Probing the pressure-temperature stability of amyloid fibrils provides new insights into their molecular properties

A number of medical disorders, including Alzheimer's disease and type II diabetes, is characterised by the deposition of amyloid fibrils in tissue. The insolubility and size of the fibrils has largely precluded the determination of their structures at high resolution. Studies probing the stability of amyloid fibrils can reveal which non-covalent interactions are important in the formation and maintenance of the fibril structure. In particular, we review here the use of high hydrostatic pressure and high temperature as perturbation techniques. In general, small aggregates formed early in the assembly process can be dissociated by high pressure, but mature amyloid fibrils are highly pressure stable. This finding suggests that a temporal transition occurs during which side chain packing and hydrogen bond formation are optimised, whereas the hydrophobic effect and electrostatic interactions play a dominant role in the early stages of the aggregation. High temperatures, however, can disrupt most aggregates. Though the observed stability of amyloid fibrils is not unique to these structures, the notion that amyloid fibrils can represent the global minimum in free energy is supported by this type of investigations. Some implications regarding the nature of toxic species, associated with at least many of the amyloid disorders, and recently proposed structural models are discussed.     

Genome comparison provides molecular insights into the phylogeny of the reassigned new genus Lysinibacillus

Background

Lysinibacillus sphaericus (formerly named Bacillus sphaericus) is incapable of polysaccharide utilization and some isolates produce active insecticidal proteins against mosquito larvae. Its taxonomic status was changed to the genus Lysinibacillus in 2007 with some other organisms previously regarded as members of Bacillus. However, this classification is mainly based on physiology and phenotype and there is limited genomic information to support it.

Results

In this study, four genomes of L. sphaericus were sequenced and compared with those of 24 representative strains belonging to Lysinibacillus and Bacillus. The results show that Lysinibacillus strains are phylogenetically related based on the genome sequences and composition of core genes. Comparison of gene function indicates the major difference between Lysinibacillus and the two Bacillus species is related to metabolism and cell wall/membrane biogenesis. Although L. sphaericus mosquitocidal isolates are highly conserved, other Lysinibacillus strains display a large heterogeneity. It was observed that mosquitocidal toxin genes in L. sphaericus were in close proximity to genome islands (GIs) and mobile genetic elements (MGEs). Furthermore, different copies and varying genomic location of the GIs containing binA/binB was observed amongst the different isolates. In addition, a plasmid highly similar to pBsph, but lacking the GI containing binA/binB, was found in L. sphaericus SSII-1.

Conclusions

Our results confirm the taxonomy of the new genus Lysinibacillus at the genome level and suggest a new species for mosquito-toxic L. sphaericus. Based on our findings, we hypothesize that (1) Lysinibacillus strains evolved from a common ancestor and the mosquitocidal L. sphaericus toxin genes were acquired by horizontal gene transfer (HGT), and (2) capture and loss of plasmids occurs in the population, which plays an important role in the transmission of binA/binB.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1359-x) contains supplementary material, which is available to authorized users.Keyword: Lysinibacillus, Bacillus, Lysinibacillus sphaericus, Genome, Phylogeny     

Spatiotemporal dynamics of glycolytic waves provides new insights into the interactions between immobilized yeast cells and gels

The immobilization of cells or enzymes is a promising tool for the development of biosensors, yet the interactions between the fixative materials and the cells are not fully understood, especially with respect to their impact on both cell metabolism and cell-to-cell signaling. We show that the spatiotemporal dynamics of waves of metabolic synchronization of yeast cells provides a new criterion to distinguish the effect of different gels on the cellular metabolism, which otherwise could not be detected. Cells from the yeast Saccharomyces carlsbergensis were immobilized into agarose gel, silica gel (TMOS), or a mixture of TMOS and alginate. We compared these immobilized cells with respect to their ability to generate temporal, intracellular oscillations in glycolysis as well as propagating, extracellular synchronization waves. While the temporal dynamics, as measured by the period and the number of oscillatory cycles, was similar for all three immobilized cell populations, significant differences have been observed with respect to the shape of the waves, wave propagation direction and velocity in the three gel matrices used.     

Analysis of codon:anticodon interactions within the ribosome provides new insights into codon reading and the genetic code structure.

Although the decoding rules have been largely elucidated, the physical-chemical reasons for the "correctness" of codon:anticodon duplexes have never been clear. In this work, on the basis of the available data, we propose that the correct codon:anticodon duplexes are those whose formation and interaction with the ribosomal decoding center are not accompanied by uncompensated losses of hydrogen and ionic bonds. Other factors such as proofreading, base-base stacking and aminoacyl-tRNA concentration contribute to the efficiency and accuracy of aminoacyl-tRNA selection, and certainly these factors are important; but we suggest that analyses of hydrogen and ionic bonding alone provides a robust first-order approximation of decoding accuracy. Thus our model can simplify predictions about decoding accuracy and error. The model can be refined with data, but is already powerful enough to explain all of the available data on decoding accuracy. Here we predict which duplexes should be considered correct, which duplexes are responsible for virtually all misreading, and we suggest an evolutionary scheme that gave rise to the mixed boxes of the genetic code.     

