Abstract 359: Investigation of the Protein Interactions of the Shear Responsive Protein, Palladin, and Its Presence in Endothelial-Derived Microparticles

Abstract

Palladin, an actin filament associated protein, plays an important role in cell cytoskeletal regulation, with particular regards to the formation and stablilization of stress fibres. The protein is part of the myotilin-myopalladin-palladin family, but unlike myotilin and myopalladin, which only appear in striated muscle, Palladin has been observed in various cell types. The protein is encoded by the PallD gene, which through alternative splicing, results in Palladin existing in multiple different isoforms. While a number of these isoforms have been documented in cell types, it is the 90kDa isoform which is most ubiquitously expressed. Physiological mechanotransduction such as cyclic strain or shear stress, experienced by the vasculature, has been shown to regulate expression of many genes that play an important role in the patho-physiology of cardiovascular disease (CVD), by altering cell fate and function. We have previously documented the presence of the 90kDa isoform of Palladin in Human Aortic Endothelial Cells (HAECs), along with the investigation of gene expression in response to strain and shear at different time points by utilising Western Blotting and Real Time PCR techniques. It has been shown that gene expression increases at early time points of mechanic stimuli, followed by a reduction of expression after chronic exposure. We also observe the presence of Palladin in Endothelial Microparticles, released under inflammatory conditions. We hypothesize that, following the inflammatory response, the cell compensates for excess protein production through microparticle secretion. Mass Spectrometric analysis of Immunoprecipited Palladin from HAECs appears to show an interaction with Drebrin (developmentally regulated brain protein), which itself binds to F-actin and is involved in cell migration. While Drebrin is mainly expressed in the brain, it has also been located in heart and muscle. We investigate here also the interactions between Palladin and Drebrin. It appears that Drebrin expression decreases as Palladin expression increases following shear stress, indicative that the two proteins compete with one another in binding to actin.