| The importance of correct protein maturation and refolding, along with efficient protein degradation, is a necessity for cell survival. However, the appearance of insoluble protein aggregates during a variety of disorders, including alcoholic liver disease (ALD), suggests impairment of these pathways. Also associated with ALD is a chronic state of oxidative stress, leading to an overabundance of reactive free radical species. While these free radicals have demonstrated cytotoxic properties, many of the aldehydic products of radical-initiated lipid peroxidation are cytotoxic as well. The lipid peroxidation product 4-hydroxynonenal (4-HNE) is recognized as an abundant and highly toxic compound, and can interfere with protein function. It is therefore the objective of the work presented here to characterize the effects of 4-HNE on protein quality control mechanisms such as protein chaperoning and ubiquitin-mediated protein degradation, thus testing the hypothesis that 4-HNE has a detrimental effect on these pathways. Initial studies, conducted following the observation that decreased proteolysis occurs during progressive stages of ALD, measured the rate of 26S proteasome-mediated protein degradation after modification of the substrate protein with 4-HNE. Using equine liver alcohol dehydrogenase (ADH) as a model substrate, it is demonstrated here that mild modification of this enzyme results in its rapid polyubiquitination and degradation. However, ADH which has been modified to a greater extent by 4-HNE is resistant to ubiquitination, suggesting a concentration-dependent effect of 4-HNE adducts on the proteasome, mediated through substrate modification. To further characterize the involvement of 4-HNE in the development of ALD, a proteomic approach was applied to cellular fractions isolated from the livers of rats ingesting an ethanol diet, demonstrating modification of multiple molecular chaperones by 4-HNE. Specifically, the heat shock proteins Hsp70 and Hsp90, as well the endoplasmic reticulum stress response chaperone, protein disulfide isomerase, were modified by 4-HNE. Further work with each of these proteins demonstrated the potential for 4-HNE to disrupt their respective chaperoning capabilities. It is thus demonstrated by the work presented here that 4-HNE possesses the ability to modify proteins directly involved in protein degradation and chaperoning, further characterizing a role for lipid peroxidation in the progression of ALD. |
