Progress 10/01/08 to 09/30/13
Outputs
Target Audience: Scientific community in the areas of biochemistry, molecular biology, cell biology, microbiology, and neuroscience reached by publications and presentations. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Graduate students who have been involved in our studies obtained advanced training in the area of biochemistry, cell, and molecular biology, which contributed to their success in completing the Ph.D. and M.S. degrees at KSU. How have the results been disseminated to communities of interest? The results were disseminated in the form of publications, presentations, and Ph.D. and M.S. theses. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Within the goal “Mechanism of protein disaggregation mediated by ClpB”, we achieved a change in fundamental knowledge that resulted in publication of results. Specifically, we completed the biochemical characterization of two ClpB variants from two pathogenic microorganisms: Plasmodium falciparum (the malaria parasite) and Ehrlichia chaffeensis (the agent causing human and animal monocytic ehrlichiosis). We discovered that there are significant mechanistic differences between the molecular chaperone ClpB from those pathogenic microorganisms and from a laboratory strain of Escherichia coli, which has been previously extensively characterized in our laboratory. Interestingly, ClpB from Ehrlichia chaffeensis (EhClpB) showed a distinct linkage between nucleotide binding and interactions with aggregated substrates, which was accompanied by the capability of EhClpB to disaggregate substrates without assistance of the co-chaperones from the DnaK/DnaJ family. Importantly, the expression of EhClpB was strongly upregulated in Ehrlichia immediately following the infection of mammalian cells. Our studies on ClpB from pathogens will have a positive impact on understanding the mechanisms of host-pathogen interactions including the pathogen response to host-induced stress. In the long-term, our studies may contribute to the development of novel antimicrobials with broad specificity that may positively affect the animal and human health and improve the safety of the food supply. Within the goal “Biochemical characterization of the torsin sub-family of AAA+ ATPases” we achieved a change in fundamental knowledge that resulted in a publication submitted to the Journal of Biological Chemistry and in another manuscript in preparation. Specifically, we completed a microarray-based study of the gene expression changes in the fruit fly Drosophila melanogaster induced by the loss of Drosophila torsin protein. We found that the genes of several heat shock proteins are upregulated upon the torsin loss, which suggests that torsin plays a role in controlling the cellular stress levels in Drosophila cells. We also completed a collaborative study (with Dr. Jeffrey Brodsky, University of Pittsburgh) on the processing and stability of torsinA in the yeast expression system. We discovered that the stability of torsinA is supported by the molecular chaperone BiP. Our studies on torsin will contribute to understanding the cellular stress-response and quality-control mechanisms and may have a broad long-term significance in multiple areas of bio-medical and bio-agricultural sciences.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Zhang, T., Kedzierska-Mieszkowska, S., Liu, H., Cheng, C., Ganta, R. R. and Zolkiewski, M. 2013. Aggregate-reactivation activity of the molecular chaperone ClpB from Ehrlichia chaffeensis. PLoS ONE. 8:e62454.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Park, S., Li, X., Kim, H. M., Singh, C. R., Tian, G., Hoyt, M. A., Lovell, S., Battaile, K. P., Zolkiewski, M., Coffino, P., Roelofs, J., Cheng, Y., and Finley, D. 2013. Reconfiguration of the proteasome during chaperone-mediated assembly. Nature. 497:512516.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Ngansop, F., Li, H., Zolkiewska, A., and Zolkiewski, M. 2013. Biochemical characterization of the apicoplast-targeted AAA+ ATPase ClpB from Plasmodium falciparum. Biochem. Biophys. Res. Comm. 439:191-195.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Our laboratory performs basic biochemical research on the mechanism of energy-dependent protein-remodeling factors. In 2012, we completed investigation of the role of the flexible linker region of bacterial ClpB in aggregate reactivation with a publication in Proteins. We also published an invited review article on ClpB in the Archives of Biochemistry and Biophysics. We completed a pilot project on ClpB from a zoonotic pathogen Ehrlichia chaffeensis and submitted a manuscript to the Journal of Biological Chemistry. Submission of an NIH grant proposal on Ehrlichia ClpB is planned for 2013. We continued our work aimed at testing the processing and stability of the human protein torsinA in yeast cells (in collaboration with Dr. Jeffrey Brodsky from the University of Pittsburgh). The publication on torsinA in yeast is planned in 2013. We completed the project aimed at analyzing gene expression profiles in the torsin loss-of-function model of Drosophila melanogaster with a publication planned in 2013. We initiated a new line of research on a ClpB ortholog from the malaria parasite Plasmodium falciparum. PARTICIPANTS: Michal Zolkiewski, PI; Hui-Chuan Wu, Graduate Student; Ting Zhang, Graduate Student; Yan Wu, Graduate Student; Fabrice Ngansop, Graduate Student. Collaborators: Paul Smith, Kansas State University; Roman Ganta, Kansas State University; Maureen Gorman, Kansas State University; Yoonseong Park, Kansas State University; Kristin Michel, Kansas State University; Jeffrey Brodsky, University of Pittsburgh; Sabina Kedzierska-Mieszkowska, University of Gdansk, Poland TARGET AUDIENCES: Scientific community in the areas of biochemistry, molecular biology, cell biology, microbiology, neuroscience. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We determined that ClpB from Ehrlichia chaffeensis reactivates aggregated protein in cooperation with the DnaK/DnaJ/GrpE system or, for selected substrates, without the need of DnaK/DnaJ/GrpE. We discovered that ClpB from E. chaffeensis links the nucleotide and substrate binding in a distinct way that has not been previously observed for similar proteins. We determined that human torsinA does not induce significant ER stress in yeast cells, nor does it help ameliorate the ER stress. We performed microarray analysis of the gene expression patterns induced by the loss of torsin in Drosophila cultured cells and in the whole organism. Full scientific impact of these studies will become evident as the work continues and is completed with publications.
