Función de los proteasomas en la degradación intracelular de proteínas en células de mamífero.

Zusammenfassung

The results demonstrate that different regulatory complexes and subpopulations of proteasomes have different distributions within mammalian cells. They were mainly found in the cytosol but were also present in the nucleus and associated with the endoplasmic reticulum. The 19S regulatory complexes were found to be relatively enriched in endoplasmic reticulum fractions from rat liver and the PA28 regulatory complexes were found to be localized predominantly in the cytoplasm. The contributions of the main proteolytic pathways to the degradation of long-lived and short-lived proteins in human fibroblasts grown under different conditions were also investigated. The effects of various commonly used pharmacological inhibitors of protein degradation were first analysed in detail. By choosing specific inhibitors of lysosomes and proteasomes, it was observed that both pathways accounted together for 80% or more of the degradation of cell proteins. With lysosomal inhibitors, it was found that serum withdrawal or amino-acid deprivation strongly stimulated macroautophagy but not other lysosomal pathways, whereas confluent conditions had no effect on macroautophagy and slightly activated other lysosomal pathways. With proteasomal inhibitors, it was found that, in exponentially growing cells in the absence of serum, activity of the ubiquitin-proteasome pathway increases, whereas under confluent conditions the contribution of proteasomes to degradation decreases, especially in cells deprived of amino acids. Interestingly, in confluent cells, the levels of two components of the 19 S regulatory complex and those of an interchangeable beta-subunit decreased. This was associated with a marked increase in the levels of components of PA28-immunoproteasomes. A mutation (C358Y) in the low-density lipoprotein receptor (LDLR), which is the most prevalent in a sample of familial hypercholesterolemia patients from Valencia (Spain) was finally investigated. Post-translational processing of the mutant LDLR precursor to the mature form was apparently retarded and part of this precursor was degraded by a lysosomal pathway. Also, the mature form of the mutant LDLR undergoes rapid degradation when compared to the wild type LDLR. The mature forms of wild-type LDLR and mutant C358Y are mainly degraded by proteasomes and lysosomes, respectively. Thus, a single mutation in the LDLR changes the degradative pathway of this protein.