Proteasome Inhibitors in Tissue Culture

The study of proteasome activity in tissue culture cells is now greatly facilitated by the availability of a suitable reporter construct, involving the green fluorescent protein (GFP) (17). This was accomplished by fusing a CLl-degron sequence to the carboxyl terminal of GFP Inhibition of proteasome activity leads to an increase of the reporter protein which is detectable by monitoring the fluorescence of the cells.

Acute inhibition of the proteasome with high levels of proteasome inhibitors abolishing all or most of the proteasome activity in primary neurons or neuronal cell lines will lead inevitably to the death of cells. Preceding cell death accumulation of ubiquitinated proteins and heat-shock proteins is detectable. Proteasome inhibitors also cause a transient and combined up-regulation of all mammalian 26S proteasome subunit mRNAs. This increase in transcription of the proteasome mRNAs results in up-regulation of all the proteasome subunits with a concomitant de novo assembly of proteasomes (18). Notably, pre-treatment of neo-cortical neuronal cultures with sub-toxic concentrations of proteasome inhibitors reduced neuronal susceptibility to oxidative stress (19). This cytopro-tective result was attributed to up-regulation of core proteasome subunits and the ensuing increase in proteasome activity rather than to up-regulation of heat shock proteins (19).

Chronic administration of low concentrations of proteasome inhibitors that do not induce neuronal death was shown to increase the levels of protein oxidation, protein insolubility and DNA as well as RNA oxidation (20,21). RNA appeared to be more affected than DNA and both the 18S and 28S ribo-somal RNA levels were significantly decreased following proteasome inhibition. Furthermore, chronic low-level proteasome inhibition seems to activate the lysosomal system manifested by an increase in macroautophagy. A microarray analysis of neuronal cells subjected to chronic proteasome inhibition revealed a limited (less than 0.8%) alteration in gene expression some of the genes being relevant to aging, AD and PD. However, the vast majority of genes altered by chronic proteasome inhibition have not been characterized yet, suggesting that impaired proteasome function affects neuronal homeostasis by still uncharac-terized mechanisms (22).

It is important to keep in mind that pharmacological inhibitors of the pro-teasome should be used with caution in cell studies in which proteins are expressed from a heterologous promoter. As expected, the proteasome inhibitors MG132, ALLN and lactacystin induced the accumulation of transfected parkin or a-synu-clein driven by the cytomegalovirus (CMV) promoter in PC12 cells. However, the high levels of parkin or a-synuclein detected upon treatment with proteasome inhibitors was found to be associated with increased protein synthesis rather than degradation (23). These data suggest that the proteasome inhibitors increase CMV-driven transcription in a non-specific manner. Proteasome inhibitors may stabilize transcription and translation factors or activate transcription pathways regulating the CMV promoter (23).

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