Contributions of glycosaminoglycan binding and clustering to the biological uptake of the nonamphipathic cell-penetrating peptide WR9

Many cell-penetrating peptides (CPPs) bind to glycosaminoglycans (GAG) located on the extracellular side of biological tissues. CPP binding to the cell surface is intimately associated with clustering of surface molecules and is usually followed by uptake into the cell interior. We have investigated the uptake mechanism by comparing CPPs which bind, but cannot induce, GAG clustering with those which do induce GAG clustering. We have synthesized the tryptophan-labeled CPP nona-l-arginine (WR(9)) and its monodispersely PEGylated derivate (PEG(27)-WR(9)) and have compared them with respect to glycan binding, glycan clustering, and their uptake into living cells. Both CPPs bind to the GAG heparin with high affinity (K(D) ～ 100 nM), but the PEGylation prevents the GAG clustering. Thus, it is possible to uncouple and analyze the contributions of GAG binding and GAG clustering to the biological CPP uptake. The uptake of PEG-WR(9) into CH-K1 cells is confined to intracellular vesicles, where colocalization with transferrin attests to an endocytic uptake. Transfection experiments with plasmid DNA for GFP revealed poor GFP expression, suggesting that endocytic uptake of PEG-WR(9) is compromised by insufficient release from endocytic vesicles. In contrast, WR(9) shows two uptake routes. At low concentration (<5 μM), WR(9) uptake occurs mainly through endocytosis. At higher concentration, WR(9) uptake is greatly enhanced, showing a diffuse spreading over the entire cytoplasm and nucleus-a phenomenon termed "transduction". Transduction of WR(9) leads to a higher GFP expression as compared to PEG-WR(9) endocytosis but also damages the plasma membrane as evidenced by SYTOX Green staining. The results suggest that GAG binding without and with GAG clustering induce two different pathways of CPP uptake.     

The maize high-lysine mutant opaque7 is defective in an acyl-CoA synthetase-like protein

Maize (Zea mays) has a large class of seed mutants with opaque or nonvitreous endosperms that could improve the nutritional quality of our food supply. The phenotype of some of them appears to be linked to the improper formation of protein bodies (PBs) where zein storage proteins are deposited. Although a number of genes affecting endosperm vitreousness have been isolated, it has been difficult to clone opaque7 (o7), mainly because of its low penetrance in many genetic backgrounds. The o7-reference (o7-ref) mutant arose spontaneously in a W22 inbred, but is poorly expressed in other lines. We report here the isolation of o7 with a combination of map-based cloning and transposon tagging. We first identified an o7 candidate gene by map-based cloning. The putative o7-ref allele has a 12-bp in-frame deletion of codons 350-353 in a 528-codon-long acyl-CoA synthetase-like gene (ACS). We then confirmed this candidate gene by generating another mutant allele from a transposon-tagging experiment using the Activator/Dissociation (Ac/Ds) system in a W22 background. The second allele, isolated from ～1 million gametes, presented a 2-kb Ds insertion that resembles the single Ds component of double-Ds, McClintock's original Dissociation element, at codon 496 of the ACS gene. PBs exhibited striking membrane invaginations in the o7-ref allele and a severe number reduction in the Ds-insertion mutant, respectively. We propose a model in which the ACS enzyme plays a key role in membrane biogenesis, by taking part in protein acylation, and that altered PBs render the seed nonvitreous.     

Labeling and enzyme studies of the central carbon metabolism in Metallosphaera sedula

Metallosphaera sedula (Sulfolobales, Crenarchaeota) uses the 3-hydroxypropionate/4-hydroxybutyrate cycle for autotrophic carbon fixation. In this pathway, acetyl-coenzyme A (CoA) and succinyl-CoA are the only intermediates that can be considered common to the central carbon metabolism. We addressed the question of which intermediate of the cycle most biosynthetic routes branch off. We labeled autotrophically growing cells by using 4-hydroxy[1-¹⁴C]butyrate and [1,4-¹³C₁]succinate, respectively, as precursors for biosynthesis. The labeling patterns of protein-derived amino acids verified the operation of the proposed carbon fixation cycle, in which 4-hydroxybutyrate is converted to two molecules of acetyl-CoA. The results also showed that major biosynthetic flux does not occur via acetyl-CoA, except for the formation of building blocks that are directly derived from acetyl-CoA. Notably, acetyl-CoA is not assimilated via reductive carboxylation to pyruvate. Rather, our data suggest that the majority of anabolic precursors are derived from succinyl-CoA, which is removed from the cycle via oxidation to malate and oxaloacetate. These C₄ intermediates yield pyruvate and phosphoenolpyruvate (PEP). Enzyme activities that are required for forming intermediates from succinyl-CoA were detected, including enzymes catalyzing gluconeogenesis from PEP. This study completes the picture of the central carbon metabolism in autotrophic Sulfolobales by connecting the autotrophic carbon fixation cycle to the formation of central carbon precursor metabolites.Sulfolobales (Crenarchaeota) comprise extreme thermoacidophiles from volcanic areas that grow best at a pH of around 2 and a temperature of 60 to 90°C (32, 33). Most Sulfolobales can grow chemoautotrophically on sulfur, pyrite, or H₂ under microaerobic conditions, which also applies to Metallosphaera sedula (31), the organism studied here. Its genome has been sequenced (2). Some species of the Sulfolobales secondarily returned to a facultative anaerobic or even strictly anaerobic life style (33), and some laboratory strains appear to have lost their ability to grow autotrophically (8). Autotrophic representatives of the Sulfolobales use a 3-hydroxypropionate/4-hydroxybutyrate cycle (in short, hydroxypropionate/hydroxybutyrate cycle) for autotrophic carbon fixation (Fig. ​(Fig.1)1) (6-8, 38). The enzymes of this cycle are oxygen tolerant, which predestines the cycle for the lifestyle of the aerobic Crenarchaeota (8). The presence of genes coding for key enzymes of the hydroxypropionate/hydroxybutyrate cycle in the mesophilic aerobic “marine group I” Crenarchaeota suggests that these abundant marine archaea use a similar autotrophic carbon fixation mechanism (6, 24, 68) (for a review of autotrophic carbon fixation in Archaea, see reference 7).Open in a separate windowFIG. 1.Proposed 3-hydroxypropionate/4-hydroxybutyrate cycle functioning in autotrophic carbon fixation in Sulfolobales and its relation to the central carbon metabolism, as studied in this work for Metallosphaera sedula. The situation may be similar in other Sulfolobales and possibly in autotrophic marine Crenarchaeota. Enzymes: 1, acetyl-CoA/propionyl-CoA carboxylase; 2, malonyl-CoA reductase (NADPH); 3, malonic semialdehyde reductase (NADPH); 4, 3-hydroxypropionate-CoA ligase (AMP forming); 5, 3-hydroxypropionyl-CoA dehydratase; 6, acryloyl-CoA reductase (NADPH); 7, acetyl-CoA/propionyl-CoA carboxylase; 8, methylmalonyl-CoA epimerase; 9, methylmalonyl-CoA mutase; 10, succinyl-CoA reductase (NADPH); 11, succinic semialdehyde reductase (NADPH); 12, 4-hydroxybutyrate-CoA ligase (AMP forming); 13, 4-hydroxybutyryl-CoA dehydratase; 14 and 15, crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase (NAD⁺); 16, acetoacetyl-CoA β-ketothiolase; 17, succinyl-CoA synthetase (ADP forming); 18, succinic semialdehyde dehydrogenase; 19, succinate dehydrogenase (natural electron acceptor unknown); 20, fumarate hydratase; 21, malate dehydrogenase; 22, malic enzyme; 23, PEP carboxykinase (GTP); 24, pyruvate:water dikinase (ATP); 25, enolase; 26, phosphoglycerate mutase; 27, phosphoglycerate kinase; 28, glyceraldehyde 3-phosphate dehydrogenase; 29, triosephosphate isomerase; 30, fructose 1,6-bisphosphate aldolase/phosphatase; 31, (si)-citrate synthase; 32, aconitase; 33, isocitrate dehydrogenase.In the cycle, one molecule of acetyl-coenzyme A (CoA) is formed from two molecules of bicarbonate. The key carboxylating enzyme is a bifunctional biotin-dependent acetyl-CoA/propionyl-CoA carboxylase (10, 11, 36, 38, 48, 49). In Bacteria and Eukarya, acetyl-CoA carboxylase catalyzes the first step in fatty acid biosynthesis. However, archaea do not contain fatty acids, and therefore acetyl-CoA carboxylase obviously plays a different metabolic role. The hydroxypropionate/hydroxybutyrate cycle can be divided into two parts. The first transforms acetyl-CoA and two bicarbonate molecules via 3-hydroxypropionate to succinyl-CoA, and the second converts succinyl-CoA via 4-hydroxybutyrate to two acetyl-CoA molecules. In brief, the product of the acetyl-CoA carboxylase reaction, malonyl-CoA, is reduced via malonic semialdehyde to 3-hydroxypropionate, which is further reductively converted to propionyl-CoA. Propionyl-CoA is carboxylated to (S)-methylmalonyl-CoA by the same carboxylase as that that carboxylates acetyl-CoA (11, 36). (S)-Methylmalonyl-CoA is isomerized to (R)-methylmalonyl-CoA, followed by carbon rearrangement to succinyl-CoA catalyzed by coenzyme B₁₂-dependent methylmalonyl-CoA mutase.Succinyl-CoA then is converted into two molecules of acetyl-CoA via succinic semialdehyde, 4-hydroxybutyrate, 4-hydroxybutyryl-CoA, crotonyl-CoA, 3-hydroxyacetyl-CoA, and acetoacetyl-CoA. This reaction sequence apparently is common to the autotrophic Crenarchaeota, as it also is used by autotrophic Crenarchaeota of the orders Thermoproteales and Desulfurococcales, which use a dicarboxylate/4-hydroxybutyrate cycle for autotrophic carbon fixation (8, 34, 55, 56) (also see the accompanying work [57]).From the list of intermediates of the hydroxypropionate/hydroxybutyrate cycle, acetyl-CoA and succinyl-CoA are the only intermediates considered common to the central carbon metabolism. In this work, we addressed the question of which intermediate of the cycle most biosynthetic routes branch off, and we came to the conclusion that succinyl-CoA serves as the main precursor for cellular carbon. This requires one turn of the cycle to regenerate the CO₂ acceptor and to generate one extra molecule of acetyl-CoA from two molecules of bicarbonate. Acetyl-CoA plus another two bicarbonate molecules are converted by an additional half turn of the cycle to succinyl-CoA. This strategy differs from that of the anaerobic pathways, in which acetyl-CoA is reductively carboxylated to pyruvate, and from there the other precursors for building blocks ultimately are derived (discussed in reference 7).     

