Site-directed Mutational Analysis Research Articles

The Role of Key Amino Acids of the Human Fe(II)/2OG-Dependent Dioxygenase ALKBH3 in Structural Dynamics and Repair Activity toward Methylated DNA.

Non-heme dioxygenases of the AlkB family hold a unique position among enzymes that repair alkyl lesions in nucleic acids. These enzymes activate the Fe(II) ion and molecular oxygen through the coupled decarboxylation of the 2-oxoglutarate co-substrate to subsequently oxidize the substrate. ALKBH3 is a human homolog of E. coli AlkB, which displays a specific activity toward N1-methyladenine and N3-methylcytosine bases in single-stranded DNA. Due to the lack of a DNA-bound structure of ALKBH3, the basis of its substrate specificity and structure-function relationships requires further exploration. Here we have combined biochemical and biophysical approaches with site-directed mutational analysis to elucidate the role of key amino acids in maintaining the secondary structure and catalytic activity of ALKBH3. Using stopped-flow fluorescence spectroscopy we have shown that conformational dynamics play a crucial role in the catalytic repair process catalyzed by ALKBH3. A transient kinetic mechanism, which comprises the steps of the specific substrate binding, eversion, and anchoring within the DNA-binding cleft, has been described quantitatively by rate and equilibrium constants. Through CD spectroscopy, we demonstrated that replacing side chains of Tyr143, Leu177, and His191 with alanine results in significant alterations in the secondary structure content of ALKBH3 and decreases the stability of mutant proteins. The bulky side chain of Tyr143 is critical for binding the methylated base and stabilizing its flipped-out conformation, while its hydroxyl group is likely involved in facilitating the product release. The removal of the Leu177 and His191 side chains substantially affects the secondary structure content and conformational flexibility, leading to the complete inactivation of the protein. The mutants lacking enzymatic activity exhibit a marked decrease in antiparallel β-strands, offset by an increase in the helical component.

METTL3-induced lncRNA GBAP1 promotes hepatocellular carcinoma progression by activating BMP/SMAD pathway

BackgroundHepatocellular carcinoma (HCC) is one of the most common and challenging cancers in the world. N6-methyladenosine (m6A) modification and long non-coding RNAs (lncRNAs) play critical roles in the progression of HCC. However, there are few reports on genome-wide screening and functional annotations of m6A-methylated lncRNAs in HCC.MethodsThe expression levels of m6A methyltransferase METTL3 and the association with the prognosis in HCC were determined by RT-qPCR, public dataset platforms. Then, RNA-seq, Pearson correlation analysis, MeRIP-qPCR, RNA half-life assay, gene site-directed mutation, RIP assay and RT-qPCR analysis were employed to determine the downstream target of METTL3 in HCC. Subsequently, the expression levels and roles of lncRNA glucosylceramidase beta pseudogene 1 (GBAP1) in HCC were determined by Kaplan–meier curves, RT-qPCR, in vitro functional experiments and in vivo tumorigenesis and lung metastasis models. Then, the downstream target and pathway of GBAP1 were explored by GO biological process, KEGG pathway enrichment, luciferase reporter assay, RIP assay and rescue experiments and so on.ResultsMETTL3 was upregulated in HCC and closely related to HCC prognosis. And METTL3 induced GBAP1 expression by acting as the m6A writer of GBAP1 and IGF2BP2 worked as its m6A reader. Clinically, GBAP1 expression was significantly associated with tumor size, venous infiltration, TNM stage and prognosis of HCC, Functionally, GBAP1 promoted HCC metastasis and growth both in vitro and in vivo. Furthermore, GBAP1 acted as the molecular sponge for miR-22-3p to increase the expression of bone morphogenetic protein receptor type 1A (BMPR1A), which then activated BMP/SMAD pathway in HCC cells.ConclusionsOur findings demonstrated that METTL3-induced GBAP1 promoted migration, invasion and proliferation of HCC cells via GBAP1/miR-22-3p/BMPR1A/SMAD axis. GBAP1 could be a potential prognosis indicator and therapeutic target for HCC.

