Emerging and re-emerging RNA viruses occasionally cause epidemics and pandemics worldwide, such as the on-going outbreak of the novel coronavirus SARS-CoV-2. Herein, we identified two potent inhibitors of human DHODH, S312 and S416, with favorable drug-likeness and pharmacokinetic profiles, which all showed broad-spectrum antiviral effects against various RNA viruses, including influenza A virus, Zika virus, Ebola virus, and particularly against SARS-CoV-2. Notably, S416 is reported to be the most potent inhibitor so far with an EC of 17 nmol/L and an SI value of 10,505.88 in infected cells. Our results are the first to validate that DHODH is an attractive host target through high antiviral efficacy in vivo and low virus replication in DHODH knock-out cells. This work demonstrates that both S312/S416 and old drugs (Leflunomide/Teriflunomide) with dual actions of antiviral and immuno-regulation may have clinical potentials to cure SARS-CoV-2 or other RNA viruses circulating worldwide, no matter such viruses are mutated or not.
Animals ; Antiviral Agents ; pharmacology ; therapeutic use ; Betacoronavirus ; drug effects ; physiology ; Binding Sites ; drug effects ; Cell Line ; Coronavirus Infections ; drug therapy ; virology ; Crotonates ; pharmacology ; Cytokine Release Syndrome ; drug therapy ; Drug Evaluation, Preclinical ; Gene Knockout Techniques ; Humans ; Influenza A virus ; drug effects ; Leflunomide ; pharmacology ; Mice ; Mice, Inbred BALB C ; Orthomyxoviridae Infections ; drug therapy ; Oseltamivir ; therapeutic use ; Oxidoreductases ; antagonists & inhibitors ; metabolism ; Pandemics ; Pneumonia, Viral ; drug therapy ; virology ; Protein Binding ; drug effects ; Pyrimidines ; biosynthesis ; RNA Viruses ; drug effects ; physiology ; Structure-Activity Relationship ; Toluidines ; pharmacology ; Ubiquinone ; metabolism ; Virus Replication ; drug effects

As a crucial signaling molecule, calcium plays a critical role in many physiological and pathological processes by regulating ion channel activity. Recently, one study resolved the structure of the transient receptor potential melastatin 2 (TRPM2) channel from Nematostella vectensis (nvTRPM2). This identified a calcium-binding site in the S2-S3 loop, while its effect on channel gating remains unclear. Here, we investigated the role of this calcium-binding site in both nvTRPM2 and human TRPM2 (hTRPM2) by mutagenesis and patch-clamp recording. Unlike hTRPM2, nvTRPM2 cannot be activated by calcium alone. Moreover, the inactivation rate of nvTRPM2 was decreased as intracellular calcium concentration was increased. In addition, our results showed that the four key residues in the calcium-binding site of S2-S3 loop have similar effects on the gating processes of nvTRPM2 and hTRPM2. Among them, the mutations at negatively charged residues (glutamate and aspartate) substantially decreased the currents of nvTRPM2 and hTRPM2. This suggests that these sites are essential for calcium-dependent channel gating. For the charge-neutralizing residues (glutamine and asparagine) in the calcium-binding site, our data showed that glutamine mutating to alanine or glutamate did not affect the channel activity, but glutamine mutating to lysine caused loss of function. Asparagine mutating to aspartate still remained functional, while asparagine mutating to alanine or lysine led to little channel activity. These results suggest that the side chain of glutamine has a less contribution to channel gating than does asparagine. However, our data indicated that both glutamine mutating to alanine or glutamate and asparagine mutating to aspartate accelerated the channel inactivation rate, suggesting that the calcium-binding site in the S2-S3 loop is important for calcium-dependent channel inactivation. Taken together, our results uncovered the effect of four key residues in the S2-S3 loop of TRPM2 on the TRPM2 gating process.
Animals ; Asparagine/physiology* ; Binding Sites ; Calcium/metabolism* ; Glutamine/physiology* ; HEK293 Cells ; Humans ; Ion Channel Gating/physiology* ; Sea Anemones ; TRPM Cation Channels/physiology*

