Ptosis evaluation through annexin V analysis (G) and caspase 3/7 activity detection (H). (I and J) TRAF6 suppresses ROS production by regulating p53. Mud crabs had been cotreated with TRAF6 siRNA and p53 siRNA for 48 h, followed by ROS measurement using a fluorescence microscope (I) and microplate reader (J). Data represent the mean 6 SD from triplicate assays (, P , 0.01).As a result, the protein level of p53 is tightly controlled in cells. MDM2 was the initial identified p53 E3 ubiquitin ligase (33), followed by PIRH2 (ring finger and CHY zinc finger domain containing 1) (34), HUWE1, WWP1 (WW domain-containing E3 ubiquitinprotein ligase 1) (35), and COP1 (constitutively photomorphogenic 1) (36), which possess fundamental roles within the ubiquitin-dependent p53 protein degradation pathway. Having said that, current study indicated that p53 ubiquitination only requires an individual E3 ubiquitin ligase (37); nevertheless, no matter whether there exist other mechanisms needs to beMarch 2022 Volume 96 Situation 6 e02029-21 jvi.asm.orgGong et al.Journal of VirologyFIG 6 HUWE1 and TRAF6 serve as negative regulators for apoptosis and ROS signals by suppressing p53. (A) The influence of WSSV infection on HUWE1 and TRAF6 mRNA expression levels. Mud crabs were challenged with WSSV for 24 h, then the mRNA levels of HUWE1 and TRAF6 had been detected by qPCR. (B) The influence of WSSV infection on HUWE1 and TRAF6 protein levels in mud crabs. (C) The effects of HUWE1 and TRAF6 silencing on WSSV replication. Mud crabs challenged with WSSV infection had been coinjected with HUWE1 siRNA or TRAF6 siRNA for 48 h, after which WSSV copy numbers had been measured. (D and E) HUWE1 and TARF6 promote WSSV infection by suppressing apoptosis signals in mud crabs. Mud crabs challenged with WSSV infection had been coinjected with HUWE1 siRNA or TRAF6 siRNA for 48 h, followed by apoptosis evaluation by way of annexin V analysis (D) and caspase 3/7 activity detection (E). (F and G) HUWE1 and TARF6 market WSSV infection by inhibiting ROS production in mud crabs. Mud crabs challenged with WSSV were coinjected with HUWE1 siRNA or TRAF6 siRNA for 48 h, followed by ROS measurement employing a fluorescence microscope (F) and microplate reader (G). Asterisks indicate substantial differences (, P , 0.01).explored. In this study, we identified that both HUWE1 and TRAF6 could serve as p53 E3 ubiquitin ligases and synchronously mediate p53 ubiquitination in mud crabs.NAMPT Protein Formulation To the most effective of our expertise, that is the initial report to confirm that p53 could be simultaneously regulated by two E3 ubiquitin ligases.PDGF-DD Protein custom synthesis This indicates the diversity of the p53 ubiquitination regulatory approach and offers novel insights in to the innate immunity of invertebrates.PMID:24103058 The functions and regulatory mechanisms of p53 (including the E3 ligase-mediated p53 ubiquitination) happen to be extensively investigated in the past handful of decades (38).March 2022 Volume 96 Problem 6 e02029-21 jvi.asm.orgp53 Ubiquitination Contributes to WSSV InfectionJournal of VirologyFIG 7 The proposed schematic diagram for p53 ubiquitination-mediated apoptosis, ROS, and regulation of virus invasion in mud crabs. During WSSV infection, the expression of HUWE1 and TRAF6 was suppressed: thus, the ubiquitination of p53 was interrupted, which further led to the accumulation of p53 protein, which sooner or later activated apoptosis and ROS signals to cope with virus invasion in the mud crabs.The research has shown that p53 ubiquitination doesn’t merely bring about protein degradation. For example, the MDM2-mediated p53.
