Unraveling the Polysaccharide Biosynthesis Potential of Ganoderma lucidum: A Chromosome-Level Assembly Using Hi-C Sequencing.

Ganoderma lucidum exhibits the ability to synthesize a diverse range of biologically active molecules with significant pharmaceutical potential, including xylomannan and fucogalactan, which have demonstrated antitumor activity. However, there exists considerable intra-species variability in the capacity to produce these metabolites at high concentrations, likely reflecting the high genomic diversity observed from a limited number of strains sequenced to date. We employed high-throughput shotgun sequencing to obtain the complete genome sequence of G. lucidum strain 5.1, which is distinguished by its remarkable xylomannan synthesis capabilities. Through the utilization of semi-automatic reordering based on conformation capture (Hi-C) data, we substantially enhanced the assembly process, resulting in the generation of 12 chromosome-level scaffolds with a cumulative length of 39 Mbp. By employing both de novo and homology-based approaches, we performed comprehensive annotation of the genome, thereby identifying a diverse repertoire of genes likely involved in polysaccharide biosynthesis. The genome sequence generated in this study serves as a valuable resource for elucidating the molecular mechanisms underlying the medicinal potential of Ganoderma species, discovering novel pharmaceutically valuable compounds, and elucidating the ecological mechanisms of the species. Furthermore, the chromosome contact map obtained for the first time for this species extends our understanding of 3D fungal genomics and provides insights into the functional and structural organization within the fungal kingdom.

The Strategies of Development of New Non-Toxic Inhibitors of Amyloid Formation.

In recent years, due to the aging of the population and the development of diagnostic medicine, the number of identified diseases associated with the accumulation of amyloid proteins has increased. Some of these proteins are known to cause a number of degenerative diseases in humans, such as amyloid-beta (Aß) in Alzheimer's disease (AD), α-synuclein in Parkinson's disease (PD), and insulin and its analogues in insulin-derived amyloidosis. In this regard, it is important to develop strategies for the search and development of effective inhibitors of amyloid formation. Many studies have been carried out aimed at elucidating the mechanisms of amyloid aggregation of proteins and peptides. This review focuses on three amyloidogenic peptides and proteins-Aß, α-synuclein, and insulin-for which we will consider amyloid fibril formation mechanisms and analyze existing and prospective strategies for the development of effective and non-toxic inhibitors of amyloid formation. The development of non-toxic inhibitors of amyloid will allow them to be used more effectively for the treatment of diseases associated with amyloid.

Characteristics of bacterial and yeast microbiomes in spontaneous and mixed-fermentation beer and cider.

The production of experimental beer and cider products has increased, worldwide. The complex microbiomes found in these beverages affect their organoleptic qualities and chemical compositions and can have diverse impacts on human health. The total diversity of a microbiome can be elucidated through the use of high-throughput sequencing and comprehensive data analysis tools. We analysed the bacterial and yeast microbiomes found in mixed and spontaneously fermented beers (n = 14) and unpasteurised apple ciders (n = 6), using high-throughput 16S rRNA and internal transcribed spacer (ITS) sequencing. The ratio of bacteria to yeast was measured using quantitative polymerase chain reaction (qPCR), and short-chain organic acids were analysed using high-performance liquid chromatography (HPLC). An upgraded version of the Knomics-Biota system was used to analyse the data. The microbiomes included both starter microorganisms and those that originate from the production environment and the raw materials. In addition to the common Saccharomyces and Brettanomyces, the yeast diversity included many non-conventional species. The bacterial community in beer was dominated by Lactobacillus species, whereas these communities were more diverse in cider. Lactobacillus acetotolerans was prevalent in wild ales, whereas Candida ethanolica was prevalent in cask-matured beverages. We observed complex patterns of subspecies-level yeast diversity across beer styles, breweries, and countries. Our study represents an exploratory analysis of non-conventional beer and cider microbiomes and metabolomes, which contributes information necessary to develop improved quality control processes and may drive innovative product development in experimental and artisanal brewing.

Is there codon usage bias for poly-Q stretches in the human proteome?

We have analyzed codon usage for poly-Q stretches of different lengths for the human proteome. First, we have obtained that all long poly-Q stretches in Protein Data Bank (PDB) belong to the disordered regions. Second, we have found the bias for codon usage for glutamine homo-repeats in the human proteome. In the cases when the same codon is used for poly-Q stretches only CAG triplets are found. Similar results are obtained for human proteins with glutamine homo-repeats associated with diseases. Moreover, for proteins associated with diseases (from the HraDis database), the fraction of proteins for which the same codon is used for glutamine homo-repeats is less (22%) than for proteins from the human proteome (26%). We have demonstrated for poly-Q stretches in the human proteome that in some cases (28) the splicing sites correspond to the homo-repeats and in 11 cases, these sites appear at the C -terminal part of the homo-repeats with statistical significance 10 -8 .

