A SPRY1 domain cardiac ryanodine receptor variant associated with short-coupled torsade de pointes

International audienceIdiopathic ventricular fibrillation (IVF) causes sudden death in young adult patients without structural or ischemic heart disease. Most IVF cases are sporadic and some patients present with short-coupled torsade de pointes, the genetics of which are poorly understood. A man who had a first syncope at the age of 35 presented with frequent short-coupled premature ventricular beats with bursts of polymorphic ventricular tachycardia and then died suddenly. By exome sequencing, we identified three rare variants: p.I784F in the SPRY1 of the ryanodine receptor 2 (RyR2), p.A96S in connexin 40 (Cx40), reported to affect electrical coupling and cardiac conduction, and a nonsense p.R244X in the cardiac-specific troponin I-interacting kinase (TNNI3K). We assessed intracellular Ca2+ handling in WT and mutant human RYR2 transfected HEK293 cells by fluorescent microscopy and an enhanced store overload-induced Ca2+ release in response to cytosolic Ca2+ was observed in RyR2-I784F cells. In addition, crystal structures and thermal melting temperatures revealed a conformational change in the I784F-SPRY1 domain compared to the WT-domain. The novel RyR2-I784F variant in SPRY1 domain causes a leaky channel under non-stress conditions. The presence of several variants affecting Ca2+ handling and cardiac conduction suggests a possible oligogenic origin for the ectopies originating from Purkinje fibres

Discovery of Potential Species-Specific Green Insecticides Targeting the Lepidopteran Ryanodine Receptor

Ryanodine receptors (RyRs) are homotetrameric intracellular calcium (Ca2+) release channels responsible for excitation-contraction coupling of muscle cells. Diamide insecticides specifically act on RyRs of Lepidoptera and Coleoptera pests and are safe for nontargeted organisms, generating big worldwide sales. Despite their popularity, several devastating agricultural pests have been reported to be resistant to them because of mutations in a small transmembrane region of their RyRs, hinting a binding pocket nearby. A potential solution to overcome resistance is to develop new insecticides targeting different binding sites in pest RyRs. Based on a high-resolution crystal structure of diamondback moth (DBM) RyR N-terminal domain (NTD) determined by our group, we carried out extensive structure-based insecticide screening targeting the intersubunit interface. We identified eight lead compounds that selectively target the open conformation of DBM RyR, which are predicted to act as channel activators similar to diamide insecticides. Binding mode analysis shows selective binding to a hydrophobic pocket of DBM NTD-A but not to the pocket of its mammalian counterpart. We tested three available compounds on the HEK293 cell lines stably expressing DBM or mammalian RyR, one of which shows good potency and selectivity against DBM RyR. The insecticidal effect of the compound was also confirmed using fruit flies. The detailed binding mode, toxicity, absorption, distribution, metabolism, and excretion, and reactivity of the compound were predicted by bioinformatic methods. Together, our study lays a foundation for developing a new class of selective RyR-targeting insecticides to control both wild-type and resistant pests.</p

Characterization of Six Diamide Insecticides on Ryanodine Receptor: Resistance and Species Selectivity

Ryanodine receptor (RyR) has been used as an insecticide target to control many destructive agricultural pests. The effectiveness of these insecticides has been limited by the spread of resistance mutations identified in pest RyRs, but the detailed molecular impacts of the individual mutations on the activity of different diamide compounds have not been fully explored. We created five HEK293 cell lines stably expressing wild type rabbit RyR1, wild type Spodoptera frugiperda RyR (Sf RyR), or Sf RyR carrying different resistance mutations, including G4891E, G4891E/I4734M, and Y4867F, respectively. R-CEPIA1er, a genetically encoded fluorescent protein, was also introduced in these cell lines to report the Ca2+ concentration in the endoplasmic reticulum. We systematically characterized the activities of six commercial diamide insecticides against different RyRs using the time-lapse fluorescence assay. Among them, cyantraniliprole (CYAN) displayed the highest activity against all three resistant Sf RyRs. The good performance of CYAN was confirmed by the toxicity assay using gene-edited Drosophila expressing the mutant RyRs, in which CYAN showed the lowest LD50 value for the double resistant mutant. In addition, we compared their acitivty between mammalian and insect RyRs and found that flubendiamide has the best insect-selectivity. The mechanism of the anti-resistance property and selectivity of the compounds was proposed based on the structural models generated by homology modeling and molecular docking. Our findings provide insights into the mechanism of insect resistance and guidance for developing effective RyR agonists that can selectively target resistant pests

SPOP-containing complex regulates SETD2 stability and H3K36me3-coupled alternative splicing.

