ENA transporters are a group of P-type ATPases that are characterized by actively moving Na
or K
out of the cell against their concentration gradient. The existence of these transporters was ...initially attributed to some fungi, although more recently they have also been identified in mosses, liverworts, and some protozoa. Given the current increase in the number of organisms whose genomes are completely sequenced, we set out to expand our knowledge about the existence of ENA in organisms belonging to other phylogenetic groups. For that, a hidden Markov model profile was constructed to identify homologous sequences to ENA proteins in protein databases. This analysis allowed us to identify the existence of ENA-type ATPases in the most primitive groups of fungi, as well as in other eukaryotic organisms not described so far. In addition, this study has allowed the identification of a possible new group of P-ATPases, initially proposed as ENA but which maintain phylogenetic distances with these proteins. Finally, this work has also addressed this study of the structure of ENA proteins, which remained unknown due to the lack of crystallographic data. For this purpose, a 3D structure prediction of the NcENA1 protein of the fungus
was performed using AlphaFold2 software v2.3.1. From this structure, the electrostatic potential of the protein was analyzed. With all these data, the protein regions and the amino acids involved in the transport of Na
or K
ions across the membrane were proposed for the first time. Targeted mutagenesis of some of these residues has confirmed their relevant participation in the transport function of ENA proteins.
This work analyses the genetic variation and evolutionary patterns of recessive resistance loci involved in matching-allele (MA) host-pathogen interactions, focusing on the pvr2 resistance gene to ...potyviruses of the wild pepper Capsicum annuum glabriusculum (chiltepin). Chiltepin grows in a variety of wild habitats in Mexico, and its cultivation in home gardens started about 25 years ago. Potyvirus infection of Capsicum plants requires the physical interaction of the viral VPg with the pvr2 product, the translation initiation factor eIF4E1. Mutations impairing this interaction result in resistance, according to the MA model. The diversity of pvr2/eIF4E1 in wild and cultivated chiltepin populations from six biogeographical provinces in Mexico was analysed in 109 full-length coding sequences from 97 plants. Eleven alleles were found, and their interaction with potyvirus VPg in yeast-two-hybrid assays, plus infection assays of plants, identified six resistance alleles. Mapping resistance mutations on a pvr2/eIF4E1 model structure showed that most were around the cap-binding pocket and strongly altered its surface electrostatic potential, suggesting resistance-associated costs due to functional constraints. The pvr2/eIF4E1 phylogeny established that susceptibility was ancestral and resistance was derived. The spatial structure of pvr2/eIF4E1 diversity differed from that of neutral markers, but no evidence of selection for resistance was found in wild populations. In contrast, the resistance alleles were much more frequent, and positive selection stronger, in cultivated chiltepin populations, where diversification of pvr2/eIF4E1 was higher. This analysis of the genetic variation of a recessive resistance gene involved in MA host-pathogen interactions in populations of a wild plant show that evolutionary patterns differ according to the plant habitat, wild or cultivated. It also demonstrates that human management of the plant population has profound effects on the diversity and the evolution of the resistance gene, resulting in the selection of resistance alleles.
ENA transporters are a group of P-type ATPases that are characterized by actively moving Nasup.+ or Ksup.+ out of the cell against their concentration gradient. The existence of these transporters ...was initially attributed to some fungi, although more recently they have also been identified in mosses, liverworts, and some protozoa. Given the current increase in the number of organisms whose genomes are completely sequenced, we set out to expand our knowledge about the existence of ENA in organisms belonging to other phylogenetic groups. For that, a hidden Markov model profile was constructed to identify homologous sequences to ENA proteins in protein databases. This analysis allowed us to identify the existence of ENA-type ATPases in the most primitive groups of fungi, as well as in other eukaryotic organisms not described so far. In addition, this study has allowed the identification of a possible new group of P-ATPases, initially proposed as ENA but which maintain phylogenetic distances with these proteins. Finally, this work has also addressed this study of the structure of ENA proteins, which remained unknown due to the lack of crystallographic data. For this purpose, a 3D structure prediction of the NcENA1 protein of the fungus Neurospora crassa was performed using AlphaFold2 software v2.3.1. From this structure, the electrostatic potential of the protein was analyzed. With all these data, the protein regions and the amino acids involved in the transport of Nasup.+ or Ksup.+ ions across the membrane were proposed for the first time. Targeted mutagenesis of some of these residues has confirmed their relevant participation in the transport function of ENA proteins.
