Increased functional diversity of plant K+ channels by preferential heteromerization of the shaker-like subunits AKT2 and KAT2

Assembly of plant Shaker subunits as heterotetramers, increasing channel functional diversity, has been reported. Here we focus on a new interaction, between AKT2 and KAT2 subunits. The assembly as AKT2/KAT2 heterotetramers is demonstrated by (i) a strong signal in two-hybrid tests with intracytoplasmic C-terminal regions, (ii) the effect of KAT2 on AKT2 subunit targeting in tobacco cells, (iii) the complete inhibition of AKT2 currents by co-expression with a dominant-negative KAT2 subunit in Xenopus oocytes, and reciprocally, and (iv) the appearance, upon co-expression of wild-type AKT2 and KAT2 subunits, of new channel functional properties that cannot be explained by the co-existence of two kinds of homotetrameric channels. In particular, the instantaneous current, characteristic of AKT2, displayed new functional features when compared with those of AKT2 homotetramers: activation by external acidification (instead of inhibition) and weak inhibition by calcium. Single channel current measurements in oocytes co-expressing AKT2 and KAT2 revealed a strong preference for incorporation of subunits into heteromultimers and a diversity of individual channels. In planta, these new channels, which may undergo specific regulations, are likely to be formed in guard cells and in the phloem, where they could participate in the control of membrane potential and potassium fluxes.     

Phosphorylation of RhoB by CK1 impedes actin stress fiber organization and epidermal growth factor receptor stabilization

RhoB is a small GTPase implicated in cytoskeletal organization, EGF receptor trafficking and cell transformation. It is an immediate-early gene, regulated at many levels of its biosynthetic pathway. Herein we show that the serine/threonine protein kinase CK1 phosphorylates RhoB in vitro but not RhoA or RhoC. With the use of specific CK1 inhibitors, IC261 and D4476, we show that the kinase phosphorylates also RhoB in HeLa cells. Mass spectrometry analysis demonstrates that RhoB is monophosphorylated by CK1, in its C-terminal end, on serine 185. The substitution of Ser185 by Ala dramatically inhibited the phosphorylation of RhoB in cultured cells. Lastly we show that the inhibition of CK1 activates RhoB and promotes RhoB dependent actin fiber formation and EGF-R level. Our data provide the first demonstration of RhoB phosphorylation and indicate that this post-translational maturation would be a novel critical mechanism to control the RhoB functions.     

Effect of In Vitro and In Vivo Anakinra on Cytokines Production in Schnitzler Syndrome

IL-1 receptor antagonist anakinra is usually highly efficient in Schnitzler syndrome (SS), a rare inflammatory condition associating urticaria, fever, and IgM monoclonal gammopathy. In this study, we aimed to assess lipopolysaccharide (LPS)-induced production of inflammatory cytokines by peripheral blood mononuclear cells (PBMCs) before and after 1 month of anakinra in patients with SS. LPS-induced production of IL-1β, IL-6 and TNFα was assessed by enzyme-linked immunosorbent assay with and without anakinra in vitro, and before and after 1 month (in vivo condition) of treatment in 2 patients with SS. Spontaneous production of IL-1β, IL-6 and TNF-α by PBMCs was similar in the patients and the healthy controls and was almost undetectable. Stimulation with LPS caused a higher release of cytokines from the patients than from the healthy controls. Before in vivo anakinra start, in vitro adjunction of anakinra reduced the high LPS-induced production of IL-1β and TNFα in both patients and of IL-6 in one patient. After 1 month of treatment with anakinra, while the patients had dramatically improved, there was also a marked reduction in LPS-induced cytokines production, which was almost normalized in one patient. This study shows an abnormal LPS-induced inflammatory cytokines production in SS, which can be decreased or even normalized by in vitro and in vivo anakinra.     

