Coronavirus disease 2019 (COVID-19) was caused by a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 utilizes human angiotensin converting enzyme 2 (hACE2) as the cellular receptor of its spike glycoprotein (SP) to gain entry into cells. Consequently, we focused on the potential of repurposing clinically available drugs to block the binding of SARS-CoV-2 to hACE2 by utilizing a novel artificial-intelligence drug screening approach. Based on the structure of S-RBD and hACE2, the pharmacophore of SARS-CoV-2-receptor-binding-domain (S-RBD) -hACE2 interface was generated and used to screen a library of FDA-approved drugs. A total of 20 drugs were retrieved as S-RBD-hACE2 inhibitors, of which 16 drugs were identified to bind to S-RBD or hACE2. Notably, tannic acid was validated to interfere with the binding of S-RBD to hACE2, thereby inhibited pseudotyped SARS-CoV-2 entry. Experiments involving competitive inhibition revealed that tannic acid competes with S-RBD and hACE2, whereas molecular docking proved that tannic acid interacts with the essential residues of S-RBD and hACE2. Based on the known antiviral activity and our findings, tannic acid might serve as a promising candidate for preventing and treating SARS-CoV-2 infection.

Lung cancer is among the most common malignancies with a poor 5-year survival rate reaching only 16%. Thus, new effective treatment modalities and drugs are urgently needed for the treatment of this malignancy. In this study, we conducted the first investigation of the effects of Biochanin A on lung cancer and revealed the mechanisms underlying its potential anticancer effects. Biochanin A decreased cell viability in a time-dependent and dose-dependent manner and suppressed colony formation in A549 and 95D cells. In addition, Biochanin A induced S phase arrest and apoptosis and decreased mitochondrial membrane potential (ΔΨm) in A549 and 95D cells in a dose-dependent manner. Our results of subcutaneous xenograft models showed that the growth of Biochanin A group was significantly inhibited compared with that of control groups. Finally, P21, Caspase-3, and Bcl-2 were activated in Biochanin A-treated cells and Biochanin A-treated xenografts which also demonstrated that Biochanin A induced cell cycle arrest and apoptosis in lung cancer cells by regulating expression of cell cycle-related proteins and apoptosis-related proteins. In conclusion, this study suggests that Biochanin A inhibits the proliferation of lung cancer cells and induces cell cycle arrest and apoptosis mainly by regulating cell cycle-related protein expression and activating the Bcl-2 and Caspase-3 pathways, thus suggesting that Biochanin A may be a promising drug to treat lung cancer.

DNA methylation patterns in plants are dynamically regulated by DNA methylation and active DNA demethylation in response to both environmental changes and development of plant. Beginning with the removal of methylated cytosine by ROS1/DME family of 5-methylcytosine DNA glycosylases, active DNA demethylation in plants occurs through base excision repair. So far, many components involved in active DNA demethylation remain undiscovered. Through a forward genetic screening of Arabidopsis mutants showing DNA hypermethylation at the EPF2 promoter region, we identified the conserved iron-sulfur cluster assembly protein MET18. MET18 dysfunction caused DNA hypermethylation at more than 1000 loci as well as the silencing of reporter genes and some endogenous genes. MET18 can directly interact with ROS1 in vitro and in vivo. ROS1 activity was reduced in the met18 mutant plants and point mutation in the conserved Fe-S cluster binding motif of ROS1 disrupted its biological function. Interestingly, a large number of DNA hypomethylated loci, especially in the CHH context, were identified from the met18 mutants and most of the hypo-DMRs were from TE regions. Our results suggest that MET18 can regulate both active DNA demethylation and DNA methylation pathways in Arabidopsis.

The tubulin colchicine binding site has been recognized as an attractive drug target to combat cancer, but none of the candidate drugs have been approved for medical treatment. We recently identified a structurally distinct small molecule S-40 as an oral potent tubulin destabilizing agent. Crystal structure analysis of S-40 in a complex with tubulin at a resolution of 2.4 Å indicated that S-40 occupies all 3 zones in the colchicine pocket with interactions different from known microtubule inhibitors, presenting unique effects on assembly and curvature of tubulin dimers. S-40 overcomes paclitaxel resistance and lacks neurotoxicity, which are the main obstacles limiting clinical applications of paclitaxel. Moreover, S-40 harbors the ability to inhibit growth of cancer cell lines as well as patient-derived organoids, induce mitotic arrest and cell apoptosis. Xenograft mouse models of human prostate cancer DU145, non-small cell lung cancer NCI-H1299 and paclitaxel-resistant A549 were strongly restrained without apparent side effects by S-40 oral administration once daily. These findings provide evidence for the development of S-40 as the next generation of orally effective microtubule inhibitors for cancer therapy.

