 5 acylated host proteins were found to be uniquely modified in IAV-infected cells. Characterization of these modified host proteins by protein functional association analysis revealed their involvement in metabolic pathways, mRNA splicing and cytoskeleton organization which are manipulated by IAV during infection. The functional enrichment analysis revealed the modified cellular proteins to be involved in multiple virus-related processes.
Acetylation was the second most abundant modification identified in our study. Host histone deacetylases (HDACs) and histone acetyltransferases (HATs) are the central regulators of acetylation; where HATs add and HDACs remove the acetyl group from a target protein. Our lab has discovered and been studying the anti-IAV properties of HDACs. Therefore, we endeavoured to identify the potential involvement of HATs in IAV infection. An initial RNA interference screening with 18 HATs revealed that the depletion of 6 HATs reduced IAV progeny release. Among them, N-alpha-acetyltransferase 60 (Naa60), an N-terminal acetyltransferase that possesses the unique capacity of acetylating proteins both co- and post-translationally, was chosen for further investigation. By employing the RNA interference and overexpression strategies, this study demonstrated that Naa60 plays a pro-viral role and is a component of host antiviral response. The silencing of Naa60 expression impaired IAV progeny release by 50% and conversely, the ectopic Naa60 expression augmented IAV progeny release by 2.3 fold. Mechanistically, the IAV-induced expression of interferon (IFN) α was increased and decreased with the depletion and ectopic expression of Naa60, respectively. Furthermore, the knockdown of Naa60 resulted in increased phosphorylation of transcription factor, STAT1 as well as the expression of certain interferon-stimulate genes (ISGs) such as viperin and IFITM3, in IAV-infected cells. Conversely, the ectopic expression of Naa60 correlated with the above data and reduced IAV-induced IFNα and ISGs expression. Finally, the variations in the relative abundances of N-terminally acetylated peptides from viral M1, NP and multiple host proteins in the absence of Naa60 indicated that the acetyltransferase activity of Naa60 has implications in IAV infection.
This PhD study provides a basic framework for future research on the functional significance of protein modifications in the IAV life cycle. Protein modifications with trackable functional roles might serve as a target for novel antiviral strategies. Additionally, the proviral properties and involvement of Naa60 in IAV-induced host innate antiviral response identified in this study represents an exciting prospect for future investigation into targeting Naa60 as a drug target to combat IAV infection
A proteomic and molecular approach to decipher the repertoire of protein modifications during influenza A virus infection
No full textInfluenza virus is an important human respiratory pathogen that continues to have a major impact on global public health. The ever-evolving nature of influenza virus has restricted the development of a universal vaccine as well as the efficacy of currently available vaccines. In the absence of a universal vaccine, antiviral drugs are serving as the first line of defense against novel influenza virus strains. However, the emergence of drug-resistant variants of influenza virus has made the available drugs almost ineffective. Therefore, there is an undeniable need for developing universal and long-lasting antiviral strategies. Exploring new approaches such as targeting critical factors for influenza virus infection might lead to the development of novel antiviral strategies. There is growing evidence that influenza exploits host protein modification machinery to proliferate and escape the host innate immune response. Therefore, a detailed profiling of the protein modification events during influenza virus infection is required.
Protein modifications occurring co- or post-translationally regulate the structure and function of a protein in various ways. Here, we used global mass spectrometry approach in association with bioinformatics analysis to identify and characterize the modifications of viral and host proteins in uninfected and IAV-infected human lung epithelial cells. To the best of our knowledge, this is the first time such a comprehensive approach has been employed in influenza virus proteomics. We identified 8 viral proteins: matrix protein (M1), nucleoprotein (NP), two non-structural proteins (NS1, NS2) and haemagglutinin (HA), modified by methylation, acetylation, allysine and/or ubiquitination. Three viral proteins, M1, NP and PA showed multiple modifications on the same amino acid residue, indicating potential crosstalk among protein modifications. The majority of modified amino acids exhibited sequence homology across IAV subtypes and strains, suggesting the fundamental importance of these modifications in IAV biology. Furthermore, the location of modified amino acids in structural context suggested the functional significance of modified sites in multiple steps of IAV life cycle. In addition, a total of 116 methylated, 103 acetylated, 29 allysine modified, 2 ubiquitinated and 5 acylated host proteins were found to be uniquely modified in IAV-infected cells. Characterization of these modified host proteins by protein functional association analysis revealed their involvement in metabolic pathways, mRNA splicing and cytoskeleton organization which are manipulated by IAV during infection. The functional enrichment analysis revealed the modified cellular proteins to be involved in multiple virus-related processes.
