Mobility Shift Experiments Research Articles

5-Aminolevulinic acid improves cold resistance through regulation of SlMYB4/SlMYB88-SlGSTU43 module to scavenge reactive oxygen species in tomato.

Cold stress severely affects the growth and quality of tomato. 5-Aminolevulinic acid (ALA) can effectively improve tomato's cold stress tolerance. In this study, a tomato glutathione S-transferase gene, SlGSTU43, was identified. Results showed that ALA strongly induced the expression of SlGSTU43 under cold stress. SlGSTU43-overexpressing lines showed increased resistance to cold stress through an enhanced ability to scavenge reactive oxygen species. On the contrary, slgstu43 mutant lines were sensitive to cold stress, and ALA did not improve their cold stress tolerance. Thus, SlGSTU43 is a key gene in the process of ALA improving tomato cold tolerance. Through yeast library screening, SlMYB4 and SlMYB88 were preliminarily identified as transcription factors that bind to the SlGSTU43 promoter. Electrophoretic mobility shift, yeast one-hybrid, dual luciferase, and chromatin immunoprecipitation assays experiments verified that SlMYB4 and SlMYB88 can bind to the SlGSTU43 promoter. Further experiments showed that SlMYB4 and SlMYB88 are involved in the process of ALA-improving tomato's cold stress tolerance and they positively regulate the expression of SlGSTU43. The findings provide new insights into the mechanism by which ALA improves cold stress tolerance. SlGSTU43, as a valuable gene, could be added to the cold-responsive gene repository. Subsequently, it could be used in genetic engineering to enhance the cold tolerance of tomato.

Case-only design identifies interactions of genetic risk variants at SIGLEC5 and PLG with the lncRNA CTD-2353F22.1 implying the importance of periodontal wound healing for disease aetiology.

The basis of phenotypic variation of periodontitis is genetic variability. Disease relevant effects of individual risk alleles are considered to result from genetic interactions. We investigated gene × gene (G×G) interactions of suggestive periodontitis susceptibility alleles. We used the case-only design and investigated single-nucleotide polymorphism (SNPs) that showed associations in our recent genome-wide association study (GWAS) and GWAS meta-analysis with p < 5 × 10-6 . CRISPR-dCas9 gene activation followed by RNA-sequencing and gene-set enrichment analyses elucidated differentially expressed genes and gene networks. With the databases of SNPInspector and Transfac professional, luciferase reporter gene assays and antibody electrophoretic mobility shift experiments, we analysed allele-specific effects on transcription factor binding. SNPs at the genes sialic acid binding Ig-like lectin 5 (SIGLEC5) and plasminogen (PLG) showed G×G interactions with rs1122900 at the long non-coding RNA (lncRNA) CTD-2353F22. Associated chromatin cis-activated CTD-2353F22.1 6.5-fold (p=.003), indicating CTD-2353F22.1 as target gene of this interaction. CTD-2353F22.1 regulated GADD45A (padj < 4.9 × 10-11 , log2 fold change (FC)=-0.55), THBS1, SERPINE1 and Tissue Factor F3 (padj < 5 × 10-7 , log2 FC ≥ -0.35) and the gene set "angiogenesis" (area under the curve=0.71, padj =8.2 × 10-5 ). rs1122900 effect C-allele decreased reporter gene activity (5.5-fold, p=.0003) and PRDM14 binding (76%). CTD-2353F22.1 mediates interaction of SIGLEC5 and PLG, together with genes that function in periodontal wound healing.

Characterization of a small tRNA-binding protein that interacts with the archaeal proteasome complex.

