Ryoo Lab Research
In the Ryoo Lab at NYU Langone, we study how cells cope with stress. We are particularly interested in stress caused by misfolded proteins in the endoplasmic reticulum (ER) and amino acid deprivation. These conditions specifically activate signaling pathways that are referred to as the unfolded protein response (UPR) and the integrated stress response (ISR).
Defects in these signaling pathways can cause cellular dysfunction and various degenerative diseases, whereas appropriate activation of these pathways can enhance our resistance to those conditions. We use various genetic, cellular, and biochemical tools to investigate how these signaling pathways are regulated and to identify their physiological roles.
Endoplasmic Reticulum Stress Response in Eye Development and Disease
Chronic ER stress is associated with a wide variety of diseases. To understand the basis of these diseases, we have been focusing on a fly model for the human disease called autosomal dominant retinitis pigmentosa. Mutations in the rhodopsin gene most frequently underlie this disease. Interestingly, fruit flies also have rhodopsin-1 mutant alleles that show similar symptoms—retinal degeneration in old individuals. Using genetic and cell biological tools, we helped establish that these rhodopsin-1 alleles encode proteins that fail to fold properly in the ER. As a result, the UPR branch mediated by IRE1 and XBP1 genes are activated in the afflicted fly retina and serve to protect those cells from degeneration (Ryoo et al., 2007, EMBO J).
Our laboratory has carried out a number of Drosophila genetic screens and identified several new factors that affect the course of retinal degeneration and regulate the UPR in this disease model. Through a gene overexpression screen, we identified hrd1 as a strong suppressor of retinal degeneration. hrd1 encodes a ubiquitin ligase that integrates into the ER membrane and suppresses the toxicity associated with mutant rhodopsin-1 by promoting its degradation (Kang and Ryoo, 2009, Proc Natl Acad Sci).
In addition, we identified highroad, a carboxypeptidase, that is required for mutant rhodopsin-1 degradation. Interestingly, we find evidence that highroad is induced by retinoic acids, and this prompts us to hypothesize that retinoic acid signaling is involved in the degradation of mutant rhodopsins in photoreceptors (Huang et al., 2018, Cell Rep). We have also performed RNAi screens to identify CDK5 as an important kinase that promotes cell death in this disease model (Kang et al., 2012, Nat Cell Biol).
More recently, we began studying why UPR mediating genes are developmentally essential, even without exposure to exogenously imposed stress. For example, Ire1 mutants fail to survive beyond the first instar larval stage, and Xbp1 mutants die during the second instar larval stage. We recently demonstrated that there is physiological ER stress that requires the help of these UPR mediators for resolution, and without IRE1 activating its downstream target XBP1, certain types of normally developing tissues show signs of stress (Huang et al., 2017, J Cell Sci).
Integrated Stress Response in Drosophila
Excessive protein misfolding in the ER and amino acid deprivation commonly activates the ISR stress response pathway. These pathways are initiated by stress activated kinases PERK and GCN2, which commonly phosphorylate the translational initiation factor eIF2a, thereby reducing overall protein synthesis rate in stressed cells. Intriguingly, the mRNA encoding the transcription factor ATF4 has unique properties that block its translation in unstressed cells, but paradoxically stimulate its translation when global translation is attenuated in response to eIF2a phosphorylation. Such ATF4 induction in stressed cells allow coupling of stress-imposed translational suppression with activation of a gene transcription program.
We recently found that the ATF4-mediated ISR signaling activates the transcription of yet another translational inhibitor, 4E-BP, and this axis allows Drosophila to better resist retinal degeneration caused by rhodopsin-1 mutation, and allow lifespan extension upon reduction of yeast content in food (Kang et al., 2017, J Cell Biol). Moreover, we found that the ATF4/4E-BP axis helps to boost innate immune response in Drosophila.
Interestingly, 4E-BP induction in response to bacterial infection still allows a robust translation of antimicrobial peptide transcripts as the 5'UTR of those mRNAs contain internal ribosome entry sites (IRES). Based on our finding that ATF4 induces 4E-BP transcription, we generated a 4E-BP-dsRed transgene that robustly reports physiological ATF4 activity in live flies. Using this as a tool, we have identified noncanonical initiation factors eIF2D and DENR as factors required for ATF4 signaling (Vasudevan et al., 2020, Nat Commun). In the Drosophila larval fat body, we find that the amino acid deprivation-activated kinase GCN2 is responsible for the physiological activation of the ATF4-4E-BP axis. We are currently screening for additional regulators of this pathway, with a particular focus to understand how cells sense amino acid deprivation.