Acetylation was the second most abundant modification identified in our study. Host histone deacetylases (HDACs) and histone acetyltransferases (HATs) are the central regulators of acetylation; where HATs add and HDACs remove the acetyl group from a target protein. Our lab has discovered and been studying the anti-IAV properties of HDACs. Therefore, we endeavoured to identify the potential involvement of HATs in IAV infection. An initial RNA interference screening with 18 HATs revealed that the depletion of 6 HATs reduced IAV progeny release. Among them, N-alpha-acetyltransferase 60 (Naa60), an N-terminal acetyltransferase that possesses the unique capacity of acetylating proteins both co- and post-translationally, was chosen for further investigation. By employing the RNA interference and overexpression strategies, this study demonstrated that Naa60 plays a pro-viral role and is a component of host antiviral response. The silencing of Naa60 expression impaired IAV progeny release by 50% and conversely, the ectopic Naa60 expression augmented IAV progeny release by 2.3 fold. Mechanistically, the IAV-induced expression of interferon (IFN) α was increased and decreased with the depletion and ectopic expression of Naa60, respectively. Furthermore, the knockdown of Naa60 resulted in increased phosphorylation of transcription factor, STAT1 as well as the expression of certain interferon-stimulate genes (ISGs) such as viperin and IFITM3, in IAV-infected cells. Conversely, the ectopic expression of Naa60 correlated with the above data and reduced IAV-induced IFNα and ISGs expression. Finally, the variations in the relative abundances of N-terminally acetylated peptides from viral M1, NP and multiple host proteins in the absence of Naa60 indicated that the acetyltransferase activity of Naa60 has implications in IAV infection.
This PhD study provides a basic framework for future research on the functional significance of protein modifications in the IAV life cycle. Protein modifications with trackable functional roles might serve as a target for novel antiviral strategies. Additionally, the proviral properties and involvement of Naa60 in IAV-induced host innate antiviral response identified in this study represents an exciting prospect for future investigation into targeting Naa60 as a drug target to combat IAV infection
Transcriptomic and Proteomic Approaches to the Analyses of Orf Virus and its Ankyrin Repeat Proteins
No full textIn common with other poxviruses, orf virus (ORFV) is believed to induce drastic changes in the host intracellular environment through expression of an arsenal of virulence factors. The ankyrin repeat (ANK) proteins are examples of such factors. ANK proteins are ubiquitous in eukaryotes and participate in protein-protein interactions. ANK proteins in viruses are rare with the exception of poxvirus. Many poxviral ANK proteins also carry an F-box motif which mediates interaction with the cellular SCF1 ubiquitin ligase complexes. It has been proposed that ORFV ANK proteins recruit substrate proteins to SCF1 complexes for subsequent ubiquitination. This study aimed to identify the binding partners of ORFV ANK proteins and changes in the cell transcriptome and proteome early in ORFV infection so as to assist in elucidating the functions of the ORFV ANK proteins. This study harnessed the power of high throughput next generation sequencing (RNA-seq) and both qualitative and quantitative mass spectrometry.
This study presents a characterisation of the ORFV ANK proteins during ORFV infection, including their timing of expression, localisation, and interaction with SCF1 components. Additionally, the use of proteasome inhibitor confirmed the importance of a function ubiquitin proteasome system to ORFV replication.
Transcriptomic studies revealed dramatic changes in host gene expression early in ORFV infection of HeLa cells, including genes involved in cell cycle related processes. These processes were highlighted in studies using an ORFV deleted of its proposed cell cycle regulator gene, PACR. Additionally, infection with a recombinant ORFV deleted of the gene encoding one of its ANK proteins (ORFV ANK008KO) revealed involvement of ANK008 in functions like cellular replication, apoptosis initiation, transcription regulation, and angiogenesis.
Mass spectrometry was used to identify proteins co-precipitating with 3 of the 5 ORFV ANK proteins when these viral proteins were expressed from their natural promoters, during infection with recombinant ORFVs. Co-precipitates of each ORV ANK protein included components of the SCF1 complex, as predicted. Further analyses revealed evidence of wider links between both ORFV ANK008 and ANK129 and the ubiquitin proteasome pathway. ANK129 also showed evidence of possible activities in cellular proliferation.
