Proteasomes degrade most proteins in mammalian cells and are established targets of anti-cancer 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 co-targets of anti-neoplastic agents are not well defined. Here we describe the development of novel, 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 co-inhibition 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 co-targets of anti-cancer drugs.

Antibody affinity maturation occurs in germinal centres (GC) where B cells cycle between the light zone (LZ) and the dark zone. In the LZ GC B cells bearing immunoglobulins with the highest affinity for antigen receive positive selection signals from T helper cells that promotes their rapid proliferation. Here we show that the RNA binding protein PTBP1 is necessary for the progression of GC B cells through late S-phase of the cell cycle and for affinity maturation. PTBP1 is required for the proper expression of the c-MYC-dependent gene program induced in GC B cells receiving T cell help and directly regulates the alternative splicing and abundance of transcripts increased during positive selection to promote proliferation.

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 ␤5-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.The ubiquitin-proteasome pathway is essential in the maintenance of protein homeostasis in all eukaryotic cells and is involved in the regulation of numerous biologic processes. Proteasome inhibition causes apoptosis of malignant cells (1, 2). The proteasome inhibitor bortezomib (Velcade, PS-341) is used for the treatment of multiple myeloma and mantle cell lymphoma. Four other proteasome inhibitors are at different stages of clinical trials (3-6).The 26 S proteasome is a large (1.6 -2.4 MDa), hollow cylindrical, and multifunctional particle that consists of a 20 S proteolytic core and one or two 19 S regulatory complexes. Each eukaryotic 20 S core particle has three pairs of proteolytic sites with distinct substrate specificities (7-11). The ␤5 proteolytic sites are "chymotrypsin-like," and the ␤2 sites are "trypsin-like." The ␤1 sites cleave after acidic residues (Glu and Asp) and are referred to as "post-acidic," "post-glutamate peptide hydrolase," or "caspase-like." Tissues of the immune system also express immunoproteasomes, in which ␤5, ␤1, and ␤2 catalytic subunits are replaced by their major histocompatibility complex (MHC) locus-encoded counterparts LMP7 (␤5i), LMP2 (␤1i), and MECL-1 (␤2i).The chymotrypsin-like sites have long been considered the only suitable targets for anti-neoplastic agents and are the primary targets of all these agents. However, our recent work indicates that cytotoxicity of proteasome inhibitors correlates poorly with exclusive inhibition of the chymotrypsin-like sites and that co-inhibition of other sites is usually needed to achieve maximal cytotoxicity (12). In this regard, we have considered it of interest to determine whether inhibitors with increased specificity for ␤5 display decreased cytotoxicity.Many structural classes of proteasome inhibitors are known (2, 13). The majority of these are N-terminally capped short peptides (2-4 residues) with an electrophilic trap at the C terminus (e.g. aldehydes, boronates, epoxyketones, and vinyl sulfones). This electrophile reacts with the catalytic N-terminal threonines ...

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.