Substrates enter the proteasome core particle (CP) through a channel that opens upon association with the regulatory particle (RP). Using yeast mutants, we show that channel opening is mediated by the ATPase domain of Rpt2, one of six ATPases in the RP. To test whether degradation products exit through this channel, we analyzed their size distribution. Their median length from an open-channel CP mutant was 40% greater than that from the wild-type. Thus, channel opening may enhance the yield of peptides long enough to function in antigen presentation. These experiments demonstrate that gating of the RP channel controls both substrate entry and product release, and is specifically regulated by an ATPase in the base of the RP.

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 ef®ciency 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-g-induced b-subunits and are more ef®cient 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 speci®city of immunoproteasomes in¯uence 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-ginduced enzyme leucine aminopeptidase).

IntroductionMultiple myeloma (MM) is a frequent and still incurable plasma cell malignancy, causing 2% of all cancer deaths. In recent years, treatment of MM has improved remarkably. For example, the proteasome inhibitor (PI) bortezomib (PS-341) proved effective even in the context of heavily pretreated, relapsed, and refractory MM, 1-3 although more than 50% of patients fail to respond to second-line treatment. 4 The molecular bases of different individual responsiveness to bortezomib remain unclear. Age (Ͻ 65 years) and extent of bone marrow plasma cell infiltration (Ͻ 50%) are the conventional factors for successful treatment identified so far. [5][6][7] Identifying the molecular bases underlying PI sensitivity would provide the framework for their improved clinical application.Bortezomib targets the proteasome, a 2.4-MDa multicatalytic protease complex ubiquitously expressed in eukaryotic cells. 1,8 Crucial for degrading proteins involved in cell cycle, angiogenesis, adhesion, cytokine production, and apoptosis, 3,9,10 proteasome inhibition can affect tumor cell growth via direct and indirect mechanisms (eg, by blocking interactions with endothelial and bone cells). 8,11 Proteasomes also dismantle damaged and misfolded/unfolded proteins, which are potentially harmful for the cell. 8 As a result, proteasome impairment causes buildup of polyubiquitinated proteins and eventual cell death. 3 Proteasomes also degrade a significant proportion of newly synthesized proteins in mammalian cells (rapidly degraded polypeptides [RDPs]). 12 Thus, increased protein synthesis or other metabolic unbalances could increase proteasome workload.We recently showed that plasma cell differentiation in vitro, ex vivo, and in vivo entails a dramatic decrease in proteasome expression and activity, correlating with increased sensitivity to PIs. 13,14 Indeed, PIs reduce antibody (Ab) responses in vivo. 14,15 Moreover, inducible expression of orphan Ig-chains sensitizes nonlymphoid tumor cells to PI-induced toxicity. 13 In MM cells (MMCs), the levels of both Ig synthesis and retention correlate with apoptotic sensitivity to PIs, and manipulating Ig synthesis alters sensitivity. 16,17 Altogether, these data suggest that the exquisite sensitivity of certain MMCs to PIs could stem from decreased proteasomal capacity, increased proteasomal workload, or both (ie, an adverse load-versus-capacity ratio).In this study, we exploited MM lines with differential apoptotic sensitivity to PIs to address if proteasome expression and degradative workload vary among different clones, and defined their role in The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on May 9, 2018. by guest www.bloodjournal.org From determining apoptotic sensitivity to PIs. Moreover, using primary patient-derived MMCs, we revealed ...

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After few days of intense immunoglobulin (Ig) secretion, most plasma cells undergo apoptosis, thus ending the humoral immune response. We asked whether intrinsic factors link plasma cell lifespan to Ig secretion. Here we show that in the late phases of plasmacytic differentiation, when antibody production becomes maximal, proteasomal activity decreases. The excessive load for the reduced proteolytic capacity correlates with accumulation of polyubiquitinated proteins, stabilization of endogenous proteasomal substrates (including Xbp1s, IjBa, and Bax), onset of apoptosis, and sensitization to proteasome inhibitors (PI). These events can be reproduced by expressing Ig-l chain in nonlymphoid cells. Our results suggest that a developmental program links plasma cell death to protein production, and help explaining the peculiar sensitivity of normal and malignant plasma cells to PI.