SWATH quantitative mass spectrometry was used in pairwise quantitative comparisons of mock-infected, ORFV-infected, and ORFV ANK008KO-infected HeLa cells. This approach built on the model that ORFV ANKs may promote the proteasomal degradation of specific cellular proteins. The data identified a group of cellular proteins whose abundance appears to be directly influenced by ANK008. For example, macrophage migration inhibition factor (MIF), an important regulator of innate immunity, was reduced during ORFV infection and ANK008 at very least contributed to that reduction. Network and pathway analyses of these proteins revealed links to cell cycle, cellular metabolism and apoptosis, reflecting results of the transcriptome analyses. The SWATH data from ORFV infection support PACR’s involvement in cell cycle in a manner that provides ORFV with an environment rich in nucleotides. Additionally, links to nucleotide metabolism were also made for ANK008 in both the qualitative and quantitative mass spectrometry, suggesting a novel function for ANK008.
Finally, the transcriptomic and proteomic data were combined to provide a comprehensive understanding of the temporal expression of all ORFV genes. This study presents a robust examination of ORFV-host interactions and the functions of ORFV ANK proteins. The results provide numerous exciting areas to pursue in future research
Transcriptomic and Proteomic Approaches to the Analyses of Orf Virus and its Ankyrin Repeat Proteins
No full textIn common with other poxviruses, orf virus (ORFV) is believed to induce drastic changes in the host intracellular environment through expression of an arsenal of virulence factors. The ankyrin repeat (ANK) proteins are examples of such factors. ANK proteins are ubiquitous in eukaryotes and participate in protein-protein interactions. ANK proteins in viruses are rare with the exception of poxvirus. Many poxviral ANK proteins also carry an F-box motif which mediates interaction with the cellular SCF1 ubiquitin ligase complexes. It has been proposed that ORFV ANK proteins recruit substrate proteins to SCF1 complexes for subsequent ubiquitination. This study aimed to identify the binding partners of ORFV ANK proteins and changes in the cell transcriptome and proteome early in ORFV infection so as to assist in elucidating the functions of the ORFV ANK proteins. This study harnessed the power of high throughput next generation sequencing (RNA-seq) and both qualitative and quantitative mass spectrometry.
This study presents a characterisation of the ORFV ANK proteins during ORFV infection, including their timing of expression, localisation, and interaction with SCF1 components. Additionally, the use of proteasome inhibitor confirmed the importance of a function ubiquitin proteasome system to ORFV replication.
Transcriptomic studies revealed dramatic changes in host gene expression early in ORFV infection of HeLa cells, including genes involved in cell cycle related processes. These processes were highlighted in studies using an ORFV deleted of its proposed cell cycle regulator gene, PACR. Additionally, infection with a recombinant ORFV deleted of the gene encoding one of its ANK proteins (ORFV ANK008KO) revealed involvement of ANK008 in functions like cellular replication, apoptosis initiation, transcription regulation, and angiogenesis.
Mass spectrometry was used to identify proteins co-precipitating with 3 of the 5 ORFV ANK proteins when these viral proteins were expressed from their natural promoters, during infection with recombinant ORFVs. Co-precipitates of each ORV ANK protein included components of the SCF1 complex, as predicted. Further analyses revealed evidence of wider links between both ORFV ANK008 and ANK129 and the ubiquitin proteasome pathway. ANK129 also showed evidence of possible activities in cellular proliferation.
SWATH quantitative mass spectrometry was used in pairwise quantitative comparisons of mock-infected, ORFV-infected, and ORFV ANK008KO-infected HeLa cells. This approach built on the model that ORFV ANKs may promote the proteasomal degradation of specific cellular proteins. The data identified a group of cellular proteins whose abundance appears to be directly influenced by ANK008. For example, macrophage migration inhibition factor (MIF), an important regulator of innate immunity, was reduced during ORFV infection and ANK008 at very least contributed to that reduction. Network and pathway analyses of these proteins revealed links to cell cycle, cellular metabolism and apoptosis, reflecting results of the transcriptome analyses. The SWATH data from ORFV infection support PACR’s involvement in cell cycle in a manner that provides ORFV with an environment rich in nucleotides. Additionally, links to nucleotide metabolism were also made for ANK008 in both the qualitative and quantitative mass spectrometry, suggesting a novel function for ANK008.
Finally, the transcriptomic and proteomic data were combined to provide a comprehensive understanding of the temporal expression of all ORFV genes. This study presents a robust examination of ORFV-host interactions and the functions of ORFV ANK proteins. The results provide numerous exciting areas to pursue in future research
YB-1 oncoprotein in cancer and drug resistance
No full textY-box-binding protein 1 (YB-1) is a biomarker that is predictive of poor prognosis in cancer. Various molecular functions of YB-1 in cancer have been proposed, including the transcriptional regulation of gene expression. YB-1 also binds to RNA transcripts to influence gene expression.