The proteasome system allows the elimination of functional or structurally impaired proteins. This includes the degradation of nascent peptides. In Archaea, how the proteasome complex interacts with the translational machinery remains to be described. Here, we characterized a small orphan protein, Q9UZY3 (UniProt ID), conserved in Thermococcales. The protein was identified in native pull‐down experiments using the proteasome regulatory complex (proteasome‐activating nucleotidase [PAN]) as bait. X‐ray crystallography and small‐angle X‐ray scattering experiments revealed that the protein is monomeric and adopts a β‐barrel core structure with an oligonucleotide/oligosaccharide‐binding (OB)‐fold, typically found in translation elongation factors. Mobility shift experiment showed that Q9UZY3 displays transfer ribonucleic acid (tRNA)‐binding properties. Pull‐downs, co‐immunoprecipitation and isothermal titration calorimetry (ITC) studies revealed that Q9UZY3 interacts in vitro with PAN. Native pull‐downs and proteomic analysis using different versions of Q9UZY3 showed that the protein interacts with the assembled PAN–20S proteasome machinery in Pyrococcus abyssi (Pa) cellular extracts. The protein was therefore named Pbp11, for Proteasome‐Binding Protein of 11 kDa. Interestingly, the interaction network of Pbp11 also includes ribosomal proteins, tRNA‐processing enzymes and exosome subunits dependent on Pbp11's N‐terminal domain that was found to be essential for tRNA binding. Together these data suggest that Pbp11 participates in an interface between the proteasome and the translational machinery.

Hypoxia-induced circWSB1 promotes breast cancer progression through destabilizing p53 by interacting with USP10

BackgroundHypoxia has long been considered as a hallmark of solid tumors and is closely associated with tumor progression. Circular RNAs (circRNAs) have been identified as a critical modulator in various cancers. However, the connections between hypoxia and circRNAs are largely unknown.MethodsHere, we investigated the expression profile of circRNAs in breast cancer (BC) MCF-7 cells under hypoxia and normoxia using microarray. We identified a novel hypoxia-responsive circRNA named circWSB1, whose expression pattern, potential diagnostic value and prognostic significance were assessed by qRT-PCR and in situ hybridization. Loss- and gain-of-function investigations in vivo and in vitro were performed to determine the biological functions of circWSB1. Mechanistically, chromatin immunoprecipitation and dual luciferase reporter assays were carried out to analyze the biogenesis of circWSB1. Furthermore, biotin-labeled RNA pull-down, mass spectrometry, RNA immunoprecipitation, fluorescent in situ hybridization, RNA electrophoretic mobility shift, deletion-mapping, co-immunoprecipitation assays and rescue experiments were applied to investigate the interaction between circWSB1 and Ubiquitin-specific peptidase 10 (USP10) as well as the relationship between USP10 and p53.ResultsWe found that the expression of circWSB1 was significantly upregulated in BC tissues and correlated with poor clinical outcomes, which might serve as an independent prognostic factor for BC patients. Ectopic expression of circWSB1 promoted the proliferation of BC cell in vitro and in vivo. Mechanistically, circWSB1 was transcriptionally upregulated by HIF1α in response to hypoxia and could competitively bind to deubiquitinase USP10 to prevent the access of p53 to USP10 in BC cells, leading to degradation of p53 and tumor progression of BC.ConclusionsTaken together, our findings disclose a novel mechanism that hypoxia-inducible circWSB1 could interact with USP10 to attenuate USP10 mediated p53 stabilization and promote the progression of BC, providing an alternative prognostic biomarker and therapeutic target for BC.

Transcription factor FTZ-F1 regulates mosquito cuticular protein CPLCG5 conferring resistance to pyrethroids in Culex pipiens pallens

