Cell Discovery最新文献

Cyclic nucleotide-gated ion channels (CNGCs) in plants mediate Ca²⁺ influx in response to environmental changes. Among numerous plant CNGCs, Medicago truncatula CNGC15a/b/c (MtCNGC15) is localized to the nuclear envelope. The opening and closing cycle of MtCNGC15 is tightly associated with the Ca²⁺ oscillation in symbiosis. However, the molecular mechanism underlying MtCNGC15 activity regulation remains unclear. In this study, we present the structures of MtCNGC15 in its apo form and in the presence of CaM. The apo MtCNGC15b exhibits a flexible cytoplasmic domain (CPD), whereas binding of the MtCaM inhibits Ca²⁺ currents and stabilizes the highly dynamic CPD. Furthermore, the activity of MtCNGC15b seems to be independent of cGMP. The hypothetical binding pocket for cGMP is occupied by an arginine residue. These findings elucidate the structural basis for the activity regulation of nuclear localized MtCNGC15.

Human glucose-6-phosphatase catalytic subunit 1 (hG6PC1) is a key enzyme in glucose metabolism, governing the final common step of gluconeogenesis and glycogenolysis, and directly regulating energy homeostasis. Aberrant mutations in G6PC1 directly cause glycogen storage disease type 1a, which is characterized by chronic hypoglycemia and glycogen accumulation. Additionally, abnormal G6PC1 function leads to increased fasting blood glucose. Consequently, it is a critical target for treating glucose metabolism disorders. In this study, we determine the cryo-EM structures of G6PC1 in both the partially open and fully open states, in either the apo form or in complex with the substrates G6P or F6P and the product phosphate. These structures offer distinct insights into the mechanism of hydrolysis and induced-fit, providing a structural foundation for the diagnostic analysis of disease-causing mutations in G6PC1. Moreover, we propose a potential mechanism by which phosphatidylserine regulates G6PC1 activity, providing a novel perspective on its role and implications.

Mushroom's specialized secondary metabolites possess important pharmacological activities, but their biosynthetic pathway elucidation is extremely challenging, not to mention reprogramming of their biosynthetic networks to target metabolites. By taking Ganoderma lucidum, a famous traditional medicinal mushroom, as a lead example, here we decoded the biosynthetic networks of type II ganoderic acids (TIIGAs), a group of its main bioactive metabolites by studying the coordinated gene expression in G. lucidum, identifying endogenous or heterologous enzymes capable of C22 hydroxylation, configuration conversion of C3 hydroxyl group, and acetylation on C3, C15 and C22 hydroxyl groups. Notably, we revealed the catalytic mechanism of the C22 hydroxylase CYP512W6, and an unexpected bifunctional acetyltransferase GlAT that is required to transfer acetyl groups to C15 and C22. Using a fluorescence-guided integration method, we achieved efficient biosynthesis of significant TIIGAs applicable to industrial fermentation. After introducing all the identified enzymes to baker's yeast, we observed that biosynthesis of downstream TIIGAs was severely impeded, and dredged the metabolic block by temporally regulating the expression of acetyltransferases. By reprogramming of the biosynthetic networks of TIIGAs, we were able to produce over 30 TIIGAs, exhibiting 1-4 orders of magnitude higher titers or efficiencies than those from farmed mushrooms. The work enables the access to valuable TIIGAs, facilitates their widespread application, and sheds light on research of other mushroom products.