In the present study, the status of YB-1 as a biomarker was confirmed by immunohistochemistry using two antibodies against YB-1. However, the prognostic sensitivity of these two antibodies differed. The observed difference in antibody affinity was most likely due to the tertiary structure or protein-protein interactions (PPI) associated with various functions of YB-1 in situ.
To gain further insights into the molecular functions and potential mechanisms of YB-1 in cancer biology the state of phosphorylation of YB-1 and the PPI were investigated in the cytoplasm and nucleus of two cancer cell lines. The YB-1 from the cytoplasm and nucleus of the cell lines was extensively phosphorylated. These experiments identified >250 proteins. These binding partners confirmed the multifunctionality of YB-1 as the proteins that co-purify with YB-1 participate in glycolysis, RNA splicing, RNA stabilization, translation, mitochondrial localisation, and chromosomal association. These data suggest that the bulk of YB-1 function may be explained by non-transcriptional mechanisms.
Mechanisms of drug resistance were also investigated. Depleting YB-1, using siRNA duplexes, reduced MDA-MB231 cell growth and increased cell death. The loss of YB-1 sensitised MDA-MB231 cells to cisplatin exposure by increasing cell death. Cisplatin exposure altered the distribution of YB-1 protein to perinuclear spots and to foci in the nucleus of many cells.
The molecular basis of YB-1 mediated cisplatin resistance was analysed by examining the alterations of YB-1 PPI during cisplatin exposure using co-immunoprecipitation of YB-1 binding partners and mass spectrometry-based protein identification. Quantitative analyses of the co-immunoprecipitated proteins from MDA-MB231 cells indicated that a subset of the proteins, such as TRIM28 and FAM120A, increased markedly after 48 and 96 hours of cisplatin exposure. The chromosomal proteins that interacted with YB-1 were disproportionately affected by cisplatin exposure.
The importance of FAM120A, TRIM28, and C1QBP, three YB-1 binding partners identified here, during cisplatin exposure was studied. The subcellular distribution of FAM120A was most similar that of YB-1 in MDA-MB231 cells. Depleting YB-1 or FAM120A, but not TRIM28 or C1QBP, sensitised MDA-MB231 cells to cisplatin exposure. Depleting YB-1 alongside either FAM120A or C1QBP partially restored the growth of MDA-MB231 cells. YB-1 does not appear to participate in the repair of double-strand DNA breaks during cisplatin exposure as depleting YB-1 had no effect on the number of γH2AX foci that formed during cisplatin exposure.
This is the first report that integrates findings of protein-binding partners, state of phosphorylation, and subcellular localisation of endogenous YB-1 to understand the complex functions of YB-1. These results confirm the importance of RNA binding to the molecular function of YB-1. The interaction of YB-1 with FAM120A, a novel finding, increases during cisplatin exposure and both proteins together, via an unknown molecular pathway, confer cisplatin resistance to breast cancer cells
YB-1 oncoprotein in cancer and drug resistance
No full textY-box-binding protein 1 (YB-1) is a biomarker that is predictive of poor prognosis in cancer. Various molecular functions of YB-1 in cancer have been proposed, including the transcriptional regulation of gene expression. YB-1 also binds to RNA transcripts to influence gene expression.
In the present study, the status of YB-1 as a biomarker was confirmed by immunohistochemistry using two antibodies against YB-1. However, the prognostic sensitivity of these two antibodies differed. The observed difference in antibody affinity was most likely due to the tertiary structure or protein-protein interactions (PPI) associated with various functions of YB-1 in situ.
To gain further insights into the molecular functions and potential mechanisms of YB-1 in cancer biology the state of phosphorylation of YB-1 and the PPI were investigated in the cytoplasm and nucleus of two cancer cell lines. The YB-1 from the cytoplasm and nucleus of the cell lines was extensively phosphorylated. These experiments identified >250 proteins. These binding partners confirmed the multifunctionality of YB-1 as the proteins that co-purify with YB-1 participate in glycolysis, RNA splicing, RNA stabilization, translation, mitochondrial localisation, and chromosomal association. These data suggest that the bulk of YB-1 function may be explained by non-transcriptional mechanisms.
Mechanisms of drug resistance were also investigated. Depleting YB-1, using siRNA duplexes, reduced MDA-MB231 cell growth and increased cell death. The loss of YB-1 sensitised MDA-MB231 cells to cisplatin exposure by increasing cell death. Cisplatin exposure altered the distribution of YB-1 protein to perinuclear spots and to foci in the nucleus of many cells.