Human papillomaviruses (HPVs) including high-risk (HR) and low-risk (LR) subtypes have distinguishable variation on both genotypes and phenotypes. The co-infection of multiple HR-HPVs, headed by HPV16, is common in cervical cancer in female. Recently accumulating reports have focused on the interaction between virus and host, particularly the role of human microRNAs (miRNAs) in anti-viral defense by targeting viral genome. Here, we found a well-conserved target site of miRNAs in the genomes of most HR-HPVs, not LR-HPVs, by scanning all potential target sites of human miRNAs on 24 HPVs of unambiguous subtypes of risk. The site is targeted by two less common human miRNAs, miR-875 and miR-3144, and is located in E6 oncogene open reading frame (ORF) and overlap with the first alternative splice exon of viral early transcripts. In validation tests, miR-875 and miR-3144 were identified to suppress the target reporter activity markedly and inhibit the expression of both synthetically exogenous E6 and endogenous E6 oncogene. High level of two miRNAs can inhibit cell growth and promote apoptosis in HPV16-positive cervical cancer cells. This study provides a promising common target of miRNAs for most HR-HPVs and highlights the effects of two low expressed human miRNAs on tumour suppression.
Apoptosis ; genetics ; Base Sequence ; Binding Sites ; genetics ; Cell Line, Tumor ; Cell Proliferation ; genetics ; Female ; Gene Expression ; Host-Pathogen Interactions ; genetics ; Human papillomavirus 16 ; genetics ; physiology ; Humans ; MicroRNAs ; genetics ; Microscopy, Fluorescence ; Molecular Sequence Data ; Oncogene Proteins, Viral ; genetics ; Repressor Proteins ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Homology, Nucleic Acid ; Uterine Cervical Neoplasms ; genetics ; pathology ; virology

Binding Sites ; Crystallography, X-Ray ; Humans ; Models, Molecular ; Protein Interaction Domains and Motifs ; Protein Multimerization ; Protein Structure, Secondary ; Receptors, Metabotropic Glutamate ; chemistry ; physiology

Diverse subtypes of voltage-gated sodium channels (VGSCs) have been found throughout tissues of the brain, muscles and the heart. Neurotoxins extracted from the venom of the Asian scorpion Buthus martensi Karsch (BmK) act as sodium channel-specific modulators and have therefore been widely used to study VGSCs. α-type neurotoxins, named BmK I, BmK αIV and BmK abT, bind to receptor site-3 on VGSCs and can strongly prolong the inactivation phase of VGSCs. In contrast, β-type neurotoxins, named BmK AS, BmK AS-1, BmK IT and BmK IT2, occupy receptor site-4 on VGSCs and can suppress peak currents and hyperpolarize the activation kinetics of sodium channels. Accumulating evidence from binding assays of scorpion neurotoxins on VGSCs, however, indicate that pharmacological sensitivity of VGSC subtypes to different modulators is much more complex than that suggested by the simple α-type and β-type neurotoxin distinction. Exploring the mechanisms of possible dynamic interactions between site 3-/4-specific modulators and region- and/or species-specific subtypes of VGSCs would therefore greatly expand our understanding of the physiological and pharmacological properties of diverse VGSCs. In this review, we discuss the pharmacological and structural diversity of VGSCs as revealed by studies exploring the binding properties and cross-competitive binding of site 3- or site 4-specific modulators in VGSC subtypes in synaptosomes from distinct tissues of diverse species.
Animals ; Binding Sites ; Binding, Competitive ; Brain ; metabolism ; Heart ; physiology ; Humans ; Insect Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Insecta ; Ion Channel Gating ; drug effects ; physiology ; Kinetics ; Mammals ; Muscles ; metabolism ; Neurotoxins ; chemistry ; classification ; pharmacology ; Protein Binding ; Scorpions ; chemistry ; Sodium ; metabolism ; Sodium Channel Blockers ; pharmacology ; Sodium Channels ; classification ; genetics ; metabolism ; Synaptosomes ; drug effects ; metabolism

In this review we summarize the progress made towards understanding the role of protein-protein interactions in the function of various bioluminescence systems of marine organisms, including bacteria, jellyfish and soft corals, with particular focus on methodology used to detect and characterize these interactions. In some bioluminescence systems, protein-protein interactions involve an "accessory protein" whereby a stored substrate is efficiently delivered to the bioluminescent enzyme luciferase. Other types of complexation mediate energy transfer to an "antenna protein" altering the color and quantum yield of a bioluminescence reaction. Spatial structures of the complexes reveal an important role of electrostatic forces in governing the corresponding weak interactions and define the nature of the interaction surfaces. The most reliable structural model is available for the protein-protein complex of the Ca(2+)-regulated photoprotein clytin and green-fluorescent protein (GFP) from the jellyfish Clytia gregaria, solved by means of Xray crystallography, NMR mapping and molecular docking. This provides an example of the potential strategies in studying the transient complexes involved in bioluminescence. It is emphasized that structural studies such as these can provide valuable insight into the detailed mechanism of bioluminescence.
Animals ; Anthozoa ; physiology ; Aquatic Organisms ; physiology ; Bacteria ; metabolism ; Binding Sites ; Calcium ; metabolism ; Crystallography, X-Ray ; Fluorescence Resonance Energy Transfer ; Green Fluorescent Proteins ; metabolism ; Hydrozoa ; physiology ; Imidazoles ; metabolism ; Luciferases ; metabolism ; Luminescent Measurements ; instrumentation ; methods ; Luminescent Proteins ; metabolism ; Models, Molecular ; Protein Binding ; Pteridines ; metabolism ; Pyrazines ; metabolism ; Scyphozoa ; physiology ; Spectrometry, Fluorescence