Colorectal cancer (CRC), including both microsatellite instability (MSI) and microsatellite stability (MSS) subtypes, frequently exhibits intrinsic resistance to immunotherapy. However, the spatial tumor microenvironment (TME) and its role in distinguishing immunotherapy responders from non-responders remain poorly understood. In this study, spatial multiomics, including imaging mass cytometry (n = 50 in-house), spatial proteomics (n = 50 in-house), and spatial transcriptomics (n = 9 in-house), were employed to elucidate the spatial TME of metastatic CRC (mCRC) patients receiving immunotherapy. These methodologies were integrated with single-cell RNA sequencing (scRNA-seq), bulk RNA-seq, and bulk proteomics for comprehensive analysis and validation. A spatial immune atlas containing 314,774 cells was constructed. We found that C1QC⁺ resident tissue macrophages (RTMs) were more abundant in responders regardless of microsatellite status. Co-localization of C1QC⁺ RTMs with CD4⁺ T cells was observed in responders, and MHC-II expression facilitated their interaction. In contrast, cancer-associated fibroblasts inhibited this interaction in non-responders. Moreover, whole genome screening identified key genes involved in antigen presentation in C1QC⁺ RTMs. Hence, our study highlights the importance of spatial immune mapping in revealing the complex spatial topology of CRC and corresponding immunotherapy response.

Maintaining pH homeostasis is critical for cellular function across all living organisms. Proton-sensing G protein-coupled receptors (GPCRs), particularly GPR4, play a pivotal role in cellular responses to pH changes. Yet, the molecular mechanisms underlying their proton sensing and activation remain incompletely understood. Here we present high-resolution cryo-electron microscopy structures of GPR4 in complex with G proteins under physiological and acidic pH conditions. Our structures reveal an intricate proton-sensing mechanism driven by a sophisticated histidine network in the receptor's extracellular domain. Upon protonation of key histidines under acidic conditions, a remarkable conformational cascade is initiated, propagating from the extracellular region to the intracellular G protein-coupling interface. This dynamic process involves precise transmembrane helix rearrangements and conformational shifts of conserved motifs, mediated by strategically positioned water molecules. Notably, we discovered a bound bioactive lipid, lysophosphatidylcholine, which has positive allosteric effects on GPR4 activation. These findings provide a comprehensive framework for understanding proton sensing in GPCRs and the interplay between pH sensing and lipid regulation, offering insights into cellular pH homeostasis and potential therapies for pH-related disorders.

Prolonged sleep deprivation (Pr-SD) causes death in many species. While various mechanisms related to sleep regulation or this fatal consequence of sleep loss have been identified, the core molecular basis linking Pr-SD-induced lethality and sleep homeostasis remains unknown in mammals. A critical "point of no return (PONE)" status in Pr-SD subjects is highlighted in classic research, and characterizing PONE status could help uncover this mystery. Using a Pr-SD model and a reliable PONE status prediction method, we show that mice in PONE exhibit an inability to enter natural sleep, and significant disruptions in brain phosphoproteome, independent of deprivation time but closely linked to PONE status. Brain kinase or phosphatase dysfunction influences PONE status development and leads to corresponding sleep aberration concurrently. Daily 80-min recovery sleep significantly delays PONE onset and restores brain phosphoproteome. The harmful effects of excessive kinase activity on PONE development can be eliminated by combining recovery sleep and compensatory phosphatase expression. We conclude that sleep is crucial for maintaining brain phosphoproteome homeostasis, whose disruption may impact both Pr-SD-induced lethality and sleep regulation.

The Hepatitis B Virus (HBV) poses a significant health threat, causing millions of deaths each year. Hepatitis B surface antigen (HBsAg), the sole membrane protein on the HBV viral envelope, plays crucial roles in viral attachment to host cells and serves as the target for neutralizing antibodies (NAbs). Despite its functional and therapeutic significance, the mechanisms by which NAbs recognize HBsAg remain elusive. Here, we found that HBsAg proteins exist in distinct subtypes and are recognized by different groups of antibodies. Cryo-electron microscopy (Cryo-EM) structures of HBsAg dimers in complex with NAb Fab fragments reveal that the antigenic loop (AGL) of these distinct HBsAg types share a common structural core comprised of four β-strands. However, their surface structures exhibit unexpected polymorphism due to distinct disulfide bond linkages within the AGL region. This structural polymorphism determines the recognition of HBsAg by different groups of NAbs.