Identification of Potent Zika Virus NS5 RNA-Dependent RNA Polymerase Inhibitors Combining Virtual Screening and Biological Assays.

The Zika virus (ZIKV) epidemic poses a significant threat to human health globally. Thus, there is an urgent need for developing effective anti-ZIKV agents. ZIKV non-structural protein 5 RNA-dependent RNA polymerase (RdRp), a viral enzyme for viral replication, has been considered an attractive drug target. In this work, we screened an anti-infection compound library and a natural product library by virtual screening to identify potential candidates targeting RdRp. Then, five selected candidates were further applied for RdRp enzymatic analysis, cytotoxicity, and binding examination by SPR. Finally, posaconazole (POS) was confirmed to effectively inhibit both RdRp activity with an IC50 of 4.29 μM and the ZIKV replication with an EC50 of 0.59 μM. Moreover, POS was shown to reduce RdRp activity by binding with the key amino acid D666 through molecular docking and site-directed mutation analysis. For the first time, our work found that POS could inhibit ZIKV replication with a stronger inhibitory activity than chloroquine. This work also demonstrated fast anti-ZIKV screening for inhibitors of RdRp and provided POS as a potential anti-ZIKV agent.

Identification of B-Cell Epitopes on Capsid Protein Reveals Two Potential Neutralization Mechanisms in Red-Spotted Grouper Nervous Necrosis Virus.

Nervous necrosis virus (NNV), a formidable pathogen in marine and freshwater fish, has inflicted enormous financial tolls on the aquaculture industry worldwide. Although capsid protein (CP) is the sole structural protein with pathogenicity and antigenicity, public information on immunodominant regions remains extremely scarce. Here, we employed neutralizing monoclonal antibodies (MAbs) specific for red-spotted grouper NNV (RGNNV) CNPgg2018 in combination with partially overlapping truncated proteins and peptides to identify two minimal B-cell epitope clusters on CP, 122GYVAGFL128 and 227SLYNDSL233. Site-directed mutational analysis confirmed residues Y123, G126, and L128 and residues L228, Y229, N230, D231, and L233 as the critical residues responsible for the direct interaction with ligand, respectively. According to homologous modeling and bioinformatic evaluation, 122GYVAGFL128 is harbored at the groove of the CP junction with strict conservation among all NNV isolates, while 227SLYNDSL233 is localized in proximity to the tip of a viral protrusion having relatively high evolutionary dynamics in different genotypes. Additionally, 227SLYNDSL233 was shown to be a receptor-binding site, since the corresponding polypeptide could moderately suppress RGNNV multiplication by impeding virion entry. In contrast, 122GYVAGFL128 seemed dedicated only to stabilizing viral native conformation and not to assisting initial virus attachment. Altogether, these findings contribute to a novel understanding of the antigenic distribution pattern of NNV and the molecular basis for neutralization, thus advancing the development of biomedical products, especially epitope-based vaccines, against NNV. IMPORTANCE NNV is a common etiological agent associated with neurological virosis in multiple aquatic organisms, causing significant hazards to the host. However, licensed drugs or vaccines to combat NNV infection are very limited to date. Toward the advancement of broad-spectrum prophylaxis and therapeutics against NNV, elucidating the diversity of immunodominant regions within it is undoubtedly essential. Here, we identified two independent B-cell epitopes on NNV CP, followed by the confirmation of critical amino acid residues participating in direct interaction. These two sites were distributed on the shell and protrusion domains of the virion, respectively, and mediated the neutralization exerted by MAbs via drastically distinct mechanisms. Our work promotes new insights into NNV antigenicity as well as neutralization and, more importantly, offers promising targets for the development of antiviral countermeasures.

