Reconstitution of RNA cap methylation reveals different features of SARS-CoV-2 and SARS-CoV methyltransferases.

Cap RNA methylations play important roles in the replication, evasion of host RNA sensor recognition, and pathogenesis. Coronaviruses possess both guanine N7- and 2'-O-ribose methyltransferases (N7-MTase and 2'-O-MTase) encoded by nonstructural protein (nsp) 14 and nsp16/10 complex, respectively. In this study, we reconstituted the two-step RNA methylations of N7-MTase and 2'-O-MTase of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro and demonstrated its common and different features in comparison with that of SARS-CoV. We revealed that the nsp16/10 2'-O-MTase of SARS-CoV-2 has a broader substrate selectivity than the counterpart of SARS-CoV and can accommodate both unmethylated and uncapped RNA substrates in a sequence-independent manner. Most intriguingly, the substrate selectivity of nsp16/10 complex is not determined by the apoenzyme of nsp16 MTase but by its cofactor nsp10. These results provide insight into the unique features of SARS-CoV-2 MTases and may help develop strategies to precisely intervene in the methylation pathway and pathogenesis of SARS-CoV-2.

A small-molecule activation mechanism that directly opens the KCNQ2 channel.

Pharmacological activation of voltage-gated ion channels by ligands serves as the basis for therapy and mainly involves a classic gating mechanism that augments the native voltage-dependent open probability. Through structure-based virtual screening, we identified a new scaffold compound, Ebio1, serving as a potent and subtype-selective activator for the voltage-gated potassium channel KCNQ2 and featuring a new activation mechanism. Single-channel patch-clamp, cryogenic-electron microscopy and molecular dynamic simulations, along with chemical derivatives, reveal that Ebio1 engages the KCNQ2 activation by generating an extended channel gate with a larger conductance at the saturating voltage (+50 mV). This mechanism is different from the previously observed activation mechanism of ligands on voltage-gated ion channels. Ebio1 caused S6 helices from residues S303 and F305 to perform a twist-to-open movement, which was sufficient to open the KCNQ2 gate. Overall, our findings provide mechanistic insights into the activation of KCNQ2 channel by Ebio1 and lend support for KCNQ-related drug development.

Ligand activation mechanisms of human KCNQ2 channel.

The human voltage-gated potassium channel KCNQ2/KCNQ3 carries the neuronal M-current, which helps to stabilize the membrane potential. KCNQ2 can be activated by analgesics and antiepileptic drugs but their activation mechanisms remain unclear. Here we report cryo-electron microscopy (cryo-EM) structures of human KCNQ2-CaM in complex with three activators, namely the antiepileptic drug cannabidiol (CBD), the lipid phosphatidylinositol 4,5-bisphosphate (PIP2), and HN37 (pynegabine), an antiepileptic drug in the clinical trial, in an either closed or open conformation. The activator-bound structures, along with electrophysiology analyses, reveal the binding modes of two CBD, one PIP2, and two HN37 molecules in each KCNQ2 subunit, and elucidate their activation mechanisms on the KCNQ2 channel. These structures may guide the development of antiepileptic drugs and analgesics that target KCNQ2.

CCDC50 promotes tumor growth through regulation of lysosome homeostasis.

The maintenance of lysosome homeostasis is crucial for cell growth. Lysosome-dependent degradation and metabolism sustain tumor cell survival. Here, we demonstrate that CCDC50 serves as a lysophagy receptor, promoting tumor progression and invasion by controlling lysosomal integrity and renewal. CCDC50 monitors lysosomal damage, recognizes galectin-3 and K63-linked polyubiquitination on damaged lysosomes, and specifically targets them for autophagy-dependent degradation. CCDC50 deficiency causes the accumulation of ruptured lysosomes, impaired autophagic flux, and superfluous reactive oxygen species, consequently leading to cell death and tumor suppression. CCDC50 expression is associated with malignancy, progression to metastasis, and poor overall survival in human melanoma. Targeting CCDC50 suppresses tumor growth and lung metastasis, and enhances the effect of BRAFV600E inhibition. Thus, we demonstrate critical roles of CCDC50-mediated clearance of damaged lysosomes in supporting tumor growth, hereby identifying a potential therapeutic target of melanoma.

Chinese herbal medicine for the treatment of cardiovascular diseases ─ Targeting cardiac ion channels.

