Comparative Laboratory Evolution of Ordered and Disordered Enzymes

Intrinsically disordered proteins are ubiquitous in nature. To assess potential evolutionary advantages and disadvantages of structural disorder under controlled laboratory conditions, we directly compared the evolvability of weakly active ordered and disordered variants of dihydrofolate reductase by genetic selection. The circularly permuted Escherichia coli enzyme, which exists as a molten globule in the absence of ligands, and a well folded deletion mutant of the Bacillus stearothermophilus enzyme served as starting points. Both scaffolds evolved at similar rates and to similar extents, reaching near-native activity after three rounds of mutagenesis and selection. Surprisingly, however, the starting structural properties of the two scaffolds changed only marginally during optimization. Although the ordered and disordered proteins accumulated distinct sets of mutations, the changes introduced likely improved catalytic efficiency indirectly in both cases by bolstering the network of dynamic conformational fluctuations that productively couple into the reaction coordinate.     

Autoxidation and mutagenicity of sodium bisulfite

An inverse correlation exists between the autoxidation of bisulfite and its mutagenicity in Salmonella. Temperature, pH, and the addition of mannitol, ethanol, or Oxoid broth affect both autoxidation and mutagenicity. A decrease in autoxidation resulted in an increase in the half-life of the parent compound, bisulfite, and its availability for uptake by the cells, leading to increased mutagenesis. The autoxidation of bisulfite is known to produce both sulfur- and oxygen-centered free radicals. The lack of mutagenicity of ammonium persulfate and peroxymonosulfate, which generate the radicals SO4- and SO5-, respectively, argues against the involvement of these oxygen-centered radicals in bisulfite mutagenesis. Inhibition of mutagenesis by the radical spin-trapping agent, DMPO, is consistent with the hypothesis that the sulfur-centered radical, SO3-, plays an important role in bisulfite mutagenesis. The mechanism of bisulfite mutagenesis suggested in this study may have relevance to other known effects attributed to bisulfite, i.e., co-carcinogenesis and immune hypersensitivity.     

Genetic analyses of yeast protein serine/threonine phosphatases

Abstract Protein phosphorylation is an important regulatory phenomenon in yeasts just as in other eukaryotic cells and controls a wide variety of cellular processes. The importance of protein phosphatases as well as protein kinases as key elements in such control is becoming increasingly clear. Over the past four years since the first yeast protein phosphatase gene was isolated, many more such genes have been described and the number of genes encoding protein phosphatase catalytic subunits in Saccharomyces cerevisiae has comfortably entered double figures. Given the genetic approaches available, yeasts offer powerful systems for addressing the cellular roles of these enzymes. This review summarises the results of genetic studies aimed at determining the functions of protein serine/threoninc phosphatases in yeast.     

Synergism between aflatoxins in covalent binding to DNA and in mutagenesis in the photoactivation system

Aflatoxins (AFs) produce singlet oxygen upon their exposure to UV (365-nm) light. Singlet oxygen in turn activates them to mutagens and DNA-binding species. DNA binding and mutagenesis by AFs were enhanced in D2O as compared to reactions in H2O, and a singlet oxygen scavenger inhibited mutagenesis. DNA photobinding of 3H-AFB1 increased in the presence of unlabeled AFB2, and the addition of AFB2 enhanced mutagenesis by AFB1 in a synergistic manner. These results are compatible with the notion that singlet oxygen, formed by one aflatoxin molecule, can readily activate another aflatoxin molecule. This may bear an environmental implication in that the weakly carcinogenic AFB2, which is often produced in nature together with AFB1, may be important in enhancing the activation of AFB1 by sunlight.     

A leucine-rich repeat assembly approach for homology modeling of the human TLR5-10 and mouse TLR11-13 ectodomains

So far, 13 groups of mammalian Toll-like receptors (TLRs) have been identified. Most TLRs have been shown to recognize pathogen-associated molecular patterns from a wide range of invading agents and initiate both innate and adaptive immune responses. The TLR ectodomains are composed of varying numbers and types of leucine-rich repeats (LRRs). As the crystal structures are currently missing for most TLR ligand-binding ectodomains, homology modeling enables first predictions of their three-dimensional structures on the basis of the determined crystal structures of TLR ectodomains. However, the quality of the predicted models that are generated from full-length templates can be limited due to low sequence identity between the target and templates. To obtain better templates for modeling, we have developed an LRR template assembly approach. Individual LRR templates that are locally optimal for the target sequence are assembled into multiple templates. This method was validated through the comparison of a predicted model with the crystal structure of mouse TLR3. With this method, we also constructed ectodomain models of human TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10 and mouse TLR11, TLR12, and TLR13 that can be used as first passes for a computational simulation of ligand docking or to design mutation experiments. This template assembly approach can be extended to other repetitive proteins.     

