Extending the Role of DDX3X in the Cellular Stress Response

DEAD-box helicase 3, X linked (DDX3X) is a host protein best characterised for its antiviral roles in the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signalling pathway. It also has crucial functions in both pro-survival stress granule and pro-death nucleotide oligomerisation domain (NOD)-like receptor pyrin domain-containing 3 (NLRP3) inflammasome formation, which occurs under cellular stress conditions. Therefore, DDX3X plays multiple roles in cell fate decisions following cellular stress. In addition, macrophage migration inhibitory factor (MIF) has been identified as an enzyme with similar roles to DDX3X in NLRP3 inflammasome formation. DDX3X is known to have both nuclear and cytoplasmic roles, though DDX3X nuclear transport pathways are poorly defined currently. Interestingly, importin 13 (IMP13) is a unique nuclear transport receptor that functions under cellular stress conditions, due to a reduced dependence on a classically vital Ras-related nuclear protein (Ran) nucleocytoplasmic gradient. While this gradient of high concentration RanGTP in the nucleus and a low concentration in the cytoplasm collapses under cellular stress, such as is caused by heat or oxidation, the exact mechanisms of collapse are poorly understood. Given the various roles of DDX3X under cellular stress, IMP13 is worth investigating as a possible nucleocytoplasmic transporter of DDX3X under these conditions. Using co-immunoprecipitation (co-IP) assays, wild-type (WT) DDX3X and WT and truncation mutants of IMP13, a novel interaction between DDX3X and IMP13 has been shown and the IMP13 interface mapped to residues 526-574. Further co-IP experiments under stress conditions will be required to fully determine when the interaction takes place, and IMP13 knockout (KO) or knockdown (KD) experiments are required to confirm whether DDX3X is a functional IMP13 cargo. Given that IMP13 utilises a Ran gradient for its function, we next investigated the effect of cellular stress conditions on Ran localisation to determine those that cause Ran gradient collapse. For the first time, the DDX3X catalytic inhibitor RK-33 was shown to collapse the Ran gradient, implicating DDX3X catalytic activity in the maintenance of the Ran gradient. Similarly, overexpression of the DDX3X catalytic mutant K230E also collapses the Ran gradient under stress conditions. Additionally, and again for the first time, the MIF tautomerase inhibitors ISO-1 and P425 were shown to collapse the Ran gradient, suggesting that MIF tautomerase activity is potentially also involved in the maintenance of the Ran gradient. Together these results indicate a potential role of DDX3X catalytic activity in maintaining the Ran gradient under conditions of cellular stress, such as is seen in viral infection, potentially enabling the antiviral or pro-cell survival functionality of both DDX3X and other related factors in viral and cellular stress response pathways. While further studies are required to establish a potential link between the DDX3X-IMP13 interaction, MIF, and modulation of the Ran gradient, as well as their functional implications for the antiviral response and cell fate, a DDX3X-IMP13 or DDX3X-Ran interaction may be useful therapeutic targets in modulating viral or cellular stress responses.