BackgroundCulex pipiens pallens poses a serious threat to human health because of its widespread distribution, high carrier capacity for several arboviruses, frequent human-biting, and growth in urban environments. Pyrethroid insecticides have been mainly used to control adult Cx. pipiens pallens during outbreaks of mosquito-borne diseases. Unfortunately, mosquitoes have developed resistance, rendering the insecticides ineffective. Cuticular resistance is the primary mechanism of pyrethroid resistance. Previously, we revealed that cuticular protein of low complexity CPLCG5 is a major cuticular protein associated with deltamethrin resistance in Cx. pipiens pallens, which is enriched in the cuticle of mosquitoes’ legs and participates in pyrethroid resistance by forming a rigid matrix. However, the regulatory mechanisms of its transcription remain unknown.ResultsFirst, qRT-PCR analysis revealed that the expression of FTZ-F1 (encoding Fushi tarazu-Factor 1) was ~ 1.8-fold higher in the deltamethrin-resistant (DR) than deltamethrin-susceptible (DS) strains at 24 h post-eclosion (PE) and ~ 2.2-fold higher in the DR strain than in the DS strain at 48 h PE. CPLCG5 and FTZ-F1 were co-expressed in the legs, indicating that they might play an essential role in the legs. Dual luciferase reporter assays and EMSA (electrophoretic mobility shift experiments) revealed that FTZ-F1 regulates the transcription of CPLCG5 by binding to the FTZ-F1 response element (− 870/− 864). Lastly, knockdown of FTZ-F1 not only affected CPLCG5 expression but also altered the cuticle thickness and structure of the legs, increasing the susceptibility of the mosquitoes to deltamethrin in vivo.ConclusionsThe results revealed that FTZ-F1 regulates the expression of CPLCG5 by binding to the CPLCG5 promoter region, altering cuticle thickness and structure, and increasing the susceptibility of mosquitoes to deltamethrin in vivo. This study revealed part of the mechanism of cuticular resistance, providing a deeper understanding of insecticide resistance.

Deducing Proton-Bound Heterodimer Association Energies from Shifts in Ion Mobility Arrival Time Distributions.

Through vapor modification of the counter-current drift gas in an atmospheric pressure drift tube ion mobility spectrometer (IMS), we demonstrate measurement of gas-phase association enthalpies and entropies for select proton-bound heterodimers formed from a phosphonic acid with 2-propanol. Previous efforts to determine gas-phase association thermodynamic properties have relied largely upon lower pressure systems and inference of the relative concentrations of m/ z isolated species. In contrast, the drift tube IMS based approach developed and applied in this study leverages the explicit gas-phase equilibrium that is established within an ion mobility drift cell. The inferred enthalpies and entropies of association are based solely upon monitoring a shift in the arrival time of an ion at different temperatures (and not on the signal intensity or on external instrument drift time calibration). We specifically report the gas-phase Gibbs free energy, enthalpy, and entropy changes for the association of 2-propanol with protonated methyl, ethyl, and propyl phosphonic acid ions (MPA, EPA, PPA) across the 100-175 °C temperature range. For all of these proton-bound heterodimers, the standard enthalpies and entropies of 2-propanol association were negative and positive, respectively. These data indicate that proton-bound heterodimer formation is both enthalpically and entropically favorable, though we find that the magnitude of the standard enthalpy change for vapor association is small (near 1 kcal/mol for all examined heterodimers). Though many prior results (largely obtained with high pressure mass-spectrometry) for other proton-bound organic heterodimer complexes show larger enthalpic favorability and an entropic barrier, our results qualitatively conform to the bulk Kelvin-Thomson-Raoult (KTR) model, which is commonly utilized in describing ion-induced nucleation of a vapor onto a soluble, nanometer scale ion. The KTR model suggests that heterodimer formation due to vapor binding to an ion should be slightly enthalpically favored (due to a larger Thomson effect than the Kelvin effect) and entropically favored because of ion solvation (Raoult's effect). The method presented in this study can be applied to any static-field ion mobility spectrometer and to a wide variety of heterodimers. Due to the ease of implementation and broad applicability, this approach may find consistent use in determining the thermodynamic properties of weakly bound gas-phase heterodimer complexes which are difficult to probe via alternative techniques. Moreover, this renewed implementation of the IMS experiment is directly compatible with soft ionization sources which will enable the characterization of vapor modifier-induced mobility shift experiments for larger molecular complexes.

Ectopic expression of the TAL effector AvrXa7 in Xanthomonas citri subsp. citri hinders citrus canker symptom formation by modulating transcriptional profile of citrus genes

Xanthomonas citri subsp. citri (Xcc) is the causal agent of citrus canker, a serious bacterial disease that affects citrus trees worldwide. The ectopic expression of TAL effector AvrXa7 in Xcc suppressed canker development. The Xcc strain expressing avrXa7 induced a yellow symptom around the inoculation site. Transcriptome analysis revealed 315 differentially expressed genes, which were categorized into several functional groups. The more interesting genes were those involved in the biosynthesis of terpene and ethylene. In particular, the linoleate 13 S-lipoxygenase gene CsLOX2-1 was found to possess the AvrXa7 binding sequence in the promoter region. The recognition of AvrXa7 to the CsLOX2-1 promoter was subsequently confirmed by yeast one-hybrid and electrophoretic mobility shift experiments. This demonstrated that the TALE effector AvrXa7 promotes CsLOX2-1 expression by directly binding to the promoter sequence. Our findings contribute a valuable clue to identifying the potential genes that can be used to prevent citrus canker.

Requirements for Septal Localization and Chromosome Segregation Activity of the DNA Translocase SftA from Bacillus subtilis

Bacillus subtilis possesses 2 DNA translocases that affect late stages of chromosome segregation: SftA separates nonsegregated DNA prior to septum closure, while SpoIIIE rescues septum-entrapped DNA. We provide evidence that SftA is associated with the division machinery via a stretch of 47 amino acids within its N-terminus, suggesting that SftA is recruited by protein-protein interactions with a component of the division machinery. SftA was also recruited to mid-cell in the absence of its first 20 amino acids, which are proposed to contain a membrane-binding motif. Cell fractionation experiments showed that SftA can be found in the cytosolic fraction, and to a minor degree in the membrane fraction, showing that it is a soluble protein in vivo. The expression of truncated SftA constructs led to a dominant sftA deletion phenotype, even at very low induction rates of the truncated proteins, indicating that the incorporation of nonfunctional monomers into SftA hexamers abolishes functionality. Mobility shift experiments and surface plasmon binding studies showed that SftA binds to DNA in a cooperative manner, and demonstrated low ATPase activity when binding to short nucleotides rather than to long stretches of DNA.

Analysis of the virulence-associated RevSR two-component signal transduction system of Clostridium perfringens

Clostridium perfringens is a Gram-positive, anaerobic, spore-forming bacterium that causes human gas gangrene (clostridial myonecrosis) and food poisoning. Early studies showed that virulence was regulated by the VirSR two-component signal transduction system. However, our identification of the RevR orphan response regulator indicated that more than one system was involved in controlling virulence. To further characterize this virulence-associated regulator, gel mobility shift experiments, coupled with DNase I footprinting, were used to identify the RevR DNA binding sequence. Bioinformatics analysis suggested that an orphan sensor histidine kinase, CPE1757 (renamed RevS), was the cognate sensor of RevR. Interaction between RevS and RevR was demonstrated by use of a bacterial two-hybrid system and validated by protein-protein interaction studies using biolayer interferometry. To assess the involvement of RevS in virulence regulation, the revS gene was inactivated by Targetron insertion. When isogenic wild-type, revS and complemented revS strains were tested in a mouse myonecrosis model, the revS mutant was found to be attenuated in virulence, which was similar to the attenuation observed previously with the revR mutant. However, transcriptional analysis of selected RevR-regulated genes in the revS mutant revealed a different pattern of expression to a revR mutant, suggesting that the RevSR system is more complex than originally thought. Taken together, the results have led to the identification and characterization of the two essential parts of a new regulatory network that is involved in the regulation of virulence in C. perfringens.

The Histone-Like Nucleoid Structuring Protein (H-NS) Is a Negative Regulator of the Lateral Flagellar System in the Deep-Sea Bacterium Shewanella piezotolerans WP3.

