In 2010 the effects of the global financial crisis were still being felt in the UK. Stock market confidence levels were low, unemployment levels were high and job opportunities were few and far ...between especially for new graduates with little work experience. Unfortunately for my partner, her recent graduation and search for job opportunities within the finance and law sector could not have come at a more unsuitable time. China however had better weathered the storm of the financial crisis and presented itself as a more familiar and fruitful climate for my partner to further develop her career. Furthermore.
Plant asparaginyl endopeptidases (AEPs) are important for the post-translational processing of seed storage proteins via cleavage of precursor proteins. Some AEPs also function as peptide bond-makers ...during the biosynthesis of several unrelated classes of cyclic peptides, namely the kalata-type cyclic peptides, PawS-Derived Peptides and cyclic knottins. These three families of gene-encoded peptides have different evolutionary origins, but all have recruited AEPs for their maturation. In the last few years, the field has advanced rapidly, with the biochemical characterization of three plant AEPs capable of peptide macrocyclization, and insights have been gained from the first AEP crystal structures, albeit mammalian ones. Although the biochemical studies have improved our understanding of the mechanism of action, the focus now is to understand what changes in AEP sequence and structure enable some plant AEPs to perform macrocyclization reactions.
An avian-origin human-infecting influenza (H7N9) virus was recently identified in China. We have evaluated the viral hemagglutinin (HA) receptor-binding properties of two human H7N9 isolates, ...A/Shanghai/1/2013 (SH-H7N9) (containing the avian-signature residue Gln²²⁶) and A/Anhui/1/2013 (AH-H7N9) (containing the mammalian-signature residue Leu²²⁶). We found that SH-H7N9 HA preferentially binds the avian receptor analog, whereas AH-H7N9 HA binds both avian and human receptor analogs. Furthermore, an AH-H7N9 mutant HA (Leu²²⁶ → Gln) was found to exhibit dual receptor-binding property, indicating that other amino acid substitutions contribute to the receptor-binding switch. The structures of SH-H7N9 HA, AH-H7N9 HA, and its mutant in complex with either avian or human receptor analogs show how AH-H7N9 can bind human receptors while still retaining the avian receptor-binding property.
Summary
Plant asparaginyl endopeptidases (AEPs) are expressed as inactive zymogens that perform maturation of seed storage protein upon cleavage‐dependent autoactivation in the low‐pH environment of ...storage vacuoles. The AEPs have attracted attention for their macrocyclization reactions, and have been classified as cleavage or ligation specialists. However, we have recently shown that the ability of AEPs to produce either cyclic or acyclic products can be altered by mutations to the active site region, and that several AEPs are capable of macrocyclization given favorable pH conditions. One AEP extracted from Clitoria ternatea seeds (butelase 1) is classified as a ligase rather than a protease, presenting an opportunity to test for loss of cleavage activity. Here, making recombinant butelase 1 and rescuing an Arabidopsis thaliana mutant lacking AEP, we show that butelase 1 retains cleavage functions in vitro and in vivo. The in vivo rescue was incomplete, consistent with some trade‐off for butelase 1 specialization toward macrocyclization. Its crystal structure showed an active site with only subtle differences from cleaving AEPs, suggesting the many differences in its peptide‐binding region are the source of its efficient macrocyclization. All considered, it seems that either butelase 1 has not fully specialized or a requirement for autocatalytic cleavage is an evolutionary constraint upon macrocyclizing AEPs.
Significance Statement
Here we produced recombinant butelase 1, said to be the world's fastest peptide ligase, and gained insight into its catalysis from its crystal structure. Working in vitro and in vivo, we show butelase 1 can still self‐mature (cleavage), but there has been some trade‐off between these two conflicting activities.
Defective epithelial barrier function in asthma Xiao, Chang, PhD; Puddicombe, Sarah M., PhD; Field, Sarah, PhD ...
