Distinct structural bases for sequence-specific DNA binding by mammalian BEN domain proteins.

The BEN domain is a recently recognized DNA binding module that is present in diverse metazoans and certain viruses. Several BEN domain factors are known as transcriptional repressors, but, overall, relatively little is known of how BEN factors identify their targets in humans. In particular, X-ray structures of BEN domain:DNA complexes are only known for Drosophila factors bearing a single BEN domain, which lack direct vertebrate orthologs. Here, we characterize several mammalian BEN domain (BD) factors, including from two NACC family BTB-BEN proteins and from BEND3, which has four BDs. In vitro selection data revealed sequence-specific binding activities of isolated BEN domains from all of these factors. We conducted detailed functional, genomic, and structural studies of BEND3. We show that BD4 is a major determinant for in vivo association and repression of endogenous BEND3 targets. We obtained a high-resolution structure of BEND3-BD4 bound to its preferred binding site, which reveals how BEND3 identifies cognate DNA targets and shows differences with one of its non-DNA-binding BEN domains (BD1). Finally, comparison with our previous invertebrate BEN structures, along with additional structural predictions using AlphaFold2 and RoseTTAFold, reveal distinct strategies for target DNA recognition by different types of BEN domain proteins. Together, these studies expand the DNA recognition activities of BEN factors and provide structural insights into sequence-specific DNA binding by mammalian BEN proteins.

Coordinated Conformational Processing of the Tumor Suppressor Protein p53 by the Hsp70 and Hsp90 Chaperone Machineries.

p53, the guardian of the genome, requires chaperoning by Hsp70 and Hsp90. However, how the two chaperone machineries affect p53 conformation and regulate its function remains elusive. We found that Hsp70, together with Hsp40, unfolds p53 in an ATP-dependent reaction. This unfolded state of p53 is susceptible to aggregation after release induced by the nucleotide exchange factor Bag-1. However, when Hsp90 and the adaptor protein Hop are present, p53 is transferred from Hsp70 to Hsp90, allowing restoration of the native state upon ATP hydrolysis. Our results suggest that the p53 conformation is constantly remodeled by the two major chaperone machineries. This connects p53 activity to stress, and the levels of free molecular chaperones are important factors regulating p53 activity. Together, our findings reveal an intricate interplay and cooperation of Hsp70 and Hsp90 in regulating the conformation of a client.

LeishIF4E2 is a cap-binding protein that plays a role in Leishmania cell cycle progression.

Leishmania encode six paralogs of the cap-binding protein eIF4E and five eIF4G candidates, forming unique complexes. Two cap-binding proteins, LeishIF4E1 and LeishIF4E2, do not bind any identified LeishIF4Gs, thus their roles are intriguing. Here, we combine structural prediction, proteomic analysis, and interaction assays to shed light on LeishIF4E2 function. A nonconserved C-terminal extension was identified through structure prediction and sequence alignment. m7 GTP-binding assays involving both recombinant and transgenic LeishIF4E2 with and without the C-terminal extension revealed that this extension functions as a regulatory gate, modulating the cap-binding activity of LeishIF4E2. The interactomes of the two LeishIF4E2 versions were investigated, highlighting the role of the C-terminal extension in binding to SLBP2. SLBP2 is known to interact with a stem-loop structure in the 3' UTRs of histone mRNAs. Consistent with the predicted inhibitory effect of SLBP2 on histone expression in Xenopus laevis, a hemizygous deletion mutant of LeishIF4E2, exhibited an upregulation of several histones. We therefore propose that LeishIF4E2 is involved in histone expression, possibly through its interaction between SLBP2 and LeishIF4E2, thus affecting cell cycle progression. In addition, cell synchronization showed that LeishIF4E2 expression decreased during the S-phase, when histones are known to be synthesized. Previous studies in T. brucei also highlighted an association between TbEIF4E2 and SLBP2, and further reported on an interaction between TbIF4E2 and S-phase-abundant mRNAs. Our results show that overexpression of LeishIF4E2 correlates with upregulation of cell cycle and chromosome maintenance proteins. Along with its effect on histone expression, we propose that LeishIF4E2 is involved in cell cycle progression.

