Reversal of aggregation using ?-breaker dipeptide containing peptides: Application to A?(1-40) self-assembly and its inhibition.
ABSTRACT: Reversion of protein or peptide aggregation is a formidable task, important in various domains of research at the interface of chemistry, medicine, and nanoscience. A novel class of dipeptides, termed as ?-breaker dipeptides (BBDPs), is identified, which can be incorporated into the self-recognizing sequences to generate a novel class of conformational switch which forms ?-sheet at an initial stage and then converts in a controlled manner to random coil at specific conditions. Incorporation of BBDPs in a well designed amyloidogenic peptides generates a special class of ?-sheet breaker peptides those undergo a chemical change at physiological condition generating a breaker element in situ. These ?-breaker peptides are shown to first incorporate into the amyloid and then disrupt it. Such conformational switches may be used to study agrregation/disaggregation process and may find many biomedical applications relevant to aggregation related disorders. Such strategy for reversion of peptide aggregation using chemical tricks may find application in material chemistry as well.
Project description:The aggregation of amyloidogenic proteins/peptides has been closely linked to the neuropathology of several important neurological disorders. In Alzheimer's disease, amyloid beta (A?) peptides and their aggregation are believed to be at least partially responsible for the etiology of Alzheimer's disease. The aggregate-inflicted cellular toxicity can be inhibited by short peptides whose sequences are homologous to segments of the A?(1-42) peptide responsible for ?-sheet stacking (referred to as the ?-sheet breaker peptides). Here, a water-soluble ferrocene (Fc)-tagged ?-sheet breaker peptide, Fc-KLVFFK(6), was used as an electrochemical probe for kinetic studies of the inhibition of the A?(1-42) fibrillation process and for determination of the optimal concentration of ?-sheet breaker peptide for efficient inhibition. Our results demonstrate that Fc-KLVFFK(6) interacts with the A? aggregates instantaneously in solution, and a sub-stoichiometric amount of Fc-KLVFFK(6) is sufficient to inhibit the formation of the A? oligomers and fibrils and to reduce the toxicity of A?(1-42). The interaction between Fc-KLVFFK(6) and A?(1-42) follows a pseudo-first-order reaction, with a rate constant of 1.89 ± 0.05 × 10(-4) s(-1). Tagging ?-sheet breaker peptides with a redox label facilitates design, screening, and rational use of peptidic inhibitors for impeding/altering A? aggregation.
Project description:In this work we present and compare the results of extensive molecular dynamics simulations of model systems comprising an Aβ1-40 peptide in water in interaction with short peptides (β-sheet breakers) mimicking the 17-21 region of the Aβ1-40 sequence. Various systems differing in the customized β-sheet breaker structure have been studied. Specifically we have considered three kinds of β-sheet breakers, namely Ac-LPFFD-NH2 and two variants thereof, one obtained by substituting the acetyl group with the sulfonic amino acid taurine (Tau-LPFFD-NH2) and a second novel one in which the aspartic acid is substituted by an asparagine (Ac-LPFFN-NH2). Thioflavin T fluorescence, circular dichroism, and mass spectrometry experiments have been performed indicating that β-sheet breakers are able to inhibit in vitro fibril formation and prevent the β sheet folding of portions of the Aβ1-40 peptide. We show that molecular dynamics simulations and far UV circular dichroism provide consistent evidence that the new Ac-LPFFN-NH2 β-sheet breaker is more effective than the other two in stabilizing the native α-helix structure of Aβ1-40. In agreement with these results thioflavin T fluorescence experiments confirm the higher efficiency in inhibiting Aβ1-40 aggregation. Furthermore, mass spectrometry data and molecular dynamics simulations consistently identified the 17-21 Aβ1-40 portion as the location of the interaction region between peptide and the Ac-LPFFN-NH2 β-sheet breaker.
