Wild-type HEK293 cells treated with tunicamycin (right panel) or mock-treated with solvent (left panel) over 2 days.
HEK293 cells expressing Fv2E-PERK treated with AP20187 (right panel) or mock-treated (left panel) over 2 days.
CELLULAR RESPONSES TO PROTEIN MISFOLDING
Organism health depends on the accuracy of the signals sent and received by constituent cells. Proteins, either secreted from the cell or embedded in the plasma membrane to monitor the environment, transmit much of this information. On the basis of these signals, cells make vital decisions - when and where to divide, migrate or change shape, differentiate, or die. Cells have evolved elaborate mechanisms to ensure the accuracy with which proteins are folded and assembled before export or transport to the cell surface. Stringent quality control is imposed by the endoplasmic reticulum (ER), a membrane-bound labyrinth of tubes and sacs, where virtually all plasma membrane and secreted proteins begin their journey to the surface. Only properly folded proteins are allowed to leave the ER; misfolded proteins are degraded. In this way, cells display or release only high-quality, functional proteins.
To maintain fidelity, the cell needs to fold proteins as they are made, and this system needs to adapt to changing environmental conditions. This feat is achieved by a set of intracellular signaling pathways, collectively termed the "unfolded protein response· (UPR), which sense when the ER has accumulated too many misfolded proteins and in turn, activate transcription of certain genes that enhance the ER's protein folding capacity as needed. UPR signaling can protect cells from ER stress by expanding the amount of ER in the cell, enhancing the degradation of misfolded proteins, and reducing the synthesis of new proteins. But if homeostasis cannot be reestablished, UPR signaling eventually induces cell death by apoptosis, an effective means of protecting the organism from rogue cells expressing dysfunctional or even toxic signaling molecules. Our research focuses on 1) understanding the molecular mechanisms by which UPR signaling protects cells or alternatively promotes cell death and 2) investigating the role of UPR signaling in the pathogenesis of human diseases associated with protein misfolding.
Characterize the molecular mechanisms of UPR signaling
The molecular gatekeepers of the UPR are ER-resident transmembrane proteins with luminal domains that monitor the quality of protein folding in the ER coupled across the ER membrane to cytosolic effector domains that transmit that information to the rest of the cell. IRE1 and PERK independently govern two key UPR signal transduction pathways. Protein misfolding concomitantly activates IRE1 and PERK, thereby obscuring insight into protective or proapoptotic contributions of these two signaling pathways toward life or death cell fates. We created artificial forms of analog, 1NM-PP1, or the FK506 derivative, AP20187. We created isogenic human cell lines bearing these chemicallv sensitized alleles which allowed us to selectively activate IRE1 or PERK signaling independent of protein misfolding. Using this strategy, we demonstrated that IRE1 signaling promoted cell survival but surprisingly, this signaling pathway was specifically shut down by persistent ER stress. By contrast, we demonstrated that sustained PERK signaling directly impaired cell viability by inhibiting proliferation and promoting apoptosis (Figure 1 ). These findings provide a molecular basis to investigate how UPR signaling promotes cell survival or cell death in response to protein misfolding. These findings lead to next level questions that we are currently studying such as: How does chronic protein misfolding attenuate IRE1 signaling? How does PERK signaling at the ER lead to activation of the intrinsic apoptosis.
Characterize the role of UPR signaling in protein folding diseases
Protein misfolding occurs in numerous human diseases including cancer, diabetes, neurodegeneration, and viral infections. Rhodopsin is a membrane protein specifically expressed by photoreceptor neurons in the eye. Rhodopsin folds and matures in the ER before export to the photoreceptor outer segment where it functions as a light sensor. Mutations in rhodopsin underlie many forms of retinitis pigmentosa, a disease in which photoreceptor cells die ultimately leading to blindness for the individual. The most common form of heritable retinitis pigmentosa in people arises from a praline to histidine mutation at amino acid 23 (P23H) in rhodopsin. P23H rhodopsin is misfolded and retained in the ER. Photoreceptors expressing P23H rhodopsin eventually die leading to blindness. We have examined transgenic animal models of retinitis pigmentosa in which P23H rhodopsin expression in photoreceptors leads to death of the photoreceptor cells and organismal blindness (Figure 2). We have demonstrated that UPR signaling pathways are abnormally dysregulated in the retinas of animals expressing P23H rhodopsin. We are using and developing genetic, chemical-genetic, and pharmacologic tools to manipulate specific UPR signaling pathways. We are evaluating the effects of activation or inhibition of individual UPR signaling pathways on the onset and progression of retinal degeneration in animals expressing misfolded rhodopsin. These approaches may be broadly applicable to other diseases associated with ER stress and protein misfolding.