Mechanism of phosphate sensing and signaling revealed by rice SPX1-PHR2 complex structure

Phosphate, a key plant nutrient, is perceived through inositol polyphosphates (InsPs) by SPX domain-containing proteins. SPX1 an inhibit the PHR2 transcription factor to maintain Pi homeostasis. How SPX1 recognizes an InsP molecule and represses transcription activation by PHR2 remains unclear. Here we show that, upon binding InsP6, SPX1 can disrupt PHR2 dimers and form a 1:1 SPX1-PHR2 complex. The complex structure reveals that SPX1 helix α1 can impose a steric hindrance when interacting with the PHR2 dimer. By stabilizing helix α1, InsP6 allosterically decouples the PHR2 dimer and stabilizes the SPX1-PHR2 interaction. In doing so, InsP6 further allows SPX1 to engage with the PHR2 MYB domain and sterically block its interaction with DNA. Taken together, our results suggest that, upon sensing the surrogate signals of phosphate, SPX1 inhibits PHR2 via a dual mechanism that attenuates dimerization and DNA binding activities of PHR2.

New insights into the evolution of SPX gene family from algae to legumes; a focus on soybean

Background SPX-containing proteins have been known as key players in phosphate signaling and homeostasis. In Arabidopsis and rice, functions of some SPXs have been characterized, but little is known about their function in other plants, especially in the legumes. Results We analyzed SPX gene family evolution in legumes and in a number of key species from algae to angiosperms. We found that SPX harboring proteins showed fluctuations in domain fusions from algae to the angiosperms with, finally, four classes appearing and being retained in the land plants. Despite these fluctuations, Lysine Surface Cluster (KSC), and the third residue of Phosphate Binding Sites (PBS) showed complete conservation in almost all of SPXs except few proteins in Selaginella moellendorffii and Papaver sumniferum, suggesting they might have different ligand preferences. In addition, we found that the WGD/segmentally or dispersed duplication types were the most frequent contributors to the SPX expansion, and that there is a positive correlation between the amount of WGD contribution to the SPX expansion in individual species and its number of EXS genes. We could also reveal that except SPX class genes, other classes lost the collinearity relationships among Arabidopsis and legume genomes. The sub- or neo-functionalization of the duplicated genes in the legumes makes it difficult to find the functional orthologous genes. Therefore, we used two different methods to identify functional orthologs in soybean and Medicago. High variance in the dynamic and spatial expression pattern of GmSPXs proved the new or sub-functionalization in the paralogs. Conclusion This comprehensive analysis revealed how SPX gene family evolved from algae to legumes and also discovered several new domains fused to SPX domain in algae. In addition, we hypothesized that there different phosphate sensing mechanisms might occur in S. moellendorffii and P. sumniferum. Finally, we predicted putative functional orthologs of AtSPXs in the legumes, especially, orthologs of AtPHO1, involved in long-distance Pi transportation. These findings help to understand evolution of phosphate signaling and might underpin development of new legume varieties with improved phosphate use efficiency.

New insights into the evolution of SPX gene family from algae to legumes; a focus on soybean

BackgroundSPX-containing proteins have been known as key players in phosphate signaling and homeostasis. In Arabidopsis and rice, functions of some SPXs have been characterized, but little is known about their function in other plants, especially in the legumes.ResultsWe analyzed SPX gene family evolution in legumes and in a number of key species from algae to angiosperms. We found that SPX harboring proteins showed fluctuations in domain fusions from algae to the angiosperms with, finally, four classes appearing and being retained in the land plants. Despite these fluctuations, Lysine Surface Cluster (KSC), and the third residue of Phosphate Binding Sites (PBS) showed complete conservation in almost all of SPXs except few proteins in Selaginella moellendorffii and Papaver sumniferum, suggesting they might have different ligand preferences. In addition, we found that the WGD/segmentally or dispersed duplication types were the most frequent contributors to the SPX expansion, and that there is a positive correlation between the amount of WGD contribution to the SPX expansion in individual species and its number of EXS genes. We could also reveal that except SPX class genes, other classes lost the collinearity relationships among Arabidopsis and legume genomes. The sub- or neo-functionalization of the duplicated genes in the legumes makes it difficult to find the functional orthologous genes. Therefore, we used two different methods to identify functional orthologs in soybean and Medicago. High variance in the dynamic and spatial expression pattern of GmSPXs proved the new or sub-functionalization in the paralogs.ConclusionThis comprehensive analysis revealed how SPX gene family evolved from algae to legumes and also discovered several new domains fused to SPX domain in algae. In addition, we hypothesized that there different phosphate sensing mechanisms might occur in S. moellendorffii and P. sumniferum. Finally, we predicted putative functional orthologs of AtSPXs in the legumes, especially, orthologs of AtPHO1 and AtPHO1;H1, involved in long-distance Pi transportation. These findings help to understand evolution of phosphate signaling and might underpin development of new legume varieties with improved phosphate use efficiency.

