Changes In Primary Structure Research Articles

Pathogenic missense MAPT mutations differentially modulate tau aggregation propensity at nucleation and extension steps

Mutations in the MAPT gene encoding tau protein lead to neurofibrillary lesion formation, neurodegeneration, and cognitive decline associated with frontotemporal lobar degeneration. While some pathogenic mutations affect MAPT introns, resulting in abnormal splicing patterns, the majority occur in the tau coding sequence leading to single amino acid changes in tau primary structure. Depending on their location within the polypeptide chain, tau missense mutations have been reported to augment aggregation propensity. To determine the mechanisms underlying mutation-associated changes in aggregation behavior, the fibrillization of recombinant pathogenic mutants R5L, G272V, P301L, V337M, and R406W prepared in a full-length four-repeat human tau background was examined in vitro as a function of time and submicromolar tau concentrations using electron microscopy assay methods. Kinetic constants for nucleation and extension phases of aggregation were then estimated by direct measurement and mathematical simulation. Results indicated that the mutants differ from each other and from wild-type tau in their aggregation propensity. G272V and P301L mutations increased the rates of both filament nucleation and extension reactions, whereas R5L and V337M increased only the nucleation phase. R406W did not differ from wild-type in any kinetic parameter. The results show that missense mutations can directly promote tau filament formation at different stages of the aggregation pathway.

Study of Microstructural Evolution and Phase’s Morphology after Partial Remelting in A356 Alloy

In this work, effective parameters of SIMA process to obtain non dendritic microstructure in A356 alloy were investigated. In addition, the effect of SIMA process on the evolution of morphology of silicon and intermetallic phases in this alloy was studied. Microstructure images obtained from optical microscopy and SEM observation showed that increase in plastic work up to 40% and then holding of samples in the semi solid state at temperature of 580oC, causes that primary dendritic structure changes to non dendritic, fine and globular structure, but optimum reheating time completely depended on initial thickness of samples. If all parameters of SIMA process are the same, the grain boundaries of thinner samples begin to wet and following globalization will be completed in shorter reheating time rather than thicker ones. Moreover, it was found that the intermetallic phases lost their angular or needle morphology and gradually changed to rounded morphology and even to globular form. Also the optimum reheating time thoroughly depends on primary casting microstructure as the finer casting microstructure begin to globalize faster than thicker one under more little stains.

Collagen structural hierarchy and susceptibility to degradation by ultraviolet radiation

Collagen type I is the most abundant extracellular matrix protein in the human body, providing the basis for tissue structure and directing cellular functions. Collagen has complex structural hierarchy, organized at different length scales, including the characteristic triple helical feature. In the present study, the relationship between collagen structure (native vs. denatured) and sensitivity to UV radiation was assessed, with a focus on changes in primary structure, changes in conformation, microstructure and material properties. A brief review of free radical reactions involved in collagen degradation is also provided as a mechanistic basis for the changes observed in the study. Structural and functional changes in the collagens were related to the initial conformation (native vs. denatured) and the energy of irradiation. These changes were tracked using SDS-PAGE to assess molecular weight, Fourier transform infrared (FTIR) spectroscopy to study changes in the secondary structure, and atomic force microscopy (AFM) to characterize changes in mechanical properties. The results correlate differences in sensitivity to irradiation with initial collagen structural state: collagen in native conformation vs. heat-treated (denatured) collagen. Changes in collagen were found at all levels of the hierarchical structural organization. In general, the native collagen triple helix is most sensitive to UV-254 nm radiation. The triple helix delays single chain degradation. The loss of the triple helix in collagen is accompanied by hydrogen abstraction through free radical mechanisms. The results received suggest that the effects of electromagnetic radiation on biologically relevant extracellular matrices (collagen in the present study) are important to assess in the context of the state of collagen structure. The results have implications in tissue remodeling, wound repair and disease progression.

Mechanisms of varicose vein formation: valve dysfunction and wall dilation

Varicose veins are a common venous disease of the lower extremity. Although the mechanisms and determinants in the development of varicosities are not clearly defined, recent clinical studies and basic science research have cast some light on possible mechanisms of the disease. In varicose veins, there are reflux and incompetent valves as well as vein wall dilation. Primary structural changes in the valves may make them 'leaky', with progressive reflux causing secondary changes in the vein wall. Alternatively, or concurrently, the valves may become incompetent secondary to structural abnormalities and focal dilation in vein wall segments near the valve junctions, and the reflux ensues as an epiphenomenon. The increase in venous pressure causes structural and functional changes in the vein wall that leads to further venous dilation. Increase in vein wall tension augments the expression/activity of matrix metalloproteinases (MMPs), which induces degradation of the extracellular matrix proteins and affect the structural integrity of the vein wall. Recent evidence also suggests an effect of MMPs on the endothelium and smooth muscle components of the vein wall and thereby causing changes in the venous constriction/relaxation properties. Endothelial cell injury also triggers leukocyte infiltration, activation and inflammation, which lead to further vein wall damage. Thus, vein wall dilation appears to precede valve dysfunction, and the MMP activation and superimposed inflammation and fibrosis would then lead to chronic and progressive venous insufficiency and varicose vein formation.

FACT and the reorganized nucleosome

FACT is an essential component of the machinery used by eukaryotic cells both to establish and to overcome the nucleosomal barrier to DNA accessibility, and it does so without hydrolyzing ATP. FACT is a transcription elongation factor, but this review stresses additional roles in DNA replication and initiation of transcription. The widely-held model that FACT functions by displacing an H2A-H2B dimer from a nucleosome is examined, and an alternative proposal is presented in which dimer loss can occur but is a secondary effect of a primary structural change induced by FACT binding which we have called "nucleosome reorganization." The structures of two domains of FACT have been determined and they reveal multiple potential interaction sites. Roles for these binding sites in FACT function and regulation are discussed.

Natural Homologous Triploidization and DNA Methylation in SARII-628, a Twin-seedling Line of Rice ( Oryza sativa)

A total of five pairs of diploid-triploid twin-seedlings (a diploid seedling and a triploid seedling emerged from a grain) were selected out from 4500 pairs of seedlings from SARII-628, a twin-seedling rice line. SSR analysis indicated that no difference between the diploid seedling and corresponding triploid seedling in a twin-seedling was found at the 310 loci, indicating that there was no obvious change in DNA primary structure. A modified AFLP technique ‘MSAP (methylation-sensitive AFLP)’ was used to analyze methylation mutation. Although no methylation mutation was noted among the five diploids, 29 methylation mutation loci were found from the corresponding triploids. This suggested that methylation mutation happened rapidly on M 0 generation after natural homologous triploidization. The mutations were classified into 10 types, including 3 increased types, 3 decreased types and 4 undecided types of methylation-degrees. The bands of 22 loci were sequenced and then those sequences were searched through website. The result showed that the methylation mutation involved into the whole rice genome and the 12 pairs of chromosomes. The mutation trend was site-related and there were different mutation loci for different triploids, which foretold that SARII-628 would have different evolution fates after natural homologous triploidization.

Selective Cysteine Protease Inhibition Contributes to Blood-feeding Success of the Tick Ixodes scapularis

Ixodes scapularis is the main vector of Lyme disease in the eastern and central United States. Tick salivary secretion has been shown as important for both blood-meal completion and pathogen transmission. Here we report a duplication event of cystatin genes in its genome that results in a transcription-regulated boost of saliva inhibitory activity against a conserved and relatively limited number of vertebrate papain-like cysteine proteases during blood feeding. We further show that the polypeptide products of the two genes differ in their binding affinity for some enzyme targets, and they display different antigenicity. Moreover, our reverse genetic approach employing RNA interference uncovered a crucial mediation in tick-feeding success. Given the role of the targeted enzymes in vertebrate immunity, we also show that host immunomodulation is implicated in the deleterious phenotype of silenced ticks making I. scapularis cystatins attractive targets for development of anti-tick vaccines.

Protect from Light: Photodegradation and Protein Biologics

The exposure of proteins to light and the ensuing chemical and physical degradation has been studied extensively for many years. The residues in proteins that undergo primary photooxidation include tryptophan, tyrosine, phenylalanine, and cysteine/cystine. While photooxidation has been recognized as a major contributor to protein degradation, the effects of photoinduced damage have not been widely studied for biopharmaceuticals. This is particularly important since photodegradation can lead to changes in primary, secondary, and tertiary structures of protein and these changes, while not definitively established, could lead to differences in long‐term stability, bioactivity, or immunogenicity. In this review we briefly describe the major pathways of photodegradation for proteins followed by a description of the limited data on photodegradation of biopharmaceuticals and methods that have been used to reduce or prevent damage. It is our intent to spur additional research in this area for increasing the safety and effectiveness of biopharmaceutical products.

