Abstract
The targeted pharmacological modulation of polymorphonuclear leukocytes (PMNs) is of major medical interest. These innate immune cells play a central role in the defense against pathogenic microorganisms. However, their excessive chemotactic recruitment into tissues after traumatic injury is detrimental due to local and systemic inflammation. Rho-GTPases, being the master regulators of the actin cytoskeleton, regulate migration and chemotaxis of PMNs, are attractive pharmacological targets. Herein, supramolecular protein complexes are assembled in a "mix-and-match" approach containing the specific Rho-inhibiting clostridial C3 enzyme and three PMN-binding peptides using an avidin platform. Selective delivery of the C3 Rho-inhibitor with these complexes into the cytosol of human neutrophil-like NB-4 cells and primary human PMNs ex vivo is demonstrated, where they catalyze the adenosine diphosphate (ADP) ribosylation of Rho and induce a characteristic change in cell morphology. Notably, the complexes do not deliver C3 enzyme into human lung epithelial cells, A549 lung cancer cells, and immortalized human alveolar epithelial cells (hAELVi), demonstrating their cell type-selectivity. The supramolecular complexes represent attractive molecular tools to decipher the role of PMNs in infection and inflammation or for the development of novel therapeutic approaches for diseases that are associated with hyperactivity and reactivity of PMNs such as post-traumatic injury.
Highlights
The biotinylated GGP (B-GGP) peptide was obtained in 40% yield after high-performance liquid chromatography (HPLC) purification with >95% purity (Figure S4, Supporting Information)
PMN-targeting supramolecular multiprotein complexes consisting of the specific Rho-inhibitor C3, Avi as molecular glue and three PMN-targeting peptides were prepared by convenient “mix-and-match” assembly using Avi/biotin technology to inhibit Rho-mediated signal transduction in PMNs. (GGP)3-Avi-C3 or (FK)3-Avi-C3 were generated, characterized and their biological mode of action was evaluated in vitro and ex vivo
We envision that our strategy provides new therapeutic avenue for diseases such as post-traumatic injury of multiple injured patients, which are associated with hyperactivity and reactivity of PMNs
Summary
After successful demonstration of the transport of the Rho inhibitor C3 into the cytosol of a human neutrophil-like cell line by both supramolecular C3-containing complexes, the effect of (GGP)3-Avi-C3 and (FK)3-Avi-C3 on biologically and medically more relevant primary human PMNs was investigated. Neither (GGP)3-Avi-C3 nor (FK)3-Avi-C3 showed similar effects on the morphology of human lung epithelial cells A549 (Figure 8A and Figure S11A, Supporting Information) or the recently established human alveolar epithelial cell line hAELVi (Figure 8C and Figure S11B, Supporting Information), a model for the air–blood barrier of the peripheral lung,[39] implicating that in these cells, the Rho-inhibitor C3 was not delivered into the cytosol by the supramolecular complexes These findings were confirmed by the biochemical evaluation of the ADP-ribosylation status of Rho of these cells (Figure 8B,D). Treatment of the A549 cells with C2INC3lim + C2IIa resulted in the expected C3-induced change in cell morphology (Figure 8A and Figure S11A, Supporting Information) and the strong ADP-ribosylation of Rho in the cytosol of the living cells by C3 (Figure 8B,D)
Sign-in/Register to view highlights & summary 
Published Version (
Free)
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 call