Nanodiscs unravel the interaction between the SecYEG channel and its cytosolic partner SecA

The translocon is a membrane-embedded protein assembly that catalyzes protein movement across membranes. The core translocon, the SecYEG complex, forms oligomers, but the protein-conducting channel is at the center of the monomer. Defining the properties of the SecYEG protomer is thus crucial to understand the underlying function of oligomerization. We report here the reconstitution of a single SecYEG complex into nano-scale lipid bilayers, termed Nanodiscs. These water-soluble particles allow one to probe the interactions of the SecYEG complex with its cytosolic partner, the SecA dimer, in a membrane-like environment. The results show that the SecYEG complex triggers dissociation of the SecA dimer, associates only with the SecA monomer and suffices to (pre)-activate the SecA ATPase. Acidic lipids surrounding the SecYEG complex also contribute to the binding affinity and activation of SecA, whereas mutations in the largest cytosolic loop of the SecY subunit, known to abolish the translocation reaction, disrupt both the binding and activation of SecA. Altogether, the results define the fundamental contribution of the SecYEG protomer in the translocation subreactions and illustrate the power of nanoscale lipid bilayers in analyzing the dynamics occurring at the membrane.     

Molecular biology of K transport across the plant cell membrane: What do we learn from comparison between plant species?

Cloning and characterizations of plant K⁺ transport systems aside from Arabidopsis have been increasing over the past decade, favored by the availability of more and more plant genome sequences. Information now available enables the comparison of some of these systems between species. In this review, we focus on three families of plant K⁺ transport systems that are active at the plasma membrane: the Shaker K⁺ channel family, comprised of voltage-gated channels that dominate the plasma membrane conductance to K⁺ in most environmental conditions, and two families of transporters, the HAK/KUP/KT K⁺ transporter family, which includes some high-affinity transporters, and the HKT K⁺ and/or Na⁺ transporter family, in which K⁺-permeable members seem to be present in monocots only. The three families are briefly described, giving insights into the structure of their members and on functional properties and their roles in Arabidopsis or rice. The structure of the three families is then compared between plant species through phylogenic analyses. Within clusters of ortologues/paralogues, similarities and differences in terms of expression pattern, functional properties and, when known, regulatory interacting partners, are highlighted. The question of the physiological significance of highlighted differences is also addressed.     

Hybrid protease inhibitors for pest and pathogen control – a functional cost for the fusion partners?

Fusion proteins integrating dual pesticidal functions have been devised over the last 10 years to improve the effectiveness and potential durability of pest-resistant transgenic crops, but little attention has been paid to the impact of the fusion partners on the actual activity of the resulting hybrids. Here we assessed the ability of the rice cysteine protease inhibitor, oryzacystatin I (OCI), to retain its protease inhibitory potency when used as a template to devise hybrid inhibitors with dual activity against papain-like proteases and carboxypeptidase A (CPA). C-terminal variants of OCI were generated by fusing to its C-terminal end: (i) the primary inhibitory site of the small CPA inhibitor potato carboxypeptidase inhibitor (PCI, amino acids 35-39); or (ii) the complete sequence of PCI (a.a. 1-39). The hybrid inhibitors were expressed in E. coli and tested for their inhibitory activity against papain, CPA and digestive cysteine proteases of herbivorous and predatory arthropods. In contrast with the primary inhibitory site of PCI, the entire PCI attached to OCI was as active against CPA as free, purified PCI. The OCI-PCI hybrids also showed activity against papain, but the presence of extra amino acids at the C terminus of OCI negatively altered its inhibitory potency against cysteine proteases. This negative effect, although not preventing dual binding to papain and CPA, was correlated with an increased binding affinity for papain presumably due to non-specific interactions with the PCI domain. These results confirm the potential of OCI and PCI for the design of fusion inhibitors with dual protease inhibitory activity, but also point out the possible functional costs associated with protein domain grafting to recipient pesticidal proteins.     

Three new BLM gene mutations associated with Bloom syndrome

Bloom's syndrome (BS) is a rare autosomal recessive disease predisposing patients to all types of cancers affecting the general population. BS cells display a high level of genetic instability, including a 10-fold increase in the rate of sister chromatid exchanges, currently the only objective criterion for BS diagnosis. We have developed a method for screening the BLM gene for mutations based on direct genomic DNA sequencing. A questionnaire based on clinical information, cytogenetic features, and family history was addressed to physicians prescribing BS genetic screening, with the aim of confirming or guiding diagnosis. We report here four BLM gene mutations, three of which have not been described before. Three of the mutations are frameshift mutations, and the fourth is a nonsense mutation. All these mutations introduce a stop codon, and may therefore be considered to have deleterious biological effect. This approach should make it possible to identify new mutations and to correlate them with clinical information.     

