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: eLife digest Cells have to be able to reliably destroy or remove molecules from their interior that they no longer need. Structures called proteasomes play a central part in this complex process by cutting up and digesting proteins. Mammals have several different types of proteasomes, each made up of several protein ‘subunits’. For example, when a cell experiences inflammation some proteasomes change some of their subunits and form an immuno-proteasome. These immuno-proteasomes tend to break down proteins more quickly than ‘standard’ proteasomes, but it was not clear how they are able to do so. Liepe et al. have now combined experiments and mathematical modelling to construct a detailed model of proteasome activity. The model shows that protein transport into and out of the proteasome chamber are the steps that limit how quickly the proteasomes can break down proteins. Furthermore, these transport processes are also to a large extent responsible for the different rates at which standard and immuno-proteasomes process proteins. Liepe et al. were also able to confirm the existence of regulatory sites within the proteasome, and describe how these are arranged. Problems that alter the rate at which proteasomes break down proteins have been linked to tumors and neurological and autoimmune diseases. Liepe et al.'s model opens up the ability to study how the proteasome's activity is affected by drugs and therefore makes it easier to investigate ways of interfering with this activity for therapeutic purposes. DOI:
    Full-text · Article · Sep 2015 · eLife Sciences
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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: eLife digest Cells have numerous methods for removing proteins that have been damaged or are no longer needed. One of these methods is carried out by a large protein complex called the proteasome. Because of its central role in maintaining protein balance, drugs that stop the proteasome functioning often kill cancer cells grown in dishes. However, these proteasome inhibitors tend not to work against most tumors in patients. Moreover, tumors that do respond to these drugs ultimately become resistant to them. Tsvetkov et al. used a genetic screen to find the mutations that allowed cancer cells to withstand exposure to proteasome inhibitors. The proteasome complex contains two types of subunits: regulatory subunits that recognize the proteins that need to be degraded; and catalytic subunits that degrade the proteins. Surprisingly, individually inactivating the genes for many different regulatory subunits provided protection against proteasome inhibitors. When the regulatory subunits were reduced, the proteasomes shifted into a state that ultimately protected the cells. This mechanism was observed to protect both yeast and human cells and may be a widespread mechanism for establishing stress-resistant states. The next challenge will be to identify the vulnerabilities of cells that have reduced regulatory subunits. Research is also needed to find out if this reduction varies from cell to cell, making some cells more able to withstand treatment. DOI:
    Full-text · Article · Sep 2015 · eLife Sciences
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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.
    Full-text · Article · May 2015 · Molecular Immunology
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