Human TP53 gene encodes the tumor suppressor p53 and, via alternative splicing, the p53β and γ isoforms. Numerous studies have shown that p53β/γ can modulate p53 functions and are critically involved in regulation of cellular response to stress conditions. However, it is not fully understood how the β and γ isoforms are regulated following splicing. Using gene targeting and RNAi, we showed that depletion of the nonsense-mediated mRNA decay (NMD) factor SMG7 or UPF1 significantly induced p53β but had minimal effect on p53γ. Sequence analysis reveals the presence of unique features – key hallmarks of NMD targets in the p53β transcript, which was further confirmed in NMD reporter gene assays. By manipulating splicing components, we found that NMD activities are crucial to control p53β levels under conditions that favor its splicing. Our data demonstrate that the NMD and alternative splicing pathways regulate p53β in a synergistic manner, and NMD plays a critical role in the determination of the p53β following its splicing. As aberrant p53β expression and dysfunctional NMD are both implicated in cancers, our studies may provide a novel insight into the regulation of p53β in tumorigenic settings.

The p53 tumor suppressor functions as a transcription factor and plays a pivotal role in regulation of cellular response to DNA damage by activating various genes including those involved in cell cycle arrest. p53 stability is essential for its function during stress response; however, the molecular mechanism for DNA damage-induced stabilization of p53 is not fully understood. In our present study, we have identified SMG7 (suppressor with morphological defects in genitalia 7), also known as EST1C, as a novel p53-binding protein. SMG7 is an mRNA surveillance factor implicated in degradation of p53 mRNA-containing nonsense mutations, yet it is completely unknown whether SMG7 regulates p53 function. Here, we show that SMG7 has a crucial role in p53-mediated response to genotoxic stress by regulating p53 stability. Using somatic gene knockout, we found that deletion of SMG7 abrogates DNA damage-induced p53 stabilization, although it exhibits minimal effect on the basal levels of p53. Importantly, loss of SMG7 impairs p53-mediated activation of p21 and cell cycle arrest following DNA damage. Pharmacological inhibition of Mdm2, a major E3 ubiquitin ligase for p53, restored p53 stability in gamma-irradiated SMG7-deficient cells. Furthermore, SMG7 physically interacts with Mdm2 and promotes ATM-mediated inhibitory phosphorylation of Mdm2 following ionizing radiation. Therefore, our present data demonstrate that SMG7 is critical for p53 function in DNA damage response, and reveal the SMG7-mediated phosphorylation of Mdm2 as a previously unknown mechanism for p53 regulation.

Identifying correlates of protection (COPs) for vaccines against lethal human (Hu) pathogens, such as Francisella tularensis (Ft), is problematic, as clinical trials are currently untenable and the relevance of various animal models can be controversial. Previously, Hu trials with the live vaccine strain (LVS) demonstrated ~80% vaccine efficacy against low dose (~50 CFU) challenge; however, protection deteriorated with higher challenge doses (~2000 CFU of SchuS4) and no COPs were established. Here, we describe our efforts to develop clinically relevant, humoral COPs applicable to high-dose, aerosol challenge with S4. First, our serosurvey of LVS-vaccinated Hu and animals revealed that rabbits (Rbs), but not rodents, recapitulate the Hu O-Ag dependent Ab response to Ft. Next, we assayed Rbs immunized with distinct S4-based vaccine candidates (S4ΔclpB, S4ΔguaBA, and S4ΔaroD) and found that, across multiple vaccines, the %O-Ag dep Ab trended with vaccine efficacy. Among S4ΔguaBA-vaccinated Rbs, the %O-Ag dep Ab in pre-challenge plasma was significantly higher in survivors than in non-survivors; a cut-off of >70% O-Ag dep Ab predicted survival with high sensitivity and specificity. Finally, we found this COP in 80% of LVS-vaccinated Hu plasma samples as expected for a vaccine with 80% Hu efficacy. Collectively, the %O-Ag dep Ab response is a bona fide COP for S4ΔguaBA-vaccinated Rb and holds significant promise for guiding vaccine trials with higher animals.

The 14-3-3-related protein SMG7 plays critical roles in regulation of DNA damage response and nonsense-mediated mRNA decay (NMD). Like 14-3-3, SMG7 engages phosphoserine-dependent protein interactions; however, the precise role of phosphorylation-mediated SMG7 binding remains unknown. Here, we show that DNA damage-induced SMG7-p53 binding requires phosphorylated Ser15 on p53, and that substitution of the conserved lysine residue K66 in the SMG7 14-3-3-like domain with the glutamic acid (E) abolishes interactions with its client proteins p53 and UPF1. Unexpectedly, loss of phosphoserine-dependent SMG7 binding does not significantly affect p53 stabilization/activation, and p53-dependent cell growth arrest or apoptosis upon DNA damage. Also surprisingly, cells expressing the SMG7 K66E-knockin mutant retain fully functional UPF1-mediated NMD. These findings are highly unusual, given that phosphorylation-mediated 14-3-3 binding has essential roles in numerous cellular signaling pathways. Thus, our studies suggest that 14-3-3-like proteins such as SMG7 likely function using additional distinct regulatory mechanisms besides phosphoserine-mediated protein interactions.

The targeting of immunogens/vaccines to specific immune cells is a promising approach for amplifying immune responses in the absence of exogenous adjuvants. However, the targeting approaches reported thus far require novel, labor-intensive reagents for each vaccine and have primarily been shown as proof-of-concept with isolated proteins and/or inactivated bacteria. We have engineered a plasmid-based, complement receptor-targeting platform that is readily applicable to live forms of multiple gram-negative bacteria, including, but not limited to, Escherichia coli, Klebsiella pneumoniae, and Francisella tularensis. Using F. tularensis as a model, we find that targeted bacteria show increased binding and uptake by macrophages, which coincides with increased p38 and p65 phosphorylation. Mice vaccinated with targeted bacteria produce higher titers of specific antibody that recognizes a greater diversity of bacterial antigens. Following challenge with homologous or heterologous isolates, these mice exhibited less weight loss and/or accelerated weight recovery as compared to counterparts vaccinated with non-targeted immunogens. Collectively, these findings provide proof-of-concept for plasmid-based, complement receptor-targeting of live gram-negative bacteria.