Ile351, Leu355 and Ile461 residues are essential for catalytic activity of bovine cytochrome P450scc (CYP11A1).

Cytochrome P450scc (CYP11A1) is a mammalian mitochondrial enzyme which catalyzes cholesterol side chain cleavage to form pregnenolone. Along with cholesterol, some other steroids including sterols with a branched side chain like ß-sitosterol are the substrates for the enzyme, but the activity towards ß-sitosterol is rather low. Modification of the catalytic site conformation could provide more effective ß-sitosterol bioconversion by the enzyme. This study was aimed to find out the amino acid residues substitution of which could modify the conformation of the active site providing possible higher enzyme activity towards ß-sitosterol. After structural and bioinformatics analysis three amino acid residues I351, L355, I461 were chosen. Molecular dynamics simulations of P450scc evidenced the stability of the wild type, double (I351A/L355A) and triple (I351A/L355A/I461A) mutants. Mutant variants of cDNA encoding P450scc with the single, double and triple mutations were obtained by site-directed mutagenesis. However, the experimental data indicate that the introduced single mutations Ile351A, Leu355A and Ile461A dramatically decrease the target catalytic activity of CYP11A1, and no activity was observed for double and triple mutants obtained. Therefore, isoleucine residues 351 and 461, and leucine residue 355 are important for the cytochrome P450scc functioning towards sterols both with unbranched (cholesterol) and branched (sitosterol) side chains.

Knomics-Biota - a system for exploratory analysis of human gut microbiota data.

BACKGROUND: Metagenomic surveys of human microbiota are becoming increasingly widespread in academic research as well as in food and pharmaceutical industries and clinical context. Intuitive tools for investigating experimental data are of high interest to researchers. RESULTS: Knomics-Biota is a web-based resource for exploratory analysis of human gut metagenomes. Users can generate and share analytical reports corresponding to common experimental schemes (like case-control study or paired comparison). Interactive visualizations and statistical analysis are provided in association with the external factors and in the context of thousands of publicly available datasets arranged into thematic collections. The web-service is available at https://biota.knomics.ru. CONCLUSIONS: Knomics-Biota web service is a comprehensive tool for interactive metagenomic data analysis.

What is Responsible for Atypical Dependence of the Rate of Amyloid Formation on Protein Concentration: Fibril-Catalyzed Initiation of New Fibrils or Competition with Oligomers?

An abnormal dependence of the rate of amyloid formation on protein concentration has been recently observed by Meisl et al. for Aß40 peptides associated with Alzheimer's disease. To explain this effect, Meisl et al. proposed a novel mechanism of fibril growth: the fibril-catalyzed initiation of fibril formation. In this paper we offer an alternative explanation of the observed anomalous kinetics: formation of metastable oligomers competing with fibril formation by decreasing the concentration of the fibril-forming free monomers. Here we show that the oligomer sizes resulting from the anomalous dependence of the fibril growth rate on protein concentration are close to the sizes of oligomers observed by electron microscopy.

Insulin and Lispro Insulin: What is Common and Different in their Behavior?

There are different insulin analogues with various pharmacokinetic characteristics, such as, rapid-acting, long-acting, or intermediate-acting analogues. Since insulin tends to form amyloid aggregates, it is of particular interest to measure characteristic times of formation of amyloid aggregates and compare those to action times for insulin and its analogues. For the study we have chosen one of the insulin analogues - insulin Lispro, which is a fast acting insulin analog. It is usually thought of amyloid aggregation as a nucleation-dependent process. We have estimated the size of the primary nucleus to be one monomer and the size of the secondary nucleus to be around zero in both insulin and Lispro insulin aggregation processes. The main structural element of insulin and Lispro insulin amyloid fibrils is a rounded ring oligomer of about 6-7 nm in diameter, about 2-3 nm in height and about 2 nm in diameter of the hole. Fibrils of several µm in length are produced due to interaction of such oligomers. The packing of ring oligomers in fibrils differs because of the difference in their orderliness. Though the initial stages of fibril formation (monomer, oligomer) are similar, the further process depends on the unique sequence of each peptide. Namely the sequence affects the final morphology of mature amyloids. These observations allow us to conclude that formation of fibrils by short peptides occurs via and by means of oligomer ring structures. Such an important issue as the nature of polymorphism of insulin amyloid fibrils has been settled by us. The role of early oligomeric aggregates in such processes as nucleation and aggregation of amyloid fibrils has been examined.