SMALL MOLECULE INDUCES TIME-DEPENDENT INHIBITION OF STAT3 DIMERIZATION AND DNA-BINDING ACTIVITY AND REGRESSES HUMAN BREAST TUMOR XENOGRAFTS.

Aberrantly-active signal transducer and activator of transcription (Stat)3 has a causal role in many human cancers and represents a validated anticancer drug target, though it has posed significant challenge to drug development. A new small molecule, JKB887, was identified through virtual library screening and is predicted to interact with Lys591, Arg609 and Pro63 in the phospho-tyrosine (pTyr)-binding pocket of the Stat3 SH2 domain. JKB887 inhibited Stat3 DNA-binding activity in vitro in a time-dependent manner, with IC50 of 2.2-4.5 µM at 30-60-min incubation. It directly disrupted both the Stat3 binding to the cognate, high-affinity pTyr (pY) peptide, GpYLPQTV-NH2 in fluorescent polarization assay with IC50 of 3.5-5.5 µM at 60-90-min incubation, and to the IL-6 receptor/gp130 or Src in treated malignant cells. Treatment with JKB887 selectively blocked constitutive Stat3 phosphorylation, nuclear translocation and transcriptional activity, Stat3-regulated gene expression, and decreased viable cell numbers, cell growth, colony formation, migration, and survival in human or mouse tumor cells. By contrast, JKB887 had minimal effects on Stat1 activity, pErk1/2MAPK, pShc, pJAK2, pSrc induction, or cells that do not harbor aberrantly-active Stat3. Additionally, JKB887 inhibited growth of human breast cancer xenografts in mice. JKB887 is a Stat3-selective inhibitor with demonstrable antitumor effects against Stat3-dependent human cancers.

Expression characteristics and inhibitory activity of a leucine-rich repeat (LRR)-only protein in the Chinese mitten crab, Eriocheir sinensis.

The leucine-rich repeat (LRR) domain is a crucial structure in a variety of immune related proteins and displays multiple immune functions. In this study, the open reading frame (ORF) of an LRR-only protein was cloned from the Chinese mitten crab, Eriocheir sinensis (EsLRRop1). The protein sequence of EsLRRop1 contained seven LRR motifs, three LRR-TYP motifs and an LRRCT motif. Tissue distribution exhibited that EsLRRop1 mainly expressed in nervous tissues including thoracic ganglion, eyestalk and brain while showed relatively lower transcriptional level in hemocyte. Based on the above expression characteristics, the responses of EsLRRop1 to the challenge of Vibrio parahaemolyticus and Staphylococcus aureus were tested. The result showed that the transcript of EsLRRop1 in thoracic ganglion and eyestalk up-regulated after being challenged with S. aureus, while it decreased post injection with V. parahaemolyticus. The transcript of EsLRRop1 in hemocytes up-regulated sharply at 3 h and decreased at 12 h and 24 h after being challenged with V. parahaemolyticus, while it decreased at 12 h and 24 h post injection with S. aureus. The recombinant protein of EsLRRop1 (His-EsLRRop1) displayed binding activities to V. alginolyticus, V. harveyi, V. parahaemolyticus, S. aureus, Corynebacterium glutamicum and Micrococcus lysodeikticus as well as lipopolysaccharide (LPS) and peptidoglycan (PGN). Moreover, the His-EsLRRop1 exhibited inhibitory activity against V. parahaemolyticus and V. harveyi with minimum inhibitory concentration (MIC) of 3.57-7.14 µM and 7.14-14.28 µM, respectively. These results provide theoretical basis for the application of EsLRRop1 in inhibiting bacteria in aquaculture practice.

Design, synthesis, and biological evaluation of 2-(2-oxoindolin-3-ylidene)hydrazinecarbothioamides as a potential EGR-1 inhibitor for targeted therapy of atopic dermatitis.

