STD and TRNOESY NMR studies on the conformation of the oncogenic protein beta-catenin containing the phosphorylated motif DpSGXXpS bound to the beta-TrCP protein

beta-TrCP is the F-box protein component of an Skp1/Cul1/F-box (SCF)-type ubiquitin ligase complex. Biochemical studies have suggested that beta-TrCP targets the oncogenic protein beta-catenin for ubiquitination and followed by proteasome degradation. To further elucidate the basis of this interaction, a complex between a 32-residue peptide from beta-catenin containing the phosphorylated motif DpSGXXpS (P-beta-Cat17-48) and beta-TrCP was studied using Saturation Transfer Difference (STD) Nuclear Magnetic Resonance (NMR) experiments. These experiments make it possible to identify the binding epitope of a ligand at atomic resolution. An analysis of STD spectra provided clear evidence that only a few of the 32 residues receive the largest saturation transfer. In particular, the amide protons of the residues in the phosphorylated motif appear to be in close contact to the amino acids of the beta-TrCP binding pocket. The amide and aromatic protons of the His24 and Trp25 residues also receive a significant saturation transfer. These findings are in keeping with a recently published x-ray structure of a shorter beta-catenin fragment with the beta-TrCP1-Skp1 complex and with the earlier findings from mutagenesis and activity assays. To better characterize the ligand-protein interaction, the bound conformation of the phosphorylated beta-catenin peptide was obtained using TRansfer Nuclear Overhauser Effect SpectroscopY (TRNOESY) experiments. Finally, we obtained the bound structure of the phosphorylated peptide showing the protons identified by STD NMR as exposed in close proximity to the molecule surface.     

Structure of a Prokaryotic Sodium Channel Pore Reveals Essential Gating Elements and an Outer Ion Binding Site Common to Eukaryotic Channels

Voltage-gated sodium channels (Na_Vs) are central elements of cellular excitation. Notwithstanding advances from recent bacterial Na_V (BacNa_V) structures, key questions about gating and ion selectivity remain. Here, we present a closed conformation of Na_VAe1p, a pore-only BacNa_V derived from Na_VAe1, a BacNa_V from the arsenite oxidizer Alkalilimnicola ehrlichei found in Mono Lake, California, that provides insight into both fundamental properties. The structure reveals a pore domain in which the pore-lining S6 helix connects to a helical cytoplasmic tail. Electrophysiological studies of full-length BacNa_Vs show that two elements defined by the Na_VAe1p structure, an S6 activation gate position and the cytoplasmic tail “neck”, are central to BacNa_V gating. The structure also reveals the selectivity filter ion entry site, termed the “outer ion” site. Comparison with mammalian voltage-gated calcium channel (Ca_V) selectivity filters, together with functional studies, shows that this site forms a previously unknown determinant of Ca_V high-affinity calcium binding. Our findings underscore commonalities between BacNa_Vs and eukaryotic voltage-gated channels and provide a framework for understanding gating and ion permeation in this superfamily.     

Acute renal dysfunction after cardiac surgery with cardiopulmonary bypass is associated with plasmatic IL6 increase

Background: Acute renal dysfunction (ARD) is common after cardiac surgery with cardiopulmonary bypass (CPB). CPB results in a sudden systemic inflammatory response. Systemic and local pro-inflammatory cytokines synthesis has been linked with sub-clinical renal injury, especially tubular lesions. Therefore, we sought to assess the systemic synthesis pro-inflammatory cytokines and its association with perioperative ARD after cardiac surgery with CPB. Methods: Sixty-two patients undergoing cardiac surgery with CPB were prospectively included. Four groups of patients were defined according to blood creatinine increase: no ARD (less than 25% increase), faint ARD (25–50% increase), moderate ARD (50–100% increase), severe ARD (more than 100% increase). Results: Within the 48 post-operative hours was ARD observed as no dysfunction (41.9%), faint (32.2%), moderate (16.1%), severe (9.6%). One patient had to undergo a dialysis. Pre-operative characteristics were homogenous between the four groups excepted the left ventricle ejection fraction. ARD was associated with a low urinary output with high sodium excretion fraction. Significant increase of IL-6 level occurred when patients underwent a severe ARD despite no significant differences for the CRP and TNF-α concentrations. Conclusion: Severe acute renal dysfunction after cardiac surgery with CPB is associated with a significant increased IL-6 systemic production.     

Ethylene Signaling Regulates Accumulation of the FLS2 Receptor and Is Required for the Oxidative Burst Contributing to Plant Immunity