Although the histone-like nucleoid structuring protein (H-NS) is well known for its involvement in the adaptation of mesophilic bacteria, such as Escherichia coli, to cold environments and high-pressure stress, an understanding of the role of H-NS in the cold-adapted benthic microorganisms that live in the deep-sea ecosystem, which covers approximately 60% of the earth's surface, is still lacking. In this study, we characterized the function of H-NS in Shewanella piezotolerans WP3, which was isolated from West Pacific sediment at a depth of 1,914 m. Anhns gene deletion mutant (WP3Δhns) was constructed, and comparative whole-genome microarray analysis was performed. H-NS had a significant influence (fold change, >2) on the expression of a variety of WP3 genes (274 and 280 genes were upregulated and downregulated, respectively), particularly genes related to energy production and conversion. Notably, WP3Δhnsexhibited higher expression levels of lateral flagellar genes than WP3 and showed enhanced swarming motility and lateral flagellar production compared to those of WP3. The DNA gel mobility shift experiment showed that H-NS bound specifically to the promoter of lateral flagellar genes. Moreover, the high-affinity binding sequences of H-NS were identified by DNase I protection footprinting, and the results support the "binding and spreading" model for H-NS functioning. To our knowledge, this is the first attempt to characterize the function of the universal regulator H-NS in a deep-sea bacterium. Our data revealed that H-NS has a novel function as a repressor of the expression of genes related to the energy-consuming secondary flagellar system and to swarming motility.

20-hydroxyecdysone mediates fat body arylphorin regulation during development of rice moth, Corcyra cephalonica

Arylphorin hexamerins are one of the major insect storage proteins involved in diverse functions during metamorphosis. However, their regulation during development is not elucidated so far. In the present study, we documented 20-hydroxyecdysone (20E)-mediated regulation of arylphorin expression in the fat body of the stored grain pest, Corcyra cephalonica. Based on the differential developmental expression and 20E-induced transcriptional as well as translational level alterations of arylphorin, we isolated the 5′ upstream region of the gene to analyze regulatory motifs. Promoter motif analysis revealed the presence of ecdysone response element (ERE). Transient transfection studies showed the functionality of the ERE. Enzyme mobility shift experiments with radiolabelled, cold and mutated probes indicate ERE-nuclear factor binding. This study is the first to report transcriptional regulation of arylphorins by 20E in lepdopteran insect species.

Crystal structure of the Mycobacterium tuberculosis transcriptional regulator Rv0302.

Mycobacterium tuberculosis is a pathogenic bacterial species, which is neither Gram positive nor Gram negative. It has a unique cell wall, making it difficult to kill and conferring resistance to antibiotics that disrupt cell wall biosynthesis. Thus, the mycobacterial cell wall is critical to the virulence of these pathogens. Recent work shows that the mycobacterial membrane protein large (MmpL) family of transporters contributes to cell wall biosynthesis by exporting fatty acids and lipidic elements of the cell wall. The expression of the Mycobacterium tuberculosis MmpL proteins is controlled by a complicated regulatory network system. Here we report crystallographic structures of two forms of the TetR-family transcriptional regulator Rv0302, which participates in regulating the expression of MmpL proteins. The structures reveal a dimeric, two-domain molecule with architecture consistent with the TetR family of regulators. Comparison of the two Rv0302 crystal structures suggests that the conformational changes leading to derepression may be due to a rigid body rotational motion within the dimer interface of the regulator. Using fluorescence polarization and electrophoretic mobility shift assays, we demonstrate the recognition of promoter and intragenic regions of multiple mmpL genes by this protein. In addition, our isothermal titration calorimetry and electrophoretic mobility shift experiments indicate that fatty acids may be the natural ligand of this regulator. Taken together, these experiments provide new perspectives on the regulation of the MmpL family of transporters.

