Structure and Topology of the Huntingtin 1–17 Membrane Anchor by a Combined Solution and Solid-State NMR Approach

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.     

Neurohumoral mechanisms of the rat heart adaptation to overload

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.     

Localization of sucrose synthase and callose in freeze-substituted secondary-wall-stage cotton fibers

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     

Synthetic prions generated in vitro are similar to a newly identified subpopulation of PrPSc from sporadic Creutzfeldt-Jakob Disease

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.     

Methionine oxidation interferes with conversion of the prion protein into the fibrillar proteinase K-resistant conformation

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.     

Membrane order perturbation in the presence of antimicrobial peptides by H solid-state NMR spectroscopy

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.     

Aspen Tension Wood Fibers Contain β-(1→4)-Galactans and Acidic Arabinogalactans Retained by Cellulose Microfibrils in Gelatinous Walls

Contractile cell walls are found in various plant organs and tissues such as tendrils, contractile roots, and tension wood. The tension-generating mechanism is not known but is thought to involve special cell wall architecture. We previously postulated that tension could result from the entrapment of certain matrix polymers within cellulose microfibrils. As reported here, this hypothesis was corroborated by sequential extraction and analysis of cell wall polymers that are retained by cellulose microfibrils in tension wood and normal wood of hybrid aspen (Populus tremula × Populus tremuloides). β-(1→4)-Galactan and type II arabinogalactan were the main large matrix polymers retained by cellulose microfibrils that were specifically found in tension wood. Xyloglucan was detected mostly in oligomeric form in the alkali-labile fraction and was enriched in tension wood. β-(1→4)-Galactan and rhamnogalacturonan I backbone epitopes were localized in the gelatinous cell wall layer. Type II arabinogalactans retained by cellulose microfibrils had a higher content of (methyl)glucuronic acid and galactose in tension wood than in normal wood. Thus, β-(1→4)-galactan and a specialized form of type II arabinogalactan are trapped by cellulose microfibrils specifically in tension wood and, thus, are the main candidate polymers for the generation of tensional stresses by the entrapment mechanism. We also found high β-galactosidase activity accompanying tension wood differentiation and propose a testable hypothesis that such activity might regulate galactan entrapment and, thus, mechanical properties of cell walls in tension wood.Contractile cell walls found in plant organs and tissues such as tendrils, contractile roots, and tension wood (TW) have remarkable functions and properties. Their traits have been most intensely studied in TW of hardwoods, where they provide negative gravitropic response capacities to stems with secondary growth, as recently reviewed by Mellerowicz and Gorshkova (2012). These properties are conferred by TW fibers, which in many species contain a so-called gelatinous cell wall layer (G-layer; Norberg and Meier, 1966; Clair et al., 2008). G-layers are formed following the deposition of xylan-type secondary cell wall layer(s) and, thus, can be considered tertiary layers (Wardrop and Dadswell, 1948). They are almost or completely devoid of xylan and lignin and have very high cellulose contents (up to 85%). However, several other polymers appear to be present in TW G-layers, according to recent chemical analyses of isolated G-layers (Nishikubo et al., 2007; Kaku et al., 2009) and immunohistochemical labeling of TW sections (Arend, 2008; Bowling and Vaughn, 2008). Notably, xyloglucan (XG) has been found in G-layers of poplar (Populus spp.) TW (Nishikubo et al., 2007) and at the boundary between secondary cell wall layers (S-layers) and G-layers (Baba et al., 2009; Sandquist et al., 2010). It is also important for tension creation (Baba et al., 2009). However, it is not detectable in mature G-layers by monoclonal antibodies or XG-binding modules (Nishikubo et al., 2007; Baba et al., 2009; Sandquist et al., 2010).Structurally similar G-layers have been also identified in phloem fibers in many fibrous crops, such as flax (Linum usitatissimum), hemp (Cannabis sativa), and ramie (Boehmeria nivea; Gorshkova et al., 2012). These fibers occur in bundles that can be isolated for biochemical analysis. G-layers in fibers from diverse sources have a very similar structure, being largely composed of cellulose (with axial microfibril orientation, high degrees of crystallinity, and large crystallite sizes) lacking xylan and lignin (Mellerowicz et al., 2001; Pilate et al., 2004; Gorshkova et al., 2010, 2012) and having high water contents (Schreiber et al., 2010). In phloem fibers, the G-layers become very prominent, reaching thicknesses up to 15 µm and occupying over 90% of the cell wall’s total cross-sectional areas (Crônier et al., 2005). Pectic β-(1→4)-galactan with complex structures has been shown to be the major matrix polysaccharide of isolated phloem fibers in flax (Gorshkova et al., 2004; Gorshkova and Morvan, 2006; Gurjanov et al., 2007). Some of it is so strongly retained within cellulose that it cannot be extracted by concentrated alkali and can only be obtained after cellulose dissolution (Gurjanov et al., 2008). Such galactan, therefore, is a prime candidate for a polymer entrapped by cellulose microfibrils during crystallization that could substantially contribute to the contractile properties of cellulose in G-layers, according to recently formulated models (Mellerowicz et al., 2008; Mellerowicz and Gorshkova 2012). Furthermore, Roach et al. (2011) have shown that trimming of β-(1→4)-galactan by β-galactosidase is important for final cellulose crystallization, the formation of G-layer structure, and, hence, the stem’s mechanical properties.There is also immunocytochemical evidence for the presence of β-(1→4)-galactan and type II arabinogalactan (AG-II) in G-layers of TW fibers (Arend, 2008; Bowling and Vaughn, 2008). In addition, high-M_r branched galactans have been isolated from TW of Fagus sylvestris (Meier, 1962) and Fagus grandifolia (Kuo and Timell, 1969), with estimated degrees of polymerization (DP) of approximately 300 and complex structure, probably including both β-(1→4) and β-(1→6) linkages, although their exact nature remains unknown. Furthermore, Gal has been identified as one of the major sugars after Glc and Xyl in hydrolysates of isolated Populus spp. G-layers (Furuya et al., 1970; Nishikubo et al., 2007), and the Gal content of cell walls is a proposed indicator of the extent of TW development in beech (Fagus spp.; Ruel and Barnoud, 1978). However, subsequent linkage analyses identified only 2- and 3,6-linked Gal in poplar TW G-layers (Nishikubo et al., 2007), while in flax fibers, 4-linked Gal is the main component (Gorshkova et al., 1996, 2004; Gurjanov et al., 2007, 2008). Thus, the type(s) of galactans present in poplar TW remains unclear, and the galactans have not been shown previously either to have a rhamnogalacturonan-I (RG-I) backbone or to be strongly retained by cellulose microfibrils, as demonstrated for flax gelatinous fibers.To improve our understanding of cell wall properties in TW and their contraction mechanism, in the study presented here, we tested aspects of the recently proposed entrapment model (Mellerowicz et al., 2008; Mellerowicz and Gorshkova, 2012). According to this model, contraction is driven by the formation of larger cellulose structures, sometimes called macrofibrils, via interactions of cellulose microfibrils in the G-layer with each other and forming inclusions containing matrix polymers. This would induce tension within cellulose through the stretching of microfibrils required to surround the inclusions. The model is compatible with available data on the structure and action of gelatinous walls, but the main assumption, that polymers are trapped inside crystalline cellulose, such as that found in flax, has not been tested previously. Therefore, we compared matrix polymers retained by cellulose microfibrils in normal wood (NW) and TW of the model hardwood species hybrid aspen (Populus tremula × Populus tremuloides) that forms TW with gelatinous fibers. For this purpose, we used a combination of sequential cell wall extractions, similar to those used previously to characterize flax gelatinous fibers (Gurjanov et al., 2008), followed by fractionation of polymers by size-exclusion chromatography, immunological analyses, and oligosaccharide profiling by polysaccharide analysis using carbohydrate gel electrophoresis (PACE). The results reveal the main polymers of cellulose-retained fractions and key differences between NW and TW. Comparison of our results and previous findings also indicates that there are both similarities and differences in the constitution of gelatinous fibers in aspen and flax. An updated model of the contractile G-layer of TW fibers based on the data is presented.     

