DNA damage can occur through diverse stimulations such as toxins, drugs, and environmental factors. To respond to DNA damage, mammalian cells induce DNA damage response (DDR). DDR signal activates a rapid signal transduction pathway, regulating the cell fate based on the damaged cell condition. Moreover, serious damaged cells have to be eliminated by the macrophage to maintain homeostasis. Because the DDR induces genomic instability followed by tumor formation, targeting the DDR signaling can be applied for the cancer therapy. Herpes virus-associated ubiquitin-specific protease (HAUSP/USP7) is one of the well-known deubiquitinating enzymes (DUBs) owing to its relevance with Mdm2-p53 complex. The involvement of HAUSP in DDR through p53 led us to investigate novel substrates for HAUSP, which is related to DDR or apoptosis. As a result, we identified annexin-1 (ANXA1) as one of the putative substrates for HAUSP. ANXA1 has numerous roles in cellular systems including anti-inflammation, damage response, and apoptosis. Several studies have demonstrated that ANXA1 can be modified in a post-translational manner by processes such as phosphorylation, SUMOylation, and ubiquitination. In addition, DNA damage gives various functions to ANXA1 such as stress response or cleavage-mediated apoptotic cell clearance. In the current study, our proteomic analysis using two-dimensional electrophoresis, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) and nano LC-MS/MS, and immunoprecipitation revealed that ANXA1 binds to HAUSP through its HAUSP-binding motif (P/AXXS), and the cleavage and damage-responsive functions of ANXA1 upon UV-induced DNA damage may be followed by HAUSP-mediated deubiquitination of ANXA1. Intriguingly, the UV-induced damage responses via HAUSP-ANXA1 interaction in HeLa cells were different from the responses shown in the Jurkat cells, suggesting that their change of roles may depend on the cell types.

Summary 3′,3′‐cyclic GMP‐AMP (cGAMP) is the third cyclic dinucleotide (CDN) to be discovered in bacteria. No activators of cGAMP signaling have yet been identified, and the signaling pathways for cGAMP have been inferred to display a narrow distribution based upon the characterized synthases, DncV and Hypr GGDEFs. Here, we report that the ubiquitous second messenger cyclic AMP (cAMP) is an activator of the Hypr GGDEF enzyme GacB from Myxococcus xanthus. Furthermore, we show that GacB is inhibited directly by cyclic di‐GMP, which provides evidence for cross‐regulation between different CDN pathways. Finally, we reveal that the HD‐GYP enzyme PmxA is a cGAMP‐specific phosphodiesterase (GAP) that promotes resistance to osmotic stress in M. xanthus. A signature amino acid change in PmxA was found to reprogram substrate specificity and was applied to predict the presence of non‐canonical HD‐GYP phosphodiesterases in many bacterial species, including phyla previously not known to utilize cGAMP signaling.

SUMMARYA newfound signaling pathway employs a GGDEF enzyme with unique activity compared to the majority of homologs associated with bacterial cyclic di-GMP signaling. This system provides a rare opportunity to study how signaling proteins natively gain distinct function. Using genetic knockouts, riboswitch reporters, and RNA-Seq, we show that GacA, the Hypr GGDEF in Geobacter sulfurreducens, specifically regulates cyclic GMP-AMP (3′,3′-cGAMP) levels in vivo to stimulate gene expression associated with metal reduction separate from electricity production. To reconcile these in vivo findings with prior in vitro results that showed GacA was promiscuous, we developed a full kinetic model combining experimental data and mathematical modeling to reveal mechanisms that contribute to in vivo specificity. A 1.4 Å-resolution crystal structure of the Geobacter Hypr GGDEF domain was determined to understand the molecular basis for those mechanisms, including key cross-dimer interactions. Together these results demonstrate that specific signaling can result from a promiscuous enzyme.3