DiANNA 1.1: an extension of the DiANNA web server for ternary cysteine classification

DiANNA is a recent state-of-the-art artificial neural network and web server, which determines the cysteine oxidation state and disulfide connectivity of a protein, given only its amino acid sequence. Version 1.0 of DiANNA uses a feed-forward neural network to determine which cysteines are involved in a disulfide bond, and employs a novel architecture neural network to predict which half-cystines are covalently bound to which other half-cystines. In version 1.1 of DiANNA, described here, we extend functionality by applying a support vector machine with spectrum kernel for the cysteine classification problem—to determine whether a cysteine is reduced (free in sulfhydryl state), half-cystine (involved in a disulfide bond) or bound to a metallic ligand. In the latter case, DiANNA predicts the ligand among iron, zinc, cadmium and carbon. Available at:

Purification and characterization of DR_2577 (SlpA) a major S-layer protein from Deinococcus radiodurans

The protein DR_2577 is a major Surface layer component of the radio-resistant bacterium Deinococcus radiodurans. In the present study DR_2577 has been purified and its oligomeric profile characterized by means of size exclusion chromatography and gel electrophoresis. DR_2577 was found to be organized into three hierarchical orders characterized by monomers, stable dimers formed by the occurrence of disulfide bonds, and hexamers resulting from a combination of dimers. The structural implications of these findings are discussed providing new elements for a more integrated model of this S-layer

CMD: A Database to Store the Bonding States of Cysteine Motifs with Secondary Structures

Computational approaches to the disulphide bonding state and its connectivity pattern prediction are based on various descriptors. One descriptor is the amino acid sequence motifs flanking the cysteine residue motifs. Despite the existence of disulphide bonding information in many databases and applications, there is no complete reference and motif query available at the moment. Cysteine motif database (CMD) is the first online resource that stores all cysteine residues, their flanking motifs with their secondary structure, and propensity values assignment derived from the laboratory data. We extracted more than 3 million cysteine motifs from PDB and UniProt data, annotated with secondary structure assignment, propensity value assignment, and frequency of occurrence and coefficiency of their bonding status. Removal of redundancies generated 15875 unique flanking motifs that are always bonded and 41577 unique patterns that are always nonbonded. Queries are based on the protein ID, FASTA sequence, sequence motif, and secondary structure individually or in batch format using the provided APIs that allow remote users to query our database via third party software and/or high throughput screening/querying. The CMD offers extensive information about the bonded, free cysteine residues, and their motifs that allows in-depth characterization of the sequence motif composition

Folding machineries displayed on a cation-exchanger for the concerted refolding of cysteine- or proline-rich proteins

<p>Abstract</p> <p>Background</p> <p><it>Escherichia coli </it>has been most widely used for the production of valuable recombinant proteins. However, over-production of heterologous proteins in <it>E. coli </it>frequently leads to their misfolding and aggregation yielding inclusion bodies. Previous attempts to refold the inclusion bodies into bioactive forms usually result in poor recovery and account for the major cost in industrial production of desired proteins from recombinant <it>E. coli</it>. Here, we describe the successful use of the immobilized folding machineries for <it>in vitro </it>refolding with the examples of high yield refolding of a ribonuclease A (RNase A) and cyclohexanone monooxygenase (CHMO).</p> <p>Results</p> <p>We have generated refolding-facilitating media immobilized with three folding machineries, mini-chaperone (a monomeric apical domain consisting of residues 191–345 of GroEL) and two foldases (DsbA and human peptidyl-prolyl <it>cis-trans </it>isomerase) by mimicking oxidative refolding chromatography. For efficient and simple purification and immobilization simultaneously, folding machineries were fused with the positively-charged consecutive 10-arginine tag at their C-terminal. The immobilized folding machineries were fully functional when assayed in a batch mode. When the refolding-facilitating matrices were applied to the refolding of denatured and reduced RNase A and CHMO, both of which contain many cysteine and proline residues, RNase A and CHMO were recovered in 73% and 53% yield of soluble protein with full enzyme activity, respectively.</p> <p>Conclusion</p> <p>The refolding-facilitating media presented here could be a cost-efficient platform and should be applicable to refold a wide range of <it>E. coli </it>inclusion bodies in high yield with biological function.</p

Genetic characterization of interleukins (IL-1α, IL-1β, IL-2, IL-4, IL-8, IL-10, IL-12A, IL-12B, IL-15 and IL-18) with relevant biological roles in lagomorphs

