Degradation of Distinct Assembly Forms of Immunoglobulin M Occurs in Multiple Sites in Permeabilized B Cells

Winitz, Dorit; Shachar, Idit; Elkabetz, Yechiel; Amitay, Raya; Samuelov, Meirav; Bar-Nun, Shoshana

doi:10.1074/jbc.271.44.27645

Cited by 19 publications

(23 citation statements)

References 62 publications

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“…4). The fact that H-L disassembly requires energy might explain why the degradation of assembled s was facilitated by cytosolic factors and reducing agents to a greater extent than free chains (26,27). Our data do not allow us to establish whether also intrachain disulfides are reduced before dislocation of Ig subunits as suggested for major histocompatibility complex class I heavy chains (42).…”

Section: Discussioncontrasting

confidence: 39%

“…For reasons that are still unclear, IgM polymerization is inefficient in B cells (24). Therefore, most s chains are retained and eventually degraded despite assembling with L chains to form s2 ⅐L 2 complexes (25)(26)(27)(28). In contrast, analogous complexes containing membrane chains ( m2 ⅐L 2 ) negotiate assembly with signaling components (Ig-␣ and Ig-␤) and are transported to the cell surface of B lymphocytes (29,30) where they act as antigen receptors.…”

Section: The Endoplasmic Reticulum (Er)mentioning

confidence: 99%

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Reduction of Interchain Disulfide Bonds Precedes the Dislocation of Ig-µ Chains from the Endoplasmic Reticulum to the Cytosol for Proteasomal Degradation

Fagioli¹,

Mezghrani²,

Sitia³

2001

Journal of Biological Chemistry

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Proteins that fail to fold or assemble in the endoplasmic reticulum (ER) are generally dislocated across the membrane to be degraded by cytosolic proteasomes. To investigate how the quality control machinery handles individual subunits that are part of covalent oligomers, we have analyzed the fate of transport-competent Ig light (L) chains that form disulfide bonds with shortlived heavy chains. When expressed alone, L chains are secreted. In cells producing excess , most L chains are retained in the ER as covalent ⅐L or 2 ⅐L 2 complexes. While chains present in these complexes are degraded by proteasomes, L chains are stable. Few L chains are secreted; most reassociate with newly synthesized chains. Therefore, interchain disulfide bonds are reduced in the ER lumen before the dislocation of chains in a site from which freed L chains can be rapidly reinserted in the assembly line. The ER can thus sustain the simultaneous formation and reduction of disulfide bonds.

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Section: Discussioncontrasting

confidence: 39%

Section: The Endoplasmic Reticulum (Er)mentioning

confidence: 99%

Reduction of Interchain Disulfide Bonds Precedes the Dislocation of Ig-µ Chains from the Endoplasmic Reticulum to the Cytosol for Proteasomal Degradation

Fagioli¹,

Mezghrani²,

Sitia³

2001

Journal of Biological Chemistry

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“…Based on its stabilization by a variety of proteasome inhibitors, we and others have shown that s is degraded by the proteasome (9,18,19). Moreover, in B lymphocytes treated with proteasome inhibitors, we were able to detect ubiquitinated species of s ( Fig.…”

Section: Resultsmentioning

confidence: 51%

“…The luminal s heavy chain exhibits developmentally regulated stability; while being a stable protein that is secreted efficiently from plasma cells, it is degraded rapidly in the earlier differentiation stages of pre-B and B cells (19,24,30). Based on its stabilization by a variety of proteasome inhibitors, we and others have shown that s is degraded by the proteasome (9,18,19).…”

Section: Resultsmentioning

confidence: 99%

“…Igs are particularly attractive because they are oligomeric proteins in the secretory pathway composed of heavy and light chains, both of which are subjected to proteasomal degradation. This is indicated by the stabilization of s chains, the heavy chains of secretory IgM (9,18,19) or of Ig light chains (8), upon proteasome inhibition. Under these conditions, s or accumulate in the microsomal fractions rather than the cytosol, implicating the proteasome in dislocation (8, 9).…”

mentioning

confidence: 99%

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Distinct Steps in Dislocation of Luminal Endoplasmic Reticulum-associated Degradation Substrates

