Building an antibody factory: a job for the unfolded protein response

Although the basic elements of the mammalian UPR are similar to those of the yeast pathway, the former is considerably more complex (Fig. 1). There are two mammalian homologs of yeast Ire1p: Ire1, which is ubiquitously expressed⁸, and Ire1, which is expressed exclusively in gut epithelium⁹. Like yeast Ire1p, both are transmembrane proteins that possess an N-terminal ER stress-sensing lumenal domain, a cytosolic kinase domain and a C-terminal endonuclease domain. Working from the other end of the response, Mori and co-workers first delineated the ER stress-responsive element (ERSE) in the promoters of mammalian ER chaperone genes and used this sequence in a one-hybrid screen to identify transcription factors that could bind in a stress-inducible manner. This screen revealed that both X box−binding protein 1 (XBP-1) and ATF6, a member of the ATF/CREB transcription factor family, bound to ERSEs¹⁰. Both and forms of ATF6 exist and are synthesized as ER-localized transmembrane preproteins with a lumenal stress-sensing module and a cytosolic transcription factor domain. During ER stress, ATF6 proteins move to the Golgi where the sequential efforts of the proteases S1P and S2P liberate the soluble transcription factor domain from the membrane¹¹. Although Atf6-null cells are not yet available for UPR analysis, current data indicate that this active form of ATF6 enters the nucleus and can upregulate transcription of ER chaperone genes¹² and Xbp1 (ref. 13). The Ire1 and ATF6 pathways converge at this point, as the ATF6-induced Xbp1 mRNA is thus far the only known substrate for the Ire1 endoribonuclease activity, which excises 26 bases from the transcript^{13, 14}. Religation of the cleaved mRNA results in a frame switch that remodels the C terminus of the spliced form of XBP-1, XBP-1(S). This change stabilizes the protein and adds a strong transactivation domain to the N-terminal DNA binding module^{13, 14}. Recent studies suggest that XBP-1(S) is responsible for the transcriptional upregulation of some ER chaperones, chaperone cofactors, components of the ER-associated degradation system (ERAD) system, and membrane biosynthesis^{15, 16}. Together, mammalian Ire1 and ATF6 fulfill the protective roles that Ire1p plays alone in yeast. This dual system could provide a mechanism for tighter control of downstream targets, for fine-tuning the magnitude of their induction, or both. In addition to processing Xbp1 mRNA, activated Ire1 can bind to TRAF2, resulting in recruitment and activation of caspase-12, an ER stress-specific inducer of apoptosis¹⁷. Caspase-12 activation occurs late in the UPR, and thus it is unclear how this Ire1-dependent activity is regulated compared to Xbp1 mRNA cleavage, an event initiated very early in the response.

Figure 1. Components of the mammalian UPR. (a) Imbalances in the ER environment that affect the normal maturation of secretory-pathway proteins are sensed via the lumenal portions of three ER-resident transmembrane glycoproteins, which are associated with the ER chaperone BiP when they are inactive. (b) Increased demands on the folding environment lead to the release of BiP from the lumenal domains and activation (star) or cleavage (red arrow) of their cytosolic domains. In the case of ATF6, the transcription factor domain (purple oval) that comprises the entire cytosolic domain is cleaved from the membrane and upregulates ER chaperone gene and Xbp1 transcription. Activation of the endoribonuclease activity at the C terminus of Ire1 (in red) induces the excision of 26 bases from the XBP-1 transcript; this, after religation, produces a remodeled XBP-1(S) protein that is a potent transcriptional activator and upregulates ER chaperones, structural proteins of the secretory pathway, components of the ERAD machinery, and membrane biosynthesis. In addition, activation of Ire1 results in recruitment of TRAF2, leading to Jun kinase activation and caspase-12 cleavage, both of which are linked to apoptosis (red). PERK is an eIF-2 kinase that inhibits cap-dependent translation. This causes a G1 cell cycle arrest, activates the antiapoptotic protein NF-B (blue) and induces the CHOP transcription factor, which in turn downregulates Bcl-2. Thus, both antiapoptotic and proapoptotic pathways are put into play. Both ATF6 and Ire1 are activated during plasma cell differentiation (beige box), but it is unclear whether all of Ire1's downstream effects are triggered. Full Figure and legend (29K)

A second mammalian ER stress-activated transmembrane kinase was identified based on its similarity to the lumenal domain of Ire1 (ref. 18). The protein kinase R−like ER kinase, PERK, is a member of the eIF-2 kinase family and serves to inhibit most cap-dependent protein synthesis early in the UPR. The inhibition is transient owing to a negative regulatory feedback loop that is initiated when translation is blocked. Paradoxically, the decrease in protein synthesis allows the ATF4 transcription factor to be translated¹⁹. ATF4 induces GADD34, the catalytic subunit of the PP1 phosphatase, which in turn dephosphorylates eIF-2, allowing translation to resume^{20, 21}. In addition to limiting the accumulation of unfolded proteins in the ER, the block in protein synthesis early in the ER stress response induces a G1 cell cycle arrest²², activation of the antiapoptotic protein NF-B²³ and induction of the proapoptotic transcription factor CHOP²⁴. Again, both cytoprotective and destructive outcomes can be initiated, and it remains unclear how these are balanced in individual cells.