Disponível em: www.ncbi.nlm.nih.gov/pmc/articles/pmid/26395994/ILs, as essential innate immune modulators, are involved in an array of biological processes. In the European rabbit (Oryctolagus cuniculus) IL-1α, IL-1β, IL-2, IL-4, IL-8, IL-10, IL-12A, IL-12B, IL-15 and IL-18 have been implicated in inflammatory processes and in the immune response against rabbit hemorrhagic disease virus and myxoma virus infections. In this study we characterized these ILs in six Lagomorpha species (European rabbit, pygmy rabbit, two cottontail rabbit species, European brown hare and American pika). Overall, these ILs are conserved between lagomorphs, including in their exon/intron structure. Most differences were observed between leporids and American pika. Indeed, when comparing both, some relevant differences were observed in American pika, such as the location of the stop codon in IL-1α and IL-2, the existence of a different transcript in IL8 and the number of cysteine residues in IL-1β. Changes at N-glycosylation motifs were also detected in IL-1, IL-10, IL-12B and IL-15. IL-1α is the protein that presents the highest evolutionary distances, which is in contrast to IL-12A where the distances between lagomorphs are the lowest. For all these ILs, sequences of human and European rabbit are more closely related than between human and mouse or European rabbit and mouse.Research Subject Categories::NATURAL SCIENCES::Biology::Cell and molecular biology::ImmunologyResearch Subject Categories::NATURAL SCIENCES::Biology::Cell and molecular biology::GeneticsFCT and North Portugal Regional Operational Programme 2007/2013 (ON.2) grants supported this work

Functionally confirmed compound heterozygous ADAM17 missense loss-of-function variants cause neonatal inflammatory skin and bowel disease 1

A disintegrin and metalloprotease 17 (ADAM17) is the major sheddase that processes more than 80 substrates, including tumour necrosis factor-α (TNFα). The homozygous genetic deficiency of ADAM17 causing a complete loss of ADAM17 expression was reported to be linked to neonatal inflammatory skin and bowel disease 1 (NISBD1). Here we report for the first time, a family with NISBD1 caused by functionally confirmed compound heterozygous missense variants of ADAM17, namely c.1699T>C (p.Cys567Arg) and c.1799G>A (p.Cys600Tyr). Both variants were detected in two siblings with clinical features of NISBD1, such as erythroderma with exudate in whole body, recurrent skin infection and sepsis and prolonged diarrhoea. In a cell-based assay using Adam10/17 double-knockout mouse embryonic fibroblasts (Adam10/17−/− mEFs) exogenously expressing each of these mutants, phorbol 12-myristate 13-acetate-stimulated shedding was strongly reduced compared with wild-type ADAM17. Thus, in vitro functional assays demonstrated that both missense variants cause the loss-of-function of ADAM17, resulting in the development of NISBD1. Our study further expands the spectrum of genetic pathology underlying ADAM17 in NISBD1 and establishes functional assay systems for its missense variants

The logic of kinetic regulation in the thioredoxin system

<p>Abstract</p> <p>Background</p> <p>The thioredoxin system consisting of NADP(H), thioredoxin reductase and thioredoxin provides reducing equivalents to a large and diverse array of cellular processes. Despite a great deal of information on the kinetics of individual thioredoxin-dependent reactions, the kinetic regulation of this system as an integrated whole is not known. We address this by using kinetic modeling to identify and describe kinetic behavioral motifs found within the system.</p> <p>Results</p> <p>Analysis of a realistic computational model of the <it>Escherichia coli </it>thioredoxin system revealed several modes of kinetic regulation in the system. In keeping with published findings, the model showed that thioredoxin-dependent reactions were adaptable (i.e. changes to the thioredoxin system affected the kinetic profiles of these reactions). Further and in contrast to other systems-level descriptions, analysis of the model showed that apparently unrelated thioredoxin oxidation reactions can affect each other via their combined effects on the thioredoxin redox cycle. However, the scale of these effects depended on the kinetics of the individual thioredoxin oxidation reactions with some reactions more sensitive to changes in the thioredoxin cycle and others, such as the Tpx-dependent reduction of hydrogen peroxide, less sensitive to these changes. The coupling of the thioredoxin and Tpx redox cycles also allowed for ultrasensitive changes in the thioredoxin concentration in response to changes in the thioredoxin reductase concentration. We were able to describe the kinetic mechanisms underlying these behaviors precisely with analytical solutions and core models.</p> <p>Conclusions</p> <p>Using kinetic modeling we have revealed the logic that underlies the functional organization and kinetic behavior of the thioredoxin system. The thioredoxin redox cycle and associated reactions allows for a system that is adaptable, interconnected and able to display differential sensitivities to changes in this redox cycle. This work provides a theoretical, systems-biological basis for an experimental analysis of the thioredoxin system and its associated reactions.</p