Project description:Alzheimer's disease (AD) is characterized by the cerebral accumulation of misfolded and aggregated amyloid-beta protein (Abeta). Disease symptoms can be alleviated, in vitro and in vivo, by 'beta-sheet breaker' pentapeptides that reduce plaque load. However the peptide nature of these compounds, made them biologically unstable and unable to penetrate membranes with high efficiency. The main goal of this study was to use computational methods to identify small molecule mimetics with better drug-like properties. For this purpose, the docked conformations of the active peptides were used to identify compounds with similar activities. A series of related beta-sheet breaker peptides were docked to solid state NMR structures of a fibrillar form of Abeta. The lowest energy conformations of the active peptides were used to design three dimensional (3D)-pharmacophores, suitable for screening the NCI database with Unity. Small molecular weight compounds with physicochemical features and a conformation similar to the active peptides were selected, ranked by docking and biochemical parameters. Of 16 diverse compounds selected for experimental screening, 2 prevented and reversed Abeta aggregation at 2-3microM concentration, as measured by Thioflavin T (ThT) fluorescence and ELISA assays. They also prevented the toxic effects of aggregated Abeta on neuroblastoma cells. Their low molecular weight and aqueous solubility makes them promising lead compounds for treating AD.
Project description:Hexanucleotide repeat expansions in C9orf72 are the most common inherited cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The expansions elicit toxicity in part through repeat-associated non-AUG (RAN) translation of the intronic (GGGGCC)n sequence into dipeptide repeat-containing proteins (DPRs). Little is known, however, about the structural characteristics and aggregation propensities of the dipeptide units comprising DPRs. To address this question, we synthesized dipeptide units corresponding to the three sense-strand RAN translation products, analyzed their structures by circular dichroism, electron microscopy and dye binding assays, and assessed their relative toxicity when applied to primary cortical neurons. Short, glycine-arginine (GR)3 dipeptides formed spherical aggregates and selectively reduced neuronal survival compared to glycine-alanine (GA)3 and glycine-proline (GP)3 dipeptides. Doubling peptide length had little effect on the structure of GR or GP peptides, but (GA)6 peptides formed ?-sheet rich aggregates that bound thioflavin T and Congo red yet lacked the typical fibrillar morphology of amyloids. Aging of (GA)6 dipeptides increased their ?-sheet content and enhanced their toxicity when applied to neurons. We also observed that the relative toxicity of each tested dipeptide was proportional to peptide internalization. Our results demonstrate that different C9orf72-related dipeptides exhibit distinct structural properties that correlate with their relative toxicity.
Project description:Fibril formation of amyloid beta peptide (Abeta) is considered to be responsible for the pathology of Alzheimer's disease (AD). The Abeta fibril is formed by a protein misfolding process in which intermolecular beta-sheet interactions become stabilized abnormally. Thus, to develop potential anti-AD drugs, we screened an in-house library to find compounds which have a profile as a beta-sheet breaker. We searched for a beta-sheet breaker profile in an in-house library of approximately 113,000 compounds. From among the screening hits, we focused on N,N'-bis(3-hydroxyphenyl)pyridazine-3,6-diamine (named RS-0406), which had been newly synthesized in our laboratory. This compound (10-100 microg ml(-1)) was found to be capable of significantly inhibiting 25 microM Abeta(1-42) fibrillogenesis and, furthermore, disassembling preformed Abeta(1-42) fibrils in vitro. 3 We then investigated the effect of RS-0406 on 111 nM Abeta(1-42)-induced cytotoxicity in primary hippocampal neurons, and found that 0.3-3 microg ml(-1) RS-0406 ameliorates the cytotoxicity. Moreover, 3 microg ml(-1) RS-0406 reversed 1 micro M Abeta(1-42)-induced impairment of long-term potentiation in hippocampal slices. 4 In this study, we have succeeded in identifying RS-0406 which has potential to inhibit Abeta(1-42) fibrillogenesis, and to protect neurons against Abeta(1-42)-induced biological toxicity in vitro. These results suggest that RS-0406 or one of the derivatives could become a therapeutic agent for AD patients.
Project description:Diabetes has emerged as a threat to the current world. More than ninety five per cent of all the diabetic population has type 2 diabetