Single hormone or synthetic agonist induces Gs/Gi coupling selectivity of EP receptors via distinct binding modes and propagating paths.

To accomplish concerted physiological reactions, nature has diversified functions of a single hormone at at least two primary levels: 1) Different receptors recognize the same hormone, and 2) different cellular effectors couple to the same hormone-receptor pair [R.P. Xiao, Sci STKE 2001, re15 (2001); L. Hein, J. D. Altman, B.K. Kobilka, Nature 402, 181-184 (1999); Y. Daaka, L. M. Luttrell, R. J. Lefkowitz, Nature 390, 88-91 (1997)]. Not only these questions lie in the heart of hormone actions and receptor signaling but also dissecting mechanisms underlying these questions could offer therapeutic routes for refractory diseases, such as kidney injury (KI) or X-linked nephrogenic diabetes insipidus (NDI). Here, we identified that Gs-biased signaling, but not Gi activation downstream of EP4, showed beneficial effects for both KI and NDI treatments. Notably, by solving Cryo-electron microscope (cryo-EM) structures of EP3-Gi, EP4-Gs, and EP4-Gi in complex with endogenous prostaglandin E2 (PGE2)or two synthetic agonists and comparing with PGE2-EP2-Gs structures, we found that unique primary sequences of prostaglandin E2 receptor (EP) receptors and distinct conformational states of the EP4 ligand pocket govern the Gs/Gi transducer coupling selectivity through different structural propagation paths, especially via TM6 and TM7, to generate selective cytoplasmic structural features. In particular, the orientation of the PGE2 ω-chain and two distinct pockets encompassing agonist L902688 of EP4 were differentiated by their Gs/Gi coupling ability. Further, we identified common and distinct features of cytoplasmic side of EP receptors for Gs/Gi coupling and provide a structural basis for selective and biased agonist design of EP4 with therapeutic potential.

Structural basis for substrate and inhibitor recognition of human multidrug transporter MRP4.

Human multidrug resistance protein 4 (hMRP4, also known as ABCC4), with a representative topology of the MRP subfamily, translocates various substrates across the membrane and contributes to the development of multidrug resistance. However, the underlying transport mechanism of hMRP4 remains unclear due to a lack of high-resolution structures. Here, we use cryogenic electron microscopy (cryo-EM) to resolve its near-atomic structures in the apo inward-open and the ATP-bound outward-open states. We also capture the PGE1 substrate-bound structure and, importantly, the inhibitor-bound structure of hMRP4 in complex with sulindac, revealing that substrate and inhibitor compete for the same hydrophobic binding pocket although with different binding modes. Moreover, our cryo-EM structures, together with molecular dynamics simulations and biochemical assay, shed light on the structural basis of the substrate transport and inhibition mechanism, with implications for the development of hMRP4-targeted drugs.

Conformational cycle of human polyamine transporter ATP13A2.

Dysregulation of polyamine homeostasis strongly associates with human diseases. ATP13A2, which is mutated in juvenile-onset Parkinson's disease and autosomal recessive spastic paraplegia 78, is a transporter with a critical role in balancing the polyamine concentration between the lysosome and the cytosol. Here, to better understand human ATP13A2-mediated polyamine transport, we use single-particle cryo-electron microscopy to solve high-resolution structures of human ATP13A2 in six intermediate states, including the putative E2 structure for the P5 subfamily of the P-type ATPases. These structures comprise a nearly complete conformational cycle spanning the polyamine transport process and capture multiple substrate binding sites distributed along the transmembrane regions, suggesting a potential polyamine transport pathway. Integration of high-resolution structures, biochemical assays, and molecular dynamics simulations allows us to obtain a better understanding of the structural basis of how hATP13A2 transports polyamines, providing a mechanistic framework for ATP13A2-related diseases.

SiFBA5, a cold-responsive factor from Saussurea involucrata promotes cold resilience and biomass increase in transgenic tomato plants under cold stress.