Hard nut to crack: Solving the disulfide linkage pattern of the Neosartorya (Aspergillus) fischeri antifungal protein 2

As a consequence of the fast resistance spreading, a limited number of drugs are available to treat fungal infections. Therefore, there is an urgent need to develop new antifungal treatment strategies. The features of a disulfide bond-stabilized antifungal protein, NFAP2 secreted by the mold Neosartorya (Aspergillus) fischeri render it to be a promising template for future protein-based antifungal drug design, which requires knowledge about the native disulfide linkage pattern as it is one of the prerequisites for biological activity. However, in the lack of tryptic and chymotryptic proteolytic sites in the ACNCPNNCK sequence, the determination of the disulfide linkage pattern of NFAP2 is not easy with traditional mass spectrometry-based methods. According to in silico predictions working with a preliminary nuclear magnetic resonance (NMR) solution structure, two disulfide isomers of NFAP2 (abbacc and abbcac) were possible. Both were chemically synthesized; and comparative reversed-phase high-performance liquid chromatography, electronic circular dichroism and NMR spectroscopy analyses, and antifungal susceptibility and efficacy tests indicated that the abbcac is the native pattern. This knowledge allowed rational modification of NAFP2 to improve the antifungal efficacy and spectrum through the modulation of the evolutionarily conserved gamma-core region, which is responsible for the activity of several antimicrobial peptides. Disruption of the steric structure of NFAP2 upon gamma-core modification led to the conclusions that this motif may affect the formation of the biologically active three-dimensional structure, and that the gamma-core modulation is not an efficient tool to improve the antifungal efficacy or to change the antifungal spectrum of NFAP2

Multidimensional Feature Engineering for Post-Translational Modification Prediction Problems

Protein sequence data has been produced at an astounding speed. This creates an opportunity to characterize these proteins for the treatment of illness. A crucial characterization of proteins is their post translational modifications (PTM). There are 20 amino acids coded by DNA after coding (translation) nearly every protein is modified at an amino acid level. We focus on three specific PTMs. First is the bonding formed between two cysteine amino acids, thus introducing a loop to the straight chain of a protein. Second, we predict which cysteines can generally be modified (oxidized). Finally, we predict which lysine amino acids are modified by the active form of Vitamin B6 (PLP/pyridoxal-5-phosphate.) Our work aims to predict the PTM\u27s from protein sequencing data. When available, we integrate other data sources to improve prediction. Data mining finds patterns in data and uses these patterns to give a confidence score to unknown PTMs. There are many steps to data mining; however, our focus is on the feature engineering step i.e. the transforming of raw data into an intelligible form for a prediction algorithm. Our primary innovation is as follows: First, we created the Local Similarity Matrix (LSM), a description of the evolutionarily relatedness of a cysteine and its neighboring amino acids. This feature is taken two at a time and template matched to other cysteine pairs. If they are similar, then we give a high probability of it sharing the same bonding state. LSM is a three step algorithm, 1) a matrix of amino acid probabilities is created for each cysteine and its neighbors from an alignment. 2) We multiply the iv square of the BLOSUM62 matrix diagonal to each of the corresponding amino acids. 3) We z-score normalize the matrix by row. Next, we innovated the Residue Adjacency Matrix (RAM) for sequential and 3-D space (integration of protein coordinate data). This matrix describes cysteine\u27s neighbors but at much greater distances than most algorithms. It is particularly effective at finding conserved residues that are further away while still remaining a compact description. More data than necessary incurs the curse of dimensionality. RAM runs in O(n) time, making it very useful for large datasets. Finally, we produced the Windowed Alignment Scoring algorithm (WAS). This is a vector of protein window alignment bit scores. The alignments are one to all. Then we apply dimensionality reduction for gains in speed and performance. WAS uses the BLAST algorithm to align sequences within a window surrounding potential PTMs, in this case PLP attached to Lysine. In the case of WAS, we tried many alignment algorithms and used the approximation that BLAST provides to reduce computational time from months to days. The performances of different alignment algorithms did not vary significantly. The applications of this work are many. It has been shown that cysteine bonding configurations play a critical role in the folding of proteins. Solving the protein folding problem will help us to find the solution to Alzheimer\u27s disease that is due to a misfolding of the amyloid-beta protein. Cysteine oxidation has been shown to play a role in oxidative stress, a situation when free radicals become too abundant in the body. Oxidative stress leads to chronic illness such as diabetes, cancer, heart disease and Parkinson\u27s. Lysine in concert with PLP catalyzes the aminotransferase reaction. Research suggests that anti-cancer drugs will potentially selectively inhibit this reaction. Others have targeted this reaction for the treatment of epilepsy and addictions