Characterization of the molecular properties of KtrC,a second RCK domain that regulates a Ktr channel in Bacillus subtilis

RCK (regulating conductance of K⁺) domains are common regulatory domains that control the activity of eukaryotic and prokaryotic K⁺ channels and transporters. In bacteria these domains play roles in osmoregulation, regulation of turgor and membrane potential and in pH homeostasis. Whole-genome sequencing unveiled RCK gene redundancy, however the biological role of this redundancy is not well understood. In Bacillus subtilis, there are two closely related RCK domain proteins (KtrA and KtrC) that regulate the activity of the Ktr cation channels. KtrA has been well characterized but little is known about KtrC. We have characterized the structural and biochemical proprieties of KtrC and conclude that KtrC binds ATP and ADP, just like KtrA. However, in solution KtrC exist in a dynamic equilibrium between octamers and non-octameric species that is dependent on the bound ligand, with ATP destabilizing the octameric ring relative to ADP. Accordingly, KtrC-ADP crystal structures reveal closed octameric rings similar to those in KtrA, while KtrC-ATP adopts an open assembly with RCK domains forming a super-helix. In addition, both KtrC-ATP and -ADP octamers are stabilized by the signaling molecule cyclic-di-AMP, which binds to KtrC with high affinity. In contrast, c-di-AMP binds with 100-fold lower affinity to KtrA. Despite these differences we show with an E. coli complementation assay that KtrC and KtrA are interchangeable and able to form functional transporters with both KtrB and KtrD. The distinctive properties of KtrC, in particular ligand-dependent assembly/disassembly, suggest that this protein has a specific physiological role that is distinct from KtrA.     

Preferred proline puckerings in cis andtrans peptide groups: Implications for collagen stability

The interplay between side-chain and main-chain conformations is a distinctive characteristic of proline residues. Here we report the results of a statistical analysis of proline conformations using a large protein database. In particular, we found that proline residues with the preceding peptide bond in the cis state preferentially adopt a down puckering. Indeed, out of 178 cis proline residues, as many as 145 (81%) are down. By analyzing the 1-4 and 1-5 nonbonding distances between backbone atoms, we provide a structural explanation for the observed trend. The observed correlation between proline puckering and peptide bond conformation suggests a new mechanism to explain the reported shift of the cis-trans equilibrium in proline derivatives. The implications of these results for the current models of collagen stability are also discussed.     

Aquaglyceroporins: Generalized metalloid channels

Background

Aquaporins (AQPs), members of a superfamily of transmembrane channel proteins, are ubiquitous in all domains of life. They fall into a number of branches that can be functionally categorized into two major sub-groups: i) orthodox aquaporins, which are water-specific channels, and ii) aquaglyceroporins, which allow the transport of water, non-polar solutes, such as urea or glycerol, the reactive oxygen species hydrogen peroxide, and gases such as ammonia, carbon dioxide and nitric oxide and, as described in this review, metalloids.

Scope of review

This review summarizes the key findings that AQP channels conduct bidirectional movement of metalloids into and out of cells.

Major conclusions

As(OH)₃ and Sb(OH)₃ behave as inorganic molecular mimics of glycerol, a property that allows their passage through AQP channels. Plant AQPs also allow the passage of boron and silicon as their hydroxyacids, boric acid (B(OH)₃) and orthosilicic acid (Si(OH)₄), respectively. Genetic analysis suggests that germanic acid (GeO₂) is also a substrate. While As(III), Sb(III) and Ge(IV) are toxic metalloids, borate (B(III)) and silicate (Si(IV)) are essential elements in higher plants.

General significance

The uptake of environmental metalloids by aquaporins provides an understanding of (i) how toxic elements such as arsenic enter the food chain; (ii) the delivery of arsenic and antimony containing drugs in the treatment of certain forms of leukemia and chemotherapy of diseases caused by pathogenic protozoa; and (iii) the possibility that food plants such as rice could be made safer by genetically modifying them to exclude arsenic while still accumulating boron and silicon. This article is part of a Special Issue entitled Aquaporins.     

Leishmania major LmACR2 is a pentavalent antimony reductase that confers sensitivity to the drug pentostam

Arsenicals and antimonials are first line drugs for the treatment of trypanosomal and leishmanial diseases. To create the active form of the drug, Sb(V) must be reduced to Sb(III). Because arsenic and antimony are related metalloids, and arsenical resistant Leishmania strains are frequently cross-resistant to antimonials, we considered the possibility that Sb(V) is reduced by a leishmanial As(V) reductase. The sequence for the arsenate reductase of Saccharomyces cerevisiae, ScAcr2p, was used to clone the gene for a homologue, LmACR2, from Leishmania major. LmACR2 was able to complement the arsenate-sensitive phenotype of an arsC deletion strain of Escherichia coli or an ScACR2 deletion strain of Saccharomyces cerevisiae. Transfection of Leishmania infantum with LmACR2 augmented Pentostam sensitivity in intracellular amastigotes. LmACR2 was purified and shown to reduce both As(V) and Sb(V). This is the first report of an enzyme that confers Pentostam sensitivity in intracellular amastigotes of Leishmania. We propose that LmACR2 is responsible for reduction of the pentavalent antimony in Pentostam to the active trivalent form of the drug in Leishmania.     