Cardiovascular disease (CVD) remains the leading cause of morbidity and mortality, imposing an increasing global health burden. Cardiac ion channels (voltage-gated NaV, CaV, KVs, and others) synergistically shape the cardiac action potential (AP) and control the heartbeat. Dysfunction of these channels, due to genetic mutations, transcriptional or post-translational modifications, may disturb the AP and lead to arrhythmia, a major risk for CVD patients. Although there are five classes of anti-arrhythmic drugs available, they can have varying levels of efficacies and side effects on patients, possibly due to the complex pathogenesis of arrhythmias. As an alternative treatment option, Chinese herbal remedies have shown promise in regulating cardiac ion channels and providing anti-arrhythmic effects. In this review, we first discuss the role of cardiac ion channels in maintaining normal heart function and the pathogenesis of CVD, then summarize the classification of Chinese herbal compounds, and elaborate detailed mechanisms of their efficacy in regulating cardiac ion channels and in alleviating arrhythmia and CVD. We also address current limitations and opportunities for developing new anti-CVD drugs based on Chinese herbal medicines.

The regulation of NLRP3 inflammasome activation by CCDC50-mediated autophagy.

The assembly of the NLRP3 inflammasome can be initiated by a wide range of stimuli including exogenous infection as well as endogenous damage. Therefore, the tight regulation of the NLRP3 inflammasome is crucial for the host to resist microbial invasion and maintain homeostasis. Our recent work has identified a negative regulator of NLRP3-mediated inflammation, namely CCDC50 (coiled-coil domain containing protein 50). CCDC50 can be induced by NLRP3 agonists and then functions as a macroautophagy/autophagy cargo receptor to recognize K63-polyubiquitinated NLRP3 and deliver it to MAP1LC3/LC3-conjugated phagophores for degradation. CCDC50 inhibits the polymerization of NLRP3 and the recruitment of PYCARD/ASC, consequently suppressing the assembly of inflammasomes. ccdc50-knockout mice are more susceptible to dextran-sulfate (DSS)-induced colitis and exhibit more severe gut inflammation with elevated NLRP3 inflammasome activity, suggesting a protective role of CCDC50 in the pathology and progression of inflammatory bowel disease (IBD). Our finding reveals a function of autophagy-related proteins in the regulation of NLRP3-mediated inflammation, thus demonstrating the intricate crosstalk between autophagy and inflammation.

CDK4/6 Inhibition Enhances Oncolytic Virus Efficacy by Potentiating Tumor-Selective Cell Killing and T-cell Activation in Refractory Glioblastoma.

Glioblastoma (GBM) is among the most aggressive human cancers. Although oncolytic virus (OV) therapy has been proposed as a potential approach to treat GBM, it frequently fails because GBM cells are usually nonpermissive to OV. Here, we describe a dual-step drug screen for identifying chemical enhancers of OV in GBM. From a high-throughput screen of 1416 FDA-approved drugs, an inhibitor of CDK4/6 was identified as the top enhancer, selectively increasing potency of two OV strains, VSVΔ51 and Zika virus. Mechanistically, CDK4/6 inhibition promoted autophagic degradation of MAVS, resulting in impaired antiviral responses and enhanced tumor-selective replication of VSVΔ51 in vitro and in vivo. CDK4/6 inhibition cooperated with VSVΔ51 to induce severe DNA damage stress and amplify oncolysis. In GBM xenograft models, combined treatment with CDK4/6 inhibitor and VSVΔ51 significantly inhibited tumor growth and prolonged the survival of tumor-bearing mice. Further investigation revealed that CDK4/6 inhibitor and VSVΔ51 synergistically induced immunogenic cell death and boosted antitumor immunity. Together, this study features a promising approach of treating aggressive GBM through the combination of CDK4/6 inhibitor with OV. SIGNIFICANCE: This study proposes inhibition of cyclin-dependent kinases as a clinically translatable combinatorial strategy to enhance the efficacy of oncolytic virotherapy in GBM.

A benzodiazepine activator locks Kv7.1 channels open by electro-mechanical uncoupling.

Loss-of-function mutations in Kv7.1 often lead to long QT syndrome (LQTS), a cardiac repolarization disorder associated with arrhythmia and subsequent sudden cardiac death. The discovery of agonistic IKs modulators may offer a new potential strategy in pharmacological treatment of this disorder. The benzodiazepine derivative (R)-L3 potently activates Kv7.1 channels and shortens action potential duration, thus may represent a starting point for drug development. However, the molecular mechanisms underlying modulation by (R)-L3 are still unknown. By combining alanine scanning mutagenesis, non-canonical amino acid incorporation, voltage-clamp electrophysiology and fluorometry, and in silico protein modelling, we show that (R)-L3 not only stimulates currents by allosteric modulation of the pore domain but also alters the kinetics independently from the pore domain effects. We identify novel (R)-L3-interacting key residues in the lower S4-segment of Kv7.1 and observed an uncoupling of the outer S4 segment with the inner S5, S6 and selectivity filter segments.

CCDC50 suppresses NLRP3 inflammasome activity by mediating autophagic degradation of NLRP3.

