Extended peptide-based inhibitors efficiently target the proteasome and reveal overlapping specificities of the catalytic β-subunits

AbstractBackground: The 26S proteasome is responsible for most cytosolic proteolysis, and is an important protease in major histocompatibility complex class I-mediated antigen presentation. Constitutively expressed proteasomes from mammalian sources possess three distinct catalytically active species, β1, β2 and β5, which are replaced in the γ-interferon-inducible immunoproteasome by a different set of catalytic subunits, β1i, β2i and β5i, respectively. Based on preferred cleavage of short fluorogenic peptide substrates, activities of the proteasome have been assigned to individual subunits and classified as ‘chymotryptic-like’ (β5), ‘tryptic-like’ (β2) and ‘peptidyl-glutamyl peptide hydrolyzing’ (β1). Studies with protein substrates indicate a far more complicated, less strict cleavage preference. We reasoned that inhibitors of extended size would give insight into the extent of overlapping substrate specificity of the individual activities and subunits.Results: A new class of proteasome inhibitors, considerably extended in comparison with the commonly used fluorescent substrates and peptide-based inhibitors, has been prepared. Application of the safety catch resin allowed the generation of the target compounds using a solid phase protocol. Evaluation of the new compounds revealed a set of highly potent proteasome inhibitors that target all individual active subunits with comparable affinity, unlike the other inhibitors described to date. Modification of the most active compound, adamantane-acetyl-(6-aminohexanoyl)3-(leucinyl)3-vinyl-(methyl)-sulfone (AdaAhx3L3VS), itself capable of proteasome inhibition in living cells, afforded a new set of radio- and affinity labels.Conclusions: N-terminal extension of peptide vinyl sulfones has a profound influence on both their efficiency and selectivity as proteasome inhibitors. Such extensions greatly enhance inhibition and largely obliterate selectivity towards the individual catalytic activities. We conclude that for the interaction with larger substrates, there appears to be less discrimination of different substrate sequences for the catalytic activities than is normally assumed based on the use of small peptide-based substrates and inhibitors. The compounds described here are readily accessible synthetically, and are more potent inhibitors in living cells than their shorter peptide vinyl sulfone counterparts

Activity of immunoproteasome inhibitor ONX-0914 in acute lymphoblastic leukemia expressing MLL–AF4 fusion protein

Proteasome inhibitors bortezomib and carfilzomib are approved for the treatment of multiple myeloma and mantle cell lymphoma and have demonstrated clinical efficacy for the treatment of acute lymphoblastic leukemia (ALL). The t(4;11)(q21;q23) chromosomal translocation that leads to the expression of MLL–AF4 fusion protein and confers a poor prognosis, is the major cause of infant ALL. This translocation sensitizes tumor cells to proteasome inhibitors, but toxicities of bortezomib and carfilzomib may limit their use in pediatric patients. Many of these toxicities are caused by on-target inhibition of proteasomes in non-lymphoid tissues (e.g., heart muscle, gut, testicles). We found that MLL–AF4 cells express high levels of lymphoid tissue-specific immunoproteasomes and are sensitive to pharmacologically relevant concentrations of specific immunoproteasome inhibitor ONX-0914, even in the presence of stromal cells. Inhibition of multiple active sites of the immunoproteasomes was required to achieve cytotoxicity against ALL. ONX-0914, an inhibitor of LMP7 (ß5i) and LMP2 (ß1i) sites of the immunoproteasome, and LU-102, inhibitor of proteasome ß2 sites, exhibited synergistic cytotoxicity. Treatment with ONX-0914 significantly delayed the growth of orthotopic ALL xenograft tumors in mice. T-cell ALL lines were also sensitive to pharmacologically relevant concentrations of ONX-0914. This study provides a strong rationale for testing clinical stage immunoproteasome inhibitors KZ-616 and M3258 in ALL

Site-Specific Proteasome Inhibitors

Proteasome is a multi-subunit protein degradation machine, which plays a key role in the maintenance of protein homeostasis and, through degradation of regulatory proteins, in the regulation of numerous cell functions. Proteasome inhibitors are essential tools for biomedical research. Three proteasome inhibitors, bortezomib, carfilzomib, and ixazomib are approved by the FDA for the treatment of multiple myeloma; another inhibitor, marizomib, is undergoing clinical trials. The proteolytic core of the proteasome has three pairs of active sites, β5, β2, and β1. All clinical inhibitors and inhibitors that are widely used as research tools (e.g., epoxomicin, MG-132) inhibit multiple active sites and have been extensively reviewed in the past. In the past decade, highly specific inhibitors of individual active sites and the distinct active sites of the lymphoid tissue-specific immunoproteasome have been developed. Here, we provide a comprehensive review of these site-specific inhibitors of mammalian proteasomes and describe their utilization in the studies of the biology of the active sites and their roles as drug targets for the treatment of different diseases