Structural diversity in dinuclear copper(I) halide complexes of 2,4-dithiouracil: Synthesis, crystal structures, induction of DNA damage and oxidative stress mediated by ROS

2,4-Dithiouracil (dtucH2) reacts with [CuX(PPh3)]4 (X=Cl, Br, I) to give dinuclear complexes with different bridging motifs, namely doubly bridged [Cu2(μ-Cl)(μ-S,S-dtucH)(PPh3)4] (1), single bridged [Cu2Br2(μ-S,S-dtucH2)(PPh3)4] (2) and triply bridged [Cu2(μ-I)2(μ-S,S-dtucH2)(PPh3)2] (3). In the structure of 1 the two copper(I) centers are joined by one bridging chlorine atom as well as a 2,4-dithiouracil anion acting as an S,S-donor, while compound 3 contains two bridging iodine atoms besides the neutral bridging 2,4-dithiouracil ligand. The structure of 2 consists of two CuBr(PPh3)2 sub-units joined by the S-atoms of a neutral dtucH2. The biological properties of the new complexes were evaluated in relevance to their binding with calf-thymus double stranded DNA (dsCTDNA) in vitro monitored by mobility shift experiments in agarose gel electrophoresis, and their ability to induce oxidative stress in bacteria Escherichia coli. The experiments showed that these complexes are able to interact with dsCTDNA and to cause DNA damage, and are also able to induce oxidative stress in bacteria. Because of the relevance of these properties, these complexes are regarded as a very promising starting point for biological studies in living systems.

Elimination of polyamine N-acetylation and regulatory engineering improved putrescine production by Corynebacterium glutamicum

Corynebacterium glutamicum has been engineered for production of the polyamide monomer putrescine or 1,4-diaminobutane. Here, N-acetylputrescine was shown to be a significant by-product of putrescine production by recombinant putrescine producing C. glutamicum strains. A systematic gene deletion approach of 18 (putative) N-acetyltransferase genes revealed that the cg1722 gene product was responsible for putrescine acetylation. The encoded enzyme was purified and characterized as polyamine N-acetyltransferase. The enzyme accepted acetyl-CoA and propionyl-CoA as donors for acetylation of putrescine and other diamines as acceptors, but showed highest catalytic efficiency with the triamine spermidine and the tetraamine spermine and, hence, was named SnaA. Upon deletion of snaA in the putrescine producing strain PUT21, no acteylputrescine accumulated, but about 41% more putrescine as compared to the parent strain. Moreover, a transcriptome approach identified increased expression of the cgmAR operon encoding a putative permease and a transcriptional TetR-family repressor upon induction of putrescine production in C. glutamicum PUT21. CgmR is known to bind to cgmO upstream of cgmAR and gel mobility shift experiments with purified CgmR revealed that putrescine and other diamines perturbed CgmR-cgmO complex formation, but not migration of free cgmO DNA. Deletion of the repressor gene cgmR resulted in expression changes of a number of genes and increased putrescine production of C. glutamicum PUT21 by 19% as compared to the parent strain. Overexpression of the putative transport gene cgmA increased putrescine production by 24% as compared to the control strain. However, cgmA overexpression in PUT21ΔsnaA did not further improve putrescine production, hence, the beneficial effects of both targets were not synergistic at the highest described yield of 0.21gg−1.

Cobalt(II) complexes with the antimicrobial drug enrofloxacin: Structure, antimicrobial activity, DNA- and albumin-binding

The cobalt(II) complexes with the quinolone antimicrobial agent enrofloxacin (Herx) in the presence of the nitrogen-donor heterocyclic ligands pyridine (py), 2,2′-bipyridylamine (bipyam), 1,10-phenanthroline (phen), 2,2′-bipyridine (bipy) or the oxygen-donor ligand N,N-dimethylformamide (DMF) were synthesized and characterized. The crystal structures of complexes [Co(erx)2(py)2]·MeOH·6H2O and [Co(erx)2(bipyam)]·4.5MeOH·1.25H2O were determined by X-ray crystallography. The deprotonated enrofloxacinato ligands are bidentately bound to cobalt(II) ion through the pyridone oxygen and a carboxylato oxygen. The antimicrobial activity of the complexes was tested against five different microorganisms (Escherichia coli XL1, Xanthomonas campestris ATCC 33013, Staphylococcus aureus ATCC 29213, Bacillus cereus ATCC 11778 and Bacillus subtilis ATCC 6633) and the complexes were more active than free Herx. The binding of the complexes to calf-thymus DNA (CT DNA) was monitored by spectroscopic techniques, viscosity measurements, cyclic voltammetry and by mobility shift experiments in agarose gel electrophoresis with CT DNA and plasmid DNA (pDNA). All complexes exhibited a preference for binding to the supercoiled structure of the pDNA. The ability of the complexes to displace ethidium bromide (EB) from the EB–DNA complex was also investigated. The experiments indicated intercalation as the most possible mode and the DNA-binding strength of the complexes were also calculated. The complexes bind to human or bovine serum albumin protein exhibiting relatively high binding constant values.

