Highlights d Patched structure reveals a hydrophobic conduit with sterollike contents d Patched mediates Hedgehog-reversible reduction of inner leaflet cholesterol activity d Hydrophobic conduit is essential for cholesterol effect and Smoothened suppression d Inner leaflet cholesterol likely mediates Hedgehog-Patched regulation of Smoothened

Hedgehog signalling is fundamental to embryonic development and postnatal tissue regeneration1. Aberrant postnatal Hedgehog signalling leads to several malignancies, including basal cell carcinoma and paediatric medulloblastoma2. Hedgehog proteins bind to and inhibit the transmembrane cholesterol transporter Patched-1 (PTCH1), which permits activation of the seven-transmembrane transducer Smoothened (SMO) via a mechanism that is poorly understood. Here we report the crystal structure of active mouse SMO bound to both the agonist SAG21k and to an intracellular binding nanobody that stabilizes a physiologically relevant active state. Analogous to other G protein-coupled receptors, the activation of SMO is associated with subtle motions in the extracellular domain, and larger intracellular changes. In contrast to recent models3–5, a cholesterol molecule that is critical for SMO activation is bound deep within the seven-transmembrane pocket. We propose that the inactivation of PTCH1 by Hedgehog allows a transmembrane sterol to access this seven-transmembrane site (potentially through a hydrophobic tunnel), which drives the activation of SMO. These results—combined with signalling studies and molecular dynamics simulations—delineate the structural basis for PTCH1 -SMO regulation, and suggest a strategy for overcoming clinical resistance to SMO inhibitors.

Hedgehog signaling specifies tissue patterning and renewal, and pathway components are commonly mutated in certain malignancies. Although central to ensuring appropriate pathway activity in all Hedgehog-responsive cells, how the transporter-like receptor Patched1 regulates the seven-transmembrane protein Smoothened remains mysterious, partially due to limitations in existing tools and experimental systems. Here we employ direct, real-time, biochemical and physiology-based approaches to monitor Smoothened activity in cellular and in vitro contexts. Patched1-Smoothened coupling is rapid, dynamic, and can be recapitulated without cilium-specific proteins or lipids. By reconstituting purified Smoothened in vitro, we show that cholesterol within the bilayer is sufficient for constitutive Smoothened activation. Cholesterol effects occur independently of the lipid-binding Smoothened extracellular domain, a region that is dispensable for Patched1-Smoothened coupling. Finally, we show that Patched1 specifically requires extracellular Na to regulate Smoothened in our assays, raising the possibility that a Na gradient provides the energy source for Patched1 catalytic activity. Our work suggests a hypothesis wherein Patched1, chemiosmotically driven by the transmembrane Na gradient common to metazoans, regulates Smoothened by shielding its heptahelical domain from cholesterol, or by providing an inhibitor that overrides this cholesterol activation.

Plant microRNAs (miRNAs) have been shown to play critical roles in regulating gene expression at the post-transcriptional level. In this study, we employed high throughput sequencing combined with computational analysis to survey miRNAomes from the seedlings of rice under normal conditions and treatments of H2O2 that result in oxidative stress. Comparison of the miRNAomes and subsequent northern blot analysis identified seven miRNA families differentially expressed under H2O2 stress. Predicted and experimentally validated targets of these H2O2-responsive miRNAs are involved in different cellular responses and metabolic processes including transcriptional regulation, nutrient transport, auxin homeostasis, cell proliferation and programmed cell death. This indicates that diverse miRNAs form a complex regulatory network to coordinate plants’ responses under oxidative stress. In addition, we also discovered 32 new miRNAs in the seedlings of rice. Interestingly, of these new miRNAs, miR3981 was originally found to be a putative exonic miRNA located in the exon of AK106348, suggesting that plants may also use some exons as an miRNA source. This study is the first genome-wide investigation of H2O2-regulated miRNAs in plants and broadens our perspectives on the important regulatory roles of miRNAs in plant oxidative stress and physiological adaption.

Hedgehog protein signals mediate tissue patterning and maintenance via binding to and inactivation of their common receptor Patched, a twelve-transmembrane protein that otherwise would suppress activity of the seven-transmembrane protein, Smoothened. Loss of Patched function, the most common cause of basal cell carcinoma, permits unregulated activation of Smoothened and of the Hedgehog pathway. A cryo-EM structure of the Patched protein reveals striking transmembrane domain similarities to prokaryotic RND transporters. The extracellular domain mediates association of Patched monomers in an unusual dimeric architecture that implies curvature in the associated membrane. A central conduit with cholesterol-like contents courses through the extracellular domain and resembles that used by other RND proteins to transport substrates, suggesting Patched activity in cholesterol transport. Patched expression indeed reduces cholesterol activity in the inner leaflet of the plasma membrane, in a manner antagonized by Hedgehog stimulation and with implications for regulation of Smoothened.

Iron overload causes progressive organ damage and is associated with arthritis, liver damage, and heart failure. Elevated iron levels are present in 1%-5% of individuals; however, iron overload is undermonitored and underdiagnosed. Genetic factors affecting iron homeostasis are emerging. Individuals with hereditary xerocytosis, a rare disorder with gain-of-function (GOF) mutations in mechanosensitive PIEZO1 ion channel, develop age-onset iron overload. We show that constitutive or macrophage expression of a GOF Piezo1 allele in mice disrupts levels of the iron regulator hepcidin and causes iron overload. We further show that PIEZO1 is a key regulator of macrophage phagocytic activity and subsequent erythrocyte turnover. Strikingly, we find that E756del, a mild GOF PIEZO1 allele present in one-third of individuals of African descent, is strongly associated with increased plasma iron. Our study links macrophage mechanotransduction to iron metabolism and identifies a genetic risk factor for increased iron levels in African Americans.

Silkworm silk is a promising natural biopolymer for textile and biomedical applications for its remarkable flexibility, excellent biocompatibility and controllable biodegradability. The functionalization of silks makes them more versatile for flexible displays and visible bioscaffolds. However, fluorescent silks are normally fabricated through unstable physical absorption or complicated chemical reactions under harsh conditions. Herein, we developed a simple strategy for preparing fluorescent silks. Five aggregation‐induced emission luminogens (AIEgens) with activated alkynes were synthesized by rational molecular design, and then reacted with silk fibers through facile metal‐free click bioconjugation. The resulting conjugates show bright full‐color emissions and high stability. A white light‐emitting silk was fabricated by simultaneous bioconjugation with red‐, green‐ and blue‐emissive AIEgens. The red‐emissive AIEgen‐functionalized silks were successfully applied for long‐term cell tracking and two‐photon bioimaging, demonstrating great potential for tissue engineering and bioscaffold monitoring.

By mimicking the layered structure of nacre, we successfully fabricated a nano-laminar coating on high-voltage insulators. The coating is demonstrated to achieve a higher flashover strength by effectively dissipating surface charge under dc stress.