Mechanism of UCP activation by reactive aldehydes

Free radicals (ROS) have been implicated in the aetiology of many pathological states such as metabolic, cardiovascular and inflammatory failures. Understanding ROS action mechanisms is crucial for the development of potent therapeutic strategies in the treatment of these diseases. Uncoupling proteins (UCP) are thought to be involved in ROS regulation by transporting protons through the inner mitochondrial membrane, thereby diminishing transmembrane potential. On the other hand, they have been proposed to be regulated by ROS. In our previous project (Po-524/3, German Research Foundation) we have shown that the reactive aldehyde 4-hydroxy-2-nonenal (HNE) leads to an increase of UCP1 activity only if fatty acids (FA) are present. The main goal of the present project is the evaluation of mechanisms leading to FA-mediated uncoupling protein activation in the presence of HNE. We will test two main hypotheses concerning the main mechanism of the proton transport mediated by HNE: (i) reactive aldehyde influences lipid membrane parameters, leading to the facilitation of fatty acid transport (e.g. membrane fluidity, dipole or surface membrane potential) or/and (ii) HNE binding to the protein leads to transient proton channel formation due to a conformational change of the protein. We shall use two complementary experimental systems, bilayer lipid membranes reconstituted with uncoupling proteins and primary neuronal cell cultures. We will investigate the influence of other biologically important reactive aldehydes such as malondialdehyde, 4-oxo-2-nonenal, 4-hydroxy-2-hexenal and 4-oxo-2-hexenal on the protein transport function and evaluate possible mechanisms. From the results we expect to gain mechanistic insights into the controversially discussed regulation of UCP1 and UCP2 by HNE and other reactive aldehydes, which is important for drug design.

Free radicals (ROS) have been implicated in the aetiology of many pathological states such as metabolic, cardiovascular and inflammatory failures. Understanding ROS action mechanisms is crucial for the development of potent therapeutic strategies in the treatment of these diseases. Uncoupling proteins (UCP) are thought to be involved in ROS regulation by transporting protons through the inner mitochondrial membrane, thereby diminishing the transmembrane potential. On the other hand, they have been proposed to be regulated by ROS. First we have shown that the reactive aldehyde 4-hydroxy-2-nonenal (HNE) leads to an increase of UCP1 activity only if fatty acids (FA) are present. To investigate the detailed RA action mechanism we used two complementary experimental systems ???bilayer lipid membranes reconstituted with uncoupling proteins and cells or cell lines expressing UCPs (neuroblastoma cell line, primary neurons, etc). We revealed that the activation of UCPs occurred only in PE-containing membranes due to formation of HNE- and ONE-adducts. These adducts influence lipid membrane parameters (membrane fluidity, surface membrane potential and lateral pressure), leading to the activation of UCP-mediated proton transport. We excluded the hypothesis that HNE binding to UCP1 leads to transient proton channel formation due to a conformational change of the protein. Our results stress for the first time the role of particular lipid modification for the activity regulation of mitochondrial membrane proteins and largely contribute to the understanding of the controversially discussed mechanism of RA actions in cells. This is very important for future attempts of drug design.