Alexei F. Kisselev

Certain Pairs of Ubiquitin-conjugating Enzymes (E2s) and Ubiquitin-Protein Ligases (E3s) Synthesize Nondegradable Forked Ubiquitin Chains Containing All Possible Isopeptide Linkages

It is generally assumed that a specific ubiquitin ligase (E3) links protein substrates to polyubiquitin chains containing a single type of isopeptide linkage, and that chains composed of linkages through Lys48, but not through Lys63, target proteins for proteasomal degradation. However, when we carried out a systematic analysis of the types of ubiquitin (Ub) chains formed by different purified E3s and Ub-conjugating enzymes (E2s), we found, using Ub mutants and mass spectrometry, that the U-box E3, CHIP, and Ring finger E3s, MuRF1 and Mdm2, with the E2, UbcH5, form a novel type of Ub chain that contains all seven possible linkages, but predominantly Lys48, Lys63, and Lys11 linkages. Also, these heterogeneous chains contain forks (bifurcations), where two Ub molecules are linked to the adjacent lysines at Lys6 + Lys11, Lys27 + Lys29, or Lys29 + Lys33 on the preceding Ub molecule. However, the HECT domain E3s, E6AP and Nedd4, with the same E2, UbcH5, form homogeneous chains exclusively, either Lys48 chains (E6AP) or Lys63 chains (Nedd4). Furthermore, with other families of E2s, CHIP and MuRF1 synthesize homogeneous Ub chains on the substrates. Using the dimeric E2, UbcH13/Uev1a, they attach Lys63 chains, but with UbcH1 (E2–25K), MuRF1 synthesizes Lys48 chains on the substrate. We then compared the capacity of the forked heterogeneous chains and homogeneous chains to support proteasomal degradation. When troponin I was linked by MuRF1 to a Lys48-Ub chain or, surprisingly, to a Lys63-Ub chain, troponin I was degraded rapidly by pure 26S proteasomes. However, when linked to the mixed forked chains, troponin I was degraded quite poorly, and its polyUb chain, especially the forked linkages, was disassembled slowly by proteasome-associated isopeptidases. Because these Ring finger and U-box E3s with UbcH5 target proteins for degradation in vivo, but Lys63 chains do not, cells probably contain additional factors that prevent formation of such nondegradable Ub-conjugates and that protect proteins linked to Lys63-Ub chains from proteasomal degradation.

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

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 substrates 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.

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.

Monitoring Activity and Inhibition of 26S Proteasomes with Fluorogenic Peptide Substrates

Eukaryotic proteasomes have three different types of active sites: two chymotrypsin-like, two trypsin-like, and two caspase-like (also termed PGPH) sites that differ in their specificity toward model fluorogenic peptide substrates. The chymotrypsin-like site is often considered the most important in protein breakdown, and the only one whose activity has to be assayed in order to assess the capacity of proteasomes to degrade proteins. However, recent results indicate that either trypsin-like or caspase-like sites also have to be inhibited in order to reduce breakdown of most proteins by 50%. Thus, the activities of all three types of active sites have to be assayed in order to evaluate the state of the proteasome inside cells or the potency of inhibitors. This chapter describes assays of purified 26S proteasomes with fluorogenic peptide substrates, including new substrates of the caspase- and trypsin-like sites. A novel assay of proteasome activity in crude cell extracts that allows rapid evaluation of the state of the proteasomes in cells treated with inhibitors is also described.

Selective Inhibitor of Proteasome's Caspase-like Sites Sensitizes Cells to Specific Inhibition of Chymotrypsin-like Sites

Proteasomes degrade most proteins in mammalian cells and are established targets of anticancer drugs. All eukaryotic proteasomes have three types of active sites: chymotrypsin-like, trypsin-like, and caspase-like. Chymotrypsin-like sites are the most important in protein degradation and are the primary target of most proteasome inhibitors. The biological roles of trypsin-like and caspase-like sites and their potential as cotargets of antineoplastic agents are not well defined. Here we describe the development of site-specific inhibitors and active-site probes of chymotrypsin-like and caspase-like sites. Using these compounds, we show that cytotoxicity of proteasome inhibitors does not correlate with inhibition of chymotrypsin-like sites and that coinhibition of either trypsin-like and/or caspase-like sites is needed to achieve maximal cytotoxicity. Thus, caspase-like and trypsin-like sites must be considered as cotargets of anticancer drugs.