Atopic dermatitis is a chronic inflammatory skin disease characterized by intense itching and frequent skin barrier dysfunctions. EGR-1 is a transcription factor that aggravates the pathogenesis of atopic dermatitis by promoting the production of various inflammatory cytokines. Three 2-(2-oxoindolin-3-ylidene)hydrazinecarbothioamides (IT21, IT23, and IT25) were identified as novel inhibitors of EGR-1 DNA-binding activity. In silico docking experiments were performed to elucidate the binding conditions of the EGR-1 zinc-finger (ZnF) DNA-binding domain. Electrophoretic mobility shift assays confirmed the targeted binding effect on the EGR-1 ZnF DNA-binding domain, leading to dose-dependent dissociation of the EGR-1-DNA complex. At the functional cellular level, IT21, IT23, and IT25 effectively reduced mRNA expression of TNFα-induced EGR-1-regulated inflammatory genes, particularly in HaCaT keratinocytes inflamed by TNFα. In the in vivo efficacy study, IT21, IT23, and IT25 demonstrated the potential to alleviate atopic dermatitis-like skin lesions in the ear skin of BALB/c mice. These findings suggest that targeting the EGR-1 ZnF DNA-binding domain with 2-(2-oxoindolin-3-ylidene)hydrazinecarbothioamide derivatives (IT21, IT23, and IT25) could serve as lead compounds for the development of potential therapeutic agents against inflammatory skin disorders, including atopic dermatitis.

Cla4A, a Novel Regulator of Gene Expression Networks Required for Asexual and Insect-Pathogenic Lifecycles of Beauveria bassiana.

Cla4, an orthologous p21-activated kinase crucial for non-entomopathogenic fungal lifestyles, has two paralogs (Cla4A/B) functionally unknown in hypocrealean entomopathogens. Here, we report a regulatory role of Cla4A in gene expression networks of Beauveria bassiana required for asexual and entomopathogenic lifecycles while Cla4B is functionally redundant. The deletion of cla4A resulted in severe growth defects, reduced stress tolerance, delayed conidiation, altered conidiation mode, impaired conidial quality, and abolished pathogenicity through cuticular penetration, contrasting with no phenotype affected by cla4B deletion. In ∆cla4A, 5288 dysregulated genes were associated with phenotypic defects, which were restored by targeted gene complementation. Among those, 3699 genes were downregulated, including more than 1300 abolished at the transcriptomic level. Hundreds of those downregulated genes were involved in the regulation of transcription, translation, and post-translational modifications and the organization and function of the nuclear chromosome, chromatin, and protein-DNA complex. DNA-binding elements in promoter regions of 130 dysregulated genes were predicted to be targeted by Cla4A domains. Samples of purified Cla4A extract were proven to bind promoter DNAs of 12 predicted genes involved in multiple stress-responsive pathways. Therefore, Cla4A acts as a novel regulator of genomic expression and stability and mediates gene expression networks required for insect-pathogenic fungal adaptations to the host and environment.

Using Biotinylated Iron-Responsive Element to Analyze the Activity of Iron Regulatory Proteins.

Iron regulatory proteins (IRP1 and IRP2) are the master regulators of mammalian iron homeostasis. They bind to the iron-responsive elements (IREs) of the transcripts of iron-related genes to regulate their expression, thereby maintaining cellular iron availability. The primary method to measure the IRE-binding activity of IRPs is the electrophoresis mobility shift assay (EMSA). This method is particularly useful for evaluating IRP1 activity, since IRP1 is a bifunctional enzyme and its protein levels remain similar during conversion between the IRE-binding protein and cytosolic aconitase forms. Here, we exploited a method of using a biotinylated-IRE probe to separate IRE-binding IRPs followed by immunoblotting to analyze the IRE-binding activity. This method allows for the successful measurement of IRP activity in cultured cells and mouse tissues under various iron conditions. By separating IRE-binding IRPs from the rest of the lysates, this method increases the specificity of IRP antibodies and verifies whether a band represents an IRP, thereby revealing some previously unrecognized information about IRPs. With this method, we showed that the S711-phosphorylated IRP1 was found only in the IRE-binding form in PMA-treated Hep3B cells. Second, we found a truncated IRE-binding IRP2 isoform that is generated by proteolytic cleavage on sites in the 73aa insert region of the IRP2 protein. Third, we found that higher levels of SDS, compared to 1-2% SDS in regular loading buffer, could dramatically increase the band intensity of IRPs in immunoblots, especially in HL-60 cells. Fourth, we found that the addition of SDS or LDS to cell lysates activated protein degradation at 37 °C or room temperature, especially in HL-60 cell lysates. As this method is more practical, sensitive, and cost-effective, we believe that its application will enhance future research on iron regulation and metabolism.

Design, Synthesis, and Characterization of Novel Thiazolidine-2,4-Dione-Acridine Hybrids as Antitumor Agents.

This study focuses on the synthesis and structural characterization of new compounds that integrate thiazolidine-2,4-dione, acridine moiety, and an acetamide linker, aiming to leverage the synergistic effects of these pharmacophores for enhanced therapeutic potential. The newly designed molecules were efficiently synthesized through a multi-step process and subsequently transformed into their hydrochloride salts. Comprehensive spectroscopic techniques, including nuclear magnetic resonance (NMR), high-resolution mass spectrometry (HRMS), infrared (IR) spectroscopy, and elemental analysis, were employed to determine the molecular structures of the synthesized compounds. Biological evaluations were conducted to assess the therapeutic potential of the new compounds. The influence of these derivatives on the metabolic activity of various cancer cell lines was assessed, with IC50 values determined via MTT assays. An in-depth analysis of the structure-activity relationship (SAR) revealed intriguing insights into their cytotoxic profiles. Compounds with electron-withdrawing groups generally exhibited lower IC50 values, indicating higher potency. The presence of the methoxy group at the linking phenyl ring modulated both the potency and selectivity of the compounds. The variation in the acridine core at the nitrogen atom of the thiazolidine-2,4-dione core significantly affects the activity against cancer cell lines, with the acridin-9-yl substituent enhancing the compounds' antiproliferative activity. Furthermore, compounds in their hydrochloride salt forms demonstrated better activity against cancer cell lines compared to their free base forms. Compounds 12c·2HCl (IC50 = 5.4 ± 2.4 µM), 13d (IC50 = 4.9 ± 2.9 µM), and 12f·2HCl (IC50 = 4.98 ± 2.9 µM) demonstrated excellent activity against the HCT116 cancer cell line, and compound 7d·2HCl (IC50 = 4.55 ± 0.35 µM) demonstrated excellent activity against the HeLa cancer cell line. Notably, only a few tested compounds, including 7e·2HCl (IC50 = 11.00 ± 2.2 µM), 7f (IC50 = 11.54 ± 2.06 µM), and 7f·2HCl (IC50 = 9.82 ± 1.92 µM), showed activity against pancreatic PATU cells. This type of cancer has a very high mortality due to asymptomatic early stages, the occurrence of metastases, and frequent resistance to chemotherapy. Four derivatives, namely, 7e·2HCl, 12d·2HCl, 13c·HCl, and 13d, were tested for their interaction properties with BSA using fluorescence spectroscopic studies. The values for the quenching constant (Ksv) ranged from 9.59 × 104 to 10.74 × 104 M-1, indicating a good affinity to the BSA protein.

Design of single-domain VHH antibodies to increase the binding activity in SPR amine coupling.

Single-domain antibodies, or VHH, nanobodies, are attractive tools in biotechnology and pharmaceuticals due to their favorable biophysical properties. Single-domain antibodies have potential for use in sensing materials to detect antigens, and in this paper, we propose a generic design strategy of single-domain antibodies for the highly efficient use of immobilized antibodies on a sensing substrate. Amine coupling was used to immobilize the single-domain antibodies on the substrate through a robust covalent bond. First, for two model single-domain antibodies with lysines at four highly conserved positions (K48, K72, K84, and K95), we mutated the lysines to alanine and measured the binding activity of the mutants (the percentage of immobilized antibodies that can bind antigen) using surface plasmon resonance. The two model single-domain antibodies tended to have higher binding activities when K72, which is close to the antigen binding site, was mutated. Adding a Lys-tag to the C-terminus of single-domain antibodies also increased the binding activity. We also mutated the lysine for another model single-domain antibodies with the lysine in a different position than the four residues mentioned above and measured the binding activity. Thus, single-domain antibodies immobilized in an orientation accessible to the antigen tended to have a high binding activity, provided that the physical properties of the single-domain antibodies themselves (affinity and structural stability) were not significantly reduced. Specifically, the design strategy of single-domain antibodies with high binding activity included mutating the lysine at or near the antigen binding site, adding a Lys-tag to the C-terminus, and mutating a residue away from the antigen binding site to lysine. It is noteworthy that mutating K72 close to the antigen binding site was more effective in increasing the binding activity than Lys-tag addition, and immobilization at the N-terminus close to the antigen binding site did not have such a negative effect on the binding activity compared to immobilization at the K72.

A C-type lectin with a single carbohydrate-recognition domain (CRD) containing unique QPN/WDD motifs from Tegillarca granosa is involved in the innate immune defense.

C-type lectins (CTLs), a superfamily of Ca2+-dependent carbohydrate-recognition proteins, serve as pattern recognition receptors (PRRs) in the immune response of many species. However, little is currently known about the CTLs of the commercially and ecologically important bivalve species, blood clam (Tegillarca granosa). In this study, a CTL (designated as TgCTL-1) with a single carbohydrate-recognition domain (CRD) containing unique QPN/WDD motifs was identified in the blood clam through transcriptome and whole-genome searching. Multiple alignment and phylogenetic analysis strongly suggested that TgCTL-1 was a new member of the CTL superfamily. Expression analysis demonstrated that TgCTL-1 was highly expressed in the hemocytes and visceral mass of the clam under normal condition. In addition, the expression of TgCTL-1 was shown to be significantly up-regulated upon pathogen challenge. Moreover, the recombinant TgCTL-1 (rTgCTL-1) displayed agglutinating and binding activities against both the gram-positive and gram-negative bacteria tested in a Ca2+-dependent manner. Furthermore, it was found that the in vitro phagocytic activity of hemocytes was significantly enhanced by rTgCTL-1. In general, our results showed that TgCTL-1 was an inducible acute-phase secretory protein, playing crucial roles in recognizing, agglutinating, and binding to pathogenic bacteria as well as modulating phagocytic activity of hemocytes in the innate immune defense of blood clam.

A novel C-type lectin (SpccCTL) suppresses MCRV replication by binding viral protein and regulating antiviral peptides in Scylla paramamosain.

Pattern recognition receptors (PRRs) play a crucial role in the recognition and activation of innate immune responses against invading microorganisms. This study characterizes a novel C-type lectin (CTL), SpccCTL. The cDNA sequence of SpccCTL has a full length of 1744 bp encoding a 338-amino acid protein. The predicted protein contains a signal peptide, a coiled-coil (CC) domain, and a CLECT domain. It shares more than 50 % similarity with a few CTLs with a CC domain in crustaceans. SpccCTL is highly expressed in gills and hemocytes and upregulated after MCRV challenge, suggesting that it may be involved in antiviral immunity. Recombinant SpccCTL (rSpccCTL) as well as two capsid proteins of MCRV (VP11 and VP12) were prepared. Pre-incubating MCRV virions with rSpccCTL significantly suppresses the proliferation of MCRV in mud crabs, compared with the control (treatment with GST protein), and the survival rate of mud crabs is also significantly decreased. Knockdown of SpccCTL significantly facilitates the proliferation of MCRV in mud crabs. These results reveal that SpccCTL plays an important role in antiviral immune response. GST pull-down assay result shows that rSpccCTL interacts specifically with VP11, but not to VP12. This result is further confirmed by a Co-IP assay. In addition, we found that silencing SpccCTL significantly inhibits the expression of four antimicrobial peptides (AMPs). Considering that these AMPs are members of anti-lipopolysaccharide factor family with potential antiviral activity, they are likely involved in immune defense against MCRV. Taken together, these findings clearly demonstrate that SpccCTL can recognize MCRV by binding viral capsid protein VP11 and regulate the expression of certain AMPs, suggesting that SpccCTL may function as a potential PRR playing an essential role in anti-MCRV immunity of mud crab. This study provides new insights into the antiviral immunity of crustaceans and the multifunctional characteristics of CTLs.