Reactive oxygen species (ROS) are potent signal molecules rapidly generated in response to stress. Detection of pathogen-associated molecular patterns induces a transient apoplastic ROS through the function of the NADPH respiratory burst oxidase homologs D (RbohD). However, little is known about the regulation of pathogen-associated molecular pattern-elicited ROS or its role in plant immunity. We investigated ROS production triggered by bacterial flagellin (flg22) in Arabidopsis (Arabidopsis thaliana). The oxidative burst was diminished in ethylene-insensitive mutants. Flagellin Sensitive2 (FLS2) accumulation was reduced in etr1 and ein2, indicating a requirement of ethylene signaling for FLS2 expression. Multiplication of virulent bacteria was enhanced in Arabidopsis lines displaying altered ROS production at early but not late stages of infection, suggesting an impairment of preinvasive immunity. Stomatal closure, a mechanism used to reduce bacterial entry into plant tissues, was abolished in etr1, ein2, and rbohD mutants. These results point to the importance of flg22-triggered ROS at an early stage of the plant immune response.A rapid and transient increase in reactive oxygen species (ROS), termed an “oxidative burst,” is often associated with responses to abiotic and biotic stresses and could trigger changes in stomatal aperture or programmed cell death in defense against pathogens (Kwak et al., 2003; Torres and Dangl, 2005). ROS production can occur extracellularly through activities of plasma membrane-resident NADPH oxidases (Kangasjärvi et al., 2005; Torres and Dangl, 2005). In plants, Rboh proteins, which are homologs of mammalian NADPH oxidase 2, were shown to be the predominant mediators of apoplastic ROS production (Torres et al., 1998; Galletti et al., 2008). Respiratory burst oxidase homologs D and F (RbohD and RbohF) were identified by mutation to be the responsible oxidases in Arabidopsis (Arabidopsis thaliana) defense responses (Torres et al., 2002). While most ROS generated in response to avirulent Pseudomonas syringae bacteria and Hyaloperonospora oomycete pathogens depend on RbohD function, the induced cell death response by these pathogens appears to be mostly regulated by RbohF. Cell death provoked upon infection with the necrotizing fungus Alternaria, however, is under the control of RbohD (Pogány et al., 2009). The contribution of NADPH oxidases to plant immunity was also described in barley (Hordeum vulgare) and tobacco (Nicotiana benthamiana), where resistance to powdery mildew fungi and the oomycete Phytophthora infestans, respectively, was dependent on Rboh functions (Yoshioka et al., 2003; Trujillo et al., 2006).An early layer of active plant defense is mediated by pattern recognition receptors, which sense microbes according to conserved constituents, so-called pathogen-associated molecular patterns (PAMPs). These initiate a plethora of defense responses referred to as PAMP-triggered immunity (Boller and Felix, 2009). The Arabidopsis receptor kinase Flagellin Sensitive2 (FLS2) recognizes and physically interacts with flg22, the elicitor-active epitope of bacterial flagellin (Felix et al., 1999; Gomez-Gomez and Boller, 2000; Chinchilla et al., 2006). FLS2 is plasma membrane localized and expressed throughout the plant (Robatzek et al., 2006). FLS2 requires the receptor kinase BRI1-Associated Kinase1 (BAK1), which forms a heteromeric complex upon flg22 binding (Chinchilla et al., 2007). Subsequently, a rapid and transient flg22-stimulated oxidative burst occurs that is dependent on RbohD (Zhang et al., 2007). In addition, flg22 triggers early responses, such as ethylene biosynthesis, activation of mitogen-activated protein (MAP) kinase cascades, and changes in gene expression (Felix et al., 1999; Asai et al., 2002; Zipfel et al., 2004). Late flg22 responses include the accumulation of salicylic acid (SA), callose deposition, and an arrest of seedling growth (Gomez-Gomez et al., 1999; Mischina and Zeier, 2007). This collectively contributes to plant immunity (Zipfel et al., 2004; Melotto et al., 2006).Little is known about the regulatory components of FLS2-activated early flg22 responses and their relevance in plant resistance to pathogens. Here, we investigated flg22-triggered ROS production in Arabidopsis seedlings and have identified ethylene signaling as a critical component of the oxidative burst in response to flg22, partly through promoting the accumulation of FLS2. We further provide evidence that the flg22-triggered oxidative burst is required for resistance to bacterial infection at the point of pathogen entry through stomata.     

Genetic regulation of life cycle transitions in the brown alga Ectocarpus

The life cycle of an organism is one of its most elemental features, underpinning a broad range of phenomena including developmental processes, reproductive fitness, mode of dispersal and adaptation to the local environment. Life cycle modification may have played an important role during the evolution of several eukaryotic groups, including the terrestrial plants. Brown algae are potentially interesting models to study life cycle evolution because this group exhibits a broad range of different life cycles. Currently, life cycle studies are focused on the emerging brown algal model Ectocarpus. Two life cycle mutants have been described in this species, both of which cause the sporophyte generation to exhibit gametophyte characteristics. The ouroboros mutation is particularly interesting because it induces complete conversion of the sporophyte generation into a functional, gamete-producing gametophyte, a class of mutation that has not been described so far in other systems. Analysis of Ectocarpus life cycle mutants is providing insights into several life-cycle-related processes including parthenogenesis, symmetric/asymmetric initial cell divisions and sex determination.     

Tryptophan Depletion and the Kinase GCN2 Mediate IFN-γ-Induced Autophagy

IFN-γ is a master regulator of the immune responses that occur in the transplanted kidney, acting both on the immune system and on the graft itself. The cellular responses to IFN-γ are complex, and emerging evidence suggests that IFN-γ may regulate autophagic functions. Conversely, autophagy modulates innate and adaptive immune functions in various contexts. In this study, we identify a novel mechanism by which IFN-γ activates autophagy in human kidney epithelial cells and provide new insights into how autophagy regulates immune functions in response to IFN-γ. Our results indicate that IFN-γ promotes tryptophan depletion, activates the eIF2α kinase general control nonderepressible-2 (GCN2), and leads to an increase in the autophagic flux. Further, tryptophan supplementation and RNA interference directed against GCN2 inhibited IFN-γ-induced autophagy. This process is of functional relevance because autophagy regulates the secretion of inflammatory cytokines and growth factors by human kidney epithelial cells in response to IFN-γ. These findings assign to IFN-γ a novel function in the regulation of autophagy, which, in turn, modulates IFN-γ-induced secretion of inflammatory cytokines.