Multiple BH3 Mimetics Antagonize Antiapoptotic MCL1 Protein by Inducing the Endoplasmic Reticulum Stress Response and Up-regulating BH3-only Protein NOXA

BH3 mimetics are small molecules designed or discovered to mimic the binding of BH3-only proteins to the hydrophobic groove of antiapoptotic BCL2 proteins. The selectivity of these molecules for BCL2, BCL-XL, or MCL1 has been established in vitro; whether they inhibit these proteins in cells has not been rigorously investigated. In this study, we used a panel of leukemia cell lines to assess the ability of seven putative BH3 mimetics to inhibit antiapoptotic proteins in a cell-based system. We show that ABT-737 is the only BH3 mimetic that inhibits BCL2 as assessed by displacement of BAD and BIM from BCL2. The other six BH3 mimetics activate the endoplasmic reticulum stress response inducing ATF4, ATF3, and NOXA, which can then bind to and inhibit MCL1. In most cancer cells, inhibition of one antiapoptotic protein does not acutely induce apoptosis. However, by combining two BH3 mimetics, one that inhibits BCL2 and one that induces NOXA, apoptosis is induced within 6 h in a BAX/BAK-dependent manner. Because MCL1 is a major mechanism of resistance to ABT-737, these results suggest a novel strategy to overcome this resistance. Our findings highlight a novel signaling pathway through which many BH3 mimetics inhibit MCL1 and suggest the potential use of these agents as adjuvants in combination with various chemotherapy strategies.

Specific Cell-Permeable Inhibitor of Proteasome Trypsin-like Sites Selectively Sensitizes Myeloma Cells to Bortezomib and Carfilzomib

Proteasomes degrade the majority of proteins in mammalian cells, are involved in the regulation of multiple physiological functions, and are established targets of anticancer drugs. The proteasome has three types of active sites. Chymotrypsin-like sites are the most important for protein breakdown and have long been considered the only suitable targets for antineoplastic drugs; however, our recent work demonstrated that inhibitors of caspase-like sites sensitize malignant cells to inhibitors of the chymotrypsin-like sites. Here, we describe the development of specific cell-permeable inhibitors and an activity-based probe of the trypsin-like sites. These compounds selectively sensitize multiple myeloma cells to inhibitors of the chymotrypsin-like sites, including antimyeloma agents bortezomib and carfilzomib. Thus, trypsin-like sites are cotargets for anticancers drugs. Together with inhibitors of chymotrypsin- and caspase-like sites developed earlier, we provide the scientific community with a complete set of tools to separately modulate proteasome active sites in living cells.

Activity-Based Profiling Reveals Reactivity of the Murine Thymoproteasome-Specific Subunit β5t

Epithelial cells of the thymus cortex express a unique proteasome particle involved in positive T cell selection. This thymoproteasome contains the recently discovered beta5t subunit that has an uncharted activity, if any. We synthesized fluorescent epoxomicin probes that were used in a chemical proteomics approach, entailing activity-based profiling, affinity purification, and LC-MS identification, to demonstrate that the beta5t subunit is catalytically active in the murine thymus. A panel of established proteasome inhibitors showed that the broad-spectrum inhibitor epoxomicin blocks the beta5t activity and that the subunit-specific antagonists bortezomib and NC005 do not inhibit beta5t. We show that beta5t has a substrate preference distinct from beta5/beta5i that might explain how the thymoproteasome generates the MHC class I peptide repertoire needed for positive T cell selection.

Nature of Pharmacophore Influences Active Site Specificity of Proteasome Inhibitors

Proteasomes degrade most proteins in mammalian cells and are established targets of anti-cancer drugs. The majority of proteasome inhibitors are composed of short peptides with an electrophilic functionality (pharmacophore) at the C terminus. All eukaryotic proteasomes have three types of active sites as follows: chymotrypsin-like, trypsin-like, and caspase-like. It is widely believed that active site specificity of inhibitors is determined primarily by the peptide sequence and not the pharmacophore. Here, we report that active site specificity of inhibitors can also be tuned by the chemical nature of the pharmacophore. Specifically, replacement of the epoxyketone by vinyl sulfone moieties further improves the selectivity of β5-specific inhibitors NC-005, YU-101, and PR-171 (carfilzomib). This increase in specificity is likely the basis of the decreased cytotoxicity of vinyl sulfone-based inhibitors to HeLa cells as compared with that of epoxyketone-based inhibitors.

Incorporation of Non-natural Amino Acids Improves Cell Permeability and Potency of Specific Inhibitors of Proteasome Trypsin-like Sites.

Proteasomes degrade the majority of proteins in mammalian cells by a concerted action of three distinct pairs of active sites. The chymotrypsin-like sites are targets of antimyeloma agents bortezomib and carfilzomib. Inhibitors of the trypsin-like site sensitize multiple myeloma cells to these agents. Here we describe systematic effort to develop inhibitors with improved potency and cell permeability, yielding azido-Phe-Leu-Leu-4-aminomethyl-Phe-methyl vinyl sulfone (4a, LU-102), and a fluorescent activity-based probe for this site. X-ray structures of 4a and related inhibitors complexed with yeast proteasomes revealed the structural basis for specificity. Nontoxic to myeloma cells when used as a single agent, 4a sensitized them to bortezomib and carfilzomib. This sensitizing effect was much stronger than the synergistic effects of histone acetylase inhibitors or additive effects of doxorubicin and dexamethasone, raising the possibility that combinations of inhibitors of the trypsin-like site with bortezomib or carfilzomib would have stronger antineoplastic activity than combinations currently used clinically.