Effects of the Fusion Protein PTD4–Profilin I on Trabecular Meshwork Cells and Outflow Facility

Abstract

Abstract: : Purpose: Fusion proteins with a protein transduction domain (PTD) are able to cross biological membranes. This technology has been used successfully in a wide variety of cells to introduce proteins, DNA, liposomes and drugs. We have evaluated a PTD4–Profilin I construct in trabecular meshwork (TM) cells and trabecular outflow facility. Profilin I is a nucleotid exchange protein that binds monomeric actin and regulates actin polimerization. Since previous studies have shown that latrunculin A increases aqueous humor (AH) outflow, we have studied the regulation of actin cytoskeleton on TM cells and outflow facility. Methods: pRSETA–m–Profilin I (control) and pRSETA–PTD4–Profilin I constructs were created and expressed in bacteria. Purified proteins were detected by western blotting and tested by immunocytochemistry in bovine TM primary cultures. Outflow facility studies were performed in bovine anterior segments perfused at constant pressure. Results: Western blot revealed two proteins of 20 and 25 KDa corresponding to Profilin I and PTD4–Profilin I. Immunocytochemistry showed a time and concentration–dependent transduction of PTD4–Profilin I (10–120min; 1–10µM) in TM cells. Confocal microscopy confirmed the protein presence in the cell cytoplasm that was not seen when control Profilin I was used. Actin filament staining showed that the effects of PTD4–Profilin I in the actin cytoskeleton were weaker compared with latrunculin A. In perfused anterior segments, 2µM PTD4–Profilin I significantly increased trabecular outflow facility in a reversible manner while control Profilin I did not induce any effect. Latrunculin A (2µM), as a positive control, also increased outflow facility significantly. Conclusions: This work proves the applicability of protein transduction technology to TM tissue, as a tool to study its physiology and a possible drug–delivery method. Moreover, results indicate that regulation of actin cytoskeleton by Profilin I or other actin binding proteins may be a physiological mechanism to modulate AH outflow.

Keywords: trabecular meshwork • cytoskeleton • protein purification and characterization