S-acylation: an orchestrator of the life cycle and function of membrane proteins.

S-acylation is a covalent post-translational modification of proteins with fatty acids, achieved by enzymatic attachment via a labile thioester bond. This modification allows for dynamic control of protein properties and functions in association with cell membranes. This lipid modification regulates a substantial portion of the human proteome and plays an increasingly recognized role throughout the lifespan of affected proteins. Recent technical advancements have propelled the S-acylation field into a 'molecular era', unveiling new insights into its mechanistic intricacies and far-reaching implications. With a striking increase in the number of studies on this modification, new concepts are indeed emerging on the roles of S-acylation in specific cell biology processes and features. After a brief overview of the enzymes involved in S-acylation, this viewpoint focuses on the importance of S-acylation in the homeostasis, function, and coordination of integral membrane proteins. In particular, we put forward the hypotheses that S-acylation is a gatekeeper of membrane protein folding and turnover and a regulator of the formation and dynamics of membrane contact sites.

Recent progress in the structural study of ion channels as insecticide targets.

Ion channels, many expressed in insect neural and muscular systems, have drawn huge attention as primary targets of insecticides. With the recent technical breakthroughs in structural biology, especially in cryo-electron microscopy (cryo-EM), many new high-resolution structures of ion channel targets, apo or in complex with insecticides, have been solved, shedding light on the molecular mechanism of action of the insecticides and resistance mutations. These structures also provide accurate templates for structure-based insecticide screening and rational design. This review summarizes the recent progress in the structural studies of 5 ion channel families: the ryanodine receptor (RyR), the nicotinic acetylcholine receptor (nAChR), the voltage-gated sodium channel (VGSC), the transient receptor potential (TRP) channel, and the ligand-gated chloride channel (LGCC). We address the selectivity of the channel-targeting insecticides by examining the conservation of key coordinating residues revealed by the structures. The possible resistance mechanisms are proposed based on the locations of the identified resistance mutations on the 3D structures of the target channels and their impacts on the binding of insecticides. Finally, we discuss how to develop "green" insecticides with a novel mode of action based on these high-resolution structures to overcome the resistance.

Ryanodine Receptor as Insecticide Target.

The ryanodine receptor (RyR) is one of the primary targets of commercial insecticides. The diamide insecticide family, including flubendiamide, chlorantraniliprole, cyantraniliprole, etc., targets insect RyRs and can be used to control a wide range of destructive agricultural pests. The diamide insecticides are highly selective against lepidopteran and coleopteran pests with relatively low toxicity for non-target species, such as mammals, fishes, and beneficial insects. However, recently mutations identified on insect RyRs have emerged and caused resistance in several major agricultural pests throughout different continents. This review paper summarizes the recent findings on the structure and function of insect RyRs as insecticide targets. Specifically, we examine the structures of RyRs from target and non-target species, which reveals the molecular basis for insecticide action and selectivity. We also examine the structural and functional changes of RyR caused by the resistance mutations. Finally, we examine the progress in RyR structure-based insecticide design and discuss how this might help the development of a new generation of green insecticides.

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.

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.