Subunit specific inhibitors of proteasomes and their potential for immunomodulation

Specialized variants of the constitutive 20S proteasome in the immune system like the immunoproteasomes and the thymoproteasome contain active site-bearing subunits which differ in their cleavage priorities and substrate binding pockets. The immunoproteasome plays a crucial role in antigen processing and for the differentiation of pro-inflammatory T helper cells which are involved in the pathogenesis of autoimmunity. Selective inhibitors of the immunoproteasome and constitutive proteasome have recently been generated which interfere with the development and progression of autoimmune diseases. Here we describe these inhibitors and their therapeutic potential as predicted from preclinical models

Binding of hydrophobic peptides to several non-catalytic sites promotes peptide hydrolysis by all active sites of 20 S proteasomes: Evidence for peptide-induced channel opening in the alpha-rings

The eukaryotic 20 S proteasome contains the following 6 active sites: 2 chymotrypsin-like, 2 trypsin-like, and 2 caspase-like. We previously showed that hydrophobic peptide substrates of the chymotrypsin-like sites allosterically stimulate peptide hydrolysis by the caspase-like sites and their own cleavage. More thorough analysis revealed that these peptides also stimulate peptide hydrolysis by the trypsin-like site. This general activation by hydrophobic peptides occurred even if the chymotrypsin-like sites were occupied by a covalent inhibitor and was highly cooperative, with an average Hill coefficient of 7. Therefore, this stimulation of peptide hydrolysis at all active sites occurs upon binding of hydrophobic peptides to several non-catalytic sites. The stimulation by hydrophobic peptides was not observed in the yeast Delta N alpha 3 mutant 20 S proteasomes, in 20 S-PA26 complexes, or SDS-activated proteasomes and was significantly lower in 26 S proteasomes, all of which appear to have the gated channel in the alpha-rings in an open conformation and hydrolyze peptides at much faster rates than 20 S proteasomes. Also the hydrophobic peptides altered K(m), V(max) of active sites in a similar fashion as PA26 and the Delta N alpha 3 mutation. The activation by hydrophobic peptides was decreased in K(+)-containing buffer, which favors the closed state of the channels. Therefore, hydrophobic peptides stimulate peptide hydrolysis most likely by promoting the opening of the channels in the alpha-rings. During protein breakdown, this peptide-induced channel opening may function to facilitate the release of products from the proteasome

Proteasome Inhibitors: An Expanding Army Attacking a Unique Target

Proteasomes are large, multisubunit proteolytic complexes presenting multiple targets for therapeutic intervention. The 26S proteasome consists of a 20S proteolytic core and one or two 19S regulatory particles. The 20S core contains three types of active sites. Many structurally diverse inhibitors of these active sites, both natural product and synthetic, have been discovered in the last two decades. One, bortezomib, is used clinically for treatment of multiple myeloma, mantle cell lymphoma, and acute allograft rejection. Five more recently developed proteasome inhibitors are in trials for treatment of myeloma and other cancers. Proteasome inhibitors also have activity in animal models of autoimmune and inflammatory diseases, reperfusion injury, promote bone and hair growth, and can potentially be used as anti-infectives. In addition, inhibitors of ATPases and deubiquitinases of 19S regulatory particles have been discovered in the last decade

26S proteasomes and immunoproteasomes produce mainly N-extended versions of an antigenic peptide

Protein degradation by proteasomes is the source of most antigenic peptides presented on MHC class I molecules. To determine whether proteasomes generate these peptides directly or longer precursors, we developed new methods to measure the efficiency with which 26S and 20S particles, during degradation of a protein, generate the presented epitope or potential precursors. Breakdown of ovalbumin by the 26S and 20S proteasomes yielded the immunodominant peptide SIINFEKL, but produced primarily variants containing 1–7 additional N-terminal residues. Only 6–8% of the times that ovalbumin molecules were digested was a SIINFEKL or an N-extended version produced. Surprisingly, immunoproteasomes which contain the interferon-γ-induced β-subunits and are more efficient in antigen presentation, produced no more SIINFEKL than proteasomes. However, the immunoproteasomes released 2–4 times more of certain N-extended versions. These observations show that the changes in cleavage specificity of immunoproteasomes influence not only the C-terminus, but also the N-terminus of potential antigenic peptides, and suggest that most MHC-presented peptides result from N-terminal trimming of larger proteasome products by aminopeptidases (e.g. the interferon-γ-induced enzyme leucine aminopeptidase)