Proteotypic profiling of LHCI from Chlamydomonas reinhardtii provides new insights into structure and function of the complex

We used isotope dilution MS to measure the stoichiometry of light‐harvesting complex I (LHCI) proteins with the photosystem I (PSI) core complex in the green alga Chlamydomonas reinhardtii. Proteotypic peptides served as quantitative markers for each of the nine gene products (Lhca1–9) and for PSI subunits. The quantitative data revealed that the LHCI antenna of C. reinhardtii contains about 7.5 ± 1.4 subunits. It further demonstrated that the thylakoid LHCI population is heterogeneously composed and that several lhca gene products are not present in 1:1 stoichiometries with PSI. When compared with vascular plants, LHCI of C. reinhardtii possesses a lower proportion of proteins potentially contributing to far‐red fluorescence emission. In general, the strategy presented is universally applicable for exploring subunit stoichiometries within the C. reinhardtii proteome.     

Pressure provides new insights into protein folding, dynamics and structure

Hydrostatic pressure is a powerful tool for studying protein folding, and the dynamics and structure of folding intermediates. Recently, pressure techniques have opened two important fronts to aid our understanding of how polypeptides fold into highly structured conformations. The first advance is the stabilization of folding intermediates, making it possible to characterize their structures and dynamics by different methodologies. Kinetic studies under pressure constitute the second advance, promising detailed appraisal and understanding of protein folding landscapes. The combination of these two approaches enables dissection of the roles of packing and cavities in folding, and in assembly of multimolecular structures such as protein-DNA complexes and viruses. The study of aggregates and amyloids, derived from partially folded intermediates at the junction between productive and off-pathway folding, have also been studied, promising better understanding of diseases associated with protein misfolding.     

Virus-host interactions: new insights from the small RNA world

RNA silencing has a known role in the antiviral responses of plants and insects. Recent evidence, including the finding that the Tat protein of human immunodeficiency virus (HIV) can suppress the host's RNA-silencing pathway and may thus counteract host antiviral RNAs, suggests that RNA-silencing pathways could also have key roles in mammalian virus-host interactions.     

Ancient DNA provides new insights into the history of south Siberian Kurgan people

To help unravel some of the early Eurasian steppe migration movements, we determined the Y-chromosomal and mitochondrial haplotypes and haplogroups of 26 ancient human specimens from the Krasnoyarsk area dated from between the middle of the second millennium BC. to the fourth century AD. In order to go further in the search of the geographic origin and physical traits of these south Siberian specimens, we also typed phenotype-informative single nucleotide polymorphisms. Our autosomal, Y-chromosomal and mitochondrial DNA analyses reveal that whereas few specimens seem to be related matrilineally or patrilineally, nearly all subjects belong to haplogroup R1a1-M17 which is thought to mark the eastward migration of the early Indo-Europeans. Our results also confirm that at the Bronze and Iron Ages, south Siberia was a region of overwhelmingly predominant European settlement, suggesting an eastward migration of Kurgan people across the Russo-Kazakh steppe. Finally, our data indicate that at the Bronze and Iron Age timeframe, south Siberians were blue (or green)-eyed, fair-skinned and light-haired people and that they might have played a role in the early development of the Tarim Basin civilization. To the best of our knowledge, no equivalent molecular analysis has been undertaken so far.     

Conditional switching of VEGF provides new insights into adult neovascularization and pro-angiogenic therapy

To gain insight into neovascularization of adult organs and to uncover inherent obstacles in vascular endothelial growth factor (VEGF)-based therapeutic angiogenesis, a transgenic system for conditional switching of VEGF expression was devised. The system allows for a reversible induction of VEGF specifically in the heart muscle or liver at any selected schedule, thereby circumventing embryonic lethality due to developmental misexpression of VEGF. Using this system, we demonstrate a progressive, unlimited ramification of the existing vasculature. In the absence of spatial cues, however, abnormal vascular trees were produced, a consequence of chaotic connections with the existing network and formation of irregularly shaped sac-like vessels. VEGF also caused a massive and highly disruptive edema. Importantly, premature cessation of the VEGF stimulus led to regression of most acquired vessels, thus challenging the utility of therapeutic approaches relying on short stimulus duration. A critical transition point was defined beyond which remodeled new vessels persisted for months after withdrawing VEGF, conferring a long-term improvement in organ perfusion. This novel genetic system thus highlights remaining problems in the implementation of pro-angiogenic therapy.     

