BiPPred: Combined sequence‐ and structure‐based prediction of peptide binding to the Hsp70 chaperone BiP

Substrate binding to Hsp70 chaperones is involved in many biological processes, and the identification of potential substrates is important for a comprehensive understanding of these events. We present a multi‐scale pipeline for an accurate, yet efficient prediction of peptides binding to the Hsp70 chaperone BiP by combining sequence‐based prediction with molecular docking and MMPBSA calculations. First, we measured the binding of 15mer peptides from known substrate proteins of BiP by peptide array (PA) experiments and performed an accuracy assessment of the PA data by fluorescence anisotropy studies. Several sequence‐based prediction models were fitted using this and other peptide binding data. A structure‐based position‐specific scoring matrix (SB‐PSSM) derived solely from structural modeling data forms the core of all models. The matrix elements are based on a combination of binding energy estimations, molecular dynamics simulations, and analysis of the BiP binding site, which led to new insights into the peptide binding specificities of the chaperone. Using this SB‐PSSM, peptide binders could be predicted with high selectivity even without training of the model on experimental data. Additional training further increased the prediction accuracies. Subsequent molecular docking (DynaDock) and MMGBSA/MMPBSA‐based binding affinity estimations for predicted binders allowed the identification of the correct binding mode of the peptides as well as the calculation of nearly quantitative binding affinities. The general concept behind the developed multi‐scale pipeline can readily be applied to other protein‐peptide complexes with linearly bound peptides, for which sufficient experimental binding data for the training of classical sequence‐based prediction models is not available. Proteins 2016; 84:1390–1407. © 2016 Wiley Periodicals, Inc.     

Formation, maintenance and consequences of the imprint at the mating-type locus in fission yeast

Mating-type switching in the fission yeast Schizosaccharomyces pombe is initiated by a strand-specific imprint located at the mating-type (mat1) locus. We show that the imprint corresponds to a single-strand DNA break (SSB), which is site- but not sequence-specific. We identified three novel cis-acting elements, involved in the formation and stability of the SSB. One of these elements is essential for a replication fork pause next to mat1 and interacts in vivo with the Swi1 protein. Another element is essential for maintaining the SSB during cell cycle progression. These results suggest that the DNA break appears during the S-phase and is actively protected against repair. Consequently, during the following round of replication, a polar double-strand break is formed. We show that when the replication fork encounters the SSB, the leading-strand DNA polymerase is able to synthesize DNA to the edge of the SSB, creating a blunt-ended recombination intermediate.     

Changes of the power coefficient in the 'metabolism-mass' relationship in the evolutionary process of animals

The power coefficient k decreases along evolution in an allometric relationship between the oxygen consumption rate and the body mass of animals. This theoretical study investigated the role of the power coefficient k and its behavior along evolution. The animals were organized in three groups according to the values of the power coefficient k as follows: (I) from unicellular Prokaryotes to Eukaryotes; (II) from Mytilus and Annelida to Pisces; (III) from Reptilia to Mammals and Aves. At the beginning of each animal group (stage), the value of k was close to 0.9-1.0 and at the end of the stage it was close to 0.67-0.70. Exponential sharp increase of the power coefficient k was observed during the biological transition from Protozoa to simply organized Metazoa and in the transition from Poikylothermic to Homothermic organisms (e.g. from Pisces to Reptilia). Also, when using the periodogram regression analysis, a cyclic (periodic) pattern in this increase was observed (i.e. period T approximately 8-11 units, P<0.05). It was postulated that the power coefficient k, as with the coefficient a, might represent the increase of complexity of animal organization within each group.     

Relationship between type and age of the inoculum cultures and betalains biosynthesis by Beta vulgaris hairy root culture

From a study of the relationship between the type and age of the inocula, and the growth and biosynthesis of betalains in a Beta vulgaris hairy root culture, the best results were achieved with a 14 d inoculum grown in submerged culture giving 42 mg betalains (16 mg betacyanins and 26 betaxanthins) and 1.5 g dry biomass in 40 ml medium.     

Direct somatic embryogenesis and plant regeneration of carnation (Dianthus caryophyllus L.)

Conditions for efficient direct somatic embryogenesis and plant regeneration of leaf explants from carnation cultivars Lena (SIM group) and Bulgarian spray cultivars Nasslada, Yanita, Regina and Line 84 were established. Murashige and Skoog (MS) liquid medium supplemented with 1 mg/l 2,4-dichlorophenoxyacetic acid and 0.2 mg/l 6-benzylaminopurine was used for direct induction of embryoids without an additional callus phase. The first globular structures were observed after 20 days of cultivation. Their further development to the torpedo stage was correlated with the presence of polyethylene glycol (PEG 6000). Somatic embryo maturation was promoted by casein hydrolysate (1000 mg/l) in MS liquid media. The percentage conversion of embryos and polyembryos to whole plants varied between 10 and 75% among studied cultivars. Plantlets regenerated by this procedure were morphologically identical to the donor material and developed normally in a greenhouse. Received: 29 November 1996 / Revision received: 28 April 1997 / Accepted: 28 May 1998     

Biological and Structural Basis for Aha1 Regulation of Hsp90 ATPase Activity in Maintaining Proteostasis in the Human Disease Cystic Fibrosis