DNA methylation affects nuclear organization, histone modifications, and linker histone binding but not chromatin compaction

DNA methylation has been implicated in chromatin condensation and nuclear organization, especially at sites of constitutive heterochromatin. How this is mediated has not been clear. In this study, using mutant mouse embryonic stem cells completely lacking in DNA methylation, we show that DNA methylation affects nuclear organization and nucleosome structure but not chromatin compaction. In the absence of DNA methylation, there is increased nuclear clustering of pericentric heterochromatin and extensive changes in primary chromatin structure. Global levels of histone H3 methylation and acetylation are altered, and there is a decrease in the mobility of linker histones. However, the compaction of both bulk chromatin and heterochromatin, as assayed by nuclease digestion and sucrose gradient sedimentation, is unaltered by the loss of DNA methylation. This study shows how the complete loss of a major epigenetic mark can have an impact on unexpected levels of chromatin structure and nuclear organization and provides evidence for a novel link between DNA methylation and linker histones in the regulation of chromatin structure.

Specific Human Leukocyte Antigen Alleles Associated With HIV-1 Infection in an Indian Population

Since the beginning of the AIDS epidemic evidence has accumulated that genetic factors are involved in the pathogenesis of the disease. The role of host genes in the course of HIV-1 infection has been examined in different populations and among all major risk groups. Human leukocyte antigen (HLA) associations with HIV progression correlate well with the interaction between the pathogen and host. Most HLA polymorphisms are concentrated in the region that determines specificity for foreign peptide presentation. Three models have been proposed to explain maintenance of the HLA polymorphism: (1) balancing selection where alleles confer resistance to one disease but confer susceptibility to another; (2) heterozygote advantage where a unique HLA type increases the breadth of peptide recognition and thereby immune defense against infectious organisms; and (3) frequency-dependent selection where a pathogen evolves to escape an efficient immune response mediated by common alleles in the population butremains susceptible to responses mediated by low-frequency alleles. Each of these mechanisms has probably been operative under various host or environmental circumstances. Any change in primary structure may throttle a neutral allele into a deleterious one. HLA associations with HIV disease may be particularly apparent on investigation because the recent epidemic has not had enough time to show diminished frequencies of deleterious alleles. The paucity of data on the association of HLA alleles with the predominant HIV-1 subtype C in India motivated us to undertake the present study. (excerpt)

Easy Monitoring, by Flow FFF, of the Behavior of Nanoparticles Formed from Copolymers of Sulfopropylmethacrylate and Methylmethacrylate

The size and size distribution of nanoparticles are generally important for the characterization of these materials, but are particularly needed to determine their suitability for specific applications. When nanoparticles are obtained by micelle formation from synthetic copolymers their size and structure depend on the copolymer composition and charge. Minor changes in the macromolecule primary structure may lead to dramatic changes in the aggregation behavior, hence in the nanoparticle size and size distribution. While particle diameter may be accurately measured by various sizing methods, determination of the size distribution requires a methodology which combines an adequate resolving power with size measurement capabilities. Flow FFF has shown to be able to perform both tasks in a single run. Using a very small amount of sample it yields accurate size distribution curves of most various colloidal systems. For this reason it was used here for the analysis of two copolymers of sulfopropyl methacrylate and methylmethacrylate differing for the copolymerization process. Size analysis of these samples showed a dramatic difference in their capability of forming micellar aggregates as well as in the characteristic response to the solution ionic strength.

Pax6 and eye development in Arthropoda

The arthropod compound eye is one of the three main types of eyes observed in the animal kingdom. Comparison of the eyes seen in Insecta, Crustacea, Myriapoda and Chelicerata reveals considerable variation in terms of overall cell number, cell positioning, and photoreceptor rhabdomeres, yet, molecular data suggest there may be unexpected similarities. We review here the role of Pax6 in eye development and evolution and the relationship of Pax6 with other retinal determination genes and signaling pathways. We then discuss how the study of changes in Pax6 primary structure, in the gene networks controlled by Pax6 and in the relationship of Pax6 with signaling pathways may contribute to our insight into the relative role of conserved molecular-genetic mechanisms and emergence of evolutionary novelty in shaping the ommatidial eyes seen in the Arthropoda.

Kinetic and proteomic analyses of S-nitrosoglutathione-treated hexokinase A: consequences for cancer energy metabolism

Mammalian hexokinase (HXK) is found at the outer mitochondrial membrane, exposed to mitochondrial oxygen- and nitrogen-radicals. Given the important role of this enzyme in metabolic pathways and diseases, the effect of S-nitrosoglutathione (GSNO) on HXK A structure and activity was studied. To focus on the catalytic domain, yeast HXK A was used because it has a significant homology to the mammalian domain that contains both the regulatory and catalytic sites. Biologically relevant [GSNO]/[HXK] caused a significant decrease in V(max) with glucose (but not with fructose), along with oxidation of 5 Met and nitration of 4 Tyr. Preincubation of HXK with glucose abrogated the effect of GSNO whereas fructose was ineffective. These results are interpreted by considering the tight binding of glucose to the enzyme as opposed to that of fructose. The segment comprised from amino acids 304 to 306 contained the most modifications. Given that this sequence is highly conserved in HXK from various species, a decline in activity is expected when a high-affinity substrate is presented. Considering that changes in primary structure are envisioned at high [GSNO]/[HXK] ratios, like those present under normal conditions, it could be hypothesized that the high concentration of hexokinase present in fast growing tumors may serve not only to sustain high glycolysis rates, but also to minimize protein damage that might result in activity decline, compromising energy metabolism.

