Abstract
The regulated degradation of damaged or misfolded proteins, as well as down-regulation of key signaling proteins, within eukaryotic and bacterial cells is catalyzed primarily by large, ATP-dependent multimeric proteolytic complexes, termed proteasomes. Inhibition of proteasomal activity affects a wide variety of physiological and pathological processes, and was found to be particularly effective for cancer therapy. We report here on the development of a novel high throughput assay for proteasome inhibition using a unique, highly sensitive live-cell screening, based on the cytoplasm-to-nucleus translocation of a fluorescent proteasome inhibition reporter (PIR) protein, consisting of nuclear localization signal-deficient p53 derivative. We further show here that mdm2, a key negative regulator of p53 plays a key role in the accumulation of PIR in the nucleus upon proteasome inhibition. Using this assay, we have screened the NCI Diversity Set library, containing 1,992 low molecular weight synthetic compounds, and identified four proteasome inhibitors. The special features of the current screen, compared to those of other approaches are discussed.
Highlights
The proteasome is the major proteolytic complex, responsible, in eukaryotic cells, for the degradation of a multitude of cellular proteins
The cytoplasm-tonucleus transport of this p53 mutant was attenuated by additional triple mutation in the bipartite Nuclear Localization Signal (NLS) in which three consecutive lysine residues were replaced with alanines (K319A, K320A, and K321A), as described by O’Keefe et al [29]
In agreement with previous reports, under normal cell culture conditions, this point mutation leads to cytoplasmic localization of the proteasome inhibition reporter (PIR) protein in H1299 cells [29,30], confirming that the three basic residues at position 319–321 are part of a nuclear localization signal
Summary
The proteasome is the major proteolytic complex, responsible, in eukaryotic cells, for the degradation of a multitude of cellular proteins. This multi-protein complex, present in both the cytoplasm and the nucleus, catalyzes the ATP-dependent proteolysis of short-lived regulatory proteins, as well as the rapid elimination of damaged and abnormal proteins [1,2]. The 26S proteasome is a large complex of ,2.5 MDa. Based on biochemical analyses, this complex can be dissociated into two functionally distinct subcomplexes, the 20S core particle (CP) which is the proteolytic component, and the 19S regulatory particle (RP), that is responsible for recognizing, unfolding, and translocating polyubiquitinated substrates into the 20S CP, where they are degraded. In all three active b-subunits, proteolytic activity is associated with their N-terminal threonine residue, which acts as a nucleophile in peptide-bond hydrolysis
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