Nuclear accumulation of MLKL induces necroptosis in cardiomyocytes: potential implication in Doxorubicin-induced cardiotoxicity

Abstract Background The treatment with doxorubicin, a powerful chemotherapeutic agent, has been shown to be associated with an increased risk of lethal heart failure. Although various types of cell death pathway such as apoptosis and ferroptosis have been shown to be involved in the development of doxorubicin-induced cardiotoxicity, DIC, the involvement of necroptosis, a novel programmed necrosis induced by translocation of activated mixed lineage kinase domain-like protein, MLKL, to plasma membrane, remains unclear. Purpose The aim of this study was to determine whether necroptosis is involved in the development of DIC. Methods and results DIC was induced in C57BL/6J mice by intraperitoneal injection of doxorubicin at a dose of 10 mg/kg 3 times for a week. Eight days after the commencement of injection, echocardiographic analyses showed that left ventricular ejection fraction assessed by echocardiography was significantly lower in the doxorubicin-treated mice than in the vehicle-treated mice (44.0±13.7 vs. 70.5±3.7%), indicating the development of DIC. Immunoblot analysis showed that MLKL protein level was higher by 1.6 fold in the doxorubicin-treated mice than in the vehicle-treated mice. Interestingly, immunohistochemical analysis showed that signals of phospho-Ser345-MLKL, an activated form of MLKL, was found in the nuclei in addition to cytosol and intercalated discs of cardiomyocytes in the doxorubicin-treated mice. To get novel insight into significance of nuclear MLKL accumulation, a leucine-rich nuclear export signal (NES) spanning amino acids 280–284 of rat MLKL was identified by site-directed mutation analyses, and H9c2 cells, cultured rat cardiomyoblasts, were transfected with expression constructs for nucleus-directed MLKL (FLAG-mtNES-MLKL) or its wild type (FLAG-WT-MLKL). Percentage of FLAG-positive cells stained with Zombie Red, a fluorescent dye that is non-permeant to live cells, was higher in FLAG-mtNES-MLKL-transfected cells than in FLAG-WT-MLKL-transfected cells (80.0±3.5% vs. 6.3±1.3%, p<0.05), whereas percentage of cells immunostained with cleaved caspase-3 to FLAG-positive cells was similar in the two groups. The effect of the MLKL mutant on necroptosis was attenuated by treatment with GppNHp, an inhibitor of Ran-mediated nuclear protein import. Conclusion Nuclear accumulation of MLKL induces necroptosis in cardiomyocytes, which may contribute to progression of DIC. Funding Acknowledgement Type of funding sources: None.

Molecular Basis of Complement C1q Collagen-Like Region Interaction with the Immunoglobulin-Like Receptor LAIR-1

The immune system homeostasis relies on a tight equilibrium of interconnected stimulatory and inhibitory signals. Disruption of this balance is characteristic of autoimmune diseases such as systemic lupus erythematosus (SLE). Aside from activating the classical complement pathway and enhancing pathogens and apoptotic cells phagocytosis, C1q has been recently shown to play an important role in immune modulation and tolerance by interacting with several inhibitory and stimulatory immune receptors. Due to its functional organization into collagen-like (CLR) and globular (GR) regions and its multimeric nature, C1q is able to interact simultaneously with several of these receptors and locally congregate pro- and anti-inflammatory signals, thus modulating the immune response. Leukocyte associated immunoglobulin-like (Ig-like) receptor 1 (LAIR-1), a ubiquitous collagen receptor expressed in many immune cell types, has been reported to interact with the CLR of C1q. In this study, we provide new insights into the molecular and structural determinants underlying C1q/LAIR-1 interaction. Recombinant LAIR-1 extracellular Ig-like domain was produced and tested for its interaction with C1q. A molecular dissection of C1q combined with competition assays reveals that LAIR-1 interacts with C1q’s CLR through a binding site close but different from the one of its associated C1r2s2 proteases tetramer. On the other side, we identified LAIR-1 residues involved in C1q interaction by site-directed mutational analysis. All together, these results lead to propose a possible model for C1q interaction with LAIR-1 and will contribute to the fundamental understanding of C1q-mediated immune tolerance.