In vitro antiviral activity of persimmon-derived tannin against avian influenza viruses.

Tannins derived from natural plant sources are known to provide many health benefits to humans and animals. Among the various tannins, those derived from persimmon (Diospyros kaki) have exhibited strong inactivating effects against pathogens that induce diseases in humans. However, few studies have focused on the antiviral effects of persimmon tannin against pathogen-induced diseases in animals. In this study, we investigated the antiviral effects of persimmon tannin against various avian influenza viruses revealing that tannin at a concentration of 1.0 mg ml-1 reduced viral infectivity in >6.0-log scale against all tested avian influenza viruses. In addition, this persimmon tannin concentration effectively inhibited the receptor binding and membrane fusion abilities of viral hemagglutinin (HA), which play important roles in avian influenza virus infection. These results suggest that persimmon tannin inactivates the HA of avian influenza viruses and reduces their infectivity. Persimmon tannin is a safer natural substance than the currently used chemical compound related to antiviral substance. When inactivation of the viruses which are present in environmental water such as roosting water of wild birds will be needed, persimmon tannin is expected to become an antiviral resource that may prevent the spread of several avian influenza virus subtypes.

The RNA-Binding and RNA-Melting Activities of the Multifunctional Protein Nucleobindin 1.

Nucleobindin 1 (NUCB1) is a ubiquitous multidomain protein that belongs to the EF-hand Ca2+-binding superfamily. NUCB1 interacts with Galphai3 protein, cyclooxygenase, amyloid precursor protein, and lipids. It is involved in stress response and human diseases. In addition, this protein is a transcription factor that binds to the DNA E-box motif. Using surface plasmon resonance and molecular beacon approaches, we first showed the RNA binding and RNA melting activities of NUCB1. We suggest that NUCB1 could induce local changes in structured RNAs via binding to the GGAUAU loop sequence. Our results demonstrate the importance of the multidomain structure of NUCB1 for its RNA-chaperone activity in vitro.

Cheminformatics and Machine Learning Approaches to Assess Aquatic Toxicity Profiles of Fullerene Derivatives.

Fullerene derivatives (FDs) are widely used in nanomaterials production, the pharmaceutical industry and biomedicine. In the present study, we focused on the potential toxic effects of FDs on the aquatic environment. First, we analyzed the binding affinity of 169 FDs to 10 human proteins (1D6U, 1E3K, 1GOS, 1GS4, 1H82, 1OG5, 1UOM, 2F9Q, 2J0D, 3ERT) obtained from the Protein Data Bank (PDB) and showing high similarity to proteins from aquatic species. Then, the binding activity of 169 FDs to the enzyme acetylcholinesterase (AChE)-as a known target of toxins in fathead minnows and Daphnia magna, causing the inhibition of AChE-was analyzed. Finally, the structural aquatic toxicity alerts obtained from ToxAlert were used to confirm the possible mechanism of action. Machine learning and cheminformatics tools were used to analyze the data. Counter-propagation artificial neural network (CPANN) models were used to determine key binding properties of FDs to proteins associated with aquatic toxicity. Predicting the binding affinity of unknown FDs using quantitative structure-activity relationship (QSAR) models eliminates the need for complex and time-consuming calculations. The results of the study show which structural features of FDs have the greatest impact on aquatic organisms and help prioritize FDs and make manufacturing decisions.

First identification of Nocardia seriolae GapA adhesion function and its three B-cell epitopes with cell-binding activity.

