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1.
The very amino-terminal domain of the huntingtin protein is directly located upstream of the protein’s polyglutamine tract, plays a decisive role in several important properties of this large protein and in the development of Huntington’s disease. This huntingtin 1–17 domain is on the one hand known to markedly increase polyglutamine aggregation rates and on the other hand has been shown to be involved in cellular membrane interactions. Here, we determined the high-resolution structure of huntingtin 1–17 in dodecyl phosphocholine micelles and the topology of its helical domain in oriented phosphatidylcholine bilayers. Using two-dimensional solution NMR spectroscopy the low-energy conformations of the polypeptide were identified in the presence of dodecyl phosphocholine detergent micelles. In a next step a set of four solid-state NMR angular restraints was obtained from huntingtin 1–17 labeled with 15N and 2H at selected sites. Of the micellar ensemble of helical conformations only a limited set agrees in quantitative detail with the solid-state angular restraints of huntingtin 1–17 obtained in supported planar lipid bilayers. Thereby, the solid-state NMR data were used to further refine the domain structure in phospholipid bilayers. At the same time its membrane topology was determined and different motional regimes of this membrane-associated domain were explored. The pronounced structural transitions of huntingtin 1–17 upon membrane-association result in a α-helical conformation from K6 to F17, i.e., up to the very start of the polyglutamine tract. This amphipathic helix is aligned nearly parallel to the membrane surface (tilt angle ∼77°) and is characterized by a hydrophobic ridge on one side and an alternation of cationic and anionic residues that run along the hydrophilic face of the helix. This arrangement facilitates electrostatic interactions between huntingtin 1–17 domains and possibly with the proximal polyglutamine tract. 相似文献
2.
Localization of sucrose synthase and callose in freeze-substituted secondary-wall-stage cotton fibers 总被引:2,自引:0,他引:2
Summary. Methods for cryogenic fixation, freeze substitution, and embedding were developed to preserve the cellular structure and
protein localization of secondary-wall-stage cotton (Gossypium hirsutum L.) fibers accurately for the first time. Perturbation by specimen handling was minimized by freezing fibers still attached
to a seed fragment within 2 min after removal of seeds from a boll still attached to the plant. These methods revealed native
ultrastructure, including numerous active Golgi bodies, multivesicular bodies, and proplastids. Immunolocalization in the
context of accurate structure was accomplished after freeze substitution in acetone only. Quantitation of immunolabeling identified
sucrose synthase both near the cortical microtubules and plasma membrane and in a proximal exoplasmic zone about 0.2 μm thick.
Immunolabeling also showed that callose (β-1,3-glucan) was codistributed with sucrose synthase within this exoplasmic zone.
Similar results were obtained from cultured cotton fibers. The distribution of sucrose synthase is consistent with its having
a dual role in cellulose and callose synthesis in secondary-wall-stage cotton fibers.
Received August 19, 2002; accepted November 12, 2002; published online June 13, 2003
RID="*"
ID="*" Correspondence and reprints: Department of Biological Sciences, Texas Tech University, Lubbock, TX 79409-3131, U.S.A.
E-mail: candace.haigler@ttu.edu 相似文献
3.
Bocharova OV Breydo L Salnikov VV Gill AC Baskakov IV 《Protein science : a publication of the Protein Society》2005,14(5):1222-1232
In recent studies, the amyloid form of recombinant prion protein (PrP) encompassing residues 89-230 (rPrP 89-230) produced in vitro induced transmissible prion disease in mice. These studies showed that unlike "classical" PrP(Sc) produced in vivo, the amyloid fibrils generated in vitro were more proteinase-K sensitive. Here we demonstrate that the amyloid form contains a proteinase K-resistant core composed only of residues 152/153-230 and 162-230. The PK-resistant fragments of the amyloid form are similar to those observed upon PK digestion of a minor subpopulation of PrP(Sc) recently identified in patients with sporadic Creutzfeldt-Jakob disease (CJD). Remarkably, this core is sufficient for self-propagating activity in vitro and preserves a beta-sheet-rich fibrillar structure. Full-length recombinant PrP 23-230, however, generates two subpopulations of amyloid in vitro: One is similar to the minor subpopulation of PrP(Sc), and the other to classical PrP(Sc). Since no cellular factors or templates were used for generation of the amyloid fibrils in vitro, we speculate that formation of the subpopulation of PrP(Sc) with a short PK-resistant C-terminal region reflects an intrinsic property of PrP rather than the influence of cellular environments and/or cofactors. Our work significantly increases our understanding of the biochemical nature of prion infectious agents and provides a fundamental insight into the mechanisms of prions biogenesis. 相似文献
4.
Breydo L Bocharova OV Makarava N Salnikov VV Anderson M Baskakov IV 《Biochemistry》2005,44(47):15534-15543
In recent studies, we developed a protocol for in vitro conversion of full-length mouse recombinant PrP (Mo rPrP23-230) into amyloid fibrils [Bocharova et al. (2005) J. Mol. Biol. 346, 645-659]. Because amyloid fibrils produced from recombinant Mo PrP89-230 display infectivity [Legname et al. (2004) Science 305, 673-676], polymerizatiom of rPrPs in vitro represents a valuable model for elucidating the mechanism of prion conversion. Unexpectedly, when the same conversion protocol was used for hamster (Ha) rPrP23-231, we experienced substantial difficulties in forming fibrils. While searching for potential reasons of our failure to produce fibrils, we probed the effect of methionine oxidation in rPrP. We found that oxidation of methionines interferes with the formation of rPrP fibrils and that this effect is more profound for Ha than for Mo rPrP. To minimize the level of spontaneous oxidation, we developed a new protocol for rPrP purification, in which highly amyloidogenic Ha rPrP with minimal levels of oxidized residues was produced. Furthermore, our studies revealed that oxidation of methionines in preformed fibrils inhibited subsequent maturation of fibrils into proteinase K-resistant PrP(Sc)-like conformation (PrP-res). Our data are consistent with the proposition that conformational changes within the central region of the protein (residues 90-140) are essential for adopting PrP-res conformation and demonstrate that methionine oxidation interferes with this process. These studies provide new insight into the mechanism of prion polymerization, solve a long-standing practical problem in producing PrP-res fibrils from full-length PrP, and may help in identifying new genetic and environmental factors that modulate prion disease. 相似文献
5.