Metal–Peptide Complexes—A Novel Class of Molecular Receptors for Electrochemical Phosphate Sensing

Amyloid-β (Aβ) peptides are crucial in the pathology of Alzheimer’s disease. On the other hand, their metal complexes possess distinctive coordination properties that could be of great importance in the selective recognition of (bio)analytes, such as anions. Here, we report a novel group of molecular receptors for phosphate anions recognition: metal–peptide complexes of Aβ peptides, which combine features of synthetic inorganic ligands and naturally occurring binding proteins. The influence of the change in the metal ion center on the coordination and redox properties of binary Cu(II)/Ni(II)-Aβ complexes, as well as the affinity of these complexes towards phosphate species, were analyzed. This approach offers the possibility of fine-tuning the receptor affinity for desired applications.

Sugar phosphate activation of the stress sensor eIF2B

The multi-subunit translation initiation factor eIF2B is a control node for protein synthesis. eIF2B activity is canonically modulated through stress-responsive phosphorylation of its substrate eIF2. The eIF2B regulatory subcomplex is evolutionarily related to sugar-metabolizing enzymes, but the biological relevance of this relationship was unknown. To identify natural ligands that might regulate eIF2B, we conduct unbiased binding- and activity-based screens followed by structural studies. We find that sugar phosphates occupy the ancestral catalytic site in the eIF2Bα subunit, promote eIF2B holoenzyme formation and enhance enzymatic activity towards eIF2. A mutant in the eIF2Bα ligand pocket that causes Vanishing White Matter disease fails to engage and is not stimulated by sugar phosphates. These data underscore the importance of allosteric metabolite modulation for proper eIF2B function. We propose that eIF2B evolved to couple nutrient status via sugar phosphate sensing with the rate of protein synthesis, one of the most energetically costly cellular processes.

The Complexities of Organ Crosstalk in Phosphate Homeostasis: Time to Put Phosphate Sensing Back in the Limelight

Phosphate homeostasis is essential for health and is achieved via interaction between the bone, kidney, small intestine, and parathyroid glands and via intricate processes involving phosphate transporters, phosphate sensors, and circulating hormones. Numerous genetic and acquired disorders are associated with disruption in these processes and can lead to significant morbidity and mortality. The role of the kidney in phosphate homeostasis is well known, although it is recognized that the cellular mechanisms in murine models and humans are different. Intestinal phosphate transport also appears to differ in humans and rodents, with recent studies demonstrating a dominant role for the paracellular pathway. The existence of phosphate sensing has been acknowledged for decades; however, the underlying molecular mechanisms are poorly understood. At least three phosphate sensors have emerged. PiT2 and FGFR1c both act as phosphate sensors controlling Fibroblast Growth Factor 23 secretion in bone, whereas the calcium-sensing receptor controls parathyroid hormone secretion in response to extracellular phosphate. All three of the proposed sensors are expressed in the kidney and intestine but their exact function in these organs is unknown. Understanding organ interactions and the mechanisms involved in phosphate sensing requires significant research to develop novel approaches for the treatment of phosphate homeostasis disorders.

FC 004PHOSPHATE FACILITATES PHOSPHATURIA BY PIT-2-MEDIATED ACTIVATION OF ERK1/2 SIGNALLING INTERNALIZING NAPI-2A FROM THE APICAL BRUSH BORDER MEMBRANE INDEPENDENT OF FGF23