Trimethylation of histone H3K36 is a chromatin mark associated with active gene expression, which has been implicated in coupling transcription with mRNA splicing and DNA damage response. SETD2 is a major H3K36 trimethyltransferase, which has been implicated as a tumor suppressor in mammals. Here, we report the regulation of SETD2 protein stability by the proteasome system, and the identification of SPOP, a key subunit of the CUL3 ubiquitin E3 ligase complex, as a SETD2-interacting protein. We demonstrate that SPOP is critically involved in SETD2 stability control and that the SPOP/CUL3 complex is responsible for SETD2 polyubiquitination both in vivo and in vitro ChIP-Seq analysis and biochemical experiments demonstrate that modulation of SPOP expression confers differential H3K36me3 on SETD2 target genes, and induce H3K36me3-coupled alternative splicing events. Together, these findings establish a functional connection between oncogenic SPOP and tumor suppressive SETD2 in the dynamic regulation of gene expression on chromatin

SPOP-containing complex regulates SETD2 stability and H3K36me3-coupled alternative splicing.

Trimethylation of histone H3K36 is a chromatin mark associated with active gene expression, which has been implicated in coupling transcription with mRNA splicing and DNA damage response. SETD2 is a major H3K36 trimethyltransferase, which has been implicated as a tumor suppressor in mammals. Here, we report the regulation of SETD2 protein stability by the proteasome system, and the identification of SPOP, a key subunit of the CUL3 ubiquitin E3 ligase complex, as a SETD2-interacting protein. We demonstrate that SPOP is critically involved in SETD2 stability control and that the SPOP/CUL3 complex is responsible for SETD2 polyubiquitination both in vivo and in vitro ChIP-Seq analysis and biochemical experiments demonstrate that modulation of SPOP expression confers differential H3K36me3 on SETD2 target genes, and induce H3K36me3-coupled alternative splicing events. Together, these findings establish a functional connection between oncogenic SPOP and tumor suppressive SETD2 in the dynamic regulation of gene expression on chromatin

SPOP-containing complex regulates SETD2 stability and H3K36me3-coupled alternative splicing

Trimethylation of histone H3K36 is a chromatin mark associated with active gene expression, which has been implicated in coupling transcription with mRNA splicing and DNA damage response. SETD2 is a major H3K36 trimethyltransferase, which has been implicated as a tumor suppressor in mammals. Here, we report the regulation of SETD2 protein stability by the proteasome system, and the identification of SPOP, a key subunit of the CUL3 ubiquitin E3 ligase complex, as a SETD2-interacting protein. We demonstrate that SPOP is critically involved in SETD2 stability control and that the SPOP/CUL3 complex is responsible for SETD2 polyubiquitination both in vivo and in vitro. ChIP-Seq analysis and biochemical experiments demonstrate that modulation of SPOP expression confers differential H3K36me3 on SETD2 target genes, and induce H3K36me3-coupled alternative splicing events. Together, these findings establish a functional connection between oncogenic SPOP and tumor suppressive SETD2 in the dynamic regulation of gene expression on chromatin

Structural basis for diamide modulation of ryanodine receptor

The diamide insecticide class is one of the top-selling insecticides globally. They are used to control a wide range of pests by targeting their ryanodine receptors (RyRs). Here, we report the highest-resolution cryo-electron microscopy (cryo-EM) structure of RyR1 in the open state, in complex with the anthranilic diamide chlorantraniliprole (CHL). The 3.2-Å local resolution map facilitates unambiguous assignment of the CHL binding site. The molecule induces a conformational change by affecting the S4–S5 linker, triggering channel opening. The binding site is further corroborated by mutagenesis data, which reveal how diamide insecticides are selective to the Lepidoptera group of insects over honeybee or mammalian RyRs. Our data reveal that several pests have developed resistance via two mechanisms, steric hindrance and loss of contact. Our results provide a foundation for the development of highly selective pesticides aimed at overcoming resistance and therapeutic molecules to treat human myopathies. [Figure not available: see fulltext.].</p