4-hydroxy-2-nonenal-mediated impairment of intracellular proteolysis during oxidative stress - Identification of proteasomes as target molecules

Oxidative stress is associated with important pathophysiological events in a variety of diseases. It has been postulated that free radicals and lipid peroxidation products generated during the process may be responsible for t hese effects because of their ability to damage cellular components such as membranes, proteins, and DNA, In the present study, we provide evidence th at oxidative stress causes a transient impairment of intracellular proteoly sis via covalent binding of 4-hydroxy-2-nonenal (HNE), a major end product of lipid peroxidation, to proteasomes. A single intraperitoneal treatment w ith the renal carcinogen, ferric nitrilotriacetate, caused oxidative stress , as monitored by accumulation of lipid peroxidation products and 8-hydroxy -2'-deoxyguanosine, in the kidney of mice. In addition, transient accumulat ion of HNE-modified proteins in the kidney was also found by competitive en zyme-linked immunosorbent assay and immunohistochemical analyses. This and the observation that the HNE-modified proteins were significantly ubiquitin ated suggested a crucial role of proteasomes in the metabolism of HNE-modif ied proteins. In vitro incubation of the kidney homogenates with HNE indeed resulted in a transient accumulation of HNE-modified proteins, whereas the proteasome inhibitor significantly suppressed the time-dependent eliminati on of HNE-modified proteins. We found that, among three proteolytic activit ies (trypsin, chymotrypsin, and peptidylglutamyl peptide hydrolase activiti es) of proteasomes, both trypsin and peptidylglutamyl peptide hydrolase act ivities in the kidney were transiently diminished in accordance with the ac cumulation of HNE-modified proteins during oxidative stress. The loss of pr oteasome activities was partially ascribed to the direct attachment of HNE to the protein, based on the detection of HNE-proteasome conjugates by an i mmunoprecipitation technique. These results suggest that HNE may contribute to the enhanced accumulation of oxidatively modified proteins via an impai rment of ubiquitin/proteasome-dependent intracellular proteolysis.