Isolation and analysis of the promoter of an anthocyanin synthase gene from purple-fleshed sweet potato tubers

Purple-fleshed sweet potato (Ipomoea batatas (L.) Lam) accumulates a large amount of anthocyanins in its tubers. Activation of anthocyanin gene expression requires transcription factors such as MYB domain, basic helix-loop-helix domain, or WD40-repeat domain-containing proteins. However, the mechanisms controlling pigmentation in underground organs remain unresolved. We used a principal component analysis to identify the most important gene in anthocyanin biosynthesis in pigmented sweet potato tubers, because this gene was the most likely to be regulated by IbMYB1. Anthocyanidin synthase was identified as the most important gene. Functional analysis of its promoter identified four MYB DNA-binding sites. In gel mobility shift experiments with recombinant IbMYB1, the IbMYB1 protein bound specifically to TAACCG box and TATCC box motifs in vitro. We conducted transient expression experiments in which various promoter fragments were used to drive expression of the LUC reporter gene. The reporter gene was strongly expressed under the control of the full-length promoter, but weakly expressed under the control of promoter fragments that lacked the MYB DNA-binding domains. This provided direct evidence that IbMYB1 activates the expression of this structural anthocyanin gene. Together, these results show that IbMYB1 is important in controlling the expression of genes in the anthocyanin biosynthetic pathway in cells.

Fine tuning of the catalytic activity of colicin E7 nuclease domain by systematic N-terminal mutations.

The nuclease domain of colicin E7 (NColE7) promotes the nonspecific cleavage of nucleic acids at its C-terminal HNH motif. Interestingly, the deletion of four N-terminal residues (446-449 NColE7 = KRNK) resulted in complete loss of the enzyme activity. R447A mutation was reported to decrease the nuclease activity, but a detailed analysis of the role of the highly positive and flexible N-terminus is still missing. Here, we present the study of four mutants, with a decreased activity in the following order: NColE7 >> KGNK > KGNG ∼ GGNK > GGNG. At the same time, the folding, the metal-ion, and the DNA-binding affinity were unaffected by the mutations as revealed by linear and circular dichroism spectroscopy, isothermal calorimetric titrations, and gel mobility shift experiments. Semiempirical quantum chemical calculations and molecular dynamics simulations revealed that K446, K449, and/or the N-terminal amino group are able to approach the active centre in the absence of the other positively charged residues. The results suggested a complex role of the N-terminus in the catalytic process that could be exploited in the design of a controlled nuclease.

The Uve1 Endonuclease Is Regulated by the White Collar Complex to Protect Cryptococcus neoformans from UV Damage

The pathogenic fungus Cryptococcus neoformans uses the Bwc1-Bwc2 photoreceptor complex to regulate mating in response to light, virulence and ultraviolet radiation tolerance. How the complex controls these functions is unclear. Here, we identify and characterize a gene in Cryptococcus, UVE1, whose mutation leads to a UV hypersensitive phenotype. The homologous gene in fission yeast Schizosaccharomyces pombe encodes an apurinic/apyrimidinic endonuclease acting in the UVDE-dependent excision repair (UVER) pathway. C. neoformans UVE1 complements a S. pombe uvde knockout strain. UVE1 is photoregulated in a Bwc1-dependent manner in Cryptococcus, and in Neurospora crassa and Phycomyces blakesleeanus that are species that represent two other major lineages in the fungi. Overexpression of UVE1 in bwc1 mutants rescues their UV sensitivity phenotype and gel mobility shift experiments show binding of Bwc2 to the UVE1 promoter, indicating that UVE1 is a direct downstream target for the Bwc1-Bwc2 complex. Uve1-GFP fusions localize to the mitochondria. Repair of UV-induced damage to the mitochondria is delayed in the uve1 mutant strain. Thus, in C. neoformans UVE1 is a key gene regulated in response to light that is responsible for tolerance to UV stress for protection of the mitochondrial genome.

