Negative regulation of syntaxin4/SNAP-23/VAMP2-mediated membrane fusion by Munc18c <i>In Vitro</i>

Background: Translocation of the facilitative glucose transporter GLUT4 from an intracellular store to the plasma membrane is responsible for the increased rate of glucose transport into fat and muscle cells in response to insulin. This represents a specialised form of regulated membrane trafficking. Intracellular membrane traffic is subject to multiple levels of regulation by conserved families of proteins in all eukaryotic cells. Notably, all intracellular fusion events require SNARE proteins and Sec1p/Munc18 family members. Fusion of GLUT4-containing vesicles with the plasma membrane of insulin-sensitive cells involves the SM protein Munc18c, and is regulated by the formation of syntaxin 4/SNAP23/VAMP2 SNARE complexes. Methodology/Principal Findings Here we have used biochemical approaches to characterise the interaction(s) of Munc18c with its cognate SNARE proteins and to examine the role of Munc18c in regulating liposome fusion catalysed by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. We demonstrate that Munc18c makes contacts with both t- and v-SNARE proteins of this complex, and directly inhibits bilayer fusion mediated by the syntaxin 4/SNAP23/VAMP2 SNARE complex. Conclusion/Significance Our reductionist approach has enabled us to ascertain a direct inhibitory role for Munc18c in regulating membrane fusion mediated by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. It is important to note that two different SM proteins have recently been shown to stimulate liposome fusion mediated by their cognate SNARE complexes. Given the structural similarities between SM proteins, it seems unlikely that different members of this family perform opposing regulatory functions. Hence, our findings indicate that Munc18c requires a further level of regulation in order to stimulate SNARE-mediated membrane fusion

Understanding the chemistry of the artificial electron acceptors PES, PMS, DCPIP and Wurster’s Blue in methanol dehydrogenase assays

Methanol dehydrogenases (MDH) have recently taken the spotlight with the discovery that a large portion of these enzymes in nature utilize lanthanides in their active sites. The kinetic parameters of these enzymes are determined with a spectrophotometric assay first described by Anthony and Zatman 55 years ago. This artificial assay uses alkylated phenazines, such as phenazine ethosulfate (PES) or phenazine methosulfate (PMS), as primary electron acceptors (EAs) and the electron transfer is further coupled to a dye. However, many groups have reported problems concerning the bleaching of the assay mixture in the absence of MDH and the reproducibility of those assays. Hence, the comparison of kinetic data among MDH enzymes of different species is often cumbersome. Using mass spectrometry, UV–Vis and electron paramagnetic resonance (EPR) spectroscopy, we show that the side reactions of the assay mixture are mainly due to the degradation of assay components. Light-induced demethylation (yielding formaldehyde and phenazine in the case of PMS) or oxidation of PES or PMS as well as a reaction with assay components (ammonia, cyanide) can occur. We suggest here a protocol to avoid these side reactions. Further, we describe a modified synthesis protocol for obtaining the alternative electron acceptor, Wurster’s blue (WB), which serves both as EA and dye. The investigation of two lanthanide-dependent methanol dehydrogenases from Methylorubrum extorquens AM1 and Methylacidiphilum fumariolicum SolV with WB, along with handling recommendations, is presented

3D stochastic bicontinuous microstructures: Generation, topology and elasticity

Motivated by recent experimental investigations of the mechanical behavior of nanoporous metal we explore an efficient and robust method for generating 3D representative volume elements (RVEs) with strikingly similar behavior. Our approach adopts Cahn's method of generating a Gaussian random field by taking a superposition of standing sinusoidal waves of fixed wavelength but random in direction and phase. In its theory part, our study describes closed-form expressions for how the solid volume fraction affects the binarization level, mean structure size, specific surface area, averages of mean and Gaussian curvature, and the scaled topological genus. Based on numerical studies we report on criteria for achieving representative realizations of the structure by proper choice of the number of waves and element size. We also show that periodic structures are readily created. We analyze the mechanical properties considering linear and infinitesimal elasticity and evaluate the residual anisotropy (which can be made small) and the effective values of the Young's modulus and Poisson's ratio. The numerical results are in excellent agreement with experimental findings for the variation of stiffness with solid fraction of nanoporous gold made by dealloying. We propose scaling relations that achieve naturally a perfect agreement with the numerical and experimental data. The scaling relation for the stiffness accounts for a percolation-to-cluster transition in the random field microstructure at a finite solid fraction. We propose that this transition is the origin of the previously reported anomalous compliance of nanoporous gold

Unintentional high density p-type modulation doping of a GaAs/AlAs core-multi-shell nanowire

Achieving significant doping in GaAs/AlAs core/shell nanowires (NWs) is of considerable technological importance but remains a challenge due to the amphoteric behavior of the dopant atoms. Here we show that placing a narrow GaAs quantum well in the AlAs shell effectively getters residual carbon acceptors leading to an \emph{unintentional} p-type doping. Magneto-optical studies of such a GaAs/AlAs core multi-shell NW reveal quantum confined emission. Theoretical calculations of NW electronic structure confirm quantum confinement of carriers at the core/shell interface due to the presence of ionized carbon acceptors in the 1~nm GaAs layer in the shell. Micro-photoluminescence in high magnetic field shows a clear signature of avoided crossings of the $n=0$ Landau level emission line with the $n=2$ Landau level TO phonon replica. The coupling is caused by the resonant hole-phonon interaction, which points to a large 2D hole density in the structure.Comment: just published in Nano Letters (http://pubs.acs.org/doi/full/10.1021/nl500818k