The molecular basis of YB-1 mediated cisplatin resistance was analysed by examining the alterations of YB-1 PPI during cisplatin exposure using co-immunoprecipitation of YB-1 binding partners and mass spectrometry-based protein identification. Quantitative analyses of the co-immunoprecipitated proteins from MDA-MB231 cells indicated that a subset of the proteins, such as TRIM28 and FAM120A, increased markedly after 48 and 96 hours of cisplatin exposure. The chromosomal proteins that interacted with YB-1 were disproportionately affected by cisplatin exposure.
The importance of FAM120A, TRIM28, and C1QBP, three YB-1 binding partners identified here, during cisplatin exposure was studied. The subcellular distribution of FAM120A was most similar that of YB-1 in MDA-MB231 cells. Depleting YB-1 or FAM120A, but not TRIM28 or C1QBP, sensitised MDA-MB231 cells to cisplatin exposure. Depleting YB-1 alongside either FAM120A or C1QBP partially restored the growth of MDA-MB231 cells. YB-1 does not appear to participate in the repair of double-strand DNA breaks during cisplatin exposure as depleting YB-1 had no effect on the number of γH2AX foci that formed during cisplatin exposure.
This is the first report that integrates findings of protein-binding partners, state of phosphorylation, and subcellular localisation of endogenous YB-1 to understand the complex functions of YB-1. These results confirm the importance of RNA binding to the molecular function of YB-1. The interaction of YB-1 with FAM120A, a novel finding, increases during cisplatin exposure and both proteins together, via an unknown molecular pathway, confer cisplatin resistance to breast cancer cells
Dissecting the proteome of lipoproteins: New biomarkers for cardiovascular diseases?
No full textAbstractProteomics has proven to be a powerful tool for the characterization of lipoproteins and has provided important insights into the biochemistry and pathophysiology of various lipoprotein classes. It has significantly contributed to the way we now see lipoproteins as complex particles not only involved in lipid transport and exchange, but also in processes such as immune response, inflammation and wound healing. Ongoing proteomics research is focussing on the identification of new candidate markers for cardiovascular disease, the leading cause of death worldwide. The ratio between good cholesterol (high density lipoprotein) and bad cholesterol (low density lipoprotein) is routinely used to estimate an individual’s risk for developing premature coronary heart disease. While statin therapy has proven effects in reducing cardiovascular events, other therapies such as resins, fibrates and niacin have failed to substantially reduce cardiovascular risk. Thus new targets and candidate biomarkers for risk assessment and for the development of alternative drugs and treatments of disease are needed. Here we review the recent findings in lipoprotein proteomics with the main emphasis on studies that differentially displayed various states of diseases and on new targeted, high throughput strategies with the capability to translate discovery findings into the clinical context of large cohort analyzes
seine Wirkungsgeschichte in der Literatur
No full textSoftcover, 175 S.Softcover, 17x24Im Jahr 2003 fand in der Göttinger Jakobikirche eine hochkarätige und deshalb zu Recht viel beachtete Vorlesungsreihe zur Wirkungsgeschichte der Bibel statt. Namhafte Vertreter bester Göttinger Philologie fanden hier ihr Publikum - interessiert, oft gelehrt, aber in seinem Interesse nicht auf eine fachwissenschaftliche Binnendiskussion beschränkt. Diese nun gedruckt vorliegende Vorlesungsreihe ist nicht nur ein Beispiel für das gute Zusammenleben von Universität und Stadt, wie es typisch für Göttingen ist. Sie ist darüberhinaus Beispiel für ein fruchtbares Gespräch zwischen Wissenschaft und Kirche, wie es auch außerhalb der akademischen Theologie möglich und nötig ist.The firms and markets of today's complex socio-economic system developed in a spontaneous process termed evolution, in just the same way as the universe, the solar system, the Earth and all that lives upon it. Darwin's theory of evolution clearly demonstrated that evolution involved increasing organization. As we began to explore the molecular basis of life and its evolution, it became equally clear that it depended on the processing and communication of information. This book develops a consistent theory of evolution in its wider sense, examining the information based laws and forces that drive it. Exploring subjects as diverse as economics and the theories of thermodynamics, the author revisits the paradox of the apparent conflict between the laws of thermodynamics and evolution to arrive at a systems theory, tracing a continuous line of evolving information sets that connect the Big-Bang to the firms and markets of our current socio-economic system
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