Knockout of the HMG domain of the porcine SRY gene causes sex reversal in gene-edited pigs

The sex-determining region on the Y chromosome (SRY) is thought to be the central genetic element of male sex development in mammals. Pathogenic modifications within the SRY gene are associated with a male-to-female sex reversal syndrome in humans and other mammalian species, including rabbits and mice. However, the underlying mechanisms are largely unknown. To understand the biological function of the SRY gene, a site-directed mutational analysis is required to investigate associated phenotypic changes at the molecular, cellular, and morphological level. Here, we successfully generated a knockout of the porcine SRY gene by microinjection of two CRISPR-Cas ribonucleoproteins, targeting the centrally located "high mobility group" (HMG), followed by a frameshift mutation of the downstream SRY sequence. This resulted in the development of genetically male (XY) pigs with complete external and internal female genitalia, which, however, were significantly smaller than in 9-mo-old age-matched control females. Quantitative digital PCR analysis revealed a duplication of the SRY locus in Landrace pigs similar to the known palindromic duplication in Duroc breeds. Our study demonstrates the central role of the HMG domain in the SRY gene in male porcine sex determination. This proof-of-principle study could assist in solving the problem of sex preference in agriculture to improve animal welfare. Moreover, it establishes a large animal model that is more comparable to humans with regard to genetics, physiology, and anatomy, which is pivotal for longitudinal studies to unravel mammalian sex determination and relevant for the development of new interventions for human sex development disorders.

Therapeutic potential of IBP as an autophagy inducer for treating lung cancer via blocking PAK1/Akt/mTOR signaling

Lung cancer is the most frequent and fatal malignancy in humans worldwide, yet novel successful drugs for control of this disease are still lacking. Ipomoea batatas polysaccharides (IBPs) have been implicated in inhibiting diverse cancer types, but their functions in mitigating lung cancer are largely unknown. In this study, we identify a role of IBP in inhibiting lung cancer proliferation. We found that IBP significantly impedes the proliferation of lung cancer cells by inducing cytostatic macroautophagy both in vitro and in vivo. Mechanistically, IBP specifically promotes ubiquitination-mediated degradation of PAK1 (p21-activated kinase 1) and blocks its downstream Akt1/mTOR signaling pathway, leading to increased autophagic flux. In lung cancer xenografts in mice, IBP-induced cytostatic autophagy suppresses tumor development. Through site-directed mutational analysis, the underlying signaling augments ubiquitination via PAK1-ubiquitin interaction. Collectively, this work unravels the molecular mechanism underpinning IBP-induced cytostatic autophagy in lung cancer and characterizes IBP as a potential therapeutic agent for lung cancer treatment.

The N-terminal residual arginine19 of influenza A virus NS1 protein is required for its nuclear localization and RNA binding

RNA binding ability and cellular distribution are important for nonstructural protein 1 (NS1) of influenza A virus to act as a viral regulatory factor to control virus life cycle. In this study, we identified that the N-terminal residues 19−21 of NS1 are a highly conserved motif depending on all the available NS1 full length sequence of H5N1 influenza A virus from NCBI database. Site-directed mutation analysis demonstrated that the R19 residue of NS1 is critical for its RNA binding and nuclear localization. Furthermore, the residue R19 of NS1 was identified to be critical for regulating M1 mRNA splicing and NS1 nuclear export. Biological analysis of the rescued viruses indicated that the R19A mutation of NS1 did not interfere the replication of H5N1 virus during infection and attenuated the virulence of H5N1 virus in mice.