Fish nocardiosis mainly caused by Nocardia seriolae (N. seriolae) is a serious threat to aquaculture. Bacterial adhesion to host cells mediated by adhesin is an initial step of pathogenesis. But it is not clear whether glyceraldehyde-3-phosphate dehydrogenase (GapA) is an adhesin of N. seriolae. Here, recombinant GapA protein (rGapA) was prokaryotic expressed, and its role in the bacterial adhesion to Ctenopharyngodon idella kidney cells was investigated by indirect immunofluorescence, protein-binding assay and adhesion inhibition assay. The results showed that an obvious green fluorescence was observed on the surface of the cells co-incubated with rGapA protein; the cytomembrane proteins of the cells pretreated with rGapA could react with anti-rGapA antibody; and the antibody significantly inhibited the adhesion ability of the bacteria. Subsequently, B-cell linear epitopes of GapA protein were identified by using a immunoinformatics approach combined with peptide ELISA and Western blot for the first time. It was found that four predicted epitopes (Ep58-69 , Ep139-150 , Ep186-197 , Ep318-329 ) could all react with anti-rGapA antibody and obviously inhibit the immunoreactivity between rGapA and anti-rGapA antibody, and they were confirmed as indeed B-cell linear epitopes of the protein. Furthermore, flow cytometry analysis found the percentage of positive cells co-incubated with FITC-labelled epitope peptides (Ep139-150 , Ep186-197 , Ep318-329 ) was significantly higher than those in the FITC-labelled Ep58-69 , unrelated control peptide and cell control. Collectively, GapA is an adhesin of N. seriolae, and epitope peptides (Ep139-150 , Ep186-197 , Ep318-329 ) possess cell-binding activity, which are potential candidates for developing a multiple epitopes-based adhesin vaccine against fish nocardiosis.

Selective Recovery of Estrogenic Endocrine Disruptors from 48 Environmental Samples Using a Substrate for Activity-Specific Concentration.

hER-MIP is a molecularly imprinted polymer (MIP) that has been shown to selectively collect human estrogen receptor (hER) binding active substances. However, environmental samples contain various chemicals depending on the location and regional differences, and the hER binding activity depends on the sample type. Thus, the general applicability of hER-MIP to actual environmental samples must be elucidated. In this study, 48 environmental samples were collected and screened with hER-MIP, and a yeast assay was performed to evaluate the adsorption characteristics of the samples according to the adsorption and elution fractions. The results showed that hER-MIP collects hER binding active substances almost selectively but does not collect constitutive androstane receptor (CAR) binding active substances selectively. CAR binding activity was detected in the adsorbed fraction because several hER binding active substances also demonstrate CAR binding activity.

OsMFT2 is involved in the regulation of ABA signaling-mediated seed germination through interacting with OsbZIP23/66/72 in rice.

Seed germination is a complex process involving various physical and biochemical cues, determined by exogenous and endogenous factors. Here, we identified a gene, OsMFT2, that negatively regulates seed germination in rice. OsMFT2 knock-out lines exhibited pre-harvest sprouting, whereas OsMFT2 overexpression lines showed delayed germination. RNA expression profiling showed that OsMFT2 was specifically expressed in seeds. Subcellular localization indicated that OsMFT2 was a nuclear protein. Exogenous abscisic acid (ABA) treatment of imbibed seeds and seedlings indicated that OsMFT2 altered ABA sensitivity during seed germination and post-germination growth. In vivo and in vitro assays showed that three bZIP transcription factors, OsbZIP23, OsbZIP66 and OsbZIP72, interacted with OsMFT2. OsbZIP23/66/72 bound to the promoter of Rab16A, a typical gene containing the ABA-responsive element, and OsMFT2 enhanced the binding to the Rab16A promoter. Moreover, several ABA-responsive genes were differentially expressed in the imbibed seeds of OsMFT2 transgenic lines and the wild type. The performance of the transgenic plants demonstrated that overexpressing OsbZIP23 rescued the pre-harvest sprouting phenotype and the decrease in ABA-signaling genes expression caused by OsMFT2 knock-out. All of these results demonstrate that OsMFT2 positively regulates ABA-responsive genes through interacting with OsbZIP23/66/72 and functions in seed germination.

Residues R192 and K225 in RNA-Binding Pocket of Tobacco Vein Banding Mosaic Virus CP Control Virus Cell-to-Cell Movement and Replication.

Potyviruses move to neighboring cells in the form of virus particles or a coat protein (CP)-containing ribonucleoprotein complex. However, the precise roles of RNA-binding residues in potyviral CP in viral cell-to-cell movement remain to be elucidated. In this study, we predicted the three-dimensional model of tobacco vein banding mosaic virus (TVBMV)-encoded CP and found nine residues presumably located in the CP RNA-binding pocket. Substitutions of the two basic residues at positions 192 and 225 (R192 and K225) with either alanine, cysteine, or glutamic acid abolished TVBMV cell-to-cell and systemic movement in Nicotiana benthamiana plants. These substitutions also reduced the replication of the mutant viruses. Results from the electrophoretic mobility shift assay showed that the RNA-binding activity of mutant CPs derived from R192 or K225 substitutions was significantly lower than that of wild-type CP. Analysis of purified virus particles showed that mutant viruses with R192 or K225 substitutions formed RNA-free virus-like particles. Mutations of R192 and K225 did not change the CP plasmodesmata localization. The wild-type TVBMV CP could rescue the deficient cell-to-cell movement of mutant viruses. Moreover, deletion of any of the other seven residues also abolished TVBMV cell-to-cell movement and reduced the CP RNA-binding activity. The corresponding nine residues in watermelon mosaic virus CP were also found to play essential roles in virus cell-to-cell movement. In conclusion, residues R192 and K225 in the CP RNA-binding pocket are critical for viral RNA binding and affect both virus replication and cell-to-cell movement.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Identification of an Intermediate Form of Ferredoxin That Binds Only Iron Suggests That Conversion to Holo-Ferredoxin Is Independent of the ISC System in Escherichia coli.

Escherichia coli [2Fe-2S]-ferredoxin and other ISC proteins encoded by the iscRSUA-hscBA-fdx-iscX (isc) operon are responsible for the assembly of iron-sulfur clusters. It is proposed that ferredoxin (Fdx) donates electrons from its reduced [2Fe-2S] center to iron-sulfur cluster biogenesis reactions. However, the underlying mechanisms of the [2Fe-2S] cluster assembly in Fdx remain elusive. Here, we report that Fdx preferentially binds iron, but not the [2Fe-2S] cluster, under cold stress conditions (≤16°C). The iron binding in Fdx is characterized by a unique absorption peak at 320 nm based on UV-visible spectroscopy. In addition, the iron-binding form of Fdx could be converted to the [2Fe-2S] cluster-bound form after transferring cold-stressed cells to normal cultivation temperatures above 25°C. In vitro experiments also revealed that Fdx could utilize bound iron to assemble the [2Fe-2S] cluster by itself. Furthermore, inactivation of the genes encoding IscS, IscU, and IscA did not limit [2Fe-2S] cluster assembly in Fdx, which was also observed by inactivating the isc or suf operon, indicating that iron-sulfur cluster biogenesis in Fdx arose from a unique pathway in E. coli Our results suggest that the intracellular assembly of [2Fe-2S] clusters in Fdx is susceptible to environmental temperatures. The iron binding form of Fdx (Fe-Fdx) is a precursor during its maturation to a cluster binding form ([2Fe-2S]-Fdx), and reassembly of the [2Fe-2S] clusters during temperature increases is not strictly reliant on other specific iron donors and scaffold proteins within the Isc or Suf system.IMPORTANCE Fdx is an electron carrier that is required for the maturation of many other iron-sulfur proteins. Its function strictly depends on its [2Fe-2S] center that bonds with the cysteinyl S atoms of four cysteine residues within Fdx. However, the assembly mechanism of the [2Fe-2S] clusters in Fdx remains controversial. This study reports that Fdx fails to form its [2Fe-2S] cluster under cold stress conditions but instead binds a single Fe atom at the cluster binding site. Moreover, when temperatures increase, Fdx can assemble clusters by itself from its iron-only binding form in E. coli cells. The possibility remains that Fdx can effectively accept clusters from multiple sources. Nevertheless, our results suggest that Fdx has a strong iron binding activity that contributes to the assembly of its own [2Fe-2S] cluster and that Fdx acts as a temperature sensor to regulate Isc system-mediated iron-sulfur cluster biogenesis.