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Your search keyword '"Cytoplasmic PrP"' showing total 3 results
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3 results on '"Cytoplasmic PrP"'

1. Development of kinomic analyses to identify dysregulated signaling pathways in cells expressing cytoplasmic PrP.

Author

Shott, Rory H., Appanah, Cathy, Grenier, Catherine, Tremblay, Guillaume, Roucou, Xavier, and Schang, Luis M.

Abstract

Background: Dysregulated protein kinase signaling is involved in the pathogenesis of many chronic diseases. However, the dysregulated signaling pathways critical to prion pathogenesis remain incompletely characterized. Global analyses of signaling pathways may be useful to better characterize these pathways. We therefore set out to develop such global assays. To this end, we used as a model cytoplasmic mutants of the cellular prion protein (PrPC), which are toxic to N2a neuroblastoma cells. We tested the global assays for their sensitivity to detect changes in signaling pathways in cells expressing cytoplasmic PrP mutants. Methods: We developed a targeted proteomics (kinomics) approach using multiplex Western blots to identify signaling pathways dysregulated in chronic neurological pathologies. We tested the approach for its potential ability to detect signaling changes in N2a cells expressing cytoplasmic PrP mutants. Results: Multiplex Western blots were designed to quantitate the expression levels of 137 protein kinases in a single membrane and using only 1.2 mg of sample. The response of the blots was sensitive and linear to changes of 6% in protein levels. Hierarchical and functional clustering of the relative expression levels identified an mTOR signaling pathway as potentially dysregulated in N2a cells expressing cytoplasmic PrP. The mTOR signaling pathway regulates global protein synthesis, which is inhibited in cells expressing cytoplasmic PrP. The levels of proteins involved in the Akt1/p70S6K branch of mTOR signaling changed in synchrony with time of cytoplasmic PrP expression. Three kinases in this pathway, Akt, p70S6K, and eIF4B were in their inactive states, as evaluated by phosphorylation of their regulatory sites. Conclusion: The results presented are consistent with the previously reported inhibition of Akt/p70S6K/eIF4B signaling as mediating pathogenesis of cytoplasmic PrP. We conclude that the kinomic analyses are sensitive and specific to detect signaling pathways dysregulated in a simple in vitro model of PrP pathogenesis. [ABSTRACT FROM AUTHOR]

Published
2014
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2. Development of kinomic analyses to identify dysregulated signaling pathways in cells expressing cytoplasmic PrP

Author

Luis M. Schang, Catherine Grenier, Xavier Roucou, Cathy Appanah, Guillaume Tremblay, and Rory H Shott

Subjects
Proteomics, Cytoplasm, animal diseases, Blotting, Western, Prion disease, Kinomics, AKT1, Biology, Protein kinase, 03 medical and health sciences, 0302 clinical medicine, Cell Line, Tumor, Virology, Neurotoxicity, Humans, PrPC Proteins, EIF4B, p70S6K, Protein kinase A, Protein kinase B, 030304 developmental biology, Cytoplasmic PrP, 0303 health sciences, Kinase, Research, Multiplex Western blots, Akt, Proteins, Cell biology, Infectious Diseases, eIF4B, Phosphorylation, Signal transduction, 030217 neurology & neurosurgery, Signal Transduction
Abstract

Background Dysregulated protein kinase signaling is involved in the pathogenesis of many chronic diseases. However, the dysregulated signaling pathways critical to prion pathogenesis remain incompletely characterized. Global analyses of signaling pathways may be useful to better characterize these pathways. We therefore set out to develop such global assays. To this end, we used as a model cytoplasmic mutants of the cellular prion protein (PrPC), which are toxic to N2a neuroblastoma cells. We tested the global assays for their sensitivity to detect changes in signaling pathways in cells expressing cytoplasmic PrP mutants. Methods We developed a targeted proteomics (kinomics) approach using multiplex Western blots to identify signaling pathways dysregulated in chronic neurological pathologies. We tested the approach for its potential ability to detect signaling changes in N2a cells expressing cytoplasmic PrP mutants. Results Multiplex Western blots were designed to quantitate the expression levels of 137 protein kinases in a single membrane and using only 1.2 mg of sample. The response of the blots was sensitive and linear to changes of 6% in protein levels. Hierarchical and functional clustering of the relative expression levels identified an mTOR signaling pathway as potentially dysregulated in N2a cells expressing cytoplasmic PrP. The mTOR signaling pathway regulates global protein synthesis, which is inhibited in cells expressing cytoplasmic PrP. The levels of proteins involved in the Akt1/p70S6K branch of mTOR signaling changed in synchrony with time of cytoplasmic PrP expression. Three kinases in this pathway, Akt, p70S6K, and eIF4B were in their inactive states, as evaluated by phosphorylation of their regulatory sites. Conclusion The results presented are consistent with the previously reported inhibition of Akt/p70S6K/eIF4B signaling as mediating pathogenesis of cytoplasmic PrP. We conclude that the kinomic analyses are sensitive and specific to detect signaling pathways dysregulated in a simple in vitro model of PrP pathogenesis. Electronic supplementary material The online version of this article (doi:10.1186/1743-422X-11-175) contains supplementary material, which is available to authorized users.

Published
2014
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3. Identification of Signaling Pathways Dysregulated during Scrapie Pathogenesis using a Kinomics Approach

Author

Shott, Rory H

Subjects
Prion diseases, Multiplex Western blots, MST1, Protein kinase, Cytoplasmic PrP, Scrapie, Pathogenesis, CaMK4, Neurotoxicity, CREB
Abstract

Abstract: Prion diseases are a family of chronic lethal neurodegenerative diseases that affect humans and animals. The conversion of the cellular prion protein (PrPC) to abnormal conformations (PrPSc) is accepted to be required for pathogenesis. However, the molecular mechanisms whereby PrP conversion ultimately results in neuronal death remains poorly understood. Reversible protein phosphorylation by protein kinases and phosphatases regulates signal transduction. Dysregulated protein kinases are involved in the pathogenesis of many chronic diseases, including the neurodegenerative Alzheimer's and Parkinson’s diseases. Consequently, protein kinases have become major therapeutic targets. Protein kinases are also involved in prion disease pathogenesis. However, the signaling pathways most critical to pathogenesis are yet unknown. In this thesis, I describe the development of a multiplex Western blot-based kinomics approach to identify signaling pathways dysregulated during prion disease pathogenesis. This approach was validated using an in vitro model of prion pathogenesis, N2a neuroblastoma cells expressing cytoplasmic PrP mutants. The application of the kinomics approach to an in vivo model, mice infected with scrapie, revealed the dysregulation of two signaling pathways involved in neuronal survival or death at different times after infection. The pro-survival CaMK4β/CREB signaling pathway was activated at pre-clinical stages, but returned to mock-infected levels prior to the onset of clinical disease and the activation of pro-death MST1 signaling. My discovery that CaMK4β/CREB and MST1 signaling is dysregulated in mice infected with scrapie expands our understanding of the molecular mechanisms of prion-mediated neuronal death and identifies potential targets to further evaluate the roles of these pathways in prion disease pathogenesis.

Published
2017

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