The characterization of DNAJC3: elucidating the function of the TPR domains
- Mutsvunguma, Lorraine Zvichapera
- Authors: Mutsvunguma, Lorraine Zvichapera
- Date: 2014
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/55874 , vital:26751
- Description: DNAJC3 is a novel member of the DNAJ family with two domains linked to co-chaperone functions, namely the tetratricopeptide repeat (TPR) and J domain. Out of the two domains, the TPR domains are the least characterized. Therefore, the aim of this study was to characterize and elucidate additional functions of DNAJC3 TPR domains through in silico, in vitro and ex vivo approaches. Through multiple sequence and structural alignment as well as electrostatic potential analysis, DNAJC3 TPR domain were found to be most similar to TPR-containing proteins with Hsp90 or Hsp70 independent functions. In vitro pull down assays illustrated that DNAJC3 TPR domains did not interact with either cytosolic Hsp90 and Hsp70 or Grp78 and Grp94 directly, however a potential indirect interaction with Grp94 and Hsp90 was observed in mammalian lysates, via pull down assays; suggesting the formation of a complex between the proteins mediated by a specific substrate. DNAJC3 TPR domains were found to bind indiscriminately to both native and heat denatured substrates in a dose dependent manner. DNAJC3 TPR domains bound to β-galactosidase with greater affinity than malate dehydrogenase (MDH), suggesting that DNAJC3 TPR domains might exhibit substrate specificity that has not been reported before. Preliminary ex vivo analysis of DNAJC3 in mammalian cells showed that induced stress conditions did not alter the cytosolic or endoplasmic reticulum (ER) localization, or levels of DNAJC3 protein, suggesting that the protein is not stress inducible. However, protein levels of DNAJC3 were dramatically reduced by Hsp90 inhibitor novobiocin at 500 μM. Transient knockdown DNAJC3 did not change the protein levels of either Grp78 or Grp94, but decreased the protein levels of Hsp70/Hsp90 organizing protein HOP. On the other hand, protein levels of DNAJC3 were increased in HOP depleted cells. In conclusion, this study was the first to experimentally demonstrate that DNAJC3 TPR domains do not interact directly with Hsp90, Hsp70, Grp78 or Grp94, and therefore DNAJC3 is unlikely to participate in traditional co-chaperone interactions with those proteins via its TPR domain. However, the J domain is known to interact with Grp78. The discovery that DNAJC3 TPR domains resemble that of TPR-containing proteins with functions independent of Hsp90 or Hsp70 suggests that DNAJC3 might link the Hsp70/Grp78 chaperone machinery to non co-chaperone related functions, which requires further analysis.
- Full Text:
- Date Issued: 2014
- Authors: Mutsvunguma, Lorraine Zvichapera
- Date: 2014
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/55874 , vital:26751
- Description: DNAJC3 is a novel member of the DNAJ family with two domains linked to co-chaperone functions, namely the tetratricopeptide repeat (TPR) and J domain. Out of the two domains, the TPR domains are the least characterized. Therefore, the aim of this study was to characterize and elucidate additional functions of DNAJC3 TPR domains through in silico, in vitro and ex vivo approaches. Through multiple sequence and structural alignment as well as electrostatic potential analysis, DNAJC3 TPR domain were found to be most similar to TPR-containing proteins with Hsp90 or Hsp70 independent functions. In vitro pull down assays illustrated that DNAJC3 TPR domains did not interact with either cytosolic Hsp90 and Hsp70 or Grp78 and Grp94 directly, however a potential indirect interaction with Grp94 and Hsp90 was observed in mammalian lysates, via pull down assays; suggesting the formation of a complex between the proteins mediated by a specific substrate. DNAJC3 TPR domains were found to bind indiscriminately to both native and heat denatured substrates in a dose dependent manner. DNAJC3 TPR domains bound to β-galactosidase with greater affinity than malate dehydrogenase (MDH), suggesting that DNAJC3 TPR domains might exhibit substrate specificity that has not been reported before. Preliminary ex vivo analysis of DNAJC3 in mammalian cells showed that induced stress conditions did not alter the cytosolic or endoplasmic reticulum (ER) localization, or levels of DNAJC3 protein, suggesting that the protein is not stress inducible. However, protein levels of DNAJC3 were dramatically reduced by Hsp90 inhibitor novobiocin at 500 μM. Transient knockdown DNAJC3 did not change the protein levels of either Grp78 or Grp94, but decreased the protein levels of Hsp70/Hsp90 organizing protein HOP. On the other hand, protein levels of DNAJC3 were increased in HOP depleted cells. In conclusion, this study was the first to experimentally demonstrate that DNAJC3 TPR domains do not interact directly with Hsp90, Hsp70, Grp78 or Grp94, and therefore DNAJC3 is unlikely to participate in traditional co-chaperone interactions with those proteins via its TPR domain. However, the J domain is known to interact with Grp78. The discovery that DNAJC3 TPR domains resemble that of TPR-containing proteins with functions independent of Hsp90 or Hsp70 suggests that DNAJC3 might link the Hsp70/Grp78 chaperone machinery to non co-chaperone related functions, which requires further analysis.
