1,720,981 research outputs found
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
Full text linkY-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
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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