Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins
Abstract
Background: The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function. Results: In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function. We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. Conclusion: Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.
- Authors:
-
- Medical College of Wisconsin, Milwaukee, WI (United States)
- Marquette Univ., Milwaukee, WI (United States)
- Concordia Univ. of Wisconsin, Mequon, WI (United States)
- Argonne National Lab. (ANL), Argonne, IL (United States)
- Medical College of Wisconsin, Milwaukee, WI (United States); CRI Developmental Vascular Biology Program, Milwaukee, WI (United States)
- Publication Date:
- Research Org.:
- Argonne National Laboratory (ANL), Argonne, IL (United States)
- Sponsoring Org.:
- USDOE
- OSTI Identifier:
- 1201767
- Grant/Contract Number:
- AC02-06CH11357
- Resource Type:
- Journal Article: Accepted Manuscript
- Journal Name:
- BMC Biochemistry (Online)
- Additional Journal Information:
- Journal Volume: 15; Journal Issue: 1; Journal ID: ISSN 1471-2091
- Publisher:
- BioMed Central
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; DUSP5; mutation; vascular anomalies; protein purification; molecular modeling
Citation Formats
Nayak, Jaladhi, Gastonguay, Adam J., Talipov, Marat R., Vakeel, Padmanabhan, Span, Elise A., Kalous, Kelsey S., Kutty, Raman G., Jensen, Davin R., Pokkuluri, Phani Raj, Sem, Daniel S., Rathore, Rajendra, and Ramchandran, Ramani. Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins. United States: N. p., 2014.
Web. doi:10.1186/s12858-014-0027-0.
Nayak, Jaladhi, Gastonguay, Adam J., Talipov, Marat R., Vakeel, Padmanabhan, Span, Elise A., Kalous, Kelsey S., Kutty, Raman G., Jensen, Davin R., Pokkuluri, Phani Raj, Sem, Daniel S., Rathore, Rajendra, & Ramchandran, Ramani. Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins. United States. https://doi.org/10.1186/s12858-014-0027-0
Nayak, Jaladhi, Gastonguay, Adam J., Talipov, Marat R., Vakeel, Padmanabhan, Span, Elise A., Kalous, Kelsey S., Kutty, Raman G., Jensen, Davin R., Pokkuluri, Phani Raj, Sem, Daniel S., Rathore, Rajendra, and Ramchandran, Ramani. 2014.
"Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins". United States. https://doi.org/10.1186/s12858-014-0027-0. https://www.osti.gov/servlets/purl/1201767.
@article{osti_1201767,
title = {Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins},
author = {Nayak, Jaladhi and Gastonguay, Adam J. and Talipov, Marat R. and Vakeel, Padmanabhan and Span, Elise A. and Kalous, Kelsey S. and Kutty, Raman G. and Jensen, Davin R. and Pokkuluri, Phani Raj and Sem, Daniel S. and Rathore, Rajendra and Ramchandran, Ramani},
abstractNote = {Background: The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function. Results: In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function. We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. Conclusion: Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.},
doi = {10.1186/s12858-014-0027-0},
url = {https://www.osti.gov/biblio/1201767},
journal = {BMC Biochemistry (Online)},
issn = {1471-2091},
number = 1,
volume = 15,
place = {United States},
year = {Thu Dec 18 00:00:00 EST 2014},
month = {Thu Dec 18 00:00:00 EST 2014}
}
Web of Science
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PROSESS: a protein structure evaluation suite and server
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