DNA-HISTONE CROSS-LINKS: FORMATION AND REPAIR

The nucleosome is a stretch of DNA wrapped around a histone octamer. Electrostatic interactions and hydrogen bonds between histones and DNA are vital for the stable organization of nucleosome core particles, and for the folding of chromatin into more compact structures, which regulate gene expression via controlled access to DNA. As a drawback of tight association, under genotoxic stress, DNA can accidentally crosslink to histone in a covalent manner, generating a highly toxic DNA-histone cross-link (DHC). DHC is a bulky lesion that can impede DNA transcription, replication, and repair, often with lethal consequences. The chemotherapeutic agent cisplatin, as well as ionizing and ultraviolet irradiations and endogenously occurring reactive aldehydes, generate DHCs by forming either stable or transient covalent bonds between DNA and side-chain amino groups of histone lysine residues. The mechanisms of DHC repair start to unravel, and certain common principles of DNA-protein cross-link (DPC) repair mechanisms that participate in the removal of cross-linked histones from DNA have been described. In general, DPC is removed via a two-step repair mechanism. First, cross-linked proteins are degraded by specific DPC proteases or by the proteasome, relieving steric hindrance. Second, the remaining DNA-peptide cross-links are eliminated in various DNA repair pathways. Delineating the molecular mechanisms of DHC repair would help target specific DNA repair proteins for therapeutic intervention to combat tumor resistance to chemotherapy and radiotherapy

NEIL3-mediated proteasomal degradation facilitates the repair of cisplatin-induced DNA damage in human cells

Abstract Anti-neoplastic effect of DNA cross-linking agents such as cisplatin, mitomycin C, and psoralen is attributed to their ability to induce DNA interstrand cross-links (ICLs), which block replication, transcription, and linear repair pathways by preventing DNA strand separation and trigger apoptosis. It is generally agreed that the Fanconi anemia (FA) pathway orchestrates the removal of ICLs by the combined actions of various DNA repair pathways. Recently, attention has been focused on the ability of the NEIL3-initiated base excision repair pathway to resolve psoralen- and abasic site-induced ICLs in an FA-independent manner. Intriguingly, overexpression of NEIL3 is associated with chemo-resistance and poor prognosis in many solid tumors. Here, using loss- and gain-of-function approaches, we demonstrate that NEIL3 confers resistance to cisplatin and participates in the removal of cisplatin–DNA adducts. Proteomic studies reveal that the NEIL3 protein interacts with the 26S proteasome in a cisplatin-dependent manner. NEIL3 mediates proteasomal degradation of WRNIP1, a protein involved in the early step of ICL repair. We propose that NEIL3 participates in the repair of ICL-stalled replication fork by recruitment of the proteasome to ensure a timely transition from lesion recognition to repair via the degradation of early-step vanguard proteins

Molecular basis of differential sensitivity of myeloma cells to clinically relevant bolus treatment with bortezomib.

The proteasome inhibitor bortezomib (Velcade) is prescribed for the treatment of multiple myeloma. Clinically achievable concentrations of bortezomib cause less than 85% inhibition of the chymotrypsin-like activity of the proteasome, but little attention has been paid as to whether in vitro studies are representative of this level of inhibition. Patients receive bortezomib as an intravenous or subcutaneous bolus injection, resulting in maximum proteasome inhibition within one hour followed by a gradual recovery of activity. In contrast, most in vitro studies use continuous treatment so that activity never recovers. Replacing continuous treatment with 1 h-pulse treatment increases differences in sensitivity in a panel of 7 multiple myeloma cell lines from 5.3-fold to 18-fold, and reveals that the more sensitive cell lines undergo apoptosis at faster rates. Clinically achievable inhibition of active sites was sufficient to induce cytotoxicity only in one cell line. At concentrations of bortezomib that produced similar inhibition of peptidase activities a different extent of inhibition of protein degradation was observed, providing an explanation for the differential sensitivity. The amount of protein degraded per number of active proteasomes correlated with sensitivity to bortezomib. Thus, (i) in vitro studies of proteasome inhibitors should be conducted at pharmacologically achievable concentrations and duration of treatment; (ii) a similar level of inhibition of active sites results in a different extent of inhibition of protein breakdown in different cell lines, and hence a difference in sensitivity