The RNA-activated protein kinase, PKR, is induced by interferon and plays a pivotal role in the innate immunity response to viral infection. PKR is synthesized in a latent state, but upon binding activating RNAs it undergoes autophosphorylation reactions that activate the kinase. Phosphorylation of the eukaryotic initiation factor eIF2a by PKR inhibits viral protein synthesis. PKR is also activated by cytokines, growth factor withdrawal and stress signals and participates in pathways regulating stress response, cellular growth and proliferation, nutrient signaling and metabolism. The broad objective of our research program is to define the molecular mechanisms that regulate the activity of PKR using quantitative biophysical and structural methods.
In Aim I we will define the interactions of PKR with complex RNA inhibitors and activators. Specifically, we will identify the structural features within the Adenovirus VAI RNA that mediate high-affinity PKR binding without activating the kinase, determine the mechanism of activation of PKR by novel single stranded-double stranded RNAs and correlate PKR dimerization and activation with the length and flexibility of a linker region in model RNAs. PKR represents an attractive target for development of novel antiviral therapies to enhance the innate immune response and in Aim 2 we will use a nonradioactive assay to screen libraries of drug-like compounds for novel PKR activators.
In Aim 3, small angle scattering methods will be used to determine the conformation of ordered forms of PKR and to develop structural model of complexes of PKR with RNA inhibitors and activators. These studies will provide the foundation for the design of therapeutic agents that target PKR for the treatment of viral infections and cancer.

Public Health Relevance

PKR plays a central role in the biological activity of interferons, which are widely employed in the treatment of viral, neoplastic and immunological diseases. However, the therapeutic effectiveness of interferons is compromised by severe side effects as well as viral resistance mechanisms that inhibit the PKR pathway. A detailed understanding of the molecular mechanism of PKR activation will help us to design new drugs that will selectively activate PKR to avoid side effects and to evade viral resistance.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI053615-09
Application #
8372394
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Dempsey, Walla L
Project Start
2003-01-01
Project End
2016-08-31
Budget Start
2012-12-01
Budget End
2013-11-30
Support Year
9
Fiscal Year
2013
Total Cost
$366,949
Indirect Cost
$131,949
Name
University of Connecticut
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
614209054
City
Storrs-Mansfield
State
CT
Country
United States
Zip Code
06269
Launer-Felty, Katherine; Cole, James L (2014) Domain interactions in adenovirus VAI RNA mediate high-affinity PKR binding. J Mol Biol 426:1285-95
Lyons, Daniel F; Lary, Jeffrey W; Husain, Bushra et al. (2013) Are fluorescence-detected sedimentation velocity data reliable? Anal Biochem 437:133-7
Anderson, Eric; Pierre-Louis, Willythssa S; Wong, C Jason et al. (2011) Heparin activates PKR by inducing dimerization. J Mol Biol 413:973-84
Wong, C Jason; Launer-Felty, Katherine; Cole, James L (2011) Analysis of PKR-RNA interactions by sedimentation velocity. Methods Enzymol 488:59-79
Cole, James L; Correia, John J; Stafford, Walter F (2011) The use of analytical sedimentation velocity to extract thermodynamic linkage. Biophys Chem 159:120-8
Cole, James L (2010) Analysis of PKR activation using analytical ultracentrifugation. Macromol Biosci 10:703-13
Launer-Felty, Katherine; Wong, C Jason; Wahid, Ahmed M et al. (2010) Magnesium-dependent interaction of PKR with adenovirus VAI. J Mol Biol 402:638-44
Anderson, Eric; Quartararo, Christine; Brown, Raymond S et al. (2010) Analysis of monomeric and dimeric phosphorylated forms of protein kinase R. Biochemistry 49:1217-25
VanOudenhove, Jennifer; Anderson, Eric; Krueger, Susan et al. (2009) Analysis of PKR structure by small-angle scattering. J Mol Biol 387:910-20
Heinicke, Laurie A; Wong, C Jason; Lary, Jeffrey et al. (2009) RNA dimerization promotes PKR dimerization and activation. J Mol Biol 390:319-38

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