Year-end Sale % OFF 
Abstract
BackgroundRegulators of G protein signaling (RGSs) accelerate GTP hydrolysis by Gα subunits and profoundly inhibit signaling by G protein-coupled receptors (GPCRs). The distinct expression patterns and pathophysiologic regulation of RGS proteins suggest that inhibitors may have therapeutic potential. We recently described a focused one-bead, one-compound (OBOC) library screen to identify peptide inhibitors of RGS4. Here we extend our observations to include another peptide with a different mechanism of action.ResultsPeptide 5nd (Tyr-Trp-c [Cys-Lys-Gly-Leu-Cys]-Lys-NH2, S-S) blocks the RGS4-Gαo interaction with an IC50 of 28 μM. It forms a covalent, dithiothreitol (DTT) sensitive adduct with a mass consistent with the incorporation of one peptide per RGS. Peptide 5nd activity is abolished by either changing its disulfide bridge to a methylene dithioether bridge, which cannot form disulfide bridges to the RGS, or by removing all cysteines from the RGS protein. However, no single cysteine in RGS4 is completely necessary or sufficient for 5nd activity.ConclusionThough it has some RGS selectivity, 5nd appears to be a partially random cysteine modifier. These data suggest that it inhibits RGS4 by forming disulfide bridges with the protein.
Highlights
Regulators of G protein signaling (RGSs) accelerate guanosine triphosphate (GTP) hydrolysis by Gα subunits and profoundly inhibit signaling by G protein-coupled receptors (GPCRs)
Since the majority of those hit peptides could bind tightly to the RGS without disrupting GTPase accelerating protein (GAP) activity, they were tested in the flow cytometry protein interaction assay (FCPIA) (Figure 1), which measures the interaction between RGS4 and Gαo
In this method we used a Luminex flow cytometer to detect the binding of Alexa Fluor 532-labeled Gαo to biotinylated RGS4 on avidin beads in the presence of AMF (AlCl3, MgCl2, NaF and GDP) to put the Gα subunit into a transition state-like conformation [7]
Summary
Peptide 5nd (Tyr-Trp-c [Cys-Lys-Gly-Leu-Cys]-Lys-NH2, S-S) blocks the RGS4-Gαo interaction with an IC50 of 28 μM. It forms a covalent, dithiothreitol (DTT) sensitive adduct with a mass consistent with the incorporation of one peptide per RGS. Peptide 5nd activity is abolished by either changing its disulfide bridge to a methylene dithioether bridge, which cannot form disulfide bridges to the RGS, or by removing all cysteines from the RGS protein. No single cysteine in RGS4 is completely necessary or sufficient for 5nd activity
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