6.
V. N. Fedorov M. M. Fateev E. V. Salnikov A. V. Sidorov 《Journal of Evolutionary Biochemistry and Physiology》2009,45(4):484-489
The present study confirms involvement of the sympathicoadrenal system in adaptation of heart to overload. Besides, at formation
of chronic heart failure (CHF) there have been revealed a rise of the histamine and serotonin levels in blood plasma and myocardium
as well as glucocorticoid hyperactivation. 相似文献
7.
The 2H solid-state NMR spectra of deuterated fatty acyl chains provide direct access to the order of the hydrophobic membrane interior. From the deuterium order parameter profiles of perdeuterated fatty acyl chains the membrane hydrophobic thickness can be calculated. Here we show data obtained from POPC, POPE and mixed POPE/POPG bilayers, representative of bacterial membranes, in the presence of cholesterol or ergosterol and antimicrobial peptaibols. Whereas sterols have a strong ordering effect also on these membranes, the peptides exhibit neutral or disordering effects. By comparing with data from the literature it becomes obvious that cationic amphipathic peptides that probably reside within the interface of phospholipid membranes tend to strongly disorder the packing of the fatty acyl chains, an effect that has been correlated to antimicrobial and DNA transfection activities. In contrast transmembrane sequences or hydrophobic peptides that probably partition deeply into the membrane tend to have only modest disordering activities. The 2H solid-state NMR approach has also been used to monitor the lateral separation of domains rich in anionic phospholipids in the presence of cationic peptides and has thereby provided important insights into their mechanisms of action. 相似文献
8.
Pathogen‐induced conditioning of the primary xylem vessels – a prerequisite for the formation of bacterial emboli by Pectobacterium atrosepticum
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V. Y. Gorshkov A. G. Daminova P. V. Mikshina O. E. Petrova M. V. Ageeva V. V. Salnikov T. A. Gorshkova Y. V. Gogolev 《Plant biology (Stuttgart, Germany)》2016,18(4):609-617
Representatives of Pectobacterium genus are some of the most harmful phytopathogens in the world. In the present study, we have elucidated novel aspects of plant–Pectobacterium atrosepticum interactions. This bacterium was recently demonstrated to form specific ‘multicellular’ structures – bacterial emboli in the xylem vessels of infected plants. In our work, we showed that the process of formation of these structures includes the pathogen‐induced reactions of the plant. The colonisation of the plant by P. atrosepticum is coupled with the release of a pectic polysaccharide, rhamnogalacturonan I, into the vessel lumen from the plant cell wall. This polysaccharide gives rise to a gel that serves as a matrix for bacterial emboli. P. atrosepticum‐caused infection involves an increase of reactive oxygen species (ROS) levels in the vessels, creating the conditions for the scission of polysaccharides and modification of plant cell wall composition. Both the release of rhamnogalacturonan I and the increase in ROS precede colonisation of the vessels by bacteria and occur only in the primary xylem vessels, the same as the subsequent formation of bacterial emboli. Since the appearance of rhamnogalacturonan I and increase in ROS levels do not hamper the bacterial cells and form a basis for the assembly of bacterial emboli, these reactions may be regarded as part of the susceptible response of the plant. Bacterial emboli thus represent the products of host–pathogen integration, since the formation of these structures requires the action of both partners. 相似文献
9.
A I Salnikov 《Prikladnaia biokhimiia i mikrobiologiia》1975,11(6):885-887
The distribution of farnesene and farnesene hydroperoxide in apples of different varieties was studied. The relationship between the content of these compounds and the pathological browning of the apple surface, scald was followed. It was shown that 1) farnesene and farnesene hydroperoxide were concentrated in the cuticle; 2) there was no distinct correlation between farnesene concentration and scald, 3) there was a direct correlation between the concentration of farnese hydroperoxide and scald development; 4) the factors that inhibited farnesene oxidation (antioxidants, low oxygen concentration, mineral oils--farnesene absorbers) slowed down the scald development during apple storage. 相似文献
10.
Aruna Kasoju M Lakshmi Narasu Charuvaka Muvva Bathula VV SubbaRao 《Bioinformation》2012,8(14):684-686
Aflatoxins are polyketide-derived secondary metabolites produced by Aspergillus spp. The toxic effects of aflatoxins have adverse
consequences for human health and agricultural economics. The aflR gene, a regulatory gene for aflatoxin biosynthesis, encodes a
protein containing a zinc-finger DNA-binding motif. AFLR-Protein three-dimensional model was generated using Robetta server.
The modeled AFLR-Protein was further optimization and validation using Rampage. In the simulations, we monitored the
backbone atoms and the C-α-helix of the modeled protein. The low RMSD and the simulation time indicate that, as expected, the
3D structural model of AFLR-protein represents a stable folding conformation. This study paves the way for generating computer
molecular models for proteins whose crystal structures are not available and which would aid in detailed molecular mechanism of
inhibition of aflatoxin. 相似文献