USER friendly DNA engineering and cloning method by uracil excision

Here we report a PCR-based DNA engineering technique for seamless assembly of recombinant molecules from multiple components. We create cloning vector and target molecules flanked with compatible single-stranded (ss) extensions. The vector contains a cassette with two inversely oriented nicking endonuclease sites separated by restriction endonuclease site(s). The spacer sequences between the nicking and restriction sites are tailored to create ss extensions of custom sequence. The vector is then linearized by digestion with nicking and restriction endonucleases. To generate target molecules, a single deoxyuridine (dU) residue is placed 6–10nt away from the 5′-end of each PCR primer. 5′ of dU the primer sequence is compatible either with an ss extension on the vector or with the ss extension of the next-in-line PCR product. After amplification, the dU is excised from the PCR products with the USER enzyme leaving PCR products flanked by 3′ ss extensions. When mixed together, the linearized vector and PCR products directionally assemble into a recombinant molecule through complementary ss extensions. By varying the design of the PCR primers, the protocol is easily adapted to perform one or more simultaneous DNA manipulations such as directional cloning, site-specific mutagenesis, sequence insertion or deletion and sequence assembly.     

Probing the interaction between p53 and the bacterial protein azurin by single molecule force spectroscopy

p53 is a human tumour suppressor which regulates multiple cellular processes, including cell growth, genomic stability and cell death. Recent works have demonstrated the bacterial redox protein azurin to enter cancer cells and induce apoptosis through p53 stabilization, resulting in a tumour growth regression. Azurin has been shown to bind p53 although many details of the complex formed by these two proteins are still poorly characterized. Here, we get insight into the kinetics of this complex formation, by exploring the interaction between p53 and azurin in their environment by single molecule force spectroscopy. To this aim, azurin has been linked to the atomic force microscope tip, whereas p53 has been immobilized onto a gold substrate. Therefore, by performing force-distance cycles we have detected specific recognition events between p53 and azurin, displaying unbinding forces of around 70 pN for an applied loading rate of 3 nN s(-1). The specificity of these events has been assessed by the significant reduction of their frequency observed after blocking the p53 sample by an azurin solution. Moreover, by measuring the rupture force as a function of the loading rate we have determined the dissociation rate constant of this complex to be approximately 0.1 s(-1). Our findings are here discussed in connection with results obtained in bulk experiments, with the aim of clarifying some molecular details of the p53-azurin complex that may help designing new anticancer strategy.     

Effect of specific inhibitors on mitochondrial DNA replication in HeLa cells

The crystal structure of an orthorhombic form of 2′-0-methyl cytidine was determined from three dimensional X-ray diffraction data. The two molecules in each asymmetric unit have C2-$\text{endo}$ C3-$\text{exo}$ puckered furanose rings. This differs from the C3-$\text{endo}$ puckering observed for cytidine (1) and it may have some relevance to the kinks that appear at the two 2′-0-methylated nucleotides in the anticodon phosphate ester backbone of the phe tRNA structure (2). This work and other studies (3,4) show that the presence of a 2′-0-methyl group does not prevent the furanose moiety from adopting its most commonly observed configurations. 2′-0-methyl nucleotides make up a small percentage of the residues in HnRNA, rRNA, tRNA and mRNA and therefore their conformational nuances are of interest.     

New advances and future directions in plant polyspermy

Plants have evolved a battery of mechanisms that potentially act as polyspermy barriers. Supernumerary sperm fusion to one egg cell has consequently long remained a hypothetical concept. The recent discovery that polyspermy in flowering plants is not lethal but generates viable triploid plants is a game changer affecting the field of developmental biology, evolution, and plant breeding. The establishment of protocols to artificially induce polyspermy together with the development of a high‐throughput assay to identify and trace polyspermic events in planta now provide powerful tools to unravel mechanisms of polyspermy regulation. These achievements are likely to open new avenues for animal polyspermy research as well, where forward genetic approaches are hampered by the fatal outcome of supernumerary sperm fusion.     

A375 melanoma cells are sensitized to cisplatin-induced toxicity by a synthetic nitro-flavone derivative 2-(4-Nitrophenyl)-4H-chromen-4-one through inhibition of PARP1

Background

Cisplatin has been extensively used in therapeutics for its broad-spectrum anticancer activity and frequently used for the treatment of solid tumors. However, it presents several side-effects and several cancers develop resistance. Combination therapy of cisplatin with poly (ADP-ribose) polymerase 1 (PARP1) inhibitors has been effective in increasing its efficacy at lower doses.

Methods and results

In this work, we have shown that the nitro-flavone derivative, 2-(4-Nitrophenyl)-4H-chromen-4-one (4NCO), can improve the sensitivity of cancer cells to cisplatin through inhibition of PARP1. The effect of 4NCO on cisplatin toxicity was studied through combination therapy in both exponential and density inhibited A375 melanoma cells. Combination index (CI) was determined from isobologram analysis. The mechanism of cell killing was assessed by lactate dehydrogenase (LDH) assay. Temporal nicotinamide adenine dinucleotide (NAD⁺) assay was done to show the inhibition of PARP1. We also performed in silico molecular modeling studies to know the binding mode of 4NCO to a modeled PARP1-DNA complex containing cisplatin-crosslinked adduct. The results from both in silico and in cellulo studies confirmed that PARP1 inhibition by 4NCO was most effective in sensitizing A375 melanoma cells to cisplatin. Isobologram analysis revealed that 4NCO reduced cell viability both in exponential and density inhibited A375 cells synergistically. The combination led to cell death through apoptosis.

Conclusion

The synthetic nitro-flavone derivative 4NCO effectively inhibited the important nuclear DNA repair enzyme PARP1 and therefore, could complement the DNA-damaging anticancer drug cisplatin in A375 cells and thus, could act as a potential adjuvant to cisplatin in melanoma therapy.

     

Enhancement of salt tolerance in corn using Azospirillum brasilense: an approach on antioxidant systems

Journal of Plant Research - Salinity has become one of the major factors limiting agricultural production. In this regard, different cost-effective management strategies such as the use of plant...