Structural and functional characterization of a recombinant sticholysin I (rSt I) from the sea anemone Stichodactyla helianthus

Sticholysins I and II (Sts I and II) are two potent cytolysins from the sea anemone Stichodactyla helianthus. These isoforms present 13 substitutions, with three non-conservative located at the N-terminus. St II is considerably more hemolytic than St I in human red blood cells, a result explained by the smaller number of negatively charged groups present at St II's N-terminus. In the present work, we have obtained a recombinant St I (rSt I), differing from the wild type in a single amino acid residue (E16Q). This pseudo-wild type is structurally similar to St I and shows a similar capacity to interact with and form pores in model membranes. This was assessed by the intrinsic fluorescence increase in the presence of liposomes, their adsorption to bilayers (measured by SPR), their concentration at the air–water interface, their interaction with lipid monolayers and their capacity to promote the release of carboxyfluorescein entrapped in liposomes. In spite of these similarities, rSt I presents a larger hemolytic activity in human red blood cells than St I, being intermediate in activity between Sts I and II. The results obtained in the present work emphasize that even the change of one single E by Q at the N-terminal segment may modify the toxin HA and show that this functional property is the most sensitive to subtle changes in the protein primary structure.

Repetitive DNA elements as mediators of genomic change in response to environmental cues

There is no logical or theoretical barrier to the proposition that organismal and cell signaling could transduce environmental signals into specific, beneficial changes in primary structure of noncoding DNA via repetitive element movement or mutation. Repetitive DNA elements, including transposons and microsatellites, are known to influence the structure and expression of protein-coding genes, and to be responsive to environmental signals in some cases. These effects may create fodder for adaptive evolution, at rates exceeding those observed for point mutations. In many cases, the changes are no doubt random, and fitness is increased through simple natural selection. However, some transposons insert at specific sites, and certain regions of the genome exhibit selectively and beneficially high mutation rates in a range of organisms. In multicellular organisms, this could benefit individuals in situations with significant potential for clonal expansion: early life stages or regenerative tissues in animals, and most plant tissues. Transmission of the change to the next generation could occur in plants and, under some circumstances, in animals.

Tracking adaptive evolution in the structure, function and molecular phylogeny of haemoglobin in non-Antarctic notothenioid fish species

With the notable exception of Antarctic icefishes, haemoglobin (Hb) is present in all vertebrates. In polar fish, Hb evolution has included adaptations with implications at the biochemical, physiological and molecular levels. Cold adaptation has been shown to be also linked to small changes in primary structure and post-translational modifications in proteins, including hydrophobic remodelling and increased flexibility. A wealth of knowledge is available on the oxygen-transport system of fish inhabiting Antarctic waters, but very little is known on the structure and function of Hb of non-Antarctic notothenioid fishes. The comparison of the biochemical and physiological adaptations between cold-adapted and non-cold-adapted species is a powerful tool to understand whether (and to what extent) extreme environments require specific adaptations or simply select for phenotypically different life styles. This study focuses on structure, function and molecular phylogeny of Hb in Antarctic and non-Antarctic notothenioid fishes. The rationale is to use the primary structure of Hb as tool of choice to gain insight into the pathways of the evolution history of α and β globins of notothenioids and also as a basis for reconstructing the phylogenetic relationships among Antarctic and non-Antarctic species.