Essential Oils of Myrtaceae Species Growing Wild in Tunisia: Chemical Variability and Antifungal Activity Against Biscogniauxia mediterranea,the Causative Agent of Charcoal Canker

The chemical composition of five Eucalyptus species and five Myrtus communis L. populations was investigated using GC/MS and GC‐FID. For Eucalyptus essential oils, 32 compounds, representing 88.56 – 96.83% of the total oil according to species, were identified. The main compounds were 1,8‐cineole, α‐pinene, p‐cymene, γ‐gurjunene, α‐aromadendrene, and β‐phellandrene. For Myrtle essential oils, 26 compounds, representing 93.13 – 98.91% of the total oil were identified. α‐Pinene, 1,8‐cineole, linalool, and myrtenyl acetate were found to be the major compounds. Principal component analysis (PCA) showed chemical differentiation between Eucalyptus species and between Myrtle populations. Biscogniauxia mediterranea, the causative agent of charcoal canker, was identified according to its morphological and molecular characteristics. Essential oils of the investigated Eucalyptus species and Myrtle populations were tested for their antifungal capacity against this fungus. The antifungal activity varied according to the essential oil composition. Biscogniauxia mediterranea exhibited powerful resistance to some essential oils including them of Eucalyptus lehmannii and Eucalyptus sideroxylon but it was very sensitive to Eucalyptus camaldulensis oil (IC₅₀ = 3.83 mg/ml) and M. communis oil from Zaghouan (IC₅₀ = 1 mg/ml). This sensitivity was found to be correlated to some essential oil compounds such as p‐cymene, carvacrol, cuminaldehyde, and linalool.     

Design,synthesis and antimalarial activity of novel bis{N-[(pyrrolo[1,2-a]quinoxalin-4-yl)benzyl]-3-aminopropyl}amine derivatives

Novel series of bis- and tris-pyrrolo[1,2-a]quinoxaline derivatives 1 were synthesized and tested for in vitro activity upon the intraerythrocytic stage of W2 and 3D7 Plasmodium falciparum strains. Biological results showed good antimalarial activity with IC₅₀ in the μM range. In attempting to investigate the large broad-spectrum antiprotozoal activities of these new derivatives, their properties toward Leishmania donovani were also investigated and revealed their selective antiplasmodial profile. In parallel, the in vitro cytotoxicity of these molecules was assessed on the human HepG2 cell line. Structure–activity relationships of these new synthetic compounds are discussed here. The bis-pyrrolo[1,2-a]quinoxalines 1n and 1p were identified as the most potent antimalarial candidates with selectivity index (SI) of 40.6 on W2 strain, and 39.25 on 3D7 strain, respectively. As the telomeres of the parasite could constitute an attractive target, we investigated the possibility of targeting Plasmodium telomeres by stabilizing the Plasmodium telomeric G-quadruplexes through a FRET melting assay by our new compounds.     

Phenotypic and Functional Characteristics of Blood Natural Killer Cells from Melanoma Patients at Different Clinical Stages

Melanomas are aggressive skin tumors characterized by high metastatic potential. Immunotherapy is a valuable alternative for metastatic melanoma patients resistant to chemotherapy. Natural Killer (NK) cells are efficient anti-tumor cytotoxic effectors. We previously showed that blood NK cells from stage IV metastatic melanoma patients display decreased NK receptors and that chemotherapy modifies the functional status of blood NK cells. To investigate the role of NK cells along melanoma progression, we have here studied NK cells from patients at different stages of the disease. First, we showed that ex vivo NK cells from certain stage III–IV patients displayed low degranulation potential. Using a dynamic label-free assay, we found that immunoselected IL-2 activated blood NK cells from patients efficiently lysed melanoma cells through NKp46 and NKG2D receptors, independently to the clinical stage. Moreover, the ex vivo phenotype of circulating NK cells from 33 patients (stage I to IV) was extensively analyzed. NK cells from patients displayed higher variability in the percentages of Natural Cytotoxicity Receptors (NCR) and Natural Killer Group 2D (NKG2D) receptor expression compared to donor NK cells. The main defect was the decreased expression of NCR1 (NKp46) by NK cells from metastatic patients. Interestingly, we found a positive correlation between the NK cell percentages of NKp46 and the duration of stage IV in melanoma patients. Finally, we showed that NK cells infiltrated primary melanomas and displayed a predominant peritumoral distribution. These results are new arguments for the development of NK-based therapies in melanoma patients.