Importance of the Different Proteolytic Sites of the Proteasome and the Efficacy of Inhibitors Varies with the Protein Substrate

Department of Molecular and Cell Biology, Harvard University, Cambridge, Massachusetts, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2006; 281(13):8582-90. DOI: 10.1074/jbc.M509043200
Source: PubMed


The relative importance of the different proteolytic sites in mammalian proteasomes in protein degradation has not been studied
systematically. Nevertheless, it is widely assumed that inhibition of the chymotrypsin-like site, the primary target of the
proteasome inhibitors used in research and cancer therapy, reflects the degree of inhibition of protein breakdown. Here we
demonstrate that selective inactivation of the chymotrypsin-like site reduced degradation of model proteins by pure 26 S proteasomes
by only 11-50% and decreased only slightly the breakdown of proteins in HeLa cells. Inactivation of the caspase-like site
decreased breakdown of model proteins by 12-22% and of the trypsin-like site by 3-35%. The relative contributions of these
different sites depended on the protein substrate, and the importance of the trypsin-like sites depended on the substrate's
content of basic residues. Simultaneous inhibition of the chymotrypsin-like and the caspase- or trypsin-like sites was needed
to reduce degradation by >50%. Thus, 1) all three types of active sites contribute significantly to protein breakdown, 2)
their relative importance varies widely with the substrate, 3) assaying the chymotrypsin-like activity overestimates the actual
reduction in protein degradation, and 4) inhibition of multiple sites is required to markedly decrease proteolysis.

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    • "Nevertheless, they provide arguably the most promising class of leads of proteasomal inhibitors (Bellavista et al., 2013; Kisselev and Groettrup, 2014). Furthermore, kinetic and structural analyses rely on their structural and biophysical characteristics (Gaczynska et al., 1993; Kisselev et al., 2002, 2003, 2006; Osmulski et al., 2009); we, too, use them here for our analysis, which is based on a representative set of such peptides. As we show below the insights gained from the short fluorogenic peptides are borne out by further analysis of polypeptides. "
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    ABSTRACT: Proteasomal protein degradation is a key determinant of protein half-life and hence of cellular processes ranging from basic metabolism to a host of immunological processes. Despite its importance the mechanisms regulating proteasome activity are only incompletely understood. Here we use an iterative and tightly integrated experimental and modelling approach to develop, explore and validate mechanistic models of proteasomal peptide-hydrolysis dynamics. The 20S proteasome is a dynamic enzyme and its activity varies over time because of interactions between substrates and products and the proteolytic and regulatory sites; the locations of these sites and the interactions between them are predicted by the model, and experimentally supported. The analysis suggests that the rate-limiting step of hydrolysis is the transport of the substrates into the proteasome. The transport efficiency varies between human standard- and immuno-proteasomes thereby impinging upon total degradation rate and substrate cleavage-site usage.
    eLife Sciences 09/2015; 4. DOI:10.7554/eLife.07545 · 9.32 Impact Factor
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    • "While proteasome function can be impaired by many factors, none can be controlled with the dosage-dependent precision of proteasome inhibitors such as bortezomib and the peptide aldehyde MG132. These inhibit both 20S and 26S proteasomes by targeting the core proteolytic catalytic activity of the 20S subunits (Kisselev et al., 2006, 2012; Goldberg, 2012). "
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    ABSTRACT: Proteasomes are central regulators of protein homeostasis in eukaryotes. Proteasome function is vulnerable to environmental insults, cellular protein imbalance and targeted pharmaceuticals. Yet, mechanisms that cells deploy to counteract inhibition of this central regulator are little understood. To find such mechanisms, we reduced flux through the proteasome to the point of toxicity with specific inhibitors and performed genome-wide screens for mutations that allowed cells to survive. Counter to expectation, reducing expression of individual subunits of the proteasome's 19S regulatory complex increased survival. Strong 19S reduction was cytotoxic but modest reduction protected cells from inhibitors. Protection was accompanied by an increased ratio of 20S to 26S proteasomes, preservation of protein degradation capacity and reduced proteotoxic stress. While compromise of 19S function can have a fitness cost under basal conditions, it provided a powerful survival advantage when proteasome function was impaired. This means of rebalancing proteostasis is conserved from yeast to humans.
    eLife Sciences 09/2015; 4. DOI:10.7554/eLife.08467 · 9.32 Impact Factor
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    • "Occasional failures in finding a peptidase responsible for the generation of proteasome inhibitor-resistant epitopes have not a unique explanation: (i) the inhibition of the proteasome may not be complete (Kisselev et al., 2006 "
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    ABSTRACT: Major histocompatibility complex class I proteins (MHC-I) load short peptides derived from proteolytic cleavage of endogenous proteins in any cell of the body, in a process termed antigen processing and presentation. When the source proteins are altered self or encoded by a pathogen, recognition of peptide/MHC-I complexes at the plasma membrane leads to CD8(+) T-lymphocyte responses that clear infections and probably underlie tumor immune surveillance. On the other hand, presentation of self peptides may cause some types of autoimmunity. The peptides that are presented determine the specificity and efficiency of pathogen clearance or, conversely, of immunopathology. In this review we highlight the growing number of peptidases which, as a by-product of their regular activity, can generate peptide epitopes for immune surveillance. These ∼20 peptidases collectively behave as a guerrilla army partnering with the regular proteasome army in generating a variety of peptides for presentation by MHC-I and thus optimally signaling infection. Copyright © 2015. Published by Elsevier Ltd.
    Molecular Immunology 05/2015; 95. DOI:10.1016/j.molimm.2015.04.014 · 2.97 Impact Factor
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