The activator of Hsp90 ATPase 1, Aha1, has been shown to participate in the Hsp90 chaperone cycle by stimulating the low intrinsic ATPase activity of Hsp90. To elucidate the structural basis for ATPase stimulation of human Hsp90 by human Aha1, we have developed novel mass spectrometry approaches that demonstrate that the N- and C-terminal domains of Aha1 cooperatively bind across the dimer interface of Hsp90 to modulate the ATP hydrolysis cycle and client activity in vivo. Mutations in both the N- and C-terminal domains of Aha1 impair its ability to bind Hsp90 and stimulate its ATPase activity in vitro and impair in vivo the ability of the Hsp90 system to modulate the folding and trafficking of wild-type and variant (ΔF508) cystic fibrosis transmembrane conductance regulator (CFTR) responsible for the inherited disease cystic fibrosis (CF). We now propose a general model for the role of Aha1 in the Hsp90 ATPase cycle in proteostasis whereby Aha1 regulates the dwell time of Hsp90 with client. We suggest that Aha1 activity integrates chaperone function with client folding energetics by modulating ATPase sensitive N-terminal dimer structural transitions, thereby protecting transient folding intermediates in vivo that could contribute to protein misfolding systems disorders such as CF when destabilized.     

Volatile metabolic profiles of cell suspension cultures of Lavandula vera,Nicotiana tabacum and Helianthus annuus,cultivated under different regimes

Cell suspension cultures of Lavandula vera (Lamiaceae), Nicotiana tabacum (Solanaceae), and Helianthus annuus (Asteraceae) were cultivated in three different ways: in shake flasks both as free suspensions and in two‐phase systems (in the presence of Amberlite XAD‐4 resin as a second phase), as well as in 3‐L stirred tank reactor, and their volatile metabolic profiles were studied using GC‐MS. A number of compounds, some of them having allelochemical and biological activities, were identified in all the three cell suspension cultures under study. Also the presence of some compounds, unusual for the intact plants, was observed. It was found that the cultivation mode strongly influences the production and the transport (secretion into the culture medium) of the low‐molecular‐mass volatile metabolites. Principal component analyses of 12 common hydrocarbons showed discrimination between the different cultivation modes (shake flasks and two‐phase systems cultivation) by first principal component (PC1) and second principal component (PC2).     

Functional amyloid--from bacteria to humans

Amyloid--a fibrillar, cross beta-sheet quaternary structure--was first discovered in the context of human disease and tissue damage, and was thought to always be detrimental to the host. Recent studies have identified amyloid fibers in bacteria, fungi, insects, invertebrates and humans that are functional. For example, human Pmel17 has important roles in the biosynthesis of the pigment melanin, and the factor XII protein of the hemostatic system is activated by amyloid. Functional amyloidogenesis in these systems requires tight regulation to avoid toxicity. A greater understanding of the diverse physiological applications of this fold has the potential to provide a fresh perspective for the treatment of amyloid diseases.     

Immunolocalization of hypochlorite-induced, catalase-bound free radical formation in mouse hepatocytes

The establishment of oxidants as mediators of signal transduction has renewed the interest of investigators in oxidant production and metabolism. In particular, H(2)O(2) has been demonstrated to play pivotal roles in mediating cell differentiation, proliferation, and death. Intracellular concentrations of H(2)O(2) are modulated by its rate of production and its rate of decomposition by catalase and peroxidases. In inflammation and infection, some of the H(2)O(2) is converted to hypochlorous acid, a key mediator of the host immune response against pathogens. In vivo HOCl production is mediated by myeloperoxidase, which uses excess H(2)O(2) to oxidize Cl(-). Mashino and Fridovich (Biochim. Biophys. Acta 956:63-69; 1988) observed that a high excess of HOCl over catalase inactivated the enzyme by mechanisms that remain unclear. The potential relevance of this as an alternative mechanism for catalase activity control and its potential impact on H(2)O(2)-mediated signaling and HOCl production compelled us to explore in depth the HOCl-mediated catalase inactivation pathways. Here, we demonstrate that HOCl induces formation of catalase protein radicals and carbonyls, which are temporally correlated with catalase aggregation. Hypochlorite-induced catalase aggregation and free radical formation that paralleled the enzyme loss of function in vitro were also detected in mouse hepatocytes treated with the oxidant. Interestingly, the novel immuno-spin-trapping technique was applied to image radical production in the cells. Indeed, in HOCl-treated hepatocytes, catalase and protein-DMPO nitrone adducts were colocalized in the cells' peroxisomes. In contrast, when hepatocytes from catalase-knockout mice were treated with hypochlorous acid, there was extensive production of free radicals in the plasma membrane. Because free radicals are short-lived species with fundamental roles in biology, the possibility of their detection and localization to cell compartments is expected to open new and stimulating research venues in the interface of chemistry, biology, and medicine.     

Synthesis and biological study of new galanthamine-peptide derivatives designed for prevention and treatment of Alzheimer’s disease

Amino Acids - The Alzheimer’s disease leads to neurodegenerative processes and affecting negatively million people worldwide. The treatment of the disease is still difficult and incomplete in...