The NLRP3-directed inflammasome complex is crucial for the host to resist microbial infection and monitor cellular damage. However, the hyperactivation of NLRP3 inflammasome is implicated in pathogenesis of inflammatory diseases, including inflammatory bowel disease (IBD). Autophagy and autophagy-related genes are closely linked to NLRP3-mediated inflammation in these inflammatory disorders. Here, we report that CCDC50, a novel autophagy cargo receptor, negatively regulates NLRP3 inflammasome assembly and suppresses the cleavage of pro-caspase-1 and interleukin 1ß (IL-1ß) release by delivering NLRP3 for autophagic degradation. Transcriptome analysis showed that knockdown of CCDC50 results in upregulation of signaling pathways associated with autoinflammatory diseases. CCDC50 deficiency leads to enhanced proinflammatory cytokine response triggered by a wide range of endogenous and exogenous NLRP3 stimuli. Ccdc50-deficient mice are more susceptible to dextran sulfate (DSS)-induced colitis and exhibit more severe gut inflammation with elevated NLRP3 inflammasome activity. These results illustrate the physiological significance of CCDC50 in the pathogenicity of inflammatory diseases, suggesting protective roles of CCDC50 in keeping gut inflammation under control.

Structural mechanisms for the activation of human cardiac KCNQ1 channel by electro-mechanical coupling enhancers.

The cardiac KCNQ1 potassium channel carries the important IKs current and controls the heart rhythm. Hundreds of mutations in KCNQ1 can cause life-threatening cardiac arrhythmia. Although KCNQ1 structures have been recently resolved, the structural basis for the dynamic electro-mechanical coupling, also known as the voltage sensor domain-pore domain (VSD-PD) coupling, remains largely unknown. In this study, utilizing two VSD-PD coupling enhancers, namely, the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) and a small-molecule ML277, we determined 2.5-3.5 Å resolution cryo-electron microscopy structures of full-length human KCNQ1-calmodulin (CaM) complex in the apo closed, ML277-bound open, and ML277-PIP2-bound open states. ML277 binds at the "elbow" pocket above the S4-S5 linker and directly induces an upward movement of the S4-S5 linker and the opening of the activation gate without affecting the C-terminal domain (CTD) of KCNQ1. PIP2 binds at the cleft between the VSD and the PD and brings a large structural rearrangement of the CTD together with the CaM to activate the PD. These findings not only elucidate the structural basis for the dynamic VSD-PD coupling process during KCNQ1 gating but also pave the way to develop new therapeutics for anti-arrhythmia.

Autophagy receptor CCDC50 tunes the STING-mediated interferon response in viral infections and autoimmune diseases.

DNA sensing and timely activation of interferon (IFN)-mediated innate immunity are crucial for the defense against DNA virus infections and the clearance of abnormal cells. However, overactivation of immune responses may lead to tissue damage and autoimmune diseases; therefore, these processes must be intricately regulated. STING is the key adaptor protein, which is activated by cyclic GMP-AMP, the second messenger derived from cGAS-mediated DNA sensing. Here, we report that CCDC50, a newly identified autophagy receptor, tunes STING-directed type I IFN signaling activity by delivering K63-polyubiquitinated STING to autolysosomes for degradation. Knockout of CCDC50 significantly increases herpes simplex virus 1 (HSV-1)- or DNA ligand-induced production of type I IFN and proinflammatory cytokines. Ccdc50-deficient mice show increased production of IFN, decreased viral replication, reduced cell infiltration, and improved survival rates compared with their wild-type littermates when challenged with HSV-1. Remarkably, the expression of CCDC50 is downregulated in systemic lupus erythematosus (SLE), a chronic autoimmune disease. CCDC50 levels are negatively correlated with IFN signaling pathway activation and disease severity in human SLE patients. CCDC50 deficiency potentiates the cGAS-STING-mediated immune response triggered by SLE serum. Thus, our findings reveal the critical role of CCDC50 in the immune regulation of viral infections and autoimmune diseases and provide insights into the therapeutic implications of CCDC50 manipulation.

Endogenous reverse transcriptase and RNase H-mediated antiviral mechanism in embryonic stem cells.