Structural and biochemical characterisation of a novel alginate lyase from Paenibacillus sp. str. FPU-7

A novel alginate lyase, PsAly, with a molecular mass of 33 kDa and whose amino acid sequence shares no significant similarity to other known proteins, was biochemically and structurally characterised from Paenibacillus sp. str. FPU-7. The maximum PsAly activity was obtained at 65 °C, with an optimum pH of pH 7–7.5. The activity was enhanced by divalent cations, such as Mg2+, Mn2+, or Co2+, and inhibited by a metal chelator, ethylenediaminetetraacetic acid. The reaction products indicated that PsAly is an endolytic enzyme with a preference for polymannuronate. Herein, we report a detailed crystal structure of PsAly at a resolution of 0.89 Å, which possesses a β-helix fold that creates a long cleft. The catalytic site was different from that of other polysaccharide lyases. Site-directed mutational analysis of conserved residues predicted Tyr184 and Lys221 as catalytic residues, abstracting from the C5 proton and providing a proton to the glycoside bond, respectively. One cation was found to bind to the bottom of the cleft and neutralise the carboxy group of the substrate, decreasing the pKa of the C5 proton to promote catalysis. Our study provides an insight into the structural basis for the catalysis of alginate lyases and β-helix polysaccharide lyases.

Identification of a Heat-Inducible Element of Cysteine Desulfurase Gene Promoter in Lentinula edodes.

Volatile organosulfur compounds are the main components that contribute to the unique aroma of dried Lentinula edodes. They are mainly generated during the hot-air drying process, and cysteine desulfurase is the key enzyme in this process. Temperature may be an essential factor of volatile organosulfur compound production by influencing the expression of the cysteine desulfurase gene. In this study, the promoter sequence of the cysteine desulfurase gene (pCS) was cloned and analyzed using bioinformatics tools. A series of 5′deletion fragments and site-directed mutations of pCS were constructed to identify the element that responds to heat stress. Six heat shock transcription factor (HSTF) binding sites were predicted by SCPD (The Promoter Database of Saccharomyces cerevisiae) and three of the binding sites were predicted by Yeastract (Yeast Search for Transcriptional Regulators and Consensus Tracking) in pCS. The results indicated that pCS was able to drive the expression of the EGFP (Enhanced Green Fluorescent Protein) gene in L. edodes. Moreover, the fluorescence intensity increased after heat stress. The changes in fluorescence intensity of different 5′deletion fragments showed that the heat response region was located between −500 bp and −400 bp in pCS. The site-directed mutation analysis further showed that the heat-inducible element was between −490 bp and −500 bp (TTTCTAGAAT) in pCS. Our results provide molecular insight for studying the formation of volatile organosulfur compounds in dried L. edodes.

Extended structure of pleiotropic DNA repair-promoting protein PprA from Deinococcus radiodurans.

Pleiotropic protein promoting DNA repair A (PprA) is a key protein facilitating the extreme radiation resistance of Deinococcus radiodurans. PprA is a unique protein to the genus Deinococcus and exists as an oligomer ranging from a tetramer to an ∼100-mer depending on protein concentrations. Here, the X-ray crystal structure of PprA was determined to clarify how PprA confers radiation resistance. The tertiary structure of dimeric PprA was elucidated by using mutants obtained with random and site-directed mutagenesis methods (W183R and A139R); these mutants have disabled DNA binding and polymerization functions. Because the mutant A139R and W183R proteins have dimeric assemblies with 2 different interfaces (Interfaces 1 and 2), the linear and oligomerized PprA model was constructed as a left-handed face-to-face periodic screw structure. In addition, the linear structure in solution was confirmed by small-angle scattering experiments. The site-directed mutational analysis identified essential basic amino acids for DNA binding. These analytical data support the hypothesis that a complex assembly of PprA molecules, which are extended and have a screw structure, surrounds and stretches the DNA strand, acting as a novel guide to colocalize the DNA strands for efficient DNA repairs.-Adachi, M., Shimizu, R., Shibazaki, C., Satoh, K., Fujiwara, S., Arai, S., Narumi, I., Kuroki, R. Extended structure of pleiotropic DNA repair-promoting protein PprA from Deinococcus radiodurans.

Negative regulation of the RLR-mediated IFN signaling pathway by duck ubiquitin-specific protease 18 (USP18).

Ubiquitin-specific protease 18 (USP18) plays an important role in regulating type I interferon (IFN) signaling in innate immunity, and has a crucial impact on the IFN therapeutic effect. Although significant progress has been made in elucidating USP18 function in mammals, the role of USP18 in ducks (duUSP18) remains poorly understood. In this study, we cloned the USP18 gene from white crested ducks by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of complementary DNA (cDNA) ends. We determined that duUSP18 cDNA contains a 52-bp 5'UTR, a 1,131-bp open reading frame and a 356-bp 3'UTR, and encodes a 376-amino acid protein. Multiple sequence alignments showed that duUSP18 shares high similarity with USP18 from other vertebrates. Overexpression of duUSP18 inhibited nuclear factor-κB (NF-κB) and interferon regulatory factor 1 (IRF1) activity, and reduced IFN-β production following 5' triphosphate double-stranded RNA (5'ppp dsRNA) or lipopolysaccharide (LPS) stimulation. duUSP18 knockdown significantly activated 5'ppp dsRNA-induced and LPS-induced NF-κB and IRF1 activation, and induced IFN-β expression in duck embryo fibroblasts. Furthermore, Quantitative real-time PCR (qRT-PCR) revealed that overexpression or knockdown of duUSP18 could alter the expression of genes related to the RLR-mediated IFN signaling pathway following the treatment with 5'ppp dsRNA. In addition, site-directed mutation analysis revealed that cysteine 66 (C66), histidine 313 (H313), and histidine 321 (H321) of duUSP18 were critical for inhibiting IFN-β activity. Taken together, these results suggest that duck USP18 plays an important role in innate immune responses against double-stranded RNA viruses in the RLR-mediated IFN signaling pathway, and that further studies are warranted to elucidate its underlying mechanisms, which could provide molecular insights into the effect of the treatment of duck diseases.

Jasmonic acid/ethylene signaling coordinates hydroxycinnamic acid amides biosynthesis through ORA59 transcription factor.

Hydroxycinnamic acid amides (HCAAs) are a class of antimicrobial metabolites involved in plant defense against necrotrophic pathogens, including Alternaria brassicicola and Botrytis cinerea. The agmatine coumaryl transferase (AtACT) is the key enzyme that catalyzes the last reaction in the biosynthesis of HCAAs, including p-coumaroylagmatine (CouAgm) and feruloylagmatine in Arabidopsis thaliana. However, the regulatory mechanism of AtACT gene expression is currently unknown. Yeast one-hybrid screening using the AtACT promoter as bait isolated the key positive regulator ORA59 that is involved in jasmonic acid/ethylene (JA/ET)-mediated plant defense responses. AtACT gene expression and HCAAs biosynthesis were synergistically induced by a combination of JA and ET. In the AtACT promoter, two GCC-boxes function equivalently for trans-activation by ORA59 in Arabidopsis protoplasts, and mutation of either GCC-box abolished AtACT mRNA accumulation in transgenic plants. Site-directed mutation analysis demonstrated that the specific Leu residue at position 228 of the ORA59 EDLL motif mainly contributed to its transcriptional activity on AtACT gene expression. Importantly, MEDIATOR25 (MED25) and ORA59 homodimer are also required for ORA59-dependent activation of the AtACT gene. These results suggest that ORA59 and two functionally equivalent GCC-boxes form the regulatory module together with MED25 that enables AtACT gene expression and HCAAs biosynthesis to respond to simultaneous activation of the JA/ET signaling pathways.