Bistability in myo-inositol utilization by Salmonella enterica serovar Typhimurium

The capability of Salmonella enterica serovar Typhimurium strain 14028 (S. Typhimurium 14028) to utilize myo-inositol (MI) is determined by the genomic island GEI4417/4436 carrying the iol genes that encode enzymes, transporters, and a repressor responsible for the MI catabolic pathway. In contrast to all bacteria investigated thus far, S. Typhimurium 14028 growing on MI as the sole carbon source is characterized by a remarkable long lag phase of 40 to 60 h. We report here that on solid medium with MI as the sole carbon source, this human pathogen exhibits a bistable phenotype characterized by a dissection into large colonies and a slow-growing bacterial background. This heterogeneity is reversible and therefore not caused by mutation, and it is not observed in the absence of the iol gene repressor IolR nor in the presence of at least 0.55% CO(2). Bistability is correlated with the activity of the iolE promoter (P(iolE)), but not of P(iolC) or P(iolD), as shown by promoter-gfp fusions. On the single-cell level, fluorescence microscopy and flow cytometry analysis revealed a gradual switch of P(iolE) from the "off" to the "on" status during the late lag phase and the transition to the log phase. Deletion of iolR or the addition of 0.1% NaHCO(3) induced an early growth start of S. Typhimurium 14028 in minimal medium with MI. The addition of ethoxyzolamide, an inhibitor of carboanhydrases, elongated the lag phase in the presence of bicarbonate. The positive-feedback loop via repressor release and positive induction by bicarbonate-CO(2) might allow S. Typhimurium 14028 to adapt to rapidly changing environments. The phenomenon described here is a novel example of bistability in substrate degradation, and, to our knowledge, is the first demonstration of gene regulation by bicarbonate-CO(2) in Salmonella.     

En route to sugar-alkaloid conjugates

After stereoselective addition of N-iodosuccinimide to glycals subsequent dehalogenation results in formation of N-glycopyranosyl succinimides. By UV irradiation both azepindiones and preferentially [5.3.1.0^2,6] tricyclic oxalactams could be obtained. Their transformation into a number of novel sugar conjugates resembling some prominent alkaloid N-pyrrol components by thiation and reduction is reported.     

Evolutionary advantage of small populations on complex fitness landscapes

Recent experimental and theoretical studies have shown that small asexual populations evolving on complex fitness landscapes may achieve a higher fitness than large ones due to the increased heterogeneity of adaptive trajectories. Here, we introduce a class of haploid three-locus fitness landscapes that allow the investigation of this scenario in a precise and quantitative way. Our main result derived analytically shows how the probability of choosing the path of the largest initial fitness increase grows with the population size. This makes large populations more likely to get trapped at local fitness peaks and implies an advantage of small populations at intermediate time scales. The range of population sizes where this effect is operative coincides with the onset of clonal interference. Additional studies using ensembles of random fitness landscapes show that the results achieved for a particular choice of three-locus landscape parameters are robust and also persist as the number of loci increases. Our study indicates that an advantage for small populations is likely whenever the fitness landscape contains local maxima. The advantage appears at intermediate time scales, which are long enough for trapping at local fitness maxima to have occurred but too short for peak escape by the creation of multiple mutants.     

Comparing environmental and genetic variance as adaptive response to fluctuating selection

Phenotypic variation within populations has two sources: genetic variation and environmental variation. Here, we investigate the coevolution of these two components under fluctuating selection. Our analysis is based on the lottery model in which genetic polymorphism can be maintained by negative frequency-dependent selection, whereas environmental variation can be favored due to bet-hedging. In our model, phenotypes are characterized by a quantitative trait under stabilizing selection with the optimal phenotype fluctuating in time. Genotypes are characterized by their phenotypic offspring distribution, which is assumed to be Gaussian with heritable variation for its mean and variance. Polymorphism in the mean corresponds to genetic variance while the width of the offspring distribution corresponds to environmental variance. We show that increased environmental variance is favored whenever fluctuations in the selective optima are sufficiently strong. Given the environmental variance has evolved to its optimum, genetic polymorphism can still emerge if the distribution of selective optima is sufficiently asymmetric or leptokurtic. Polymorphism evolves in a diagonal direction in trait space: one type becomes a canalized specialist for the more common ecological conditions and the other type a de-canalized bet-hedger thriving on the less-common conditions. All results are based on analytical approximations, complemented by individual-based simulations.     

Identification of neuronal RNA targets of TDP-43-containing ribonucleoprotein complexes

TAR DNA-binding protein 43 (TDP-43) is associated with a spectrum of neurodegenerative diseases. Although TDP-43 resembles heterogeneous nuclear ribonucleoproteins, its RNA targets and physiological protein partners remain unknown. Here we identify RNA targets of TDP-43 from cortical neurons by RNA immunoprecipitation followed by deep sequencing (RIP-seq). The canonical TDP-43 binding site (TG)(n) is 55.1-fold enriched, and moreover, a variant with adenine in the middle, (TG)(n)TA(TG)(m), is highly abundant among reads in our TDP-43 RIP-seq library. TDP-43 RNA targets can be divided into three different groups: those primarily binding in introns, in exons, and across both introns and exons. TDP-43 RNA targets are particularly enriched for Gene Ontology terms related to synaptic function, RNA metabolism, and neuronal development. Furthermore, TDP-43 binds to a number of RNAs encoding for proteins implicated in neurodegeneration, including TDP-43 itself, FUS/TLS, progranulin, Tau, and ataxin 1 and -2. We also identify 25 proteins that co-purify with TDP-43 from rodent brain nuclear extracts. Prominent among them are nuclear proteins involved in pre-mRNA splicing and RNA stability and transport. Also notable are two neuron-enriched proteins, methyl CpG-binding protein 2 and polypyrimidine tract-binding protein 2 (PTBP2). A PTBP2 consensus RNA binding motif is enriched in the TDP-43 RIP-seq library, suggesting that PTBP2 may co-regulate TDP-43 RNA targets. This work thus reveals the protein and RNA components of the TDP-43-containing ribonucleoprotein complexes and provides a framework for understanding how dysregulation of TDP-43 in RNA metabolism contributes to neurodegeneration.     

Comparative ionomics and metabolomics in extremophile and glycophytic Lotus species under salt stress challenge the metabolic pre-adaptation hypothesis

The legume genus Lotus includes glycophytic forage crops and other species adapted to extreme environments, such as saline soils. Understanding salt tolerance mechanisms will contribute to the discovery of new traits which may enhance the breeding efforts towards improved performance of legumes in marginal agricultural environments. Here, we used a combination of ionomic and gas chromatography‐mass spectrometry (GC‐MS)‐based metabolite profilings of complete shoots (pooling leaves, petioles and stems) to compare the extremophile Lotus creticus, adapted to highly saline coastal regions, and two cultivated glycophytic grassland forage species, Lotus corniculatus and Lotus tenuis. L. creticus exhibited better survival after exposure to long‐term lethal salinity and was more efficient at excluding Cl^‐ from the shoots than the glycophytes. In contrast, Na⁺ levels were higher in the extremophile under both control and salt stress, a trait often observed in halophytes. Ionomics demonstrated a differential rearrangement of shoot nutrient levels in the extremophile upon salt exposure. Metabolite profiling showed that responses to NaCl in L. creticus shoots were globally similar to those of the glycophytes, providing little evidence for metabolic pre‐adaptation to salinity. This study is the first comparing salt acclimation responses between extremophile and non‐extremophile legumes, and challenges the generalization of the metabolic salt pre‐adaptation hypothesis.