Intense transpositional activity of insertion sequences in an ancient obligate endosymbiont

The streamlined genomes of ancient obligate endosymbionts generally lack transposable elements, such as insertion sequences (IS). Yet, the genome of Wolbachia, one of the most abundant bacterial endosymbionts on Earth, is littered with IS. Such a paradox raises the question as to why there are so many ISs in the genome of this ancient endosymbiont. To address this question, we investigated IS transpositional activity in the unculturable Wolbachia by tracking the evolutionary dynamics and history of ISWpi1 elements. We show that 1) ISWpi1 is widespread in Wolbachia, being present in at least 55% of the 40 sampled strains, 2) ISWpi1 copies exhibit virtually identical nucleotide sequences both within and among Wolbachia genomes and possess an intact transposase gene, 3) individual ISWpi1 copies are differentially inserted among Wolbachia genomes, and 4) ISWpi1 occurs at variable copy numbers among Wolbachia genomes. Collectively, our results provide compelling evidence for intense ISWpi1 transpositional activity and frequent ISWpi1 horizontal transmission among strains during recent Wolbachia evolution. Thus, the genomes of ancient obligate endosymbionts can carry high loads of functional and transpositionally active transposable elements. Our results also indicate that Wolbachia genomes have experienced multiple and temporally distinct ISWpi1 invasions during their evolutionary history. Such recurrent exposition to new IS invasions may explain, at least partly, the unusually high density of transposable elements found in the genomes of Wolbachia endosymbionts.     

Mutational analysis of the zinc- and substrate-binding sites in the CphA metallo-beta-lactamase from Aeromonas hydrophila

The subclass B2 CphA (Carbapenemase hydrolysing Aeromonas) beta-lactamase from Aeromonas hydrophila is a Zn(2+)-containing enzyme that specifically hydrolyses carbapenems. In an effort to evaluate residues potentially involved in metal binding and/or catalysis (His(118), Asp(120), His(196) and His(263)) and in substrate specificity (Val(67), Thr(157), Lys(224) and Lys(226)), site-directed mutants of CphA were generated and characterized. Our results confirm that the first zinc ion is in interaction with Asp(120) and His(263), and thus is located in the 'cysteine' zinc-binding site. His(118) and His(196) residues seem to be interacting with the second zinc ion, as their replacement by alanine residues has a negative effect on the affinity for this second metal ion. Val(67) plays a significant role in the binding of biapenem and benzylpenicillin. The properties of a mutant with a five residue (LFKHV) insertion just after Val(67) also reveals the importance of this region for substrate binding. This latter mutant has a higher affinity for the second zinc ion than wild-type CphA. The T157A mutant exhibits a significantly modified activity spectrum. Analysis of the K224Q and N116H/N220G/K224Q mutants suggests a significant role for Lys(224) in the binding of substrate. Lys(226) is not essential for the binding and hydrolysis of substrates. Thus the present paper helps to elucidate the position of the second zinc ion, which was controversial, and to identify residues important for substrate binding.     

Targeting the lactate transporter MCT1 in endothelial cells inhibits lactate-induced HIF-1 activation and tumor angiogenesis

Switching to a glycolytic metabolism is a rapid adaptation of tumor cells to hypoxia. Although this metabolic conversion may primarily represent a rescue pathway to meet the bioenergetic and biosynthetic demands of proliferating tumor cells, it also creates a gradient of lactate that mirrors the gradient of oxygen in tumors. More than a metabolic waste, the lactate anion is known to participate to cancer aggressiveness, in part through activation of the hypoxia-inducible factor-1 (HIF-1) pathway in tumor cells. Whether lactate may also directly favor HIF-1 activation in endothelial cells (ECs) thereby offering a new druggable option to block angiogenesis is however an unanswered question. In this study, we therefore focused on the role in ECs of monocarboxylate transporter 1 (MCT1) that we previously identified to be the main facilitator of lactate uptake in cancer cells. We found that blockade of lactate influx into ECs led to inhibition of HIF-1-dependent angiogenesis. Our demonstration is based on the unprecedented characterization of lactate-induced HIF-1 activation in normoxic ECs and the consecutive increase in vascular endothelial growth factor receptor 2 (VEGFR2) and basic fibroblast growth factor (bFGF) expression. Furthermore, using a variety of functional assays including endothelial cell migration and tubulogenesis together with in vivo imaging of tumor angiogenesis through intravital microscopy and immunohistochemistry, we documented that MCT1 blockers could act as bona fide HIF-1 inhibitors leading to anti-angiogenic effects. Together with the previous demonstration of MCT1 being a key regulator of lactate exchange between tumor cells, the current study identifies MCT1 inhibition as a therapeutic modality combining antimetabolic and anti-angiogenic activities.     