Figure 2. Making an antibody-producing machine. The conversion of a B lymphocyte into an efficient secretory machine entails a dramatic remodeling of the cell. This includes (i) building the secretory apparatus (increased ER membrane system composed of lipids, translocon components and so on); (ii) equipping the cell with larger quantities of ribosomal subunits, mitochondria to provide energy, and components of amino acid synthesis and metabolic processes; (iii) managing the quality of the product by increasing chaperones, folding enzymes, and components of the quality control system; and (iv) upregulating transcription of immunoglobulin genes and inducing the processing of immunoglobulin heavy chain mRNA to the secreted form. Blimp-1 serves as a master regulator of all four aspects of the process, including the upregulation of Xbp1 transcription. Ire1 is required to remodel XBP-1 into XBP-1(S), an active transcription factor that induces proteins involved in building, equipping and managing the machinery for high-level immunoglobulin production. Evidence for ATF6 activation during plasma cell differentiation has been obtained, but its contributions to these processes are not currently understood. Full Figure and legend (25K)

A growing body of data indicates that the UPR activation profile is likely to vary among different types of specialized secretory cells. For example, the Ire1−XBP-1 branch of the UPR is clearly active in differentiating B cells^{14, 38}, and XBP-1(S) is essential for successful plasma cell development³⁸. In addition, ATF6 undergoes proteolytic activation during LPS-induced differentiation of the CH12 B cell lymphoma⁴². A role for ATF6 in developing plasma cells has not been established, but it is certainly possible that this factor contributes to the induction of Xbp1 (ref. 13) as well as many genes encoding ER chaperones and folding enzymes⁴³. PERK, in contrast, does not seem to be activated in the normal course of terminal B cell differentiation⁴². Notably, the PERK arm of the response is functional in B cells, as evidenced by potent induction of CHOP (also known as DDIT3 or GADD34), a PERK-dependent UPR target gene, in response to ER stress agents⁴². This raises an interesting point, because PERK seems to be essential for maximal induction of ER chaperones during ER stress^{19, 44}, and chaperones are highly upregulated during plasma cell differentiation. In sharp contrast to differentiating B cells, pancreatic tissue shows high constitutive activity of both PERK and Ire1 (refs. 45,46). PERK is essential for proper control of insulin biosynthesis, islet cell survival, and normal function of pancreatic acinar cells⁴⁵. Consistent with these data, mice homozygous for a targeted knock-in point mutation encoding the eIF2^S51A mutant protein which cannot be phosphorylated by PERK, are severely deficient in glucose metabolism²⁴. PERK also is crucial in the normal physiology of osteoblasts, which secrete the type I collagen that constitutes the matrix of compact bone⁴⁷. Thus, it seems that the physiologic UPR is not a 'one size fits all' program; instead, it is likely to be unique and customized according to the specific needs of distinct cell types.

What is the mechanistic basis for UPR variation? One possibility is that the three transducers—Ire1, ATF6 and PERK—have different thresholds for activation. To date, there is no evidence supporting this idea, but it is conceivable that such differences may not have been apparent when strong ER stress-inducing agents were used to study the response. If such a hierarchy exists, it must vary by tissue given the dissimilarity between PERK activity in B cells versus the pancreas. Another idea is that there are alternative mechanisms to activate the individual transducers independent of the load of unfolded proteins and the abundance of BiP. Although no other means of activating the transducers have been elucidated, this hypothesis remains an intriguing possibility. Interplay of BiP with various ER-resident DnaJ proteins, two of which are regulated by XBP-1 (S) (ref. 48), might provide an avenue for other, as yet undefined, signals to influence the activation status of the three UPR transducers. In the case of UPR activation in differentiating B cells, expression of immunoglobulin heavy chains is required for optimal synthesis of XBP-1(S)³⁸. These data fit well with the idea that the UPR is triggered when BiP is needed to accommodate an increased flux of nascent polypeptides. It is worth noting, however, that H-chain deficient B cells did show low, but detectable, amounts of XBP-1(S) when stimulated with LPS³⁸. Moreover, a kinetic analysis found that synthesis of XBP-1(S) precedes the massive increase in immunoglobulin translation during differentiation of the CH12 B cell lymphoma⁴². Thus, the nature of the signals that can elicit the UPR in physiologic settings may not yet be fully understood.

A final possibility is that various arms of the UPR can be specifically suppressed or inactivated. Two specific mechanisms for suppressing the PERK pathway have been identified (Fig. 3). First, p58^IPK inhibits PERK activation and eIF-2 phosphorylation^{49, 50}. p58IPK is a target of XBP-1(S)⁴⁸, raising the intriguing possibility that it abrogates PERK-mediated signaling in differentiating B cells. The second inhibitor of the PERK pathway is GADD34, which is induced as a result of eIF-2 phosphorylation and serves to dephosphorylate eIF-2²¹. Data for either of these PERK pathway antagonists in the context of plasma cell development are not yet available. For Ire1, negative regulators have not been identified in mammalian cells, but the Ptc2p phosphatase has been shown to inhibit Ire1p in yeast⁵¹.

Figure 3. Mechanisms for downregulating the PERK pathway. Prolonged inhibition of protein synthesis is detrimental to cells, and at least two UPR-induced components are involved in ensuring that the inhibition is transient. First, the dual action of ATF6 and Ire1 produce XBP-1(S), one of whose targets is p58IPK, which serves to inhibit PERK activation. Second, the arrest of protein synthesis allows translation of the ATF4 transcription factor, which induces GADD34, the regulatory subunit of the PP1 phosphatase that dephosphorylates eIF-2, thereby allowing translation to resume. It is possible that the PERK pathway is specifically repressed during plasma cell differentiation, through enhanced activity of one or both of these components. Full Figure and legend (13K)

Flexibility is likely to be a key feature of the physiologic UPR, allowing the response to appropriately fit the situation. For example, pancreatic cells must rapidly modulate protein synthesis according to sudden fluctuations in circulating glucose concentrations. As a result, the islet and acinar cells must cope with episodic high-rate synthesis of secretory-pathway proteins. It follows, therefore, that these cell types might be especially reliant on the ability of PERK to rapidly regulate translation. However, as B cells terminally differentiate, they initiate and accelerate antibody secretion, presumably to an optimal level, and then continue in this capacity until death. There is no evidence that plasma cells modulate the rate of antibody production, up or down, during their lifespan. Moreover, the differentiation process seems to prepare the cell for high-rate secretion by upregulating a wide array of metabolic processes and initiating expansion of the secretory pathway before the point of greatest immunoglobulin synthesis²⁹. Thus, a UPR-initiated, PERK-mediated repression of protein synthesis would be counterproductive for cells that are designed to maximally synthesize and secrete immunoglobulins. One could regard the PERK-mediated attenuation of translation as an 'emergency brake' that, in certain cell types under specific conditions, slows the flow of nascent polypeptides into the secretory pathway, thereby guarding against the potentially catastrophic effects of an overloaded ER. This translation 'brake' might be deliberately not in use when there is no need to balance high-rate protein production with long-term survival, as would be the case for short-lived plasma cells.

Figure 4. Interplay between transcriptional programs during plasma cell differentiation. Signaling through the BCR or LPS stimulation leads to a loss of Bcl6 (blue box), which relieves the repression on Blimp1. In turn, Blimp-1 represses Bcl6 transcription to maintain its own expression, blocks proliferation by repressing Myc and E2f1 and inducing the cyclin-dependent kinase inhibitor p18 (also known as Cdkn2c), and induces switching to the secretory form of immunoglobulin heavy chain. Additionally, Blimp-1 represses Pax5, which relieves the repression on the immunoglobulin genes, allowing their upregulation. In addition, the Blimp-1-mediated loss of Pax5 relieves the repression on Xbp1, resulting in larger amounts of unspliced XBP1(U) mRNA, as does ATF6 activation via the UPR (purple box denotes potential overlap between these two pathways). Activation of Ire1 (via UPR; orange box) induces processing of the XBP-1 mRNA to produce the remodeled form of the XBP-1 protein, XBP-1(S), which drives many of the requirements for high-level secretion. Only the PERK component of the UPR seems to remain inactive (yellow box), and this regulation might also be linked to XBP-1(S). Full Figure and legend (18K)

Other aspects of normal B cell physiology might also interface with the UPR. When membrane-bound H chains are first synthesized in early B cell development, subsequent assembly and testing of BCR complexes could conceivably increase demand on both the protein folding and degradative capacities of the ER. Thus, a careful examination is warranted of UPR status at the pro-B to pre-B and pre-B to B cell transitions, when the pre-BCR and BCR, respectively, are assembled and tested. Furthermore, signaling through the cell surface BCR mobilizes calcium from the ER, and drugs that perturb ER calcium stores are potent inducers of the UPR. Might BCR-mediated signaling interface with the UPR? If so, how might this influence the fate of antigen-stimulated B cells, activation of memory B cells and generation of antibody-secreting plasma cells?

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Competing interests statement:  The authors declare that they have no competing financial interests.