Publications
- Zolkiewski, M., Zhang, T. and Nagy, M. 2012. Aggregate reactivation mediated by the Hsp100 chaperones, Arch. Biochem. Biophys., 520: 1-6.
- Zhang, T., Ploetz, E.A., Nagy, M., Doyle, S.M., Wickner, S., Smith, P.E. and Zolkiewski, M. 2012. Flexible connection of the N-terminal domain in ClpB modulates substrate binding and the aggregate reactivation efficiency, Proteins 80: 2758-68.
- Guenther, I., Zolkiewski, M., and Kedzierska-Mieszkowska, S. 2012. Cooperation between two ClpB isoforms enhances the recovery of the recombinant beta-galactosidase from inclusion bodies, Biochem. Biophys. Res. Comm. 426: 596-600.
- An, C., Hiromasa, Y., Lovell, S., Zolkiewski, M., Tomich, J.M. and Michel, K. 2012. Biochemical characterization of SRPN6, a malaria parasite invasion marker in mosquitoes, PLoS ONE 7(11): e48689.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Our laboratory performs basic biochemical research on the mechanism of energy-dependent protein-remodeling factors. In 2011, we completed investigation of the role of the flexible linker region of bacterial ClpB in aggregate reactivation. This work, which involved a collaboration with Dr. Paul Smith from the Department of Chemistry KSU (see Participants) will be soon submitted for publication. We also completed a pilot project on ClpB from a zoonotic pathogen Ehrlichia chaffeensis. This research was performed in collaboration with Dr. Roman Ganta from the Department of Pathobiology, and will be soon submitted for publication. Submission of a joint NIH grant proposal on Ehrlichia ClpB is planned for 2012. We continued our work aimed at testing the processing and stability of the human protein torsinA in yeast cells (in collaboration with Dr. Jeffrey Brodsky from the University of Pittsburgh). This work has been funded in 2010 by the Dystonia Medical Research Foundation and the funding was extended into 2012. The publication is planned in 2012. We are close to completing the project aimed at analyzing gene expression profiles in the torsin loss-of-function model of Drosophila melanogaster, which has been funded by the KSU Arthropod Genomics Center (collaboration with Dr. Maureen Gorman and Dr. Yoonseong Park) with a publication planned in 2012. PARTICIPANTS: Michal Zolkiewski, PI; Hui-Chuan Wu, Graduate Student; Ting Zhang, Graduate Student; Yan Wu, Graduate Student. Collaborators: Paul Smith, Kansas State University; Roman Ganta, Kansas State University; Maureen Gorman, Kansas State University; Yoonseong Park, Kansas State University; Jeffrey Brodsky, University of Pittsburgh TARGET AUDIENCES: Scientific community in the areas of biochemistry, molecular biology, cell biology, microbiology, neuroscience. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We determined that ClpB from Ehrlichia chaffeensis reactivates aggregated protein in cooperation with the DnaK/DnaJ/GrpE system. We characterized the similarities and differences between the biochemical properties of E. coli and Ehrlichia ClpB. We determined that human torsinA does not induce significant ER stress in yeast cells, nor does it help ameliorate the ER stress. We performed microarray analysis of the gene expression patterns induced by the loss of torsin in Drosophila cultured cells and in the whole organism. Full scientific impact of these studies will become evident as the work continues and is completed with publications.