Structural model of amyloid fibrils for amyloidogenic peptide from Bgl2p-glucantransferase of S. cerevisiae cell wall and its modifying analog. New morphology of amyloid fibrils.

We performed a comparative study of the process of amyloid formation by short homologous peptides with a substitution of aspartate for glutamate in position 2 - VDSWNVLVAG (AspNB) and VESWNVLVAG (GluNB) - with unblocked termini. Peptide AspNB (residues 166-175) corresponded to the predicted amyloidogenic region of the protein glucantransferase Bgl2 from the Saccharomyces cerevisiae cell wall. The process of amyloid formation was monitored by fluorescence spectroscopy (FS), electron microscopy (EM), tandem mass spectrometry (TMS), and X-ray diffraction (XD) methods. The experimental study at pH3.0 revealed formation of amyloid fibrils with similar morphology for both peptides. Moreover, we found that the morphology of fibrils made of untreated ammonia peptide is not mentioned in the literature. This morphology resembles snakes lying side by side in the form of a wave without intertwining. Irrespective of the way of the peptide preparation, the rate of fibril formation is higher for AspNB than for GluNB. However, preliminary treatment with ammonia highly affected fibril morphology especially for AspNB. Such treatment allowed us to obtain a lag period during the process of amyloid formation. It showed that the process was nucleation-dependent. With or without treatment, amyloid fibrils consisted of ring-like oligomers with the diameter of about 6nm packed either directly ring-to-ring or ring-on-ring with a slight shift. We also proposed the molecular structure of amyloid fibrils for two studied peptides.

The Mechanism Underlying Amyloid Polymorphism is Opened for Alzheimer's Disease Amyloid-ß Peptide.

It has been demonstrated using Aß40 and Aß42 recombinant and synthetic peptides that their fibrils are formed of complete oligomer ring structures. Such ring structures have a diameter of about 8-9 nm, an oligomer height of about 2- 4 nm, and an internal diameter of the ring of about 3-4 nm. Oligomers associate in a fibril in such a way that they interact with each other, overlapping slightly. There are differences in the packing of oligomers in fibrils of recombinant and synthetic Aß peptides. The principal difference is in the degree of orderliness of ring-like oligomers that leads to generation of morphologically different fibrils. Most ordered association of ring-like structured oligomers is observed for a recombinant Aß40 peptide. Less ordered fibrils are observed with the synthetic Aß42 peptide. Fragments of fibrils the most protected from the action of proteases have been determined by tandem mass spectrometry. It was shown that unlike Aß40, fibrils of Aß42 are more protected, showing less ordered organization compared to that of Aß40 fibrils. Thus, the mass spectrometry data agree with the electron microscopy data and structural models presented here.

Smooth muscle titin forms in vitro amyloid aggregates.

Amyloids are insoluble fibrous protein aggregates, and their accumulation is associated with amyloidosis and many neurodegenerative diseases, including Alzheimer's disease. In the present study, we report that smooth muscle titin (SMT; 500 kDa) from chicken gizzard forms amyloid aggregates in vitro This conclusion is supported by EM data, fluorescence analysis using thioflavin T (ThT), Congo red (CR) spectroscopy and X-ray diffraction. Our dynamic light scattering (DLS) data show that titin forms in vitro amyloid aggregates with a hydrodynamic radius (Rh) of approximately 700-4500 nm. The initial titin aggregates with Rh approximately 700 nm were observed beyond first 20 min its aggregation that shows a high rate of amyloid formation by this protein. We also showed using confocal microscopy the cytotoxic effect of SMT amyloid aggregates on smooth muscle cells from bovine aorta. This effect involves the disorganization of the actin cytoskeleton and result is cell damage. Cumulatively, our results indicate that titin may be involved in generation of amyloidosis in smooth muscles.

One of the possible mechanisms of amyloid fibrils formation based on the sizes of primary and secondary folding nuclei of Aß40 and Aß42.