Diverse Misfolded Conformational Strains and Cross-seeding of Misfolded Proteins Implicated in Neurodegenerative Diseases

Numerous neurodegenerative diseases including prion, Alzheimer’s and Parkinson’s diseases are characterized by accumulation of protein aggregates in brain. Prion disease is unique in that the natively folded prion protein forms diverse misfolded aggregates with distinct molecular conformations (strains), which underlie different disease phenotypes. In addition, the conformational strains are able to self-propagate their unique conformations by recruiting normal protein monomers and converting their conformations to misfolded conformers. There is an increasing body of evidence that suggests other aggregation-prone proteins including tau and α-synuclein associated with Alzheimer’s and Parkinson’s diseases, respectively, also behave like a prion that has conformational strains with self-propagation (seeding) property. Moreover, misfolded protein aggregates can promote misfolding and aggregation of different proteins through cross-seeding, which might be associated with co-occurrence of multiple neurodegenerative diseases in the same patient. Elucidation of diverse conformational strains with self-propagation capability and of molecular basis for the cross-talk between misfolded proteins is essential to the development of effective therapeutic intervention

Nontypeable Haemophilus influenzae induces COX-2 and PGE2 expression in lung epithelial cells via activation of p38 MAPK and NF-kappa B

<p>Abstract</p> <p>Background</p> <p>Nontypeable <it>Haemophilus influenzae </it>(NTHi) is an important respiratory pathogen implicated as an infectious trigger in chronic obstructive pulmonary disease, but its molecular interaction with human lung epithelial cells remains unclear. Herein, we tested that the hypothesis that NTHi induces the expression of cyclooxygenase (COX)-2 and prostaglandin E2 (PGE2) via activation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kappa B in pulmonary alveolar epithelial cells.</p> <p>Methods</p> <p>Human alveolar epithelial A549 cells were infected with different concentrations of NTHi. The phosphorylation of p38 MAPK was detected by Western blot analysis, the DNA binding activity of NF-kappa B was assessed by electrophoretic mobility shift assay (EMSA), and the expressions of COX-1 and 2 mRNA and PGE2 protein were measured by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA), respectively. The roles of Toll-like receptor (TLR) 2 and TLR4, well known NTHi recognizing receptor in lung epithelial cell and gram-negative bacteria receptor, respectively, on the NTHi-induced COX-2 expression were investigated in the HEK293 cells overexpressing TLR2 and TLR4 <it>in vitro </it>and in the mouse model of NTHi-induced pneumonia by using TLR2 and TLR4 knock-out mice <it>in vivo</it>. In addition, the role of p38 MAPK and NF-kappa B on the NTHi-induced COX-2 and PGE2 expression was investigated by using their specific chemical inhibitors.</p> <p>Results</p> <p>NTHi induced COX-2 mRNA expression in a dose-dependent manner, but not COX-1 mRNA expression in A549 cells. The enhanced expression of PGE2 by NTHi infection was significantly decreased by pre-treatment of COX-2 specific inhibitor, but not by COX-1 inhibitor. NTHi induced COX-2 expression was mediated by TLR2 in the epithelial cell <it>in vitro </it>and in the lungs of mice <it>in vivo</it>. NTHi induced phosphorylation of p38 MAPK and up-regulated DNA binding activity of NF-kappa B. Moreover, the expressions of COX-2 and PGE2 were significantly inhibited by specific inhibitors of p38 MAPK and NF-kappa B. However, NTHi-induced DNA binding activity of NF-kappa B was not affected by the inhibition of p38 MAPK.</p> <p>Conclusion</p> <p>NTHi induces COX-2 and PGE2 expression in a p38 MAPK and NF-kappa B-dependent manner through TLR2 in lung epithelial cells <it>in vitro </it>and lung tissues <it>in vivo</it>. The full understanding of the role of endogenous anti-inflammatory PGE2 and its regulation will bring new insight to the resolution of inflammation in pulmonary bacterial infections.</p

Basolateral Sorting of Syntaxin 4 Is Dependent on Its N-terminal Domain and the AP1B Clathrin Adaptor, and Required for the Epithelial Cell Polarity

Generation of epithelial cell polarity requires mechanisms to sort plasma membrane proteins to the apical and basolateral domains. Sorting involves incorporation into specific vesicular carriers and subsequent fusion to the correct target membranes mediated by specific SNARE proteins. In polarized epithelial cells, the SNARE protein syntaxin 4 localizes exclusively to the basolateral plasma membrane and plays an important role in basolateral trafficking pathways. However, the mechanism of basolateral targeting of syntaxin 4 itself has remained poorly understood. Here we show that newly synthesized syntaxin 4 is directly targeted to the basolateral plasma membrane in polarized Madin-Darby canine kidney (MDCK) cells. Basolateral targeting depends on a signal that is centered around residues 24–29 in the N-terminal domain of syntaxin 4. Furthermore, basolateral targeting of syntaxin 4 is dependent on the epithelial cell-specific clathrin adaptor AP1B. Disruption of the basolateral targeting signal of syntaxin 4 leads to non-polarized delivery to both the apical and basolateral surface, as well as partial intercellular retention in the trans-Golgi network. Importantly, disruption of the basolateral targeting signal of syntaxin 4 leads to the inability of MDCK cells to establish a polarized morphology which suggests that restriction of syntaxin 4 to the basolateral domain is required for epithelial cell polarity