REPI is a pivotal point enzyme in plant benzylisoquinoline alkaloid metabolism as it promotes the evolution of the biosynthetic branch of morphinan alkaloids. Experimental studies of its activity led ...to the identification of two modules (DRS and DRR) that catalyze two sequential steps of the epimerization of (S)- to (R)-reticuline. Recently, special attention has been paid to its genetic characterization and evolutionary history, but no structural analyses of the REPI protein have been conducted to date. We present here a computational structural characterization of REPI with heme and NADP cofactors in the apo state and in three complexes with substrate (S)-reticuline in DRS and intermediate 1,2-dehydroreticuline in DRS and in DRR. Since no experimental structure exists for REPI, we used its AlphaFold model as a scaffold to build up these four systems, which were submitted to all-atom molecular dynamics (MD) simulations. A comparison of MD results for the four systems revealed key dynamic changes associated with cofactor and ligand binding and provided a dynamic picture of the evolution of their structures and interactions. We also explored the possible dynamic occurrence of tunnels and electrostatic highways potentially involved in alternative mechanisms for channeling the intermediate from DRS to DRR.
Unlocking the resistance to wheat lipid transfer protein Gamboa, Pedro Manuel, MD, PhD; Gonzalez, Carlos, MD, PhD; Pereira, Catia, PhD ...
Journal of allergy and clinical immunology,
11/2013, Letnik:
132, Številka:
5
Journal Article
Recenzirano
First patient: 16-year-old boy who showed urticaria, angioedema, and abdominal pain after wheat ingestion (OFC, 500 mg); specific IgE (sIgE) to rTri a 14, 66.3 KU/L; wheat, 3.89 KU/L; and rPru p 3, ...320 KU/L. ...patient: 10-year-old girl with severe abdominal pain, vomiting, and urticaria after bread ingestion (OFC, 800 mg); sIgE to rTri a 14, 4.6 KU/L; wheat, 5.68 KU/L; and rPru p, 3.86 KU/L.
REPI is a pivotal point enzyme in plant benzylisoquinoline alkaloid metabolism as it promotes the evolution of the biosynthetic branch of morphinan alkaloids. Experimental studies of its activity led ...to the identification of two modules (DRS and DRR) that catalyze two sequential steps of the epimerization of (S)- to (R)-reticuline. Recently, special attention has been paid to its genetic characterization and evolutionary history, but no structural analyses of the REPI protein have been conducted to date. We present here a computational structural characterization of REPI with heme and NADP cofactors in the apo state and in three complexes with substrate (S)-reticuline in DRS and intermediate 1,2-dehydroreticuline in DRS and in DRR. Since no experimental structure exists for REPI, we used its AlphaFold model as a scaffold to build up these four systems, which were submitted to all-atom molecular dynamics (MD) simulations. A comparison of MD results for the four systems revealed key dynamic changes associated with cofactor and ligand binding and provided a dynamic picture of the evolution of their structures and interactions. We also explored the possible dynamic occurrence of tunnels and electrostatic highways potentially involved in alternative mechanisms for channeling the intermediate from DRS to DRR.
SUMMARY
The plant immune system perceives a diversity of carbohydrate ligands from plant and microbial cell walls through the extracellular ectodomains (ECDs) of pattern recognition receptors (PRRs), ...which activate pattern‐triggered immunity (PTI). Among these ligands are oligosaccharides derived from mixed‐linked β‐1,3/β‐1,4‐glucans (MLGs; e.g. β‐1,4‐D‐(Glc)2‐β‐1,3‐D‐Glc, MLG43) and cellulose (e.g. β‐1,4‐D‐(Glc)3, CEL3). The mechanisms behind carbohydrate perception in plants are poorly characterized except for fungal chitin oligosaccharides (e.g. β‐1,4‐d‐(GlcNAc)6, CHI6), which involve several receptor kinase proteins (RKs) with LysM‐ECDs. Here, we describe the isolation and characterization of Arabidopsis thaliana mutants impaired in glycan perception (igp) that are defective in PTI activation mediated by MLG43 and CEL3, but not by CHI6. igp1–igp4 are altered in three RKs – AT1G56145 (IGP1), AT1G56130 (IGP2/IGP3) and AT1G56140 (IGP4) – with leucine‐rich‐repeat (LRR) and malectin (MAL) domains in their ECDs. igp1 harbors point mutation E906K and igp2 and igp3 harbor point mutation G773E in their kinase domains, whereas igp4 is a T‐DNA insertional loss‐of‐function mutant. Notably, isothermal titration calorimetry (ITC) assays with purified ECD‐RKs of IGP1 and IGP3 showed that IGP1 binds with high affinity to CEL3 (with dissociation constant KD = 1.19 ± 0.03 μm) and cellopentaose (KD = 1.40 ± 0.01 μM), but not to MLG43, supporting its function as a plant PRR for cellulose‐derived oligosaccharides. Our data suggest that these LRR‐MAL RKs are components of a recognition mechanism for both cellulose‐ and MLG‐derived oligosaccharide perception and downstream PTI activation in Arabidopsis.