In the presented paper, theoretical as well as electron microscopy and X-ray diffraction experimental approaches were employed for studding the process of Aß amyloid formation. Using quantitative estimates of a number of monomers which form the nuclei of amyloid fibrils the sizes of folding nuclei of amyloid fibrils for Aß40 and 42 have been determined for the first time. We have shown that the size of the primary nucleus of Aß42 peptide fibrils corresponds to 3 monomers, the size of the secondary nucleus for this peptide is 2 monomers. Applying the same analysis to Aß40 we conclude that the size of the primary nucleus is 2 monomers, and the size of the secondary nucleus is one monomer. Summation of our theoretical and experimental results has allowed us to propose a new model of the structural organization of amyloid fibrils. Our model suggests that the generation of fibrils takes place along the following simplified pathway: a monomer→a ring oligomer→a mature fibril consisting of ring oligomers. These data shed more light upon our understanding of what sizes of the oligomers could represent main targets for future therapies (tetramers for Aß42 and trimers for Aß40), and aid in the development of inhibitors of Aß40 and 42 oligomer formation.

Computational Approaches to Identification of Aggregation Sites and the Mechanism of Amyloid Growth.

This chapter describes computational approaches to study amyloid formation. The first part addresses identification of potential amyloidogenic regions in the amino acid sequences of proteins and peptides. Next, we discuss nucleation and aggregation sites in protein folding and misfolding. The last part describes up-to-date kinetic models of amyloid fibrils formation. Numerous studies show that protein misfolding is initiated by specific amino acid segments with high amyloid-forming propensity. The ability to identify and, ultimately, block such segments is very important. To this end, many prediction algorithms have been developed which vary greatly in their effectiveness. We compared the predictions for 30 proteins by using different methods and found that, at best, only 50% of residues in amyloidogenic segments were predicted correctly. The best results were obtained by using the meta-servers that combine several independent approaches, and by the method PASTA2. Thus, correct prediction of amyloidogenic segments remains a difficult task. Additional data and new algorithms that are becoming available are expected to improve the accuracy of the prediction methods, particularly if they use 3D structural information on the target proteins. At the same time, our understanding of the kinetics of fibril formation is more advanced. The current kinetic models outlined in this chapter adequately describe the key features of amyloid nucleation and growth. However, the underlying structural details are less clear, not least because of the apparently different mechanisms of amyloid fibril formation which are discussed. Ultimately, the detailed understanding of the structural basis for amyloidogenesis should help develop rational therapies to block this pathogenic process.

What handedness and angles between helices has the studied three-helical protein domain?

We have created a new server FoldHandedness. Using this server it is possible: (i) to define the regions of helices from two issues (from the PDB file and using the last version of the DSSP program), (ii) to determine the handedness for any chosen three helices and (iii) to calculate the angle and sign between the chosen pairs of the helices for large proteins and complexes of proteins with DNA or RNA.

How to determine the size of folding nuclei of protofibrils from the concentration dependence of the rate and lag-time of aggregation. II. Experimental application for insulin and LysPro insulin: aggregation morphology, kinetics, and sizes of nuclei.

Insulin is a commonly used protein for studies of amyloidogenesis. There are a few insulin analogues with different pharmacokinetic characteristics, in particular the onset and duration of action. One of them is LysPro insulin. The behavior of LysPro insulin in the process of amyloid formation has not been studied in detail yet. To quantitatively investigate the differences between insulin and LysPro insulin in the aggregation reaction, we used thioflavin T fluorescence assay, electron microscopy, X-ray diffraction methods, and theoretical modeling. Kinetic experimental data for both insulin samples demonstrated the increase of the lag-time for LysPro insulin at low concentrations of monomers, particularly at 2 and 4 mg/mL, which corresponds to the pharmaceutical concentration. However, the morphology of insulin and LysPro insulin fibrils and their X-ray diffraction patterns is identical. Mature fibrils reach 10-12 µm in length and about 3-4 nm in diameter. The obtained analytical solution allow us to determine the sizes of the primary and secondary nuclei from the experimentally obtained concentration dependences of the time of growth and the ratio of the lag-time duration to the time of growth of amyloid protofibrils. In the case of insulin and LysPro insulin, we have exponential growth of amyloid protofibrils following the "bifurcation + lateral growth" scenario. In accord with the developed theory and the experimental data, we obtained that the size of the primary nucleus is equal to one monomer and the size of the secondary nucleus is zero in both insulin and LysPro insulin.

How to determine the size of folding nuclei of protofibrils from the concentration dependence of the rate and lag-time of aggregation. I. Modeling the amyloid protofibril formation.