Molecular Docking Analysis and Biochemical Evaluation of Levansucrase from Sphingobium chungbukense DJ77.

Bacterial exopolymer Levan (β-(2,6) polyfructan) synthesized by levansucrase has attracted interest for various applications due to its low intrinsic viscosity compared with other polysaccharides. We report a novel levansucrase (Lsc) isolated from Sphingobium chunbukense DJ77 and verify its biochemical characteristics by comparative analysis of molecular docking analysis (MOE) and catalytic residue analysis. The complete sequence of the Lsc encoding gene ( lsc) was cloned under the direction of the T7 promoter and purified in an Escherichia coli BL21 (DE3) protein expression system. The enzyme activity analysis and ligand docking MOE study of S. chungbukense DJ77 Lsc revealed that Arg 77, Ser112, Arg 195, Asp196, Glu257, and Gln275 were involved in the sucrose binding and splitting as well as transfructosylation activity. A catalytic comparison of Lsc of S. chungbukense DJ77 with the results of site-directed mutational analysis indicated that Gln275 may coordinate a favorable substrate binding environment, offering broad pH resistance in the range of 5-10. The results suggest that the recombinant E. coli carrying S. chungbukense DJ77 Lsc might produce levan under the regular growth conditions with less need for pH manipulation.

SUMO polymeric chains are involved in nuclear foci formation and chromatin organization in Trypanosoma brucei procyclic forms.

SUMOylation is a post-translational modification conserved in eukaryotic organisms that involves the covalent attachment of the small ubiquitin-like protein SUMO to internal lysine residues in target proteins. This tag usually alters the interaction surface of the modified protein and can be translated into changes in its biological activity, stability or subcellular localization, among other possible outputs. SUMO can be attached as a single moiety or as SUMO polymers in case there are internal acceptor sites in SUMO itself. These chains have been shown to be important for proteasomal degradation as well as for the formation of subnuclear structures such as the synaptonemal complex in Saccharomyces cerevisiae or promyelocytic leukemia nuclear bodies in mammals. In this work, we have examined SUMO chain formation in the protozoan parasite Trypanosoma brucei. Using a recently developed bacterial strain engineered to produce SUMOylated proteins we confirmed the ability of TbSUMO to form polymers and determined the type of linkage using site-directed mutational analysis. By generating transgenic procyclic parasites unable to form chains we demonstrated that although not essential for normal growth, SUMO polymerization determines the localization of the modified proteins in the nucleus. In addition, FISH analysis of telomeres showed a differential positioning depending on the polySUMOylation abilities of the cells. Thus, our observations suggest that TbSUMO chains might play a role in establishing interaction platforms contributing to chromatin organization.

A Pilin Region Affecting Host Range of the Pseudomonas aeruginosa RNA Phage, PP7.

The host range of a phage is determined primarily by phage-receptor interaction. Here, we profiled the host range of an RNA leviphage, PP7 that requires functional type IV pilus (TFP) in order to enter into its host bacterium, Pseudomonas aeruginosa. Out of 25 twitching-proficient P. aeruginosa strains, 4 with group I pilin and 7 with group III pilin displayed PP7-resistance. The remaining 14 possessed group II pilin, which included 10 PP7-sensitive and 4 PP7-resistant strains, suggesting that only the strains with TFP consisted of a subset of group II (hence, group IIa) pilin were susceptible to PP7. The co-expression of the PAO1 (group IIa) pilin rendered all the strains susceptible to PP7, with the exception of the 4 strains with group I pilin. Moreover, the expression of PA14 (group III) and PAK (group IIb) pilin in the PAO1 pilA mutant restored the twitching motility but not the PP7-suceptibility. Site-directed and random mutation analyses of PAO1 pilin enabled us to identify a pilin mutant (G96S) that is fully functional but resistant to PP7 infection. This is due to the lack of any phage-receptor interactions, suggesting the structural properties of the β1-β2 loop in the variable region 2 of the group II pilin might be involved in PP7 infection.

