In this report, we determined that MPYS, a potent stimulator IFNβ production, is a ROS sensor. We further found that the IFNβ stimulation by MPYS can be inhibited by high levels of ROS, and ROS stimulate MPYS oxidation as manifest by formation of intermolecular disulfide bonds within MPYS homodimers. We conclude that MPYS senses the Redox state of the cell and under conditions of high oxidative stress is “turned off”, presumably by oxidation.
ROS play an important role in regulation of innate immune responses and the strength of the ROS signals determines the outcome of such responses 
. For example, low levels of H2
activate the p53 antioxidant response, whereas high levels trigger p53-dependent apoptosis 
. The effect of ROS on MPYS function likely also depends on the levels of ROS. Rotenone treatment, which produces sustained cellular ROS, may cause irreversible modification on critical cysteines in MPYS thus inhibits the MPYS-mediated IFNβ response. Interestingly, NLRX1, a negative regulator of the anti-viral response 
, triggers strong ROS production (comparable to the level triggered by TNFα) 
. Both NLRX1 and MPYS can be found in mitochondria 
. It is tempting to suggest that NLRX1 negatively regulates the IFNβ response by producing strong ROS, inhibiting MPYS activity.
MPYS contains 6 critical cysteines important for IFNβ stimulation. Among them, Cys-64, Cys-148 and C88
are targets of cellular ROS. The other two Cys are adjacent to Arg (Cys292
). The positive charge of Arg293 or Arg310 may lower the pK
a of Cys292 or Cys309 so that they may also be targets of ROS 
. The question is, then, why these cysteines are important for IFNβ stimulation. Previous studies propose that MPYS/STING functions as an adaptor protein that recruits ser/thr kinase TBK1 
. We find that TBK1 associates with both oxidized and reduced MPYS (Figure S4a
). More importantly, both the C148S and C88
mutant have normal TBK1 association (Figure S4b
). Considering the fact that the CxxC motifs are often found in the oxidoreductases and are essential for their catalysis of redox reactions 
, we hypothesize that MPYS may also have the oxidoreductase activity and TBK1 may be its substrate.
Previous over-expression studies have placed MPYS in the ER and mitochondrial outer membrane 
. The mitochondrial outer membrane is physically and physiologically connected to the ER 
. This physical link may facilitate Ca2+ 
and, we suggest, ROS communication between ER and mitochondria. We found that the ER stress inducer, Brefeldin A, also generated oxidized MPYS (Figure S2a
). Thus, MPYS may also act in ER stress sensing.
The mitochondrial intermembrane space (IMS) is connected to the cytosol by porins in the outer membrane of mitochondria which allow the diffusion of small ions like glutathione 
. Thus the environment of IMS is less oxidizing than that of the ER lumen. Recently, a group of interacting mitochondrial proteins, including MPYS, VISA, NLRX1 and most recently Mitofusin 2, have been identified as key components of the innate intracellular viral sensing pathway  
. In light of our current discovery that the MPYS is a Redox sensor, we suggest that the antiviral response also utilizes mitochondrial ROS as second messenger.
In conclusion, we have shown that the IFNβ stimulator, MPYS is a ROS sensor and its signaling function is regulated by ROS. Future studies need to be done to determine if MPYS is indeed an oxidoreductase and the in vivo biological significance of that activity.