Treponema denticola Msp-Deduced Peptide Conjugate Promotes RhoA-dependent Stress Fiber Formation

Background: P34_BSA, a BSA conjugate of a synthetic 10-mer peptide deduced from Treponema denticola major outer sheath protein (Msp), promotes stabilization of actin filaments in fibroblasts and retards cell motility. It is internalized by cells, binds and bundles actin filaments in vitro, and activates RhoA. Yet, its site and mechanism of action are not defined.  Objectives: To determine P34_BSA's mechanism of action by assessing modes of its signaling to fibroblasts. Methods: Immunofluorescence microscopy and fluorometry were used to visualize and quantify actin filaments and peptide conjugates. Western analysis was used to measure active RhoA, co-immunoprecipitated p114RhoGEF, and phosphorylated cofilin. Results: At 4˚C, P34_BSA was not taken up by the cells, but it bound to the plasma membrane and promoted stress fiber formation at ~80% capacity compared with 37˚C controls, casting doubt that cellular uptake is a critical step for its cytoskeleton-stabilizing property. In Rho G-LISA^TM and co-immunoprecipitation assays, P34_BSA was found to activate RhoA, even at 4°C, and to promote its interaction with guanosine nucleotide exchange factor p114RhoGEF. It also caused phosphorylation of cofilin. Upon RhoA inhibition, either by C3 transferase RhoA inhibitor or by transfection with a dominant negative RhoA construct, P34_BSA did not achieve the stress fiber formation seen with P34_BSA alone. By inhibiting phosphatidylinositol-3 kinase (PI 3-K) with LY294002, the P34_BSA effects were completely blocked. Depletion of cholesterol with methyl-b-cyclodextrin (MbCD) partially inhibited P34_BSA signaling via the plasma membrane to the cytoskeleton. All comparisons: P<0.05, t-test. Conclusions: Multivalent P34_BSA probably activates lipid raft components that require active PI 3-K, and it initiates the pathway to stess fiber formation through a RhoGEF and RhoA. P34_BSA may represent a novel tool to investigate RhoA-dependent processes, such as remodeling filamentous actin.

Supported by CIHR IMH-5619 and STP-53877