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151.
Kethika Kulleperuma Susan M.E. Smith Deri Morgan Boris Musset John Holyoake Nilmadhab Chakrabarti Vladimir V. Cherny Thomas E. DeCoursey Régis Pomès 《The Journal of general physiology》2013,141(4):445-465
The topological similarity of voltage-gated proton channels (HV1s) to the voltage-sensing domain (VSD) of other voltage-gated ion channels raises the central question of whether HV1s have a similar structure. We present the construction and validation of a homology model of the human HV1 (hHV1). Multiple structural alignment was used to construct structural models of the open (proton-conducting) state of hHV1 by exploiting the homology of hHV1 with VSDs of K+ and Na+ channels of known three-dimensional structure. The comparative assessment of structural stability of the homology models and their VSD templates was performed using massively repeated molecular dynamics simulations in which the proteins were allowed to relax from their initial conformation in an explicit membrane mimetic. The analysis of structural deviations from the initial conformation based on up to 125 repeats of 100-ns simulations for each system reveals structural features consistently retained in the homology models and leads to a consensus structural model for hHV1 in which well-defined external and internal salt-bridge networks stabilize the open state. The structural and electrostatic properties of this open-state model are compatible with proton translocation and offer an explanation for the reversal of charge selectivity in neutral mutants of Asp112. Furthermore, these structural properties are consistent with experimental accessibility data, providing a valuable basis for further structural and functional studies of hHV1. Each Arg residue in the S4 helix of hHV1 was replaced by His to test accessibility using Zn2+ as a probe. The two outermost Arg residues in S4 were accessible to external solution, whereas the innermost one was accessible only to the internal solution. Both modeling and experimental data indicate that in the open state, Arg211, the third Arg residue in the S4 helix in hHV1, remains accessible to the internal solution and is located near the charge transfer center, Phe150. 相似文献
152.
Plants are increasingly used as production platforms of various heterologous proteins, but rapid protein turnover can seriously limit the steady-state expression level. Little is known about specific plant proteases involved in this process. In an attempt to obtain potato (Solanum tuberosum cv Desirée) plants resistant to Colorado potato beetle (Leptinotarsa decemlineata Say) larvae, the protease inhibitor equistatin was expressed under the control of strong, light-inducible and constitutive promoters and was targeted to the secretory pathway with and without endoplasmic reticulum retention signal. All constructs yielded similar stepwise protein degradation patterns, which considerably reduced the amount of active inhibitor in planta and resulted in insufficient levels for resistance against Colorado potato beetle larvae. Affinity purification of the degradation products and N-terminal sequencing allowed the identification of the amino acid P(1)-positions (asparagine [Asn]-13, lysine-56, Asn-82, and arginine-151) that were cleaved in planta. The proteases involved in the equistatin degradation were characterized with synthetic substrates and inhibitors. Kininogen domain 3 completely inhibited equistatin degradation in vitro. The results indicate that arginine/lysine-specific and legumain-type Asn-specific cysteine proteases seriously impede the functional accumulation of recombinant equistatin in planta. General strategies to improve the resistance to proteases of heterologous proteins in plants are proposed. 相似文献
153.
VGJ phi, a novel filamentous phage of Vibrio cholerae, integrates into the same chromosomal site as CTX phi 下载免费PDF全文
Campos J Martínez E Suzarte E Rodríguez BL Marrero K Silva Y Ledón T del Sol R Fando R 《Journal of bacteriology》2003,185(19):5685-5696
We describe a novel filamentous phage, designated VGJ phi, isolated from strain SG25-1 of Vibrio cholerae O139, which infects all O1 (classical and El Tor) and O139 strains tested. The sequence of the 7,542 nucleotides of the phage genome reveals that VGJ phi has a distinctive region of 775 nucleotides and a conserved region with an overall genomic organization similar to that of previously characterized filamentous phages, such as CTX phi of V. cholerae and Ff phages of Escherichia coli. The conserved region carries 10 open reading frames (ORFs) coding for products homologous to previously reported peptides of other filamentous phages, and the distinctive region carries one ORF whose product is not homologous to any known peptide. VGJ phi, like other filamentous phages, uses a type IV pilus to infect V. cholerae; in this case, the pilus is the mannose-sensitive hemagglutinin. VGJ phi-infected V. cholerae overexpresses the product of one ORF of the phage (ORF112), which is similar to single-stranded DNA binding proteins of other filamentous phages. Once inside a cell, VGJ phi is able to integrate its genome into the same chromosomal attB site as CTX phi, entering into a lysogenic state. Additionally, we found an attP structure in VGJ phi, which is also conserved in several lysogenic filamentous phages from different bacterial hosts. Finally, since different filamentous phages seem to integrate into the bacterial dif locus by a general mechanism, we propose a model in which repeated integration events with different phages might have contributed to the evolution of the CTX chromosomal region in V. cholerae El Tor. 相似文献
154.