Journal of allergy and clinical immunology,
09/2011, Letnik:
128, Številka:
3
Journal Article
Recenzirano
Background Asthma is a complex disease involving gene and environment interactions. Although atopy is a strong predisposing risk factor for asthma, local tissue susceptibilities are required for ...disease expression. The bronchial epithelium forms the interface with the external environment and is pivotally involved in controlling tissue homeostasis through provision of a physical barrier controlled by tight junction (TJ) complexes. Objectives To explain the link between environment exposures and airway vulnerability, we hypothesized that epithelial TJs are abnormal in asthma, leading to increased susceptibility to environmental agents. Methods Localization of TJs in bronchial biopsies and differentiated epithelial cultures was assessed by electron microscopy or immunostaining. Baseline permeability and the effect of cigarette smoke and growth factor were assessed by measurement of transepithelial electrical resistance and passage of fluorescently labeled dextrans. Results By using immunostaining, we found that bronchial biopsies from asthmatic subjects displayed patchy disruption of TJs. In differentiated bronchial epithelial cultures, TJ formation and transepithelial electrical resistance were significantly lower ( P < .05) in cultures from asthmatic donors (n = 43) than from normal controls (n = 40) and inversely correlated with macromolecular permeability. Cultures from asthmatic donors were also more sensitive to disruption by cigarette smoke extract. Epidermal growth factor enhanced basal TJ formation in cultures from asthmatic subjects ( P < .01) and protected against cigarette smoke–induced barrier disruption ( P < .01). Conclusions Our results show that the bronchial epithelial barrier in asthma is compromised. This defect may facilitate the passage of allergens and other agents into the airway tissue, leading to immune activation and may thus contribute to the end organ expression of asthma.
Abstract
Decades of intense herbicide use has led to resistance in weeds. Without innovative weed management practices and new herbicidal modes of action, the unabated rise of herbicide resistance ...will undoubtedly place further stress upon food security. HMGR (3-hydroxy-3-methylglutaryl-coenzyme A reductase) is the rate limiting enzyme of the eukaryotic mevalonate pathway successfully targeted by statins to treat hypercholesterolemia in humans. As HMGR inhibitors have been shown to be herbicidal, HMGR could represent a mode of action target for the development of herbicides. Here, we present the crystal structure of a HMGR from
Arabidopsis thaliana
(AtHMG1) which exhibits a wider active site than previously determined structures from different species. This plant conserved feature enables the rational design of specific HMGR inhibitors and we develop a tolerance trait through sequence analysis of fungal gene clusters. These results suggest HMGR to be a viable herbicide target modifiable to provide a tolerance trait.
Influenza A (H7N9) virus has been causing human infections in China since February 2013, raising serious concerns of potential pandemics. Previous studies demonstrate that human infection is directly ...linked to live animal markets, and that the internal genes of the virus are derived from H9N2 viruses circulating in the Yangtze River Delta area in Eastern China. Here following analysis of 109 viruses, we show a much higher genetic heterogeneity of the H7N9 viruses than previously reported, with a total of 27 newly designated genotypes. Phylogenetic and genealogical inferences reveal that genotypes G0 and G2.6 dominantly co-circulate within poultry, with most human isolates belonging to the genotype G0. G0 viruses are also responsible for the inter- and intra-province transmissions, leading to the genesis of novel genotypes. These observations suggest the province-specific H9N2 virus gene pools increase the genetic diversity of H7N9 via dynamic reassortments and also imply that G0 has not gained overwhelming fitness and the virus continues to undergo reassortment.
Constrained, cyclic peptides encoded by plant genes represent a new generation of drug leads. Evolution has repeatedly recruited the Cys-protease asparaginyl endopeptidase (AEP) to perform their ...head-to-tail ligation. These macrocyclization reactions use the substrates amino terminus instead of water to deacylate, so a peptide bond is formed. How solvent-exposed plant AEPs macrocyclize is poorly understood. Here we present the crystal structure of an active plant AEP from the common sunflower,
. The active site contained electron density for a tetrahedral intermediate with partial occupancy that predicted a binding mode for peptide macrocyclization. By substituting catalytic residues we could alter the ratio of cyclic to acyclic products. Moreover, we showed AEPs from other species lacking cyclic peptides can perform macrocyclization under favorable pH conditions. This structural characterization of AEP presents a logical framework for engineering superior enzymes that generate macrocyclic peptide drug leads.
The neuraminidase stalk of the newly emerged H7N9 influenza virus possesses a 5-amino-acid deletion. This study focuses on characterizing the biological functions of H7N9 with varied neuraminidase ...stalk lengths. Results indicate that the 5-amino-acid deletion had no impact on virus infectivity or replication in vitro or in vivo compared to that of a virus with a full-length stalk, but enhanced virulence in mice was observed for H7N9 encoding a 19- to 20-amino-acid deletion, suggesting that N9 stalk length impacts virulence in mammals, as N1 stalk length does.
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