Small molecule chaperones have been exploited as therapeutics for the hundreds of diseases caused by protein misfolding. The most successful examples are the CFTR correctors, which transformed cystic ...fibrosis therapy. These molecules revert folding defects of the ΔF508 mutant and are widely used to treat patients. To investigate the molecular mechanism of their action, we determined cryo-electron microscopy structures of CFTR in complex with the FDA-approved correctors lumacaftor or tezacaftor. Both drugs insert into a hydrophobic pocket in the first transmembrane domain (TMD1), linking together four helices that are thermodynamically unstable. Mutating residues at the binding site rendered ΔF508-CFTR insensitive to lumacaftor and tezacaftor, underscoring the functional significance of the structural discovery. These results support a mechanism in which the correctors stabilize TMD1 at an early stage of biogenesis, prevent its premature degradation, and thereby allosterically rescuing many disease-causing mutations.
Display omitted
•Lumacaftor binds to CFTR in two confirmations•Tezacaftor and lumacafor bind to the same site on TMD1•Type 1 correctors stabilize CFTR by filling an internal cavity
Drugs used to treat cystic fibrosis act via stabilization of a single domain within CFTR that promotes global folding.
ChRmine, a recently discovered pump-like cation-conducting channelrhodopsin, exhibits puzzling properties (large photocurrents, red-shifted spectrum, and extreme light sensitivity) that have created ...new opportunities in optogenetics. ChRmine and its homologs function as ion channels but, by primary sequence, more closely resemble ion pump rhodopsins; mechanisms for passive channel conduction in this family have remained mysterious. Here, we present the 2.0 Å resolution cryo-EM structure of ChRmine, revealing architectural features atypical for channelrhodopsins: trimeric assembly, a short transmembrane-helix 3, a twisting extracellular-loop 1, large vestibules within the monomer, and an opening at the trimer interface. We applied this structure to design three proteins (rsChRmine and hsChRmine, conferring further red-shifted and high-speed properties, respectively, and frChRmine, combining faster and more red-shifted performance) suitable for fundamental neuroscience opportunities. These results illuminate the conduction and gating of pump-like channelrhodopsins and point the way toward further structure-guided creation of channelrhodopsins for applications across biology.
Display omitted
•The cryo-EM structure of a pump-like channelrhodopsin, at 2.0 Å resolution•Identification of key features distinguishing ChRmine from other channelrhodopsins•Identification of key features distinguishing ChRmine from ion pump rhodopsins•Structure-guided design of ChRmine variants for all-optical neuroscience
Structure of a pump-like channelrhodopsin, ChRmine, reveals design principles and enables the creation of faster and red-light-specific optogenetic tools.
RNA interference (RNAi) has become a widely used reverse genetic tool to study gene function in eukaryotic organisms and is being developed as a technology for insect pest management. The efficiency ...of RNAi varies among organisms. Insects from different orders also display differential efficiency of RNAi, ranging from highly efficient (coleopterans) to very low efficient (lepidopterans). We investigated the reasons for varying RNAi efficiency between lepidopteran and coleopteran cell lines and also between the Colorado potato beetle, Leptinotarsa decemlineata and tobacco budworm, Heliothis virescens. The dsRNA either injected or fed was degraded faster in H. virescens than in L. decemlineata. Both lepidopteran and coleopteran cell lines and tissues efficiently took up the dsRNA. Interestingly, the dsRNA administered to coleopteran cell lines and tissues was taken up and processed to siRNA whereas the dsRNA was taken up by lepidopteran cell lines and tissues but no siRNA was detected in the total RNA isolated from these cell lines and tissues. The data included in this paper showed that the degradation and intracellular transport of dsRNA are the major factors responsible for reduced RNAi efficiency in lepidopteran insects.
Manufacturing of adeno-associated viruses (AAV) for gene and cell therapy applications has increased significantly and spurred development of improved mammalian and insect cell-based production ...systems. We developed a baculovirus-based insect cell production system—the SGMO Helper—with a novel gene architecture and greater flexibility to modulate the expression level and content of individual Rep and Cap proteins. In addition, we incorporated modifications to the AAV6 capsid sequence that improves yield, capsid integrity, and potency. Production of recombinant AAV 6 (rAAV6) using the SGMO Helper had improved yields compared to the Bac-RepCap helper from the Kotin lab. SGMO Helper-derived rAAV6 is resistant to a previously described proteolytic cleavage unique to baculovirus-insect cell production systems and has improved capsid ratios and potency, in vitro and in vivo, compared with rAAV6 produced using Bac-RepCap. Next-generation sequencing sequence analysis demonstrated that the SGMO Helper is stable over six serial passages and rAAV6 capsids contain comparable amounts of non-vector genome DNA as rAAV6 produced using Bac-RepCap. AAV production using the SGMO Helper is scalable using bioreactors and has improved yield, capsid ratio, and in vitro potency. Our studies demonstrate that the SGMO Helper is an improved platform for AAV manufacturing to enable delivery of cutting-edge gene and cell therapies.
