Proteasome inhibitors bortezomib and carfilzomib are FDA-approved anticancer agents that have contributed to significant improvements in treatment outcomes. However, the eventual onset of acquired resistance continues to limit their clinical utility, yet a clear consensus regarding the underlying mechanisms has not been reached. Bortezomib and carfilzomib are known to target both the constitutive proteasome and the immunoproteasome, two conventional proteasome subtypes comprising distinctive sets of catalytic subunits. While it has become increasingly evident that additional, ‘intermediate’ proteasome subtypes, which harbor non-standard mixtures of constitutive proteasome and immunoproteasome catalytic subunits, represent a considerable proportion of the proteasome population in many cell types, less is known regarding their contribution to cellular responses to proteasome inhibitors. Importantly, previous studies in murine models have shown that individual proteasome subtypes differ in sensitivity to specific proteasome inhibitors. Furthermore, research efforts in our laboratory and others have revealed that proteasome catalytic subunit expression levels and activity profiles are altered when human cancer cells acquire resistance to proteasome inhibitors. We therefore hypothesized that changes in the relative abundances of individual proteasome subtypes contribute to the acquired resistance of cancer cells to bortezomib and carfilzomib. A major obstacle in testing our hypothesis was a lack of chemical probes suitable for use in identifying distinct proteasome subtypes. We addressed this by developing a series of bifunctional proteasome probes capable of crosslinking specific pairs of catalytic subunits colocalized within individual proteasome complexes and compatible with immunoblotting-based detection of the crosslinked subunit pairs. We confirmed the utility of these probes in discerning the identities of individual proteasome subtypes in a multiple myeloma cell line that abundantly expresses catalytic subunits of both the constitutive proteasome and immunoproteasome. Our findings indicate that constitutive proteasomes, immunoproteasomes, and intermediate proteasomes co-exist within these cells and support conclusions drawn from previous studies in other cell types. We also established non-small cell lung cancer cell line models of acquired bortezomib and carfilzomib resistance in which to test our hypothesis. Using immunoblotting and proteasome activity assays, we discovered that changes in the expression levels and activities of individual catalytic proteasome subunits were associated with the emergence of acquired resistance to bortezomib or carfilzomib. These changes were inhibitor-dependent and persisted after the selective pressure of the inhibitor was removed. Finally, results obtained using our bifunctional proteasome probes suggest that the altered abundance of an intermediate proteasome subtype is associated with acquired proteasome inhibitor resistance. Collectively, our results provide evidence linking changes proteasome composition with acquired proteasome inhibitor resistance and support the hypothesis that such changes are involved in resistance mechanisms to these inhibitors.

20S Proteasomen der Leber gehören ausschließlich zur Population der Intermediär-Proteasomen. Chromatographisch sind drei proteasomale Subpopulationen aufgrund unterschiedlicher Oberflächenhydrophobizität trennbar. Diese beinhalten unterschiedliche Mengen der Standard- und Immunountereinheiten und zeigen unterschiedliche spezifische Aktivitäten gegenüber kurzen fluorigenen Peptidsubstraten. Außerdem lassen sie sich deutlich anhand der Schnittfrequenzen bei Hydrolyse von Polypeptidsubstraten unterscheiden. Jede dieser Subpopulationen konnte aufgrund unterschiedlicher Oberflächenladung in bis zu 5 verschiedene 20S Proteasom-Subtypen unterteilt werden, die wiederum unterschiedliche Mengen an Standard- und Immununtereinheiten enthielten. Jeder dieser Subtypen zeigte unterschiedliche Eigenschaften bezüglich der spezifischen Aktivitäten und Hydrolysegeschwindigkeiten von Polypeptidsubstraten. Unterschiede wurden auch bezüglich ihrer Peptid-Spleiß-Aktivitäten nachgewiesen. In der vorliegenden Arbeit wurden darüber hinaus Veränderungen der Spektren proteasomaler Subtypen- und ihrer Eigenschaften im Lebergewebe von 2, 8 und 23 Monate alten Ratten nachgewiesen. Während die Gesamtmenge der Leber-Proteasomen mit steigendem Alter abnahm, nahm die Menge der Subtypen mit integrierten Immununtereinheiten zu. Gleichzeitig kam es zu einer altersabhängigen Zunahme der Hydrolysegeschwindigkeit gegenüber Polypeptide-Substraten sowie veränderten Schnitthäufigkeiten. Die altersabhängigen intramolekularen Umgestaltungen von Leberproteasomen könnten eine funktionelle Anpassung an die altersbedingten zellulären Veränderungen in Verbindung mit Veränderungen der MHC Klasse I Antigen-Präsentation darstellen. / 20S proteasomes isolated from rat liver belong to the population of intermediate type proteasomes. Three subpopulations were separated by chromatography due to their differences in surface hydrophobicity. These subpopulations contain different types of intermediate type proteasomes with standard- and immunosubunits exhibiting distinct specific activities towards short fluorogenic substrates. However, depending on the substrate their hydrolyzing activity of long polypeptide substrates was significantly different. Additional separation of the three 20S proteasome subpopulations due to their different surface charges allowed further resolution of each subpopulation to at least five 20S proteasome subtypes. The subunit composition of these subtypes varied with regard to the content of exchangeable subunits. The pattern of proteolytic activities measured with short fluorogenic peptides differed between the various subtypes as well as the ability to hydrolyze polypeptide substrate. Above all, each subtype displayed a specific pattern regarding the peptide-splice-activity. Furthermore, the present work showed age-dependent alterations in the subtype patterns, which were analyzed in livers of 2, 8 and 23 month old rats. While the total amount of proteasome declines with age, in all subtypes from aged animals standard subunits were widely replaced by immunosubunits. This resulted in a relative increase of immunosubunit-containing proteasomes, paralleled by age-dependent changes regarding the hydrolyzing activity of long polypeptide substrates. Such modifications could have implications on protein homeostasis as well as on MHC class I antigen presentation as part of the immunosenescence process.