In all eukaryotic cells, the endoplasmic reticulum (ER) is the cellular organelle specialized for folding proteins that are destined for intracellular organelles or transit to the cell surface. Our primary interest is to elucidate fundamental mechanisms and the physiological impact of intracellular signaling pathways that control protein synthesis and the fidelity of protein folding in the ER.
Upon accumulation of unfolded proteins in the ER lumen, cells activate adaptive signaling pathways collectively called the unfolded protein response (UPR). The UPR signals transient attenuation of protein synthesis to reduce the protein-folding load and transcriptional induction of genes to expand the protein-folding and protein-degradative capacities of the ER (Figure 1). These responses are mediated by three ER-localized transmembrane sensors that monitor the protein-folding status in the ER lumen: (1) PERK is protein kinase that phosphorylates the α subunit of eukaryotic translation initiation factor 2 (eIF2α) to inhibit translation initiation. (2) IRE1 is a protein kinase and endoribonuclease that mediates nonconventional splicing of XBP1 mRNA to produce an active transcription factor. (3) ATF6 is an ER-localized transcription factor that is cleaved upon ER stress to generate a cytosolic fragment that translocates to the nucleus and activates transcription. If the adaptive response is inadequate to restore proper protein folding, cells enter programmed cell death. Our recent findings demonstrate that this life or death decision does not depend on selective activation of different UPR sensors, but rather is inherent in the signaling networks and differential instability of proapoptotic mRNAs and proteins.
We use a combination of structural biology, biochemistry, cell biology, and genetics to elucidate mechanisms that govern UPR activation and signaling, and to illuminate their physiological impact on differentiated cell types. Our studies demonstrate that the UPR is essential for expansion of the secretory pathway to support cellular differentiation and physiological function. These studies, which are focused on pancreatic β cells, B lymphocytes, and hepatocytes, demonstrate that these different cell types require different UPR subpathways for cell survival, differentiation, and function.
We have demonstrated that PERK-mediated eIF2α phosphorylation in pancreatic β cells couples proinsulin synthesis with the protein-folding capacity in the ER. Mice engineered with a mutation at the eIF2α phosphorylation site, Ser51Ala, demonstrated that modest defects in UPR signaling can contribute to obesity-induced β-cell failure. Furthermore, deletion of the proapoptotic gene Chop prevented β-cell failure associated with adult-onset type 2 diabetes (Figure 2). (A grant from the National Institutes of Health provided support for the work on eIF2α phosphorylation in diabetes.)
We are using gene deletion studies to analyze the functional significance of IRE1 signaling in secretory cells. Although embryonic mice with IRE1 deletion die, our analysis demonstrated a specific requirement for IRE1 at two unique steps in B lymphocyte differentiation. First, IRE1, but not its protein kinase or endoribonuclease activities, is required to initiate recombination at the immunoglobulin heavy gene locus. Second, IRE1 endoribonuclease activity and XBP1 mRNA splicing are required for mature B cell differentiation into plasma cells to produce high levels of secreted immunoglobulins. The findings reveal an unanticipated complexity in the UPR and its connection to B cell differentiation. To further elucidate how IRE1 regulates B cell differentiation, we have initiated structural studies on the luminal domain of human IRE1 that senses unfolded proteins. The x-ray crystal structure of human IRE1 revealed that it is composed of a triangular cluster of β sheets, for which one surface is required for dimerization and UPR signaling. Future studies should provide insight into how dimerization is regulated and what transitions are required to activate kinase and endoribonuclease activities that regulate B cell differentiation.
Although there are two ATF6 genes, ATF6α and ATF6β, in the mammalian genome, their contribution to UPR signaling is unknown. Our gene deletion studies in the mouse demonstrated that neither is required for normal growth and development. ATF6α is, however, required for survival in response to ER stress because it activates transcription of genes encoding protein-folding and degradative machinery to reduce the unfolded protein burden on the ER compartment. These findings indicate that ATF6 has evolved as a mechanism to promote cell survival in response to chronic stress.
We recently identified a novel cyclic AMP response element–binding protein (CREBH) as a hepatocyte-specific basic leucine zipper–containing transcription factor with a domain structure similar to ATF6. CREBH transcription is induced by proinflammatory cytokines, and its cleavage is activated by ER stress. In contrast to ATF6, however, CREBH does not activate UPR genes, but rather is required to induce transcription of the systemic arm of the inflammatory response. These studies have identified a novel association between intracellular ER stress and inflammation that we are investigating.
To determine how the presence of unfolded protein in the ER lumen regulates hepatocyte function, we have studied coagulation factor VIII (FVIII), the protein deficient in the X chromosome–linked bleeding disorder hemophilia A. Upon synthesis, FVIII forms aggregates in the ER lumen that activate the UPR and cause cell death. Our recent findings have uncovered the surprising hypothesis that oxidative stress may be the mechanism that links protein misfolding in the ER with cell death. In our future studies, we will dissect fundamental and unique mechanisms by which protein misfolding in the ER lumen leads to oxidative stress. (Grants from the National Institutes of Health provided support for the work on FVIII expression.)
It is now evident that the UPR is involved in the etiology of a growing number of diseases, such as metabolic disease, Alzheimer's disease, and cancer. Our studies into the fundamental processes of how the ER couples the protein-folding load with the protein-folding capacity may provide new approaches for the treatment of diseases of protein misfolding.