A Human Antibody to the CD4 Binding Site of gp120 Capable of Highly Potent but Sporadic Cross Clade Neutralization of Primary HIV-1

Primary isolates of HIV-1 resist neutralization by most antibodies to the CD4 binding site (CD4bs) on gp120 due to occlusion of this site on the trimeric spike. We describe 1F7, a human CD4bs monoclonal antibody that was found to be exceptionally potent against the HIV-1 primary isolate JR-FL. However, 1F7 failed to neutralize a patient-matched primary isolate, JR-CSF even though the two isolates differ by <10% in gp120 at the protein level. In an HIV-1 cross clade panel (n = 157), 1F7 exhibited moderate breadth, but occasionally achieved considerable potency. In binding experiments using monomeric gp120s of select resistant isolates and domain-swap chimeras between JR-FL and JR-CSF, recognition by 1F7 was limited by sequence polymorphisms involving at least the C2 region of Env. Putative N-linked glycosylation site (PNGS) mutations, notably at position 197, allowed 1F7 to neutralize JR-CSF potently without improving binding to the cognate, monomeric gp120. In contrast, flow cytometry experiments using the same PNGS mutants revealed that 1F7 binding is enhanced on cognate trimeric Env. BN-PAGE mobility shift experiments revealed that 1F7 is sensitive to the diagnostic mutation D368R in the CD4 binding loop of gp120. Our data on 1F7 reinforce how exquisitely targeted CD4bs antibodies must be to achieve cross neutralization of two closely related primary isolates. High-resolution analyses of trimeric Env that show the orientation of glycans and polymorphic elements of the CD4bs that affect binding to antibodies like 1F7 are desirable to understand how to promote immunogenicity of more conserved elements of the CD4bs.

Inducible cAMP Early Repressor Regulates the Period 1 Gene of the Hepatic and Adrenal Clocks

Light, restricted feeding, and hormonal inputs may operate as time givers (zeitgebers) for the circadian clock within peripheral organs through the activation of tissue-specific signaling cascades. cAMP signaling through CREM (cAMP-responsive element modulator) and its variant ICER (inducible cAMP early repressor) is linked to the circadian regulation of pineal melatonin synthesis, although little is known about its influence in other organs. We performed experiments in the absence of light and feeding-time cues to test which core clock genes are controlled by CREM/ICER in the liver and adrenal gland. In vivo, Crem loss-of-function mutation resulted in fine-tuning of all measured adrenal clock genes (Per1/2/3, Cry1/2, Bmal1, and Rev-erbα), whereas only Per1 and Cry1 were affected in the liver. Icer expression was circadian in the adrenal gland, with peak gene expression at zeitgeber 12 and the highest protein levels at zeitgeber ∼20. The expression of both Icer and Per1 genes responded to cAMP stimuli in an immediate-early fashion. In immortal cells, forskolin induced expression of Per1 after 2 h, and de novo protein synthesis led to Per1 attenuation. We show that the de novo synthesized protein responsible for Per1 attenuation is ICER. Indeed, Per1 expression is up-regulated in cells ectopically expressing antisense Icer, and mobility shift experiments identified ICER binding to cAMP-responsive elements of the Per1 promoter. We propose that ICER acts as a noise filter for different signals that could affect transcription in the adrenal gland. Because ICER is an immediate-early repressor, the circadian nature of adrenal Icer expression could serve a role in a time-dependent gating mechanism.