Comparative proteomic analysis provides new insights into the fiber elongating process in cotton

A comparative proteomic analysis was performed to explore the mechanism of cell elongation in developing cotton fibers. The temporal changes of global proteomes at five representative development stages (5-25 days post-anthesis [dpa]) were examined using 2-D electrophoresis. Among approximately 1800 stained protein spots reproducibly detected on each gel, 235 spots were differentially expressed with significant dynamics in elongating fibers. Of these, 120 spots showed a more than 2-fold change in at least one stage point, and 21 spots appeared to be specific to developmental stages. Furthermore, 106 differentially expressed proteins were identified from mass spectrometry to match 66 unique protein species. These proteins involve different cellular and metabolic processes with obvious functional tendencies toward energy/carbohydrate metabolism, protein turnover, cytoskeleton dynamics, cellular responses and redox homeostasis, indicating a good correlation between development-dependent proteins and fiber biochemical processes, as well as morphogenesis. Newly identified proteins such as phospholipase D alpha, vf14-3-3 protein, small ras-related protein, and GDP dissociation inhibitor will advance our knowledge of the complicated regulatory network. Identification of these proteins, combined with their changes in abundance, provides a global view of the development-dependent protein changes in cotton fibers, and offers a framework for further functional research of target proteins associated with fiber development.     

Functional and molecular insights into KSRP function in mRNA decay

KSRP is a single strand nucleic acid binding protein that controls gene expression at multiple levels. In this review we focus on the recent molecular, cellular, and structural insights into the mRNA decay promoting function of KSRP. We discuss also some aspects of KSRP-dependent microRNA maturation from precursors that are related to its mRNA destabilizing function. This article is part of a Special Issue entitled: RNA Decay mechanisms.     

Analysis of the phosphoproteome of Chlamydomonas reinhardtii provides new insights into various cellular pathways

The unicellular flagellated green alga Chlamydomonas reinhardtii has emerged as a model organism for the study of a variety of cellular processes. Posttranslational control via protein phosphorylation plays a key role in signal transduction, regulation of gene expression, and control of metabolism. Thus, analysis of the phosphoproteome of C. reinhardtii can significantly enhance our understanding of various regulatory pathways. In this study, we have grown C. reinhardtii cultures in the presence of an inhibitor of Ser/Thr phosphatases to increase the phosphoprotein pool. Phosphopeptides from these cells were enriched by immobilized metal-ion affinity chromatography and analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry (MS) with MS-MS as well as neutral-loss-triggered MS-MS-MS spectra. In this way, we were able to identify 360 phosphopeptides from 328 different phosphoproteins of C. reinhardtii, thus providing new insights into a variety of cellular processes, including metabolic and signaling pathways. Comparative analysis of the phosphoproteome also yielded new functional information on proteins controlled by redox regulation (thioredoxin target proteins) and proteins of the chloroplast 70S ribosome, the centriole, and especially the flagella, for which 32 phosphoproteins were identified. The high yield of phosphoproteins of the latter correlates well with the presence of several flagellar kinases and indicates that phosphorylation/dephosphorylation represents one of the key regulatory mechanisms of eukaryotic cilia. Our data also provide new insights into certain cilium-related mammalian diseases.     

Molecular modeling of human neutral sphingomyelinase provides insight into its molecular interactions

The neutral sphingomyelinase (N-SMase) is considered a major candidate for mediating the stress-induced production of ceramide, and it plays an important role in cell-cycle arrest, apoptosis, inflammation, and eukaryotic stress responses. Recent studies have identified a small region at the very N-terminus of the 55 kDa tumour necrosis factor receptor (TNF-R55), designated the neutral sphingomyelinase activating domain (NSD) that is responsible for the TNF-induced activation of N-SMase. There is no direct association between TNF-R55 NSD and N-SMase; instead, a protein named factor associated with N-SMase activation (FAN) has been reported to couple the TNF-R55 NSD to N-SMase. Since the three-dimensional fold of N-SMase is still unknown, we have modeled the structure using the protein fold recognition and threading method. Moreover, we propose models for the TNF-R55 NSD as well as the FAN protein in order to study the structural basis of N-SMase activation and regulation. Protein-protein interaction studies suggest that FAN is crucially involved in mediating TNF-induced activation of the N-SMase pathway, which in turn regulates mitogenic and proinflammatory responses. Inhibition of N-SMase may lead to reduction of ceramide levels and hence may provide a novel therapeutic strategy for inflammation and autoimmune diseases. Molecular dynamics (MD) simulations were performed to check the stability of the predicted model and protein-protein complex; indeed, stable RMS deviations were obtained throughout the simulation. Furthermore, in silico docking of low molecular mass ligands into the active site of N-SMase suggests that His135, Glu48, Asp177, and Asn179 residues play crucial roles in this interaction. Based on our results, these ligands are proposed to be potent and selective N-SMase inhibitors, which may ultimately prove useful as lead compounds for drug development.