Nucleic acid-based systems play important roles in antiviral defense, including CRISPR/Cas that adopts RNA-guided DNA cleavage to prevent DNA phage infection and RNA interference (RNAi) that employs RNA-guided RNA cleavage to defend against RNA virus infection. Here, we report a novel type of nucleic acid-based antiviral system that exists in mouse embryonic stem cells (mESCs), which suppresses RNA virus infection by DNA-mediated RNA cleavage. We found that the viral RNA of encephalomyocarditis virus can be reverse transcribed into complementary DNA (vcDNA) by the reverse transcriptase (RTase) encoded by endogenous retrovirus-like elements in mESCs. The vcDNA is negative-sense single-stranded and forms DNA/RNA hybrid with viral RNA. The viral RNA in the heteroduplex is subsequently destroyed by cellular RNase H1, leading to robust suppression of viral growth. Furthermore, either inhibition of the RTase activity or depletion of endogenous RNase H1 results in the promotion of virus proliferation. Altogether, our results provide intriguing insights into the antiviral mechanism of mESCs and the antiviral function of endogenized retroviruses and cellular RNase H. Such a natural nucleic acid-based antiviral mechanism in mESCs is referred to as ERASE (endogenous RTase/RNase H-mediated antiviral system), which is an addition to the previously known nucleic acid-based antiviral mechanisms including CRISPR/Cas in bacteria and RNAi in plants and invertebrates.

Modulating the voltage sensor of a cardiac potassium channel shows antiarrhythmic effects.

Cardiac arrhythmias are the most common cause of sudden cardiac death worldwide. Lengthening the ventricular action potential duration (APD), either congenitally or via pathologic or pharmacologic means, predisposes to a life-threatening ventricular arrhythmia, Torsade de Pointes. IKs (KCNQ1+KCNE1), a slowly activating K+ current, plays a role in action potential repolarization. In this study, we screened a chemical library in silico by docking compounds to the voltage-sensing domain (VSD) of the IKs channel. Here, we show that C28 specifically shifted IKs VSD activation in ventricle to more negative voltages and reversed the drug-induced lengthening of APD. At the same dosage, C28 did not cause significant changes of the normal APD in either ventricle or atrium. This study provides evidence in support of a computational prediction of IKs VSD activation as a potential therapeutic approach for all forms of APD prolongation. This outcome could expand the therapeutic efficacy of a myriad of currently approved drugs that may trigger arrhythmias.

An unconventional role of an ASB family protein in NF-κB activation and inflammatory response during microbial infection and colitis.

Nuclear factor κB (NF-κB)-mediated signaling pathway plays a crucial role in the regulation of inflammatory process, innate and adaptive immune responses. The hyperactivation of inflammatory response causes host cell death, tissue damage, and autoinflammatory disorders, such as sepsis and inflammatory bowel disease. However, how these processes are precisely controlled is still poorly understood. In this study, we demonstrated that ankyrin repeat and suppressor of cytokine signaling box containing 1 (ASB1) is involved in the positive regulation of inflammatory responses by enhancing the stability of TAB2 and its downstream signaling pathways, including NF-κB and mitogen-activated protein kinase pathways. Mechanistically, unlike other members of the ASB family that induce ubiquitination-mediated degradation of their target proteins, ASB1 associates with TAB2 to inhibit K48-linked polyubiquitination and thereby promote the stability of TAB2 upon stimulation of cytokines and lipopolysaccharide (LPS), which indicates that ASB1 plays a noncanonical role to further stabilize the target protein rather than induce its degradation. The deficiency of Asb1 protects mice from Salmonella typhimurium- or LPS-induced septic shock and increases the survival of mice. Moreover, Asb1-deficient mice exhibited less severe colitis and intestinal inflammation induced by dextran sodium sulfate. Given the crucial role of ASB proteins in inflammatory signaling pathways, our study offers insights into the immune regulation in pathogen infection and inflammatory disorders with therapeutic implications.

A novel selective autophagy receptor, CCDC50, delivers K63 polyubiquitination-activated RIG-I/MDA5 for degradation during viral infection.

Autophagy is a conserved process that delivers cytosolic substances to the lysosome for degradation, but its direct role in the regulation of antiviral innate immunity remains poorly understood. Here, through high-throughput screening, we discovered that CCDC50 functions as a previously unknown autophagy receptor that negatively regulates the type I interferon (IFN) signaling pathway initiated by RIG-I-like receptors (RLRs), the sensors for RNA viruses. The expression of CCDC50 is enhanced by viral infection, and CCDC50 specifically recognizes K63-polyubiquitinated RLRs, thus delivering the activated RIG-I/MDA5 for autophagic degradation. The association of CCDC50 with phagophore membrane protein LC3 is confirmed by crystal structure analysis. In contrast to other known autophagic cargo receptors that associate with either the LIR-docking site (LDS) or the UIM-docking site (UDS) of LC3, CCDC50 can bind to both LDS and UDS, representing a new type of cargo receptor. In mouse models with RNA virus infection, CCDC50 deficiency reduces the autophagic degradation of RIG-I/MDA5 and promotes type I IFN responses, resulting in enhanced viral resistance and improved survival rates. These results reveal a new link between autophagy and antiviral innate immune responses and provide additional insights into the regulatory mechanisms of RLR-mediated antiviral signaling.