Display omitted
Ward and colleagues have developed an improved helper system for AAV manufacturing in insect cells—the SGMO Helper—with superior yields, potency, and capsid integrity to enable delivery of cutting-edge gene and cell therapies to patients.
Adequate membrane fluidity is required for a variety of key cellular processes and in particular for proper function of membrane proteins. In most eukaryotic cells, membrane fluidity is known to be ...regulated by fatty acid desaturation and cholesterol, although some cells, such as insect cells, are almost devoid of sterol synthesis. We show here that insect and mammalian cells present similar microviscosity at their respective physiological temperature. To investigate how both sterols and phospholipids control fluidity homeostasis, we quantified the lipidic composition of insect SF9 and mammalian HEK 293T cells under normal or sterol-modified condition. As expected, insect cells show minimal sterols compared with mammalian cells. A major difference is also observed in phospholipid content as the ratio of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) is inverted (4 times higher in SF9 cells). In vitro studies in liposomes confirm that both cholesterol and PE can increase rigidity of the bilayer, suggesting that both can be used by cells to maintain membrane fluidity. We then show that exogenously increasing the cholesterol amount in SF9 membranes leads to a significant decrease in PE:PC ratio whereas decreasing cholesterol in HEK 293T cells using statin treatment leads to an increase in the PE:PC ratio. In all cases, the membrane fluidity is maintained, indicating that both cell types combine regulation by sterols and phospholipids to control proper membrane fluidity.
Cytoplasmic dynein is an approximately 1.4 MDa multi‐protein complex that transports many cellular cargoes towards the minus ends of microtubules. Several in vitro studies of mammalian dynein have ...suggested that individual motors are not robustly processive, raising questions about how dynein‐associated cargoes can move over long distances in cells. Here, we report the production of a fully recombinant human dynein complex from a single baculovirus in insect cells. Individual complexes very rarely show directional movement in vitro. However, addition of dynactin together with the N‐terminal region of the cargo adaptor BICD2 (BICD2N) gives rise to unidirectional dynein movement over remarkably long distances. Single‐molecule fluorescence microscopy provides evidence that BICD2N and dynactin stimulate processivity by regulating individual dynein complexes, rather than by promoting oligomerisation of the motor complex. Negative stain electron microscopy reveals the dynein–dynactin–BICD2N complex to be well ordered, with dynactin positioned approximately along the length of the dynein tail. Collectively, our results provide insight into a novel mechanism for coordinating cargo binding with long‐distance motor movement.
Synopsis
Cargo adaptor BICD2 converts dynein from a non‐processive to a highly processive motor in presence of dynactin. This might coordinate long‐distance movement with cargo availability.
Production of the 1.4 MDa recombinant mammalian dynein complex using a single baculovirus.
The dynactin complex and the N‐terminus of the cargo adaptor BICD2 (BICD2N) are sufficient to convert dynein into a highly processive motor.
In the presence of BICD2N, dynactin forms a well‐ordered complex with the dynein tail.
Results suggest a mechanism for coupling cargo availability to the activation of long‐distance transport.
Cargo adaptor BICD2 converts dynein from a non‐processive to a highly processive motor in presence of dynactin. This might coordinate long‐distance movement with cargo availability.
Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that is localized to thousands of mammalian genes. Though important to human disease and as a drug target, how PRC2 is recruited ...remains unclear. One model invokes cis-regulatory RNA. Herein, we biochemically and functionally probe PRC2’s recognition of RNA using the X-inactivation model. We observe surprisingly high discriminatory capabilities. While SUZ12 and JARID2 subunits can bind RNA, EZH2 has highest affinity and is somewhat promiscuous. EED regulates the affinity of EZH2 for RNA, lending greater specificity to PRC2-RNA interactions. Intriguingly, while RNA is crucial for targeting, RNA inhibits EZH2’s catalytic activity. JARID2 weakens PRC2’s binding to RNA and relieves catalytic inhibition. We propose that RNA guides PRC2 to its target but inhibits its enzymatic activity until PRC2 associates with JARID2 on chromatin. Our study provides a molecular view of regulatory interactions between RNA and PRC2 at the chromatin interface.
