ABSTRACT
In two proof-of-concept studies, we established and validated the Bacterial Growth Inhibition Screen (BGIS), which explores recombinant protein toxicity in Escherichia coli as a largely overlooked and alternative means for basic characterization of functional eukaryotic protein domains. By applying BGIS, we identified an unrecognized RNA-interacting domain in the DEK oncoprotein (this study) and successfully combined BGIS with random mutagenesis as a screening tool for loss-of-function mutants of the DNA modulating domain of DEK [1]. Collectively, our findings shed new light on the phenomenon of recombinant protein toxicity in E. coli. Given the easy and rapid implementation and wide applicability, BGIS will extend the repertoire of basic methods for the identification, analysis and unbiased manipulation of proteins.
Subject(s)
Escherichia coli/drug effects , Escherichia coli/growth & development , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/toxicity , Toxicity Tests/methods , Animals , Bias , Biocatalysis , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Chromosomal Proteins, Non-Histone/toxicity , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila Proteins/toxicity , Escherichia coli/genetics , Humans , Loss of Function Mutation , Oncogene Proteins/chemistry , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Oncogene Proteins/toxicity , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Peptide Fragments/toxicity , Poly-ADP-Ribose Binding Proteins/chemistry , Poly-ADP-Ribose Binding Proteins/genetics , Poly-ADP-Ribose Binding Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/toxicity , Protein Domains/genetics , RNA/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/toxicity , Receptors, Eph Family/chemistry , Receptors, Eph Family/genetics , Receptors, Eph Family/metabolism , Receptors, Eph Family/toxicity , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Reproducibility of Results , Time Factors , Toxicity Tests/standardsABSTRACT
Spinal ephrinB-EphB signaling is involved in the modulation of pain processing. The aim of the present study was to investigate whether protein kinase C-γ (PKCγ) acts as a downstream effector in regulating spinal pain processing associated with ephrinB-EphB signaling in mice. The intrathecal injection of ephrinB2-Fc, an EphB receptor activator, caused thermal hyperalgesia and mechanical allodynia, as well as increased activation of spinal PKCγ. Knockdown of spinal PKCγ prevented the pain behaviors induced by ephrinB2-Fc. Furthermore, the intrathecal injection of EphB2-Fc, an EphB receptor blocker, suppressed formalin-induced inflammatory, chronic constriction injury (CCI)-induced neuropathic, and tibia bone cavity tumor cell implantation (TCI)-induced bone cancer pain behaviors, in addition to reducing the activation of spinal PKCγ. Finally, the intrathecal injection of MK801, an N-methyl-D-aspartate (NMDA) receptor blocker, prevented the pain behaviors and spinal PKCγ activation induced by ephrinB2-Fc. Overall, the results confirm the important role of PKCγ in the regulation of spinal pain processing associated with ephrinB-EphB signaling.