The important and diverse regulatory roles of Ca(2+) in eukaryotes are conveyed by the EF-hand containing calmodulin superfamily. However, the calcium-regulatory proteins in prokaryotes are still poorly understood. In this study, we report the three-dimensional structure of the calcium-binding protein from Streptomyces coelicolor, named CabD, which shares low sequence homology with other known helix-loop-helix EF-hand proteins. The CabD structure should provide insights into the biological role of the prokaryotic calcium-binding proteins. The unusual structural features of CabD compared with prokaryotic EF-hand proteins and eukaryotic sarcoplasmic calcium-binding proteins, including the bending conformation of the first C-terminal α-helix, unpaired ligand-binding EF-hands and the lack of the extreme C-terminal loop region, suggest it may have a distinct and significant function in calcium-mediated bacterial physiological processes, and provide a structural basis for potential calcium-mediated regulatory roles in prokaryotes.
Amino Acid Sequence ; Binding Sites ; Calcium ; physiology ; Calcium-Binding Proteins ; chemistry ; Crystallography, X-Ray ; EF Hand Motifs ; Molecular Sequence Data ; Protein Binding ; Protein Structure, Tertiary ; Sequence Alignment ; Sequence Homology, Amino Acid ; Streptomyces coelicolor ; Structural Homology, Protein ; Surface Properties

Retinoic acid-inducible gene-I (RIG-I) functions as an intracellular pattern recognition receptor (PRR) that recognizes the 5'-triphosphate moiety of single-stranded RNA viruses to initiate the innate immune response. Previous studies have shown that Lys63-linked ubiquitylation is required for RIG-I activation and the downstream anti-viral type I interferon (IFN-I) induction. Herein we reported that, RIG-I was also modified by small ubiquitin-like modifier-1 (SUMO-1). Functional analysis showed that RIG-I SUMOylation enhanced IFN-I production through increased ubiquitylation and the interaction with its downstream adaptor molecule Cardif. Our results therefore suggested that SUMOylation might serve as an additional regulatory tier for RIG-I activation and IFN-I signaling.
Adaptor Proteins, Signal Transducing ; physiology ; Base Sequence ; Binding Sites ; DEAD Box Protein 58 ; DEAD-box RNA Helicases ; chemistry ; genetics ; immunology ; physiology ; DNA Primers ; genetics ; Gene Knockdown Techniques ; HEK293 Cells ; HeLa Cells ; Humans ; Immunity, Innate ; Interferon Type I ; immunology ; physiology ; RNA Interference ; SUMO-1 Protein ; physiology ; Sendai virus ; immunology ; Signal Transduction ; Sumoylation ; Ubiquitin-Conjugating Enzymes ; antagonists & inhibitors ; genetics ; physiology

OBJECTIVETo establish a new non-radioactive method for electrophoretic mobility shift assay (EMSA) to investigate the binding between glucocorticoid induced leucine zipper (GILZ) and peroxisome proliferator-activated receptor-gamma 2 (PPARgamma2) promoter oligonucleotides.

METHODSGILZ protein prepared by prokaryotic expression was linked to PPARgamma2 promoter oligonucleotides end-labeled with IRDye 800 infrared dye. The DNA-protein complex was separated with non-denatured polyacrylamide gel and scanned with the Odyssey. Infrared Imaging System.

RESULTSOne lane of DNA-protein complex was clearly presented, and the signal intensity increased along with the increment of the protein load.

CONCLUSIONThis infrared imaging system can be used for EMSA for detecting the DNA-protein complex with high sensitivity efficiency and allows easy operation.

Binding Sites ; DNA ; chemistry ; DNA-Binding Proteins ; chemistry ; metabolism ; Electrophoretic Mobility Shift Assay ; instrumentation ; methods ; Fluorescent Dyes ; chemistry ; Gene Expression Regulation ; Humans ; Infrared Rays ; Protein Binding ; Protein Interaction Domains and Motifs ; physiology ; Proteins ; chemistry

CHO cells expressing human GPIb/IX and rabbit red blood cells coated with human von Willebrand factor (VWF) were adapted to our study on the binding probability and the detachment force of GPIb/IX and VWF. With the micropipette system, the two cells were impinged under a constant force for controlled time. When the cells were pulled apart, the deformation of RBC was recorded, and the binding score and detachment force of the proteins were determined. After the two cells were impinged into 0.5 microm for 30 s, the binding probability of the two cells carrying GPIb/IX and VWF was 15.0%. Via analyzing the deformation of red blood cells, we found out the distribution of rupture forces of cells with GPIb/IX and VWF. Therefore, we infer that the continuous distribution of the detachment force is due to the stochastic effect. The most probable value of the detachment force was 10 pN.
Animals ; Binding Sites ; Blood Platelets ; metabolism ; CHO Cells ; Cell Adhesion ; Cricetinae ; Cricetulus ; Humans ; Platelet Activation ; physiology ; Platelet Glycoprotein GPIb-IX Complex ; chemistry ; metabolism ; Rabbits ; von Willebrand Factor ; chemistry ; metabolism