The question about the size of nuclei of formation of protofibrils (which constitute mature amyloid fibrils) formed by different proteins and peptides is yet open and debatable because of the absence of solid knowledge of underlying mechanisms of amyloid formation. In this work, a kinetic model of the process of formation of amyloid protofibrils is suggested, which allows calculation of the size of the nuclei using only kinetic data. In addition to the stage of primary nucleation, which is believed to be present in many protein aggregation processes, the given model includes both linear growth of protofibrils (proceeding only at the cost of attaching of monomers to the ends) and exponential growth of protofibrils at the cost of growth from the surface, branching, and fragmentation with the secondary nuclei. Theoretically, only the exponential growth is compatible with the existence of a pronounced lag-period (which can take much more time then the growth of aggregates themselves). The obtained analytical solution allows us to determine the size of the primary and secondary nuclei from the experimentally obtained concentration dependences of the time of growth and the new parameter-the ratio Lrel of the lag-time duration to the time of growth of amyloid protofibrils.

A novel web server predicts amino acid residue protection against hydrogen-deuterium exchange.

MOTIVATION: To clarify the relationship between structural elements and polypeptide chain mobility, a set of statistical analyses of structures is necessary. Because at present proteins with determined spatial structures are much less numerous than those with amino acid sequence known, it is important to be able to predict the extent of proton protection from hydrogen-deuterium (HD) exchange basing solely on the protein primary structure. RESULTS: Here we present a novel web server aimed to predict the degree of amino acid residue protection against HD exchange solely from the primary structure of the protein chain under study. On the basis of the amino acid sequence, the presented server offers the following three possibilities (predictors) for user's choice. First, prediction of the number of contacts occurring in this protein, which is shown to be helpful in estimating the number of protons protected against HD exchange (sensitivity 0.71). Second, probability of H-bonding in this protein, which is useful for finding the number of unprotected protons (specificity 0.71). The last is the use of an artificial predictor. Also, we report on mass spectrometry analysis of HD exchange that has been first applied to free amino acids. Its results showed a good agreement with theoretical data (number of protons) for 10 globular proteins (correlation coefficient 0.73). We pioneered in compiling two datasets of experimental HD exchange data for 35 proteins. AVAILABILITY: The H-Protection server is available for users at http://bioinfo.protres.ru/ogp/ SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Prediction of loop regions in protein sequence.

We suggest an algorithm that inputs a protein sequence and outputs a decomposition of the protein chain into a regular part including secondary structures and a nonregular part corresponding to loop regions. We have analyzed loop regions in a protein dataset of 3,769 globular domains and defined the optimal parameters for this prediction: the threshold between regular and nonregular regions and the optimal window size for averaging procedures using the scale of the expected number of contacts in a globular state and entropy scale as the number of degrees of freedom for the angles phi, psi, and chi for each amino acid. Comparison with known methods demonstrates that our method gives the same results as the well-known ALB method based on physical properties of amino acids (the percentage of true predictions is 64% against 66%), and worse prediction for regular and nonregular regions than PSIPRED (Protein Structure Prediction Server) without alignment of homologous proteins (the percentage of true predictions is 73%). The potential advantage of the suggested approach is that the predicted set of loops can be used to find patterns of rigid and flexible loops as possible candidates to play a structure/function role as well as a role of antigenic determinants.

Many-atom van der Waals interactions lead to direction-sensitive interactions of covalent bonds.

Strict physical theory and numerical calculations show that a specific coupling of many-atom van der Waals interactions with covalent bonding can significantly (half as much) increase the strength of attractive dispersion interactions when the direction of interaction coincides with the direction of the covalent bond, and decrease this strength when the direction of interaction is perpendicular to the direction of the covalent bond. The energy effect is comparable to that caused by the replacement of atoms (e.g. N by C or O) in conventional pairwise van der Waals interactions. Analysis of protein structures shows that they bear an imprint of this effect. This means that many-atom van der Waals interactions cannot be ignored in refinement of protein structures, in simulations of their folding, and in prediction of their binding affinities.

Prediction of number and position of domain boundaries in multi-domain proteins by use of amino acid sequence alone.

Prediction of protein domain boundaries is an important step for the prediction of three-dimensional structure. The simple method PDP has been elaborated for prediction of the number and position of domain boundaries in multi-domain proteins by use of amino acid sequence alone. The method uses an optimized scale based on the statistics of appearance of amino acid residues at domain boundaries. Our method demonstrates promising results in comparison to other methods that do not use homologous sequences. From the database of proteins that are targets from CASP6 (Critical Assessment of Techniques for Protein Structure Prediction) our program correctly assigned the number of domains for approximately 80% of one domain proteins and approximately 50% for two-domain proteins. Our method offers three main advantages: it is very simple, it is fast, and it uses a minimal number of parameters in comparison with other methods.