Dramatic Increase in Oxidative Stress in Carbon-Irradiated Normal Human Skin Fibroblasts

Skin complications were recently reported after carbon-ion (C-ion) radiation therapy. Oxidative stress is considered an important pathway in the appearance of late skin reactions. We evaluated oxidative stress in normal human skin fibroblasts after carbon-ion vs. X-ray irradiation. Survival curves and radiobiological parameters were calculated. DNA damage was quantified, as were lipid peroxidation (LPO), protein carbonylation and antioxidant enzyme activities. Reduced and oxidized glutathione ratios (GSH/GSSG) were determined. Proinflammatory cytokine secretion in culture supernatants was evaluated. The relative biological effectiveness (RBE) of C-ions vs. X-rays was 4.8 at D₀ (irradiation dose corresponding to a surviving fraction of 37%). Surviving fraction at 2 Gy (SF2) was 71.8% and 7.6% for X-rays and C-ions, respectively. Compared with X-rays, immediate DNA damage was increased less after C-ions, but a late increase was observed at D_10% (irradiation dose corresponding to a surviving fraction of 10%). LPO products and protein carbonyls were only increased 24 hours after C-ions. After X-rays, superoxide dismutase (SOD) activity was strongly increased immediately and on day 14 at D_0% (irradiation dose corresponding to a surviving fraction of around 0%), catalase activity was unchanged and glutathione peroxidase (GPx) activity was increased only on day 14. These activities were decreased after C-ions compared with X-rays. GSH/GSSG was unchanged after X-rays but was decreased immediately after C-ion irradiation before an increase from day 7. Secretion of IL-6 was increased at late times after X-ray irradiation. After C-ion irradiation, IL-6 concentration was increased on day 7 but was lower compared with X-rays at later times. C-ion effects on normal human skin fibroblasts seemed to be harmful in comparison with X-rays as they produce late DNA damage, LPO products and protein carbonyls, and as they decrease antioxidant defences. Mechanisms leading to this discrepancy between the two types of radiation should be investigated.     

Functioning of the dimeric GABA(B) receptor extracellular domain revealed by glycan wedge scanning

The G-protein-coupled receptor (GPCR) activated by the neurotransmitter GABA is made up of two subunits, GABA(B1) and GABA(B2). GABA(B1) binds agonists, whereas GABA(B2) is required for trafficking GABA(B1) to the cell surface, increasing agonist affinity to GABA(B1), and activating associated G proteins. These subunits each comprise two domains, a Venus flytrap domain (VFT) and a heptahelical transmembrane domain (7TM). How agonist binding to the GABA(B1) VFT leads to GABA(B2) 7TM activation remains unknown. Here, we used a glycan wedge scanning approach to investigate how the GABA(B) VFT dimer controls receptor activity. We first identified the dimerization interface using a bioinformatics approach and then showed that introducing an N-glycan at this interface prevents the association of the two subunits and abolishes all activities of GABA(B2), including agonist activation of the G protein. We also identified a second region in the VFT where insertion of an N-glycan does not prevent dimerization, but blocks agonist activation of the receptor. These data provide new insight into the function of this prototypical GPCR and demonstrate that a change in the dimerization interface is required for receptor activation.