Heavy Metal Exposure Influences Double Strand Break DNA Repair Outcomes

Heavy metals such as cadmium, arsenic and nickel are classified as carcinogens. Although the precise mechanism of carcinogenesis is undefined, heavy metal exposure can contribute to genetic damage by inducing double strand breaks (DSBs) as well as inhibiting critical proteins from different DNA repair pathways. Here we take advantage of two previously published culture assay systems developed to address mechanistic aspects of DNA repair to evaluate the effects of heavy metal exposures on competing DNA repair outcomes. Our results demonstrate that exposure to heavy metals significantly alters how cells repair double strand breaks. The effects observed are both specific to the particular metal and dose dependent. Low doses of NiCl₂ favored resolution of DSBs through homologous recombination (HR) and single strand annealing (SSA), which were inhibited by higher NiCl₂ doses. In contrast, cells exposed to arsenic trioxide preferentially repaired using the “error prone” non-homologous end joining (alt-NHEJ) while inhibiting repair by HR. In addition, we determined that low doses of nickel and cadmium contributed to an increase in mutagenic recombination-mediated by Alu elements, the most numerous family of repetitive elements in humans. Sequence verification confirmed that the majority of the genetic deletions were the result of Alu-mediated non-allelic recombination events that predominantly arose from repair by SSA. All heavy metals showed a shift in the outcomes of alt-NHEJ repair with a significant increase of non-templated sequence insertions at the DSB repair site. Our data suggest that exposure to heavy metals will alter the choice of DNA repair pathway changing the genetic outcome of DSBs repair.     

Distribution in the body and functional effects of fluorocarbons used as oxygen carriers

After the infusion of perfluorochemicals (PFC) given as artificial blood gas carriers a temporary storage of the inert substances in tissues of the organism occurs. The time course of this effect was investigated in longitudinal studies in the rat and compared with changes of different organ functions, expressed as changes in the levels of serum enzymes, in the elimination of indocyanine green and in the duration of pentobarbital sleeping time which are dependent on the excretory and detoxifying functions of the liver. The intensity of the elimination of colloidal carbon was used as a measure of the activity of the reticulo endothelial system. Observation of the time course of these functional tests in comparison with the storage of PFC and changes in organ weight of liver and spleen showed that diminutions in function had a distinctly shorter duration than PFC storage, whereas the longest lasting effects were seen in organ weight increase is regarded as a compensatory effect that is associated without functional loss at the maximum of its appearance.     

An improved method for purifying 2',5'-oligoadenylate synthetases

We describe a new, rapid, and convenient procedure for purifying 2',5'-oligoadenylate synthetases, employing precipitation with ammonium sulfate, fractionation by gel filtration, rapid binding to poly(I) X poly(C) cellulose, and elution with 0.35 M KCl. Unlike previously published methods, the procedure does not require sedimentation of the enzyme at 200,000 X g. Therefore, it is more general and more likely to succeed with synthetases extracted from a variety of cells or tissues, or from different subcellular fractions. We have purified the enzymes from two sources to apparent homogeneity, about 2500-fold from the cytoplasm of HeLa cells in 40% yield and more than 400,000-fold from the cytoplasm of rabbit reticulocytes in 25% yield. The specific activity of the HeLa enzyme is about 4 times higher than reported previously. The physical and functional properties of the pure enzymes are very similar to those reported by others for preparations of 2',5'-oligoadenylate synthetase from rabbit reticulocytes, mouse L cells, and human HeLa cells. A new affinity matrix was prepared by linking periodate-oxidized poly(I) X poly(C) to a hydrazide derivative of finely divided cellulose. Poly(I) X poly(C) cellulose binds about twice as much synthetase as the corresponding amount of poly(I) X poly(C) paper and activates the bound enzyme about three times better.