Malerich JP Lam JS Hart B Fine RM Klebansky B Tanga MJ D'Andrea A 《Bioorganic & medicinal chemistry letters》2010,20(24):7454-7457
Tyrosine kinase 2 (TYK2) is required for signaling of interleukin-23 (IL-23), which plays a key role in rheumatoid arthritis. Presented is the design and synthesis of 1,2,4-triazoles, and the evaluation of their inhibitory activity against the Janus associated kinases TYK2 and JAKs 1-3. 相似文献
155.
Kejnovsky E Kubat Z Hobza R Lengerova M Sato S Tabata S Fukui K Matsunaga S Vyskot B 《Genetica》2006,128(1-3):167-175
Silene latifolia is a model dioecious plant with heteromorphic sex chromosomes. The Y chromosome is the largest in this species. Theoretical
models propose an accumulation of repetitive DNA sequences in non-recombining parts of the Y chromosome. In this study, we
isolated a BAC7H5 clone preferentially hybridizing to the Y chromosome of S. latifolia. Sequence analysis revealed that this BAC7H5 contains part of the chloroplast genome, indicating that these chloroplast sequences
have accumulated on the Y chromosome and also may contribute to its large size. We constructed Y chromosome- and X chromosome-specific
libraries and screened them to find Y- and/or X-linked copies of chloroplast sequences. Sequence analysis revealed higher
divergence of a non-genic region of the chloroplast sequences located on the Y chromosome while genic regions tested showed
only very low (max 0.9%) divergence from their chloroplast homologues. 相似文献
156.
By using a functional approach of reconstituting detergent-solubilized membrane proteins into liposomes and following their
function in patch-clamp experiments, we identified a novel mechanosensitive (MS) channel in the thermophilic cell wall-less
archaeon Thermoplasma volcanium. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the enriched protein fractions revealed a band of
approx 15 kDa comparable to MscL, the bacterial MS channel of large conductance. 20 N-terminal residues determined by protein
microsequencing, matched the sequence to an unknown open reading frame in the genome of a related species Thermoplasma acidophilum. The protein encoded by the T. acidophilum gene was cloned and expressed in Escherichia coli and reconstituted into liposomes. When examined for function, the reconstituted protein exhibited properties typical of an
MS ion channel: 1) activation by negative pressure applied to the patch-clamp pipet, 2) blockage by gadolinium, and 3) activation
by the anionic amphipath trinitrophenol. In analogy to the nomenclature used for bacterial MS channels, the MS channel of
T. acidophilum was termed MscTA. Secondary structural analysis indicated that similar to MscL, the T. acidophilum MS protein may have two transmembrane domains, suggesting that MS channels of thermophilic Archaea belong to a family of
structurally related MscL-like ion channels with two membrane-spanning regions. When the mscTA gene was expressed in the mscL
− knockout strain and the MscTA protein reconstituted into liposomes, the gating of MscTA was charaterized by very brief openings
of variable conductance. In contrast, when the mscTA gene was expressed in the wild-type mscL
+ strain of E. coli, the gating properties of the channel resembled MscL. However, the channel had reduced conductance and differed from MscL
in its kinetics and in the free energy of activation, suggesting that MscTA and MscL can form functional complexes and/or
modulate each other activity. Similar to MscL, MscTA exhibited an increase in activity in liposomes made of phospholipids
having shorter acyl chain, suggesting a role of hydrophobic mismatch in the function of prokaryotic MS channels. 相似文献
157.
