A-kinase
anchoring proteins (AKAPs) play an important role in the
spatial and temporal regulation of protein kinase A (PKA) by scaffolding
critical intracellular signaling complexes. Here we report the design
of conformationally constrained peptides that disrupt interactions
between PKA and AKAPs in an isoform-selective manner. Peptides derived
from the A Kinase Binding (AKB) domain of several AKAPs were chemically
modified to contain an all-hydrocarbon staple and target the docking/dimerization
domain of PKA-R, thereby occluding AKAP interactions. The peptides
are cell-permeable against diverse human cell lines, are highly isoform-selective
for PKA-RII, and can effectively inhibit interactions between AKAPs
and PKA-RII in intact cells. These peptides can be applied as useful
reagents in cell-based studies to selectively disrupt AKAP-localized
PKA-RII activity and block AKAP signaling complexes. In summary, the
novel hydrocarbon-stapled peptides developed in this study represent
a new class of AKAP disruptors to study compartmentalized RII-regulated
PKA signaling in cells.
Paramagnetic lanthanide ions when bound to proteins offer great potential for structural investigations that utilize solution nuclear magnetic resonance spectroscopy, magnetic resonance imaging, or optical microscopy. However, many proteins do not have native metal ion binding sites and engineering a chimeric protein to bind an ion while retaining affinity for a protein of interest represents a significant challenge. Here we report the characterization of an immunoglobulin Gbinding protein redesigned to include a lanthanide binding motif in place of a loop between two helices (Z-L2LBT). It was shown to bind Tb 31 with 130 nM affinity. Ions such as Dy 31 , Yb 31 , and Ce 31 produce paramagnetic effects on NMR spectra and the utility of these effects is illustrated by their use in determining a structural model of the metal-complexed Z-L2LBT protein and a preliminary characterization of the dynamic distribution of IgG Fc glycan positions. Furthermore, this designed protein is demonstrated to be a novel IgG-binding reagent for magnetic resonance imaging (Z-L2LBT:Gd 31 complex) and luminescence microscopy (Z-L2LBT: Tb 31 complex).
A-Kinase Anchoring Proteins (AKAPs) coordinate complex signaling events by serving as spatiotemporal modulators of cAMP-dependent protein kinase activity in cells. Although AKAPs organize a plethora of diverse pathways, their cellular roles are often elusive due to the dynamic nature of these signaling complexes. AKAPs can interact with the type I or type II PKA holoenzymes by virtue of high-affinity interactions with the R-subunits. As a means to delineate AKAP-mediated PKA signaling in cells, we sought to develop isoform-selective disruptors of AKAP signaling. Here, we report the development of conformationally constrained peptides named RI-STapled Anchoring Disruptors (RI-STADs) that target the docking/dimerization domain of the type 1 regulatory subunit of PKA. These high-affinity peptides are isoform-selective for the RI isoforms, can outcompete binding by the classical AKAP disruptor Ht31, and can selectively displace RIα, but not RIIα, from binding the dual-specific AKAP149 complex. Importantly, these peptides are cell-permeable and disrupt Type I PKA-mediated phosphorylation events in the context of live cells. Hence, RI-STAD peptides are versatile cellular tools to selectively probe anchored type I PKA signaling events.
To explore the possibility of constrained peptides to target Plasmodium-infected cells, we designed a J domain mimetic derived from Plasmodium falciparum calcium-dependent protein kinase 1 (Pf CDPK1) as a strategy to disrupt J domain binding and inhibit Pf CDPK1 activity. The J domain disruptor (JDD) peptide was conformationally constrained using a hydrocarbon staple and was found to selectively permeate segmented schizonts and colocalize with intracellular merozoites in late-stage parasites. In vitro analyses demonstrated that JDD could effectively inhibit the catalytic activity of recombinant Pf CDPK1 in the low micromolar range. Treatment of late-stage parasites with JDD resulted in a significant decrease in parasite viability mediated by a blockage of merozoite invasion, consistent with a primary effect of Pf CDPK1 inhibition. To the best of our knowledge, this marks the first use of stapled peptides designed to specifically target a Plasmodium falciparum protein and demonstrates that stapled peptides may serve as useful tools for exploring potential antimalarial agents.
Drug resistance poses a significant threat to ongoing malaria control efforts. Coupled with lack of a malaria vaccine, there is an urgent need for the development of new antimalarials with novel mechanisms of action and low susceptibility to parasite drug resistance. Protein Kinase A (PKA) has been implicated as a critical regulator of pathogenesis in malaria. Therefore, we sought to investigate the effects of disrupted PKA signaling as a possible strategy for inhibition of parasite replication. Host PKA activity is partly regulated by a class of proteins called A Kinase Anchoring Proteins (AKAPs), and interaction between HsPKA and AKAP can be inhibited by the stapled peptide Stapled AKAP Disruptor 2 (STAD-2). STAD-2 was tested for permeability to and activity against Plasmodium falciparum blood stage parasites in vitro. The compound was selectively permeable only to infected red blood cells (iRBC) and demonstrated rapid antiplasmodial activity, possibly via iRBC lysis (IC50 ≈ 1 μM). STAD-2 localized within the parasite almost immediately post-treatment but showed no evidence of direct association with PKA, indicating that STAD-2 acts via a PKA-independent mechanism. Furosemide-insensitive parasite permeability pathways in the iRBC were largely responsible for uptake of STAD-2. Further, peptide import was highly specific to STAD-2 as evidenced by low permeability of control stapled peptides. Selective uptake and antiplasmodial activity of STAD-2 provides important groundwork for the development of stapled peptides as potential antimalarials. Such peptides may also offer an alternative strategy for studying protein-protein interactions critical to parasite development and pathogenesis.
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