Biological and physicochemical factors controlling short-term variability in phytoplankton primary production and photosynthetic parameters in a macrotidal ecosystem (eastern English Channel)

Links between short-term variability of phytoplankton primary production and community structure changes have been studied rarely. This has been examined in a macrotidal ecosystem, the Baie des Veys (eastern English Channel, France), in 2003 and 2004, over the complete tidal cycle (semi-diurnal mode, 12 h). Within this area, primary production and photosynthetic parameter estimates, according to the 14C incorporation technique, were supported by an exhaustive taxonomic study and measurements of physicochemical factors to illustrate the environmental framework. Related to the river Vire discharge, daily interactions between estuarine and bay waters were demonstrated. Depth-integrated primary production P z was maximal around noon in the bay (48.7–68.0 mg C m −2 h −1) and decreased through the day in the mouth of the river. Photosynthetic parameters' variations and photoacclimation were influenced by the ecosystem variability level: short-term photoacclimation was possible in low mixing conditions. Changes in taxonomic composition according to tidal forcing led to variations in primary production levels. Large species, associated with high photosynthetic parameters, were observed in the bay, whereas small ones were present in the mouth of the river, when low primary production was measured. On a short-time scale, a positive relationship was observed between species diversity and primary production. This work emphasizes the need to focus on changes in phytoplankton community structure in order to understand short-term variability in primary production.

Unique modifications with phosphocholine and phosphoethanolamine define alternate antigenic forms of Neisseria gonorrhoeae type IV pili.

Several major bacterial pathogens and related commensal species colonizing the human mucosa express phosphocholine (PC) at their cell surfaces. PC appears to impact host-microbe biology by serving as a ligand for both C-reactive protein and the receptor for platelet-activating factor. Type IV pili of Neisseria gonorrhoeae (Ng) and Neisseria meningitidis, filamentous protein structures critical to the colonization of their human hosts, are known to react variably with monoclonal antibodies recognizing a PC epitope. However, the structural basis for this reactivity has remained elusive. To address this matter, we exploited the finding that the PilE pilin subunit in Ng mutants lacking the PilV protein acquired the PC epitope independent of changes in pilin primary structure. Specifically, we show by using mass spectrometry that PilE derived from the pilV background is composed of a mixture of subunits bearing O-linked forms of either phosphoethanolamine (PE) or PC at the same residue, whereas the wild-type background carries only PE at that same site. Therefore, PilV can influence pilin structure and antigenicity by modulating the incorporation of these alternative modifications. The disaccharide covalently linked to Ng pilin was also characterized because it is present on the same peptides bearing the PE and PC modifications and, contrary to previous reports, was found to be linked by means of 2,4-diacetamido-2,4,6-trideoxyhexose. Taken together, these findings provide new insights into Ng type IV pilus structure and antigenicity and resolve long-standing issues regarding the nature of both the PC epitope and the pilin glycan.

The nature of structural changes in nanocrystalline ZnO powders under linear heating conditions

The nature of exothermal and irreversible structural changes observed during linear heating of nanocrystalline ZnO powders was investigated using differential scanning calorimetry, X-ray, SEM and TEM analysis. Nanocrystalline ZnO powders were obtained by ultrasonic spray pyrolysis of 0.8 mol/dm 3 zinc nitrate solution and subsequent decomposition of 2.7 μm sized droplets in the temperature range up to 600 °C, F air=1.2 dm 3/min. Produced particles are characterized by uniform and submicronic size, BET=4.94 m 2/g, high phase purity and granular or circular “open” surface as the consequence of primary crystallites presence with average size of d=20 nm. The observed quasi nucleation-growth process during heating of this powder was discussed in the terms of primary crystallites structural changes.

Temperature-dependent behavior of protein-chromophore interactions: a theoretical study of a blue fluorescent antibody.

The unusual temperature-dependent excited-state dynamics of a stilbene-antibody complex reported by Simeonov et al. are explored using theoretical methods. The anomalous temperature-dependent fluorescence emission and lifetime are shown to be the result of interplay among temperature-modulated protein flexibility, the excited-state potential surface for the stilbene central twist, and changes in the stilbene charge distribution upon excitation. Stilbene is found to possess a similar geometry and orientation within the antibody binding pocket at all temperatures in the ground state and at low temperatures (approximately 240 K) in the excited state. At higher temperatures (approximately 260 K), the excited-state conformation twists around the central double bond and adopts an alternate orientation within the binding pocket. These changes result from protein side chain and loop motions that are frozen out at lower temperatures and account for the observed red shift of the fluorescence emission spectrum (a calculated shift of 3.8 kcal mol-1 compares favorably with the approximately 5 kcal.mol-1 observed experimentally). Approximately 3.0 kcal mol-1 of this stabilization is global in nature and is not attributable to specific local interactions. Local interactions between stilbene and Tyr-B39 contribute approximately 0.8 kcal mol-1 to the fluorescence shift. The primary structural change in simulations of the high temperature excited state involves a decrease in the stilbene-tyrosine distance and a relative change in orientation of the aromatic rings. We identify several nearby charged residues that contribute to the fluorescence emission shift and provide targets for mutagenesis to probe the temperature-dependent dynamics of protein-chromophore interactions.