- Full Text:
- Date Issued: 2014
Investigating the role of heat shock proteins (Hsps) 40, 70 and 90 in the life cycle of Theiler's murine encephalomyelitis virus (TMEV)
- Mutsvunguma, Lorraine Zvichapera
- Authors: Mutsvunguma, Lorraine Zvichapera
- Date: 2011
- Subjects: Heat shock proteins , Picornaviruses , Encephalomyelitis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3966 , http://hdl.handle.net/10962/d1004025 , Heat shock proteins , Picornaviruses , Encephalomyelitis
- Description: Introduction: Picornaviruses are a family of RNA viruses which are economically and clinically significant. Like many other viruses, picornaviruses utilise host cell machinery to facilitate their replication and assembly, including heat shock proteins (Hsps). The aim of this research was to investigate the role of Hsp40, Hsp70 and Hsp90 during picornavirus infection using the cardiovirus, Theiler’s murine encephalomyelitis virus (TMEV), as a study model. Methodology: Picornavirus VP1 capsid proteins were analysed by multiple sequence alignment and multiple structural comparisons. Protein domain architecture was used to analyse Hsp90 cellular and viral client proteins. Effects of Hsp90 inhibitors, novobiocin and geldanamycin, on TMEV growth in BHK-21 cells was observed over a 48hr period. Localisation of Hsp40, Hsp90 and Hsp70 in TMEV-infected BHK-21 cells was investigated by indirect immunofluorescence and confocal microscopy. Results and Discussion: VP1 proteins of picornaviruses are highly divergent within the family at the amino acid level, which might be linked to the protein’s function in determining virus tropism and antibody neutralisation. An eight-stranded anti-parallel beta-barrel structure was found conserved in the VP1 protein structures which might be linked to the highly conserved picornavirus capsid assembly process. Absence of a common protein domain between Hsp90 viral and cellular client proteins that might be functionally connected to Hsp90, suggests that Hsp90 most likely recognises surface features rather than sequence motifs/patterns. The Hsp90 inhibitors, novobiocin and geldanamycin, had a negative effect on virus growth as virus-induced cytopathic effect was not observed in treated cell after 48hrs. TMEV 2C protein was detected by Western analysis in infected cell lysates treated with geldanamycin but not novobiocin, suggesting novobiocin affects the translation or processing of TMEV 2C. Immunofluorescence analysis of TMEV-infected cells showed a relocalisation of Hsp40 into the nucleus during infection. Overlap of Hsp40 and TMEV P1 was observed in the perinuclear region, suggesting colocalisation between these proteins. Hsp70 converged around the replication complex during infection but did not overlap with TMEV 2C. Hsp90 concentrated in the region of the replication complex where it overlapped with TMEV 2C and this redistribution was found to be dependent on the stage of infection. The overlap between Hsp90 and TMEV 2C signals observed, suggested colocalisation between the two proteins. Conclusion: This study identified Hsp90, Hsp70 and Hsp40 as possible host factors required in TMEV replication.
- Full Text:
- Date Issued: 2011
- Authors: Mutsvunguma, Lorraine Zvichapera
- Date: 2011
- Subjects: Heat shock proteins , Picornaviruses , Encephalomyelitis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3966 , http://hdl.handle.net/10962/d1004025 , Heat shock proteins , Picornaviruses , Encephalomyelitis
- Description: Introduction: Picornaviruses are a family of RNA viruses which are economically and clinically significant. Like many other viruses, picornaviruses utilise host cell machinery to facilitate their replication and assembly, including heat shock proteins (Hsps). The aim of this research was to investigate the role of Hsp40, Hsp70 and Hsp90 during picornavirus infection using the cardiovirus, Theiler’s murine encephalomyelitis virus (TMEV), as a study model. Methodology: Picornavirus VP1 capsid proteins were analysed by multiple sequence alignment and multiple structural comparisons. Protein domain architecture was used to analyse Hsp90 cellular and viral client proteins. Effects of Hsp90 inhibitors, novobiocin and geldanamycin, on TMEV growth in BHK-21 cells was observed over a 48hr period. Localisation of Hsp40, Hsp90 and Hsp70 in TMEV-infected BHK-21 cells was investigated by indirect immunofluorescence and confocal microscopy. Results and Discussion: VP1 proteins of picornaviruses are highly divergent within the family at the amino acid level, which might be linked to the protein’s function in determining virus tropism and antibody neutralisation. An eight-stranded anti-parallel beta-barrel structure was found conserved in the VP1 protein structures which might be linked to the highly conserved picornavirus capsid assembly process. Absence of a common protein domain between Hsp90 viral and cellular client proteins that might be functionally connected to Hsp90, suggests that Hsp90 most likely recognises surface features rather than sequence motifs/patterns. The Hsp90 inhibitors, novobiocin and geldanamycin, had a negative effect on virus growth as virus-induced cytopathic effect was not observed in treated cell after 48hrs. TMEV 2C protein was detected by Western analysis in infected cell lysates treated with geldanamycin but not novobiocin, suggesting novobiocin affects the translation or processing of TMEV 2C. Immunofluorescence analysis of TMEV-infected cells showed a relocalisation of Hsp40 into the nucleus during infection. Overlap of Hsp40 and TMEV P1 was observed in the perinuclear region, suggesting colocalisation between these proteins. Hsp70 converged around the replication complex during infection but did not overlap with TMEV 2C. Hsp90 concentrated in the region of the replication complex where it overlapped with TMEV 2C and this redistribution was found to be dependent on the stage of infection. The overlap between Hsp90 and TMEV 2C signals observed, suggested colocalisation between the two proteins. Conclusion: This study identified Hsp90, Hsp70 and Hsp40 as possible host factors required in TMEV replication.
- Full Text:
- Date Issued: 2011
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