TGF-β signaling regulates DACT1 expression in intestinal epithelial cells

DACT1, was first identified as a Dishevelled-associated antagonist of Wnt signaling pathway. It has been reported that DACT1 functions in embryonic development and tumorigenesis. However, the regulation of DACT1 still remains unclear. We found Wnt signaling has no effect on DACT1, but TGF-β increases expression of DACT1 in intestinal epithelial cells. In addition, the minimal promoter is located in the region of −500bp to +1bp and the region between −3000bp to +1bp enhanced promoter activity. Site-directed mutation analysis was performed and indicated that potential regulatory elements was near −335bp. Our study provided the basic information for the exploration of DACT1 regulation and expression. Moreover, TGF-β inhibits Wnt signaling to enhance the function of DACT1 inhibiting Wnt signaling.

Characterization and functional analysis of the Paralichthys olivaceus prdm1 gene promoter

PR domain containing protein 1 (Prdm1) is a transcriptional repressor identified in various species and plays multiple important roles in immune response and embryonic development. However, little is known about the transcriptional regulation of the prdm1 gene. This study aims to characterize the promoter of Paralichthys olivaceus prdm1 (Po-prdm1) gene and determine the regulatory mechanism of Po-prdm1 expression. A 2000bp-long 5'-flanking region (translation initiation site designated as +1) of the Po-prdm1 gene was isolated and characterized. The regulatory elements in this fragment were then investigated and many putative transcription factor (TF) binding sites involved in immunity and multiple tissue development were identified. A 5'-deletion analysis was then conducted, and the ability of the deletion mutants to promote luciferase and green fluorescent protein (GFP) expression in a flounder gill cell line was examined. The results revealed that the minimal promoter is located in the region between -446 and -13bp, and the region between -1415 and -13bp enhanced the promoter activity. Site-directed mutation analysis was subsequently performed on the putative regulatory elements sites, and the results indicated that FOXP1, MSX and BCL6 binding sites play negative functional roles in the regulation of the Po-prdm1 expression in FG cells. In vivo analysis demonstrated that a GFP reporter gene containing 1.4kb-long promoter fragment (-1415/-13) was expressed in the head and trunk muscle fibres of transient transgenic zebrafish embryos. Our study provided the basic information for the exploration of Po-prdm1 regulation and expression.

Antiviral profiling of the capsid assembly modulator BAY41-4109 on full-length HBV genotype A-H clinical isolates and core site-directed mutants in vitro

The HBV core protein represents an attractive target for new antiviral therapies due to its multiple functions within the viral life-cycle. Here, we report the antiviral activity of the capsid assembly modulator (CAM) BAY41-4109 and two nucleos(t)ide analogues (NAs) on a diverse panel of 54 HBV clinical isolates from genotype (GT) A-H and assessed the impact of core amino acid (aa) substitutions using site-directed mutants (SDMs).The median EC50 values of BAY41-4109 across genotypes ranged from 26 nM in GT G to 215 nM in GT F irrespective of the presence of NA resistance mutations compared to 43 nM for the GT D reference construct. Combined analyses of clinical isolates and SDMs identified aa changes at positions 29, 33 and 118 led to reduced antiviral activity of BAY41-4109 with fold changes in EC50 values of 6, 46, and 9 for D29G, T33N, and Y118F, respectively. These aa substitutions are located within the CAM binding pocket, and are expected to have an effect on CAM binding based on structural modeling. Importantly aa variations at these positions were rarely (<0.3%) observed as naturally occurring in public sequence databases. NA's remained fully active against these variants.Our study demonstrated that BAY41-4109 generally remained fully active across GT A-H clinical isolates. In addition, core aa substitutions within the CAM-binding pocket replicated in vitro and variants at positions 29, 33, and 118 were identified to reduce antiviral activity.