Importance of the different proteolytic sites of the proteasome and the efficacy of inhibitors varies with the protein substrate.
ABSTRACT 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.
- SourceAvailable from: Godefridus J Peters[show abstract] [hide abstract]
ABSTRACT: BACKGROUND: In clinical and experimental settings, antibody-based anti-CD20/rituximab and small molecule proteasome inhibitor (PI) bortezomib (BTZ) treatment proved effective modalities for B cell depletion in lymphoproliferative disorders as well as autoimmune diseases. However, the chronic nature of these diseases requires either prolonged or re-treatment, often with acquired resistance as a consequence. METHODS: Here we studied the molecular basis of acquired resistance to BTZ in JY human B lymphoblastic cells following prolonged exposure to this drug and examined possibilities to overcome resistance by next generation PIs and anti-CD20/rituximab-mediated complement-dependent cytotoxicity (CDC). RESULTS: Characterization of BTZ-resistant JY/BTZ cells compared to parental JY/WT cells revealed the following features: (a) 10--12 fold resistance to BTZ associated with the acquisition of a mutation in the PSMB5 gene (encoding the constitutive beta5 proteasome subunit) introducing an amino acid substitution (Met45Ile) in the BTZ-binding pocket, (b) a significant 2--4 fold increase in the mRNA and protein levels of the constitutive beta5 proteasome subunit along with unaltered immunoproteasome expression, (c) full sensitivity to the irreversible epoxyketone-based PIs carfilzomib and (to a lesser extent) the immunoproteasome inhibitor ONX 0914. Finally, in association with impaired ubiquitination and attenuated breakdown of CD20, JY/BTZ cells harbored a net 3-fold increase in CD20 cell surface expression, which was functionally implicated in conferring a significantly increased anti-CD20/rituximab-mediated CDC. CONCLUSIONS: These results demonstrate that acquired resistance to BTZ in B cells can be overcome by next generation PIs and by anti-CD20/rituximab-induced CDC, thereby paving the way for salvage therapy in BTZ-resistant disease.Experimental hematology & oncology. 01/2013; 2(1):2.
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ABSTRACT: Aging induces alterations of tissue protein homoeostasis. To investigate one of the major systems catalysing intracellular protein degradation we have purified 20S proteasomes from rat liver of young (2 months) and aged (23 months) animals and separated them into three subpopulations containing different types of intermediate proteasomes with standard- and immuno-subunits. The smallest subpopulation ΙΙΙ and the major subpopulation Ι comprised proteasomes containing immuno-subunits β1i and β5i beside small amounts of standard-subunits, whereas proteasomes of subpopulation ΙΙ contained only β5i beside standard-subunits. In favour of a relative increase of the major subpopulation Ι, subpopulation ΙΙ and ΙΙΙ were reduced for about 55 % and 80 %, respectively, in aged rats. Furthermore, in all three 20S proteasome subpopulations from aged animals standard-active site subunits were replaced by immuno-subunits. Overall, this transformation resulted in a relative increase of immuno-subunit-containing proteasomes, paralleled by reduced activity towards short fluorogenic peptide substrates. However, depending on the substrate their hydrolysing activity of long polypeptide substrates was significantly higher or unchanged. Furthermore, our data revealed an altered MHC class I antigen-processing efficiency of 20S proteasomes from liver of aged rats. We therefore suggest that the age-related intramolecular alteration of hepatic proteasomes modifies its cleavage preferences without a general decrease of its activity. Such modifications could have implications on protein homeostasis as well as on MHC class I antigen presentation as part of the immunosenescence process.Age 05/2013; · 6.28 Impact Factor
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ABSTRACT: Bortezomib is an antitumor drug that competitively inhibits proteasome beta-1 and beta-5 subunits. While the impact of bortezomib on protein stability is known, the effect of this drug on intracellular peptides has not been previously explored. A quantitative peptidomics technique was used to examine the effect of treating human embryonic kidney 293T (HEK293T) cells with 5-500 nM bortezomib for various lengths of time (30 minutes to 16 hours), and human neuroblastoma SH-SY5Y cells with 500 nM bortezomib for 1 hour. Although bortezomib treatment decreased the levels of some intracellular peptides, the majority of peptides were increased by 50-500 nM bortezomib. Peptides requiring cleavage at acidic and hydrophobic sites, which involve beta-1 and -5 proteasome subunits, were among those elevated by bortezomib. In contrast, the proteasome inhibitor epoxomicin caused a decrease in the levels of many of these peptides. Although bortezomib can induce autophagy under certain conditions, the rapid bortezomib-mediated increase in peptide levels did not correlate with the induction of autophagy. Taken together, the present data indicate that bortezomib alters the balance of intracellular peptides, which may contribute to the biological effects of this drug.PLoS ONE 01/2013; 8(1):e53263. · 3.73 Impact Factor