Background and Aims Increased phosphate load stimulates the secretion of fibroblast growth factor (FGF) 23 in the bone leading to decreased phosphate reabsorption in the kidney. FGF23 activates FGFR1/Klotho/ERK1/2 signalling in proximal tubule cells to suppress type II sodium phosphate transporters NaPi-2a and NaPi-2c in the apical brush border membrane (BBM) resulting in lower serum phosphate levels. The type III sodium-dependent phosphate transporters PiT-1 and PiT-2 are expressed in key organs of phosphate regulation and were shown to activate ERK1/2 in osseous cells in the presence of high extracellular phosphate. Furthermore, PiT-2 was shown to be responsible for the phosphate-dependent FGF23 secretion in bone cells. Whether phosphate itself can be sensed by kidney cells and stimulate its own excretion remains unknown. The aim of our study was to examine the molecular mechanism regulating renal phosphate transport in the setting of chronic oral phosphate loading in mice and to analyse phosphate sensing as well as phosphaturic actions of phosphate itself independent of FGF23. Method First, eight-week-old male C57BL/6 wildtype mice were fed a 2% high phosphate diet (HPD) or a 0.8% normal phosphate diet (NPD). Mice were sacrificed after six months and blood and urine were collected to determine parameters of phosphate homeostasis. Kidneys were isolated to evaluate the HPD-induced regulation of phosphate transporters by qPCR, immunoblot and histological analyses. Second, murine proximal tubule (mPT) cells were stimulated with either phosphate or FGF23 in the presence or absence of Foscarnet, as an inhibitor of phosphate transporters, to verify the molecular mechanism of phosphate sensing. Results Although, HPD caused significantly elevated circulating levels of intact FGF23 which resulted in hyperphosphaturia, serum phosphate levels were still enhanced compared to NPD-fed mice. Renal Klotho protein expression was significantly reduced in HPD mice and histological staining demonstrated lower Klotho accumulation in proximal and distal tubule cells, while FGFR1 was not altered. The FGF23/Klotho/FGFR1 downstream pathway revealed neither a clear activation of the ERK1/2 signalling pathway nor induction of the transcription factor Egr-1 due to HPD. Nevertheless, NaPi-2a mRNA expression was significantly reduced in HPD-fed mice compared to NPD group and NaPi-2c was unchanged. The amount of NaPi-2a protein in isolated BBM vesicles of HPD-fed mice was lower compared to NPD and immunofluorescent staining confirmed the internalisation of NaPi-2a from the apical BBM. Among the type III sodium-dependent phosphate cotransporters, renal PiT-1 mRNA expression was not altered in HPD-fed mice, but PiT-2 was significantly increased compared to NPD group and immunofluorescent staining revealed an enhanced localization of PiT-2 on the basolateral membrane of proximal tubule cells. Stimulation of mPTs with phosphate or FGF23 increased the expression of PiT-2, induced the phosphorylation of ERK1/2 and decreased NaPi-2a in vitro. The pre-treatment with Foscarnet blunted the phosphate-mediated activation of ERK1/2 signalling pathway, but not the FGF23-induced effects, suggesting a direct phosphate transporter-regulating mechanism of high phosphate in renal proximal tubule cells. Conclusion A chronic high dietary intake of phosphate results in downregulation of renal Klotho causing hyperphosphatemia, suggesting in part a renal resistance of FGF23/Klotho signalling pathway. However, HPD-induced internalization NaPi-2a from the apical BBM pointing to an FGF23-independent mechanism regulating phosphate reabsorption. Our data indicate that in the settings of high phosphate-mediated renal resistance of FGF23, phosphate itself may stimulate its urinary secretion via PiT-2-mediated activation of ERK1/2 signalling pathway which results in NaPi-2a downregulation and hyperphosphaturia independent of FGF23.

RegX3-Mediated Regulation of Methylcitrate Cycle in Mycobacterium smegmatis

Mycobacterium tuberculosis is a global human pathogen that infects macrophages and can establish a latent infection. Emerging evidence has established the nutrients metabolism as a key point to study the pathogenesis of M. tuberculosis and host immunity. It was reported that fatty acids and cholesterol are the major nutrient sources of M. tuberculosis in the period of infection. However, the mechanism by which M. tuberculosis utilizes lipids for maintaining life activities in nutrient-deficiency macrophages is poorly understood. Mycobacterium smegmatis is fast-growing and generally used to study its pathogenic counterpart, M. tuberculosis. In this work, we found that the phosphate sensing regulator RegX3 of M. smegmatis is required for its growing on propionate and surviving in macrophages. We further demonstrated that the expression of prpR and related genes (prpDBC) in methylcitrate cycle could be enhanced by RegX3 in response to the phosphate-starvation condition. The binding sites of the promoter region of prpR for RegX3 and PrpR were investigated. In addition, cell morphology assay showed that RegX3 is responsible for cell morphological elongation, thus promoting the proliferation and survival of M. smegmatis in macrophages. Taken together, our findings revealed a novel transcriptional regulation mechanism of RegX3 on propionate metabolism, and uncovered that the nutrients-sensing regulatory system puts bacteria at metabolic steady state by altering cell morphology. More importantly, since we observed that M. tuberculosis RegX3 also binds to the prpR operon in vitro, the RegX3-mediated regulation might be general in M. tuberculosis and other mycobacteria for nutrient sensing and environmental adaptation.