Display omitted
•PRC2 binds RNA selectively with high specificity•EZH2 is somewhat promiscuous, and EED lends specificity to PRC2-RNA interactions•RNA inhibits PRC2’s histone methyltransferase activity•JARID2 attenuates RNA binding and relieves inhibition of methylation
PRC2 methylates histone H3 at thousands of sites in the genome, but how it is targeted remains unknown. Cifuentes-Rojas et al. show that RNA plays dual positive-negative roles in this process, first targeting PRC2 to specific sites but also inhibiting its methyltransferase activity until JARID2 relieves the inhibition by dissociating RNA from PRC2.
Pannexin 1 (PANX1) is an ATP-permeable channel with critical roles in a variety of physiological functions such as blood pressure regulation
, apoptotic cell clearance
and human oocyte development
. ...Here we present several structures of human PANX1 in a heptameric assembly at resolutions of up to 2.8 angström, including an apo state, a caspase-7-cleaved state and a carbenoxolone-bound state. We reveal a gating mechanism that involves two ion-conducting pathways. Under normal cellular conditions, the intracellular entry of the wide main pore is physically plugged by the C-terminal tail. Small anions are conducted through narrow tunnels in the intracellular domain. These tunnels connect to the main pore and are gated by a long linker between the N-terminal helix and the first transmembrane helix. During apoptosis, the C-terminal tail is cleaved by caspase, allowing the release of ATP through the main pore. We identified a carbenoxolone-binding site embraced by W74 in the extracellular entrance and a role for carbenoxolone as a channel blocker. We identified a gap-junction-like structure using a glycosylation-deficient mutant, N255A. Our studies provide a solid foundation for understanding the molecular mechanisms underlying the channel gating and inhibition of PANX1 and related large-pore channels.
Alternative mRNA processing is a critical mechanism for proteome expansion and gene regulation in higher eukaryotes. The SR family proteins play important roles in splicing regulation. Intriguingly, ...mammalian genomes encode many poorly characterized SR-like proteins, including subunits of the mRNA 3′-processing factor CFIm, CFIm68 and CFIm59. Here we demonstrate that CFIm functions as an enhancer-dependent activator of mRNA 3′ processing. CFIm regulates global alternative polyadenylation (APA) by specifically binding and activating enhancer-containing poly(A) sites (PASs). Importantly, the CFIm activator functions are mediated by the arginine-serine repeat (RS) domains of CFIm68/59, which bind specifically to an RS-like region in the CPSF subunit Fip1, and this interaction is inhibited by CFIm68/59 hyper-phosphorylation. The remarkable functional similarities between CFIm and SR proteins suggest that interactions between RS-like domains in regulatory and core factors may provide a common activation mechanism for mRNA 3′ processing, splicing, and potentially other steps in RNA metabolism.
Display omitted
•UGUA is a position-dependent enhancer for mRNA 3′ processing•CFIm is an enhancer-dependent activator of mRNA 3′ processing•The activator function of CFIm is mediated by its RS-like domains•mRNA 3′ processing and splicing share a common activation mechanism
Zhu et al. show that CFIm is an enhancer-dependent activator that promotes mRNA 3′-processing complex assembly. CFIm activator function requires the RS-like domains of CFIm68/59, and it involves a mechanism similar to SR protein-mediated splicing regulation, suggesting a unified activation mechanism for mRNA 3′ processing and splicing.
The spike glycoprotein （S） of recently identified Middle East respiratory syndrome coronavirus （MERS-CoV） tar- gets the cellular receptor, dipeptidyl peptidase 4 （DPP4）. Sequence comparison and ...modeling analysis have revealed a putative receptor-binding domain （RBD） on the viral spike, which mediates this interaction. We report the 3.0 X- resolution crystal structure of MERS-CoV RBD bound to the extracellular domain of human DPP4. Our results show that MERS-CoV RBD consists of a core and a receptor-binding subdomain. The receptor-binding subdomain interacts with DPP4 IS-propeller but not its intrinsic hydrolase domain. MERS-CoV RBD and related SARS-CoV RBD share a high degree of structural similarity in their core subdomains, but are notably divergent in the receptor- binding subdomain. Mutagenesis studies have identified several key residues in the receptor-binding subdomain that are critical for viral binding to DPP4 and entry into the target cell. The atomic details at the interface between MERS-CoV RBD and DPP4 provide structural understanding of the virus and receptor interaction, which can guide development of therapeutics and vaccines against MERS-CoV infection.
PDF