Galactosidase of plant fibers with gelatinous cell wall: Identification and localization

Using a comprehensive approach, we have identified a tissue-specific β-galactosidase from flax (Linum usitatissimum L.) phloem fibers forming a gelatinous cell wall. It was found that when fibers started to develop gelatinous cell wall, β-galactosidase gene expression was enhanced.. Using the antibodies against β-galactosidase, we showed that the enzyme was located in flax phloem fibers where it was detected together with tissue-specific galactan in secreted Golgi vesicles and in gelatinous secondary cell wall. Similar β-galactosidase present in gelatinous cell wall of fibers was found in plants belonging to various taxa and produced by different meristems; these data presume the identical mechanisms of gelatinous cell wall formation and an important role of β-galactosidase. The role of this enzyme in developing the supramolecular structure of gelatinous cell wall is discussed.     

Phylogenetic relationships of the algae scraping cyprinid genus Capoeta (Teleostei: Cyprinidae)

We reconstructed the matrilineal phylogeny of Asian algae-eating fishes of the genus Capoeta based on complete mitochondrial gene for cytochrome b sequences obtained from 20 species sampled from the majority of the range and 44 species of closely related barbs of the genera Barbus s. str. and Luciobarbus. The results of this study show that Capoeta forms a strongly supported monophyletic subclade nested within the Luciobarbus clade, suggesting that specialized scraping morphology appeared once in the evolutionary history of the genus. We detected three main groups of Capoeta: the Mesopotamian group, which includes three species from the Tigris-Euphrates system and adjacent water bodies, the Anatolian-Iranian group, which has the most diversified structure and encompasses many species distributed throughout Anatolian and Iranian inland waters, and the Aralo-Caspian group, which consists of species distributed in basins of the Caspian and Aral Seas, including many dead-end rivers in Central Asia and Northern Iran. The most probable origination pathway of the genus Capoeta is hypothesized to occur as a result of allopolyploidization. The origin of Capoeta was found around the Langhian-Serravallian boundary according to our molecular clock. The diversification within the genus occurred along Middle Miocene-Late Pliocene periods.