BACKGROUND: Saussurea involucrata survives in extreme arctic conditions and is very cold-resistant. This species grows in rocky, mountainous areas with elevations of 2400-4100 m, which are snow-covered year-round and are subject to freezing temperatures. S. involucrata's ability to survive in an extreme low-temperature environment suggests that it has particularly high photosynthetic efficiency, providing a magnificent model, and rich gene pool, for the analysis of plant cold stress response. Fructose-1, 6-bisphosphate aldolase (FBA) is a key enzyme in the photosynthesis process and also mediates the conversion of fructose 1, 6-bisphosphate (FBP) into dihydroxyacetone phosphate (DHAP) and glycerol triphosphate (GAP) during glycolysis and gluconeogenesis. The molecular mechanisms underlying S. involucrata's cold tolerance are still unclear; therefore, our work aims to investigate the role of FBA in plant cold-stress response. RESULTS: In this study, we identified a cold-responsive gene, SiFBA5, based on a preliminary low-temperature, genome-wide transcriptional profiling of S. involucrata. Expression analysis indicated that cold temperatures rapidly induced transcriptional expression of SiFBA5, suggesting that SiFBA5 participates in the initial stress response. Subcellular localization analysis revealed that SiFBA5 is localized to the chloroplast. Transgenic tomato plants that overexpressed SiFBA5 were generated using a CaMV 35S promoter. Phenotypic observation suggested that the transgenic plants displayed increased cold tolerance and photosynthetic efficiency in comparison with wild-type plants. CONCLUSION: Cold stress has a detrimental impact on crop yield. Our results demonstrated that SiFBA5 positively regulates plant response to cold stress, which is of great significance for increasing crop yield under cold stress conditions.

All-in-one sequencing: an improved library preparation method for cost-effective and high-throughput next-generation sequencing.

BACKGROUND: Next generation sequencing (NGS) has been widely used in biological research, due to its rapid decrease in cost and increasing ability to generate data. However, while the sequence generation step has seen many improvements over time, the library preparation step has not, resulting in low-efficiency library preparation methods, especially for the most time-consuming and labor-intensive steps: size-selection and quantification. Consequently, there can be bottlenecks in projects with large sample cohorts. RESULTS: We have described the all-in-one sequencing (AIO-seq) method, where instead of performing size-selection and quantification for samples individually, one sample one tube, up to 116 samples are pooled and analyzed in a single tube, 'All-In-One'. The AIO-seq method pools libraries based on the samples' expected data yields and the calculated concentrations of the size selected regions (target region), which can easily be obtained with the Agilent 2100 Bioanalyzer and Qubit Fluorometer. AIO-seq was applied to whole genome sequencing and RNA-seq libraries successfully, and it is envisaged that it could be applied to any type of NGS library, such as chromatin immunoprecipitation coupled with massively parallel sequencing, assays for transposase-accessible chromatin with high-throughput sequencing, and high-throughput chromosome conformation capture. We also demonstrated that for genetic population samples with low coverage sequences, like recombinant inbred lines (RIL), AIO-seq could be further simplified, by mixing the libraries immediately after PCR, without calculating the target region concentrations. CONCLUSIONS: The AIO-seq method is thus labor saving and cost effective, and suitable for projects with large sample cohorts, like those used in plant breeding or population genetics research.

De Novo Transcriptome Sequencing and the Hypothetical Cold Response Mode of Saussurea involucrata in Extreme Cold Environments.

Saussurea involucrata grows in high mountain areas covered by snow throughout the year. The temperature of this habitat can change drastically in one day. To gain a better understanding of the cold response signaling pathways and molecular metabolic reactions involved in cold stress tolerance, genome-wide transcriptional analyses were performed using RNA-Seq technologies. A total of 199,758 transcripts were assembled, producing 138,540 unigenes with 46.8 Gb clean data. Overall, 184,416 (92.32%) transcripts were successfully annotated. The 365 transcription factors identified (292 unigenes) belonged to 49 transcription factor families associated with cold stress responses. A total of 343 transcripts on the signal transduction (132 upregulated and 212 downregulated in at least any one of the conditions) were strongly affected by cold temperature, such as the CBL-interacting serine/threonine-protein kinase (CIPKs), receptor-like protein kinases, and protein kinases. The circadian rhythm pathway was activated by cold adaptation, which was necessary to endure the severe temperature changes within a day. There were 346 differentially expressed genes (DEGs) related to transport, of which 138 were upregulated and 22 were downregulated in at least any one of the conditions. Under cold stress conditions, transcriptional regulation, molecular transport, and signal transduction were involved in the adaptation to low temperature in S. involucrata. These findings contribute to our understanding of the adaptation of plants to harsh environments and the survival traits of S. involucrata. In addition, the present study provides insight into the molecular mechanisms of chilling and freezing tolerance.