Abstract P316: Cysteine-Independent Interactions of Thioredoxin-1 and Apex1: Implications for Endothelial Cell Apoptosis and Aging?

Abstract

Aging processes are associated with increased intracellular formation of reactive oxygen species. One redox regulator is the ubiquitously expressed oxidoreductase Thioredoxin-1 (Trx). It contains two active site cysteines and has anti-oxidative and anti-apoptotic properties. Trx levels are reduced in senescent endothelial cells (EC), leading to increased apoptosis sensitivity. Several functions of Trx are enhanced by its interaction with AP-endonuclease 1 (APEX1). APEX1 is a multifunctional protein and in concert with Trx-1 a reductive activator of e.g. the transcription factor AP-1, suggesting at least an interaction of Trx and APEX1 in vivo. Recent evidence suggests that also APEX1 levels are decreased in senescent cells. Therefore, the aim of this study is to understand the function of APEX1 in concert with Trx in EC. We first investigated whether APEX1, similar to Trx has anti-apoptotic properties in EC. Indeed, overexpression of APEX1 inhibited apoptosis. To analyze where the Trx/APEX1 interaction occurs, we performed fluorescence microscopy demonstrating a colocalization throughout the whole cell. To study the interaction in more detail, we carried out coimmunoprecipitations from nuclear and cytosolic extracts and demonstrated their association in both fractions. To determine whether disulfide bridges are involved in the Trx/APEX1 association, we mutated APEX1 cysteines 65, 93 and 310 previously shown to interact with cysteines in other proteins, to serines. This mutant bound Trx in the nucleus and cytosol of EC with the same efficiency as APEX1 wildtype. Moreover, a Trx mutant with serine mutations of active site cysteines (Trx(C32S/C35S)) was still able to bind to endogenous APEX1. These data demonstrate that the Trx/APEX-1 interaction is independent of disulfide bridge formation. We are currently analyzing a set of APEX1 deletion mutants for their interaction with Trx-1 and a potential loss of anti-apoptotic properties Exact delineation of the Trx/APEX1 interaction surface will in the future allow the development of small peptides containing the interaction domains of Trx or APEX1 respectively, in order to enhance transcription factor activity and bypass the loss of antioxidative capacity during aging processes.