Adrenergic stimulation of the heart initiates a signaling cascade in cardiac myocytes that increases the concentration of cAMP. Although cAMP elevation may occur over a large area of a target-organ cell, its effects are often more restricted due to local concentration of its main effector, protein kinase A (PKA), through A-kinase anchoring proteins (AKAPs). The HERG potassium channel, which produces the cardiac rapidly activating delayed rectifying K+ current (I(Kr)), is a target for cAMP/PKA regulation. PKA regulation of the current may play a role in the pathogenesis of hereditary and acquired abnormalities of the channel leading to cardiac arrhythmia. We examined the possible role for AKAP-mediated regulation of HERG channels. Here, we report that the PKA-RII-specific AKAP inhibitory peptide AKAP-IS perturbs the distribution of PKA-RII and diminishes the PKA-dependent phosphorylation of HERG protein. The functional consequence of AKAP-IS is a reversal of cAMP-dependent regulation of HERG channel activity. In further support of AKAP-mediated targeting of kinase to HERG, PKA activity was coprecipitated from HERG expressed in HEK cells. Velocity gradient centrifugation of solubilized porcine cardiac membrane proteins showed that several PKA-RI and PKA-RII binding proteins cosediment with ERG channels. A physical association of HERG with several specific AKAPs with known cardiac expression, however, was not demonstrable in heterologous cotransfection studies. These results suggest that one or more AKAP(s) targets PKA to HERG channels and may contribute to the acute regulation of I(Kr) by cAMP.

P311 is a highly conserved multifunctional protein. However, it does not belong to any established family of proteins, and its biological function has not been entirely determined. This study aims to reveal the unknown molecular and cellular function of P311. OCG (Overlapping Cluster Generator) is a clustering method used to partition a protein-protein network into overlapping clusters. Multifunctional proteins are at the intersection of relevant clusters. DAVID is an analytic tool used to extract biological meaning from a large protein list. Here we presented OD2 (OCG + DAVID + 2 human PPI datasets), a novel strategy to increase the likelihood to identify biological functions most pertinent to the multifunctional proteins. The principle of OD2 is that OCG prepares the protein lists from multifunctional protein relevant overlapping clusters, for a functional enrichment analysis by DAVID, and the similar functional enrichments, which occurs simultaneously when analyzing two human PPI datasets, are supposed to be the predicted functions. By applying OD2 to two reconstructed human PPI datasets, we supposed the function of the P311 in inflammatory responses, cell proliferation and coagulation, which were confirmed by the following biological experiments. Collectively, our study preliminarily found that P311 could play a role in inflammatory responses, cell proliferation and coagulation. Further studies are required to validate and elucidate the underlying mechanism.

Human genetic studies support that loss-of-function mutations in the SH3 domain and ankyrin repeat containing family proteins (SHANK1-3), the large synaptic scaffolding proteins enriched at the postsynaptic density of excitatory synapses, are causative for autism spectrum disorder and other neuropsychiatric disorders in humans. To better understand the in vivo functions of Shank and facilitate dissection of neuropathology associated with SHANK mutations in human, we generated multiple mutations in the Shank gene, the only member of the SHANK family in Drosophila melanogaster Both male and female Shank null mutants were fully viable and fertile with no apparent morphological or developmental defects. Expression analysis revealed apparent enrichment of Shank in the neuropils of the CNS. Specifically, Shank coexpressed with another PSD scaffold protein, Homer, in the calyx of mushroom bodies in the brain. Consistent with high expression in mushroom body calyces, Shank mutants show an abnormal calyx structure and reduced olfactory acuity. These morphological and functional phenotypes were fully rescued by pan-neuronal reexpression of Shank, and only partially rescued by presynaptic but no rescue by postsynaptic reexpression of Shank. Our findings thus establish a previously unappreciated presynaptic function of Shank.SIGNIFICANCE STATEMENT Mutations in SHANK family genes are causative for idiopathic autism spectrum disorder. To understand the neural function of Shank, a large scaffolding protein enriched at the postsynaptic densities, we examined the role of Drosophila Shank in synapse development at the peripheral neuromuscular junctions and the central mushroom body calyx. Our results demonstrate that, in addition to its conventional postsynaptic function, Shank also acts presynaptically in synapse development in the brain. This study offers novel insights into the synaptic role of Shank.