Publications
- No publications reported this period
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Our laboratory performs basic biochemical research on the mechanism of energy-dependent protein-remodeling factors. In 2010, we continued investigating the role of the flexible linker region of ClpB in aggregate reactivation. This work included experiments performed in PI laboratory as well as computer simulations performed in collaboration with Dr. Paul Smith from the Department of Chemistry KSU (see Participants). Submission of a publication on this project is planned early next year. We also initiated a new direction of research on ClpB from a human pathogen Ehrlichia chaffeensis. In collaboration with Dr. Roman Ganta from the Department of Pathobiology, we produced the recombinant E. chaffeensis ClpB and performed its initial biochemical characterization. Submission of a joint NIH grant proposal is planned for 2011. We continued our work aimed at developing a torsinA expression system in yeast (in collaboration with Dr. Jeffrey Brodsky). This work has been funded in 2010 by the Dystonia Medical Research Foundation. In 2010, we also started a new project aimed at discovering the role of torsin in Drosophila melanogaster which has been funded by the KSU Arthropod Genomics Center (collaboration with Dr. Maureen Gorman and Dr. Yoonseong Park). PARTICIPANTS: Michal Zolkiewski, PI; Hui-Chuan Wu, Graduate Student; Ting Zhang, Graduate Student. Collaborators: Paul Smith, Kansas State University; Roman Ganta, Kansas State University; Maureen Gorman, Kansas State University; Yoonseong Park, Kansas State University; Jeffrey Brodsky, University of Pittsburgh TARGET AUDIENCES: Scientific community in the areas of biochemistry, molecular biology, cell biology, microbiology. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We found that the flexible attachment of the N-terminal domain supports the chaperone activity of ClpB by controlling the efficiency of substrate binding. Moreover, the length and conformational flexibility of the linker region may have been optimized during evolution to achieve the most advantageous aggregate-removal rates. We also produced the first recombinant expression system of Ehrlichia ClpB which will be used to study the role of stress-response in the pathogen-host interactions. We developed several experimental systems for studying the role of torsin in Drosophila using RNAi technology. Full scientific impact of these studies will become evident as the work continues and is completed with publications.
Publications
- Nagy, M., Guenther, I., Akoyev, V., Barnett, M. E., Zavodszky, M. I., Kedzierska-Mieszkowska, S. and Zolkiewski, M. 2010. Synergistic cooperation between two ClpB isoforms in aggregate reactivation. J. Mol. Biol. 396:697-707.
- Gilmore, J., Urbauer, R., Minakhin, L., Akoyev, V., Zolkiewski, M., Severinov, K. and Urbauer, J. 2010. Determinants of Affinity and Activity of the Anti-Sigma Factor AsiA. Biochemistry 49:6143-6154.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Our laboratory performs basic biochemical research on the mechanism of energy-dependent protein-remodeling factors. In 2009, we finished a project (with a publication in a high-quality journal) on the synergistic functional cooperation of two isoforms of ClpB. We have been also investigating the role of the flexible linker region of ClpB in aggregate reactivation. This work included experiments performed in PI laboratory as well as computer simulations performed in collaboration with Dr. Paul Smith (see Participants). We also initiated a new line of research on the interaction between ClpB and a yeast prion-like protein Sup35 and other prion-like mammalian proteins (in collaboration with Dr. Kausik Si). Finally, we continued our work aimed at producing a torsinA expression system in yeast (in collaboration with Dr. Jeffrey Brodsky). A poster on torsinA has been presented by the graduate student Hui-Chuan Wu at the 14th Annual Midwest Stress Response and Molecular Chaperone Meeting in Evanston, Illinois. PARTICIPANTS: Michal Zolkiewski - PI, Hui-Chuan Wu - Graduate Student, Ting Zhang - Graduate Student; Collaborators: Paul Smith, Kansas State University; Jeffrey Brodsky, University of Pittsburgh; Kausik Si, Stowers Institute for Medical Research TARGET AUDIENCES: Scientific community in the areas of biochemistry, molecular biology, cell biology, microbiology. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We described how two ClpB isoforms (with different molecular weight) produce a highly effective chaperone system which is more potent in protein reactivation than each of the two isoforms separately. We proposed a molecular model which emphasizes the role of mobility of the N-terminal domains of ClpB in the protein-disaggregation activity. This result will stimulate further studies on ClpB and other AAA+ ATPases. We also found that ClpB can block aggregation of the yeast prion Sup35. This result may in the long-term help control prion-induced pathological protein aggregation in cells and tissues. Full scientific impact of these studies will become evident as the work continues.
Publications
- Nagy, M., Wu, H.-C., Liu, Z., Kedzierska-Mieszkowska, S. and Zolkiewski, M.: Walker-A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB, Protein Sci. 18, 287-293, 2009.
- Nagy, M., Guenther, I., Akoyev, V., Barnett, M. E., Zavodszky, M. I., Kedzierska-Mieszkowska, S. and Zolkiewski, M.: Synergistic cooperation between two ClpB isoforms in aggregate reactivation, J. Mol. Biol. (in press, 2009).
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: This project has been active for two months. We initiated studies aimed at discovering the biological function of torsinA. To reach this goal, we produced DNA constructs of torsinA in yeast expression plasmids. In collaboration with Dr. Jeffery Brodsky from the University of Pittsburgh, we are currently testing whether production of torsinA in yeast modifies stress-related phenotypes. PARTICIPANTS: Michal Zolkiewski, PI; Hui-Chuan Wu, Graduate Student; Ting Zhang, Graduate Student TARGET AUDIENCES: Scientific community in the areas of biochemistry, molecular biology, cell biology, microbiology PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have produced new reagents that will be used in future studies.
Publications
- No publications reported this period
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