Upon encounter with antigen, B lymphocytes differentiate into Ig-secreting plasma cells. This step involves a massive development of secretory organelles, most notably the endoplasmic reticulum. To analyze the relationship between organelle reshaping and Ig secretion, we performed a dynamic proteomics study of B lymphoma cells undergoing in vitro terminal differentiation. By clustering proteins according to temporal expression patterns, it appeared that B cells anticipate their secretory role in a multistep process. Metabolic capacity and secretory machinery expand first to accommodate the mass production of IgM that follows.

A.Mezghrani and A.Fassio contributed equally to this workIn the endoplasmic reticulum (ER), disul®de bonds are simultaneously formed in nascent proteins and removed from incorrectly folded or assembled molecules. In this compartment, the redox state must be, therefore, precisely regulated. Here we show that both human Ero1-La and Ero1-Lb (hEROs) facilitate disul®de bond formation in immunoglobulin subunits by selectively oxidizing PDI. Disul®de bond formation is controlled by hEROs, which stand at a crucial point of an electron-¯ow starting from nascent secretory proteins and passing through PDI. The redox state of ERp57, another ER-resident oxidoreductase, is not affected by over-expression of Ero1-La, suggesting that parallel and speci®c pathways control oxidative protein folding in the ER. Mutants in the Ero1-La CXXCXXC motif act as dominant negatives by limiting immunoglobulin oxidation. PDI-dependent oxidative folding in living cells can thus be manipulated by using hERO variants.

In human cells, Ero1-Lalpha and -Lbeta (hEROs) regulate oxidative protein folding by selectively oxidizing protein disulfide isomerase. Specific protein--protein interactions are probably crucial for regulating the formation, isomerization and reduction of disulfide bonds in the endoplasmic reticulum (ER). To identify molecules involved in ER redox control, we searched for proteins interacting with Ero1-Lalpha. Here, we characterize a novel ER resident protein (ERp44), which contains a thioredoxin domain with a CRFS motif and is induced during ER stress. ERp44 forms mixed disulfides with both hEROs and cargo folding intermediates. Whilst the interaction with transport-competent Ig-K chains is transient, ERp44 binds more stably with J chains, which are retained in the ER and eventually degraded by proteasomes. ERp44 does not bind a short-lived ribophorin mutant lacking cysteines. Its overexpression alters the equilibrium of the different Ero1-Lalpha redox isoforms, suggesting that ERp44 may be involved in the control of oxidative protein folding.

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 ...