There are five subfamilies of

There are five subfamilies of P-type ATPases (I-V), which transport metal ions or phospholipids across membranes (Palmgren and Nissen, 2011). Although our studies indicate that the P-type ATPase domain is important for function, how it contributes to cGMP production is currently unknown. The P-type ATPase domain could feasibly regulate GC domain activity by providing a metal cofactor such as Mg2+ or Mn2+ (Type II-like) or by promoting interaction of the two membrane-embedded CHDs by altering the surrounding lipid composition (Type IV-like). The P-type ATPase domain may also provide subcellular targeting information that positions TgGC near the site of PKG-dependent microneme Tanshinone IIA---sulfonic sodium at the apical plasma membrane. The fusion of the two domains may also promote the proper folding of TgGC needed for enzyme activity. The precise mechanism by which the P-type ATPase domain contributes to TgGC domain function warrants future investigation. In summary, our findings, taken together with previous studies, define the mechanistic basis for the initiating step of cGMP signaling in T. gondii. Previous studies implicated TgGC in relaying extracellular signals (e.g., BSA), via the second messenger cGMP, to control essential parasite processes. Here we have uncovered TgGC as the upstream regulator of cGMP signaling and provided key functional analysis of the domain structure for this enzyme class, which revealed a critical requirement for both P-type ATPase and guanylate cyclase activities in controlling microneme secretion and parasite viability. The essential nature of TgGC was also extended in an animal model of infection, in which loss of TgGC rendered T. gondii avirulent, reducing parasite expansion and dissemination and preventing lethal toxoplasmosis. Collectively, these findings suggest that the P-type ATPase domain performs an essential function to activate or control the GC domain, thus generating cGMP to activate PKG. Given the conserved nature of PKG signaling in apicomplexans, the unique domain architecture of the GC is likely to control similar essential processes in related protists.
STAR★Methods
Acknowledgments We thank Qiuling Wang for technical assistance with mouse experiments and Jennifer Barks for technical assistance with tissue culture. This work was supported in part by grants from the National Institutes of Health to L.D.S. (AI034036) and the American Heart Association (15POST22130001) to K.M.B.
Introduction Colorectal cancer (CRC) cancer is one of the leading causes of cancer-related deaths in the US and world-wide [[1], [2], [3]]. An estimated 140,250 cases of CRC will be diagnosed in the US in 2018 [3]. Identification of novel biomarkers/targets is needed for early diagnosis and to aid in the development of novel chemopreventive and therapeutic agents for CRC. Several research groups have worked to identify novel biomarkers/targets in CRC. A significant progress has been made in understanding the underlying histopathologic and molecular processes in the transition from normal epithelium to an invasive adenocarcinoma. The adenomatous polyposis coli (APC) gene is mutated in more than 80% of patients with CRC [4]. However, APC mutation and activation of the wnt/β-catenin cascade is insufficient for CRC initiation and progression. Disruption of the homeostasis of intestinal epithelial cells is associated with tumorigenesis and progression, which results from dysregulation of several signaling processes, including the guanylate cyclase (GUCY2C)/cyclic GMP/ Phosphodiesterases (PDEs) pathway. GUCY2C, a membrane-bound guanylate cyclase receptor, is present in the apical membranes of intestinal epithelial cells from the duodenum to the distal rectum [5,6]. It is a key regulator of secretions of intestinal fluid and electrolytes, and the pathophysiology of enterotoxigenic diarrhea [5,6]. Paracrine hormones, such as guanylin (found throughout the intestine) and uroguanylin (present selectively in small intestine), and the enterotoxins of diarrheagenic bacteria are cognate ligands, structurally peptides, that, when bound to GUCY2C, lead to the formation of cyclic GMP (cGMP) from GTP [5,6]. Further, the cGMP-dependent protein kinases, also called as protein kinase G (PKG), are activated as a result of formation of cGMP, and transduce the signals from GUCY2C though activation of downstream signaling mediators [7].