Significance Statement
The mechanisms of perception of newly described cell‐wall derived oligosaccharides that trigger plant immunity are poorly characterized. Here we described three Arabidopsis thaliana receptor kinases (RK) with Leucine Rich Repeat (LRR) and Malectin (MAL) domains in their ectodomains (ECDs), which are components of a novel mechanism of both cellulose and mixed‐linked β‐glucan (MLGs)‐derived oligosaccharides perception and downstream immune activation. One of these RKs binds cellulose oligosaccharides indicating that is a plant pattern recognition receptor (PRR).
Since their discovery, toxin-antitoxin (TA) systems have captivated the attention of many scientists. Recent studies have demonstrated that TA systems play a key role in phage inhibition. The aim of ...the present study was to investigate the role of the PemIK (PemK/PemI) type II TA system in phage inhibition by its intrinsic expression in clinical strains of Klebsiella pneumoniae carrying the lncL plasmid, which harbours the carbapenemase OXA-48 and the PemK/PemI TA system. Furthermore, induced expression of the system in an IPTG-inducible plasmid in a reference strain of K. pneumoniae ATCC10031 was also studied. The results showed that induced expression of the whole TA system did not inhibit phage infection, whereas overexpression of the pemK toxin prevented early infection. To investigate the molecular mechanism involved in the PemK toxin-mediated inhibition of phage infection, assays measuring metabolic activity and viability were performed, revealing that overexpression of the PemK toxin led to dormancy of the bacteria. Thus, we demonstrate that the PemK/PemI TA system plays a role in phage infection and that the action of the free toxin induces a dormant state in the cells, resulting in inhibition of phage infections.
Background Baker's asthma is a frequent occupational allergic disorder mainly caused by inhalation of cereal flours. Lipid transfer proteins (LTPs) constitute a family of plant food panallergens, but ...their role as inhalant and wheat allergens is still unclear. Objective We sought to explore the involvement of wheat LTPs in baker's asthma caused by wheat flour sensitization. Methods Forty patients with occupational asthma caused by wheat flour inhalation were studied. Wheat LTP, Tri a 14, was purified by using a 2-step chromatographic protocol and characterized by N-terminal amino acid sequencing and 3-dimensional modeling. Its reactivity was confirmed by means of IgE immunodetection, ELISA and ELISA-inhibition assays, and skin prick tests. Results Specific IgE to Tri a 14 was found in 60% of 40 individual sera from patients with baker's asthma, and the purified allergen elicited positive skin prick test reactions in 62% of 24 of these patients. Tri a 14 and peach LTP, Pru p 3, showed a sequence identity of 45%, but the low cross-reactivity between both allergens detected in several individual sera reflected great differences in their 3-dimensional IgE-binding regions. Conclusions Wheat LTP is a major inhalant allergen associated with baker's asthma caused by wheat flour sensitization. Poor cross-reactivity with its peach homolog was found in some patients. Clinical implications LTPs can be considered relevant inhalant allergens linked to respiratory disorders. LTP from wheat (Tri a 14) can be used as a helpful tool for the diagnosis of baker's asthma.
is a human pathogen that worsens the prognosis of many immunocompromised patients. Here, we annotated and compared the genomes of two lytic phages that infect clinical strains of
(vB_KpnM-VAC13 and ...vB_KpnM-VAC66) and phenotypically characterized vB_KpnM-VAC66 (time of adsorption of 12 min, burst size of 31.49 ± 0.61 PFU/infected cell, and a host range of 20.8% of the tested strains). Transmission electronic microscopy showed that vB_KpnM-VAC66 belongs to the
family. The genomic analysis of the phage vB_KpnM-VAC66 revealed that its genome encoded 289 proteins. When compared to the genome of vB_KpnM-VAC13, they showed a nucleotide similarity of 97.56%, with a 93% of query cover, and the phylogenetic study performed with other
phages showed a close common ancestor. However, there were 21 coding sequences which differed. Interestingly, the main differences were that vB_KpnM-VAC66 encoded 10 more homing endonucleases than vB_KpnM-VAC13, and that the nucleotidic and amino-acid sequences of the L-shaped tail fiber protein were highly dissimilar, leading to different three-dimensional protein predictions. Both phages differed significantly in their host range. These viruses may be useful in the development of alternative therapies to antibiotics or as a co-therapy increasing its antimicrobial potential, especially when addressing multidrug resistant (MDR) pathogens.
PDF