Elkabetz

Shapira

Rabinovich

et al. 2004

Journal of Biological Chemistry

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104

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Dislocation of endoplasmic reticulum-associated degradation (ERAD) substrates from the endoplasmic reticulum (ER) lumen to cytosol is considered to occur in a single step that is tightly coupled to proteasomal degradation. Here we show that dislocation of luminal ERAD substrates occurs in two distinct consecutive steps. The first is passage across ER membrane to the ER cytosolic face, where substrates can accumulate as ubiquitin conjugates. In vivo, this step occurs despite proteasome inhibition but requires p97/Cdc48p because substrates remain entrapped in ER lumen and are prevented from ubiquitination in cdc48 yeast strain. The second dislocation step is the release of accumulated substrates to the cytosol. In vitro, this release requires active proteasome, consumes ATP, and relies on salt-removable ERbound components, among them the ER-bound p97 and ER-bound proteasome, which specifically interact with the cytosol-facing substrates. An additional role for Cdc48p subsequent to ubiquitination is revealed in the cdc48 strain at permissive temperature, consistent with our finding that p97 recognizes luminal ERAD substrates through multiubiquitin. BiP interacts exclusively with ERAD substrates, suggesting a role for this chaperone in ERAD. We propose a model that assigns the cytosolic face of the ER as a midpoint to which luminal ERAD substrates emerge and p97/Cdc48p and the proteasome are recruited. Although p97/Cdc48p plays a dual role in dislocation and is involved both in passage of the substrate across ER membrane and subsequent to its ubiquitination, the proteasome takes part in the release of the substrate from the ER face to the cytosol en route to degradation.The endoplasmic reticulum-associated degradation (ERAD) 1 is a quality control process that selectively eliminates malfolded proteins or unassembled subunits of oligomeric proteins in the secretory pathway (1, 2). ERAD substrates are dislocated from the endoplasmic reticulum (ER) back to the cytosol via the Sec61 complex (3-6). In the cytosol, ubiquitin is conjugated to the ERAD substrates that are degraded by the proteasome (7). The proteolytically active proteasome has been implicated in the dislocation of ERAD substrates by virtue of their scarcity in cytosol of proteasome-inhibited cells (8 -11). Although stabilization of proteins in the secretory pathway by proteasome inhibitors is generally accepted as an indication for ERAD, ubiquitination of such proteins is a direct evidence for the access of the substrate to the cytosol (7). In most cases, however, accumulation of the multiubiquitinated ERAD substrates to detectable levels requires inhibition of the proteasome. If the proteasome is indeed required for dislocation (8 -11), then ubiquitination of ERAD substrates would not be observed. This applies especially to luminal ERAD substrates, as membrane ERAD substrates inherently display cytosolic domains, which may be ubiquitinated irrespective of dislocation (11-15). Therefore, it is our view that luminal ERAD substrates, whose dislocation is an ...

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An empirical modeling platform to evaluate the relative control discrete CHO cell synthetic processes exert over recombinant monoclonal antibody production process titer

McLeod

O'Callaghan

Pybus

et al. 2011

Biotech & Bioengineering

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In this study we have combined empirically derived mathematical models of intracellular Mab synthesis to quantitatively compare the degree to which individual cellular processes limit recombinant IgG(4) monoclonal antibody production by GS-CHO cells throughout a state-of-the-art industrial fed-batch culture process. Based on the calculation of a production process control coefficient for each stage of the intracellular Mab synthesis and secretion pathway, we identified the major cellular restrictions on Mab production throughout the entire culture process to be recombinant heavy chain gene transcription and heavy chain mRNA translation. Surprisingly, despite a substantial decline in the rate of cellular biomass synthesis during culture, with a concomitant decline in the calculated rate constants for energy-intensive Mab synthetic processes (Mab folding/assembly and secretion), these did not exert significant control of Mab synthesis at any stage of production. Instead, cell-specific Mab production was maintained by increased Mab gene transcription which offset the decline in cellular biosynthetic rates. Importantly, this study shows that application of this whole-process predictive modeling strategy should rationally precede and inform cell engineering approaches to increase production of a recombinant protein by a mammalian host cell--where control of productivity is inherently protein product and cell line specific.

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Degradation of Distinct Assembly Forms of Immunoglobulin M Occurs in Multiple Sites in Permeabilized B Cells

Cited by 19 publications

References 62 publications

Reduction of Interchain Disulfide Bonds Precedes the Dislocation of Ig-µ Chains from the Endoplasmic Reticulum to the Cytosol for Proteasomal Degradation

Reduction of Interchain Disulfide Bonds Precedes the Dislocation of Ig-µ Chains from the Endoplasmic Reticulum to the Cytosol for Proteasomal Degradation

Distinct Steps in Dislocation of Luminal Endoplasmic Reticulum-associated Degradation Substrates

An empirical modeling platform to evaluate the relative control discrete CHO cell synthetic processes exert over recombinant monoclonal antibody production process titer

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