Praveen Suraneni Ben Fogelson Boris Rubinstein Philippe Noguera Niels Volkmann Dorit Hanein Alex Mogilner Rong Li 《Molecular biology of the cell》2015,26(5):901-912
Cells employ protrusive leading edges to navigate and promote their migration in diverse physiological environments. Classical models of leading-edge protrusion rely on a treadmilling dendritic actin network that undergoes continuous assembly nucleated by the Arp2/3 complex, forming ruffling lamellipodia. Recent work demonstrated, however, that, in the absence of the Arp2/3 complex, fibroblast cells adopt a leading edge with filopodia-like protrusions (FLPs) and maintain an ability to move, albeit with altered responses to different environmental signals. We show that formin-family actin nucleators are required for the extension of FLPs but are insufficient to produce a continuous leading edge in fibroblasts lacking Arp2/3 complex. Myosin II is concentrated in arc-like regions of the leading edge in between FLPs, and its activity is required for coordinated advancement of these regions with formin-generated FLPs. We propose that actomyosin contraction acting against membrane tension advances the web of arcs between FLPs. Predictions of this model are verified experimentally. The dependence of myosin II in leading-edge advancement helps explain the previously reported defect in directional movement in the Arpc3-null fibroblasts. We provide further evidence that this defect is cell autonomous during chemotaxis. 相似文献
158.
Core pathways operating during methylotrophy of Bacillus methanolicus MGA3 and induction of a bacillithiol‐dependent detoxification pathway upon formaldehyde stress 下载免费PDF全文
Jonas E. N. Müller Fabian Meyer Boris Litsanov Patrick Kiefer Julia A. Vorholt 《Molecular microbiology》2015,98(6):1089-1100
Bacillus methanolicus MGA3 is a model facultative methylotroph of interest for fundamental research and biotechnological applications. Previous research uncovered a number of pathways potentially involved in one‐carbon substrate utilization. Here, we applied dynamic 13C labeling to elucidate which of these pathways operate during growth on methanol and to uncover potentially new ones. B. methanolicus MGA3 uses the assimilatory and dissimilatory ribulose monophosphate (RuMP) cycles for conversion of the central but toxic intermediate formaldehyde. Additionally, the operation of two cofactor‐dependent formaldehyde oxidation pathways with distinct roles was revealed. One is dependent on tri‐ and tetraglutamylated tetrahydrofolate (THF) and is involved in formaldehyde oxidation during growth on methanol. A second pathway was discovered that is dependent on bacillithiol, a thiol cofactor present also in other Bacilli where it is known to function in redox‐homeostasis. We show that bacillithiol‐dependent formaldehyde oxidation is activated upon an upshift in formaldehyde induced by a substrate switch from mannitol to methanol. The genes and the corresponding enzymes involved in the biosynthesis of bacillithiol were identified by heterologous production of bacillithiol in Escherichia coli. The presented results indicate metabolic plasticity of the methylotroph allowing acclimation to fluctuating intracellular formaldehyde concentrations. 相似文献
159.
Nata?a Resnik Ur?ka Repnik Mateja Erdani Kreft Kristina Sep?i? Peter Ma?ek Boris Turk Peter Verani? 《PloS one》2015,10(9)
Cholesterol content can vary distinctly between normal and cancer cells, with elevated levels in cancer cells. Here, we investigated cholesterol sequestration with methyl-β-cyclodextrin (MCD), and pore-formation with the ostreolysin A/pleurotolysin B (OlyA/PlyB) protein complex that binds to cholesterol/sphingomyelin-rich membrane domains. We evaluated the effects on viability of T24 invasive and RT4 noninvasive human urothelial cancer cells and normal porcine urothelial (NPU) cells. Cholesterol content strongly correlated with cancerous transformation, as highest in the T24 high-grade invasive urothelial cancer cells, and lowest in NPU cells. MCD treatment induced prominent cell death of T24 cells, whereas OlyA/PlyB treatment resulted in greatly decreased viability of the RT4 low-grade noninvasive carcinoma cells. Biochemical and transmission electron microscopy analyses revealed that MCD and OlyA/PlyB induce necrotic cell death in these cancer cells, while viability of NPU cells was not significantly affected by either treatment. We conclude that MCD is more toxic for T24 high-grade invasive urothelial cancer cells, and OlyA/PlyB for RT4 low-grade noninvasive urothelial cancer cells, and neither is toxic for NPU cells. The cholesterol and cholesterol/sphingomyelin-rich membrane domains in urothelial cancer cells thus constitute a selective therapeutic target for elimination of urothelial cancer cells. 相似文献
160.