Circular RNAs (circRNAs) are an intriguing class of widely prevalent endogenous RNAs, the vast majority of which have not been characterized functionally. Here, we identified a novel oncogenic circRNA originating from the back-splicing of Exon2 and Exon3 of a tumor suppressor gene, ARHGAP35 (also known as P190-A), termed as circARHGAP35. have observe that circARHGAP35 and linear ARHGAP35 have antithetical expression and functions. Interestingly, circARHGAP35 contains a 3867 nt long ORF with an m6A-modified start codon and encodes a truncated protein comprising four FF domains and lacking the Rho GAP domain. Mechanistically, circARHGAP35 protein promotes cancer cell progression by interacting with TFII-I protein in the nucleus. The RNA binding protein, HNRNPL, facilitates the formation of circARHGAP35. Clinically, circARHGAP35 is associated with poor survival in cancer patients. Our findings characterize an oncogenic circRNA and demonstrate a novel mechanism of oncogene activation in cancer by circRNA through the production of a truncated protein.

Severe Acute Respiratory Syndrome (SARS), an emerging disease characterized by atypical pneumonia, has recently been attributed to a novel coronavirus. The genome of SARS Coronavirus (SARS-CoV) has recently been sequenced, and a number of genes identified, including that of the nucleocapsid protein (N). It is noted, however, that the N protein of SARS-CoV (SARS-CoV N) shares little homology with nucleocapsid proteins of other members of the coronavirus family [Science 300 (2003) 1399; Science 300 (2003) 1394]. N proteins of other coronavirus have been reported to be involved in forming the viral core and also in the packaging and transcription of the viral RNA. As data generated from some viral systems other than coronaviruses suggested that viral N-N self-interactions may be necessary for subsequent formation of the nucleocapsid and assembly of the viral particles, we decided to investigate SARS-CoV N-N interaction. By using mammalian two-hybrid system and sucrose gradient fractionations, a homotypic interaction of N, but not M, was detected by the two-hybrid analysis. The mammalian two-hybrid assay revealed an approximately 50-fold increase in SEAP activity (measurement of protein-protein interaction) in N-N interaction compared to that observed in either M-M or mock transfection. Furthermore, mutational analyses characterized that a serine/arginine-rich motif (SSRSSSRSRGNSR) between amino acids 184 and 196 is crucial for N protein oligomerization, since deletion of this region completely abolished the N protein self-multimerization. Finally, the full-length nucleocapsid protein expressed and purified from baculovirus system was found to form different levels of higher order structures as detected by Western blot analysis of the fractionated proteins. Collectively, these results may aid us in elucidating the mechanism pertaining to formation of viral nucleocapsid core, and designing molecular approaches to intervene SARS-CoV replication.

Enolase is a highly conserved enzyme that presents in all organisms capable of glycolysis or fermentation. Its immediate product phosphoenolpyruvate is essential for other important processes like peptidoglycan synthesis and the phosphotransferase system in bacteria. Therefore, enolase inhibitors are of great interest. Here, we report that Gp60, a phage-encoded enolase inhibitor protein (PEIP) of bacteriophage SPO1 for Bacillus subtilis, is an enolase inhibitor. PEIP-expressing bacteria exhibit growth attenuation, thinner cell walls, and safranin color in Gram staining owing to impaired peptidoglycan synthesis. We solve the structure of PEIP-enolase tetramer and show that PEIP disassembles enolase by disrupting the basic dimer unit. The structure reveals that PEIP does not compete for substrate binding but induces a cascade of conformational changes that limit accessibility to the enolase catalytic site. This phage-inspired disassembly of enolase represents an alternative strategy for the development of anti-microbial drugs.

Hypoxia-inducible factor 1α (HIF-1α) is essential in hepatocellular carcinoma (HCC) glycolysis and progression. Yes-associated protein (YAP) is a powerful regulator and is overexpressed in many cancers, including HCC. The regulatory mechanism of YAP and HIF-1α in HCC glycolysis is unknown.

FEAT is an intracellular protein that potently drives tumorigenesis in vivo. It is only weakly expressed in normal human tissues, including the testis. In contrast, FEAT is aberrantly upregulated in most human cancers. The present study was designed to investigate whether FEAT is applicable to tumor immunotherapy and whether FEAT is discernible in the bloodstream as a molecular biomarker of human cancers.

The multispanning membrane protein ATG9A is a scramblase that flips phospholipids between the two membrane leaflets, thus contributing to the expansion of the phagophore membrane in the early stages of autophagy. Herein, we show that depletion of ATG9A does not only inhibit autophagy but also increases the size and/or number of lipid droplets in human cell lines and C. elegans. Moreover, ATG9A depletion blocks transfer of fatty acids from lipid droplets to mitochondria and, consequently, utilization of fatty acids in mitochondrial respiration. ATG9A localizes to vesicular-tubular clusters (VTCs) that are tightly associated with an ER subdomain enriched in another multispanning membrane scramblase, TMEM41B, and also in close proximity to phagophores, lipid droplets and mitochondria. These findings indicate that ATG9A plays a critical role in lipid mobilization from lipid droplets to autophagosomes and mitochondria, highlighting the importance of ATG9A in both autophagic and non-autophagic processes.

The identification of biomarkers that predict susceptibility to major depressive disorder and treatment response to antidepressants is a major challenge. Vortioxetine is a novel multimodal antidepressant that possesses pro-cognitive properties and differentiates from other conventional antidepressants on various cognitive and plasticity measures. The aim of the present study was to identify biological systems rather than single biomarkers that may underlie vortioxetine's treatment effects.

Early embryonic development generates precursors of all major cell types in Arabidopsis. Among these precursors, the hypophysis divides asymmetrically to form the progenitors of the quiescent center and columella stem cells. A great deal has been learnt about the mechanisms that control the asymmetric division of the hypophysis and embryogenesis at the transcriptional level; however, no evidence of regulation at the co- or post-translational level has been reported. Here, we show that mutation of the catalytic subunit (Naa10) or auxiliary subunit (Naa15) of NatA, an N-terminal acetyltransferase that catalyzes protein N-terminal acetylation, produces an embryo-lethal phenotype. In addition, Naa10 and Naa15 were found to interact physically in planta Further analysis revealed that the observed embryonic patterning defects started at the early globular stage and that the asymmetric division of the hypophysis was irregular; thus, no quiescent center progenitor cells were generated in naa10 and naa15 embryos. We further observed that the polar distributions of auxin and its efflux carrier PIN1 were disturbed in naa10 embryos. Our results suggest that NatA is required for asymmetric division of the hypophysis and early embryonic patterning in Arabidopsis, and provides a link between protein N-terminal acetylation and embryogenesis in plants.

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is bringing an unprecedented health crisis to the world. To date, our understanding of the interaction between SARS-CoV-2 and host innate immunity is still limited. Previous studies reported that SARS-CoV-2 nonstructural protein 12 (NSP12) was able to suppress interferon-β (IFN-β) activation in IFN-β promoter luciferase reporter assays, which provided insights into the pathogenesis of COVID-19. In this study, we demonstrated that IFN-β promoter-mediated luciferase activity was reduced during coexpression of NSP12. However, we could show NSP12 did not affect IRF3 or NF-κB activation. Moreover, IFN-β production induced by Sendai virus (SeV) infection or other stimulus was not affected by NSP12 at mRNA or protein level. Additionally, the type I IFN signaling pathway was not affected by NSP12, as demonstrated by the expression of interferon-stimulated genes (ISGs). Further experiments revealed that different experiment systems, including protein tags and plasmid backbones, could affect the readouts of IFN-β promoter luciferase assays. In conclusion, unlike as previously reported, our study showed SARS-CoV-2 NSP12 protein is not an IFN-β antagonist. It also rings the alarm on the general usage of luciferase reporter assays in studying SARS-CoV-2. IMPORTANCE Previous studies investigated the interaction between SARS-CoV-2 viral proteins and interferon signaling and proposed that several SARS-CoV-2 viral proteins, including NSP12, could suppress IFN-β activation. However, most of these results were generated from IFN-β promoter luciferase reporter assay and have not been validated functionally. In our study, we found that, although NSP12 could suppress IFN-β promoter luciferase activity, it showed no inhibitory effect on IFN-β production or its downstream signaling. Further study revealed that contradictory results could be generated from different experiment systems. On one hand, we demonstrated that SARS-CoV-2 NSP12 could not suppress IFN-β signaling. On the other hand, our study suggests that caution needs to be taken with the interpretation of SARS-CoV-2-related luciferase assays.

Protein transmembrane domains (TMDs) are generally hydrophobic, but our bioinformatics analysis shows that many TMDs contain basic residues at terminal regions. Physiological functions of these membrane-snorkeling basic residues are largely unclear. Here, we show that a membrane-snorkeling Lys residue in integrin αLβ2 (also known as lymphocyte function-associated antigen 1 [LFA-1]) regulates transmembrane heterodimer formation and integrin adhesion through ionic interplay with acidic phospholipids and calcium ions (Ca2+) in T cells. The amino group of the conserved Lys ionically interacts with the phosphate group of acidic phospholipids to stabilize αLβ2 transmembrane association, thus keeping the integrin at low-affinity conformation. Intracellular Ca2+ uses its charge to directly disrupt this ionic interaction, leading to the transmembrane separation and the subsequent extracellular domain extension to increase adhesion activity. This Ca2+-mediated regulation is independent on the canonical Ca2+ signaling or integrin inside-out signaling. Our work therefore showcases the importance of intramembrane ionic protein-lipid interaction and provides a new mechanism of integrin activation.

The R-loop, composed of a DNA-RNA hybrid and the displaced single-stranded DNA, regulates diverse cellular processes. However, how cellular R-loops are recognized remains poorly understood. Here, we report the discovery of the evolutionally conserved ALBA proteins (AtALBA1 and AtALBA2) functioning as the genic R-loop readers in Arabidopsis. While AtALBA1 binds to the DNA-RNA hybrid, AtALBA2 associates with single-stranded DNA in the R-loops in vitro. In vivo, these two proteins interact and colocalize in the nucleus, where they preferentially bind to genic regions with active epigenetic marks in an R-loop-dependent manner. Depletion of AtALBA1 or AtALBA2 results in hypersensitivity of plants to DNA damaging agents. The formation of DNA breaks in alba mutants originates from unprotected R-loops. Our results reveal that the AtALBA1 and AtALBA2 protein complex is the genic R-loop reader crucial for genome stability in Arabidopsis.

We studied structural and immunological properties of the SARS-CoV M (membrane) protein, based on comparative analyses of sequence features, phylogenetic investigation, and experimental results. The M protein is predicted to contain a triple-spanning transmembrane (TM) region, a single N-glycosylation site near its N-terminus that is in the exterior of the virion, and a long C-terminal region in the interior. The M protein harbors a higher substitution rate (0.6% correlated to its size) among viral open reading frames (ORFs) from published data. The four substitutions detected in the M protein, which cause non-synonymous changes, can be classified into three types. One of them results in changes of pI (isoelectric point) and charge, affecting antigenicity. The second changes hydrophobicity of the TM region, and the third one relates to hydrophilicity of the interior structure. Phylogenetic tree building based on the variations of the M protein appears to support the non-human origin of SARS-CoV. To investigate its immunogenicity, we synthesized eight oligopeptides covering 69.2% of the entire ORF and screened them by using ELISA (enzyme-linked immunosorbent assay) with sera from SARS patients. The results confirmed our predictions on antigenic sites.

Endosomal trafficking and amyloidogenic cleavage of amyloid precursor protein (APP) is believed to play a role in the neurodegeneration observed in Alzheimer's disease (AD). Recent evidence has suggested that packaging and secretion of APP and its amyloidogenic cleaved products into small extracellular vesicles (EVs) may facilitate uptake of these neurotoxic factors during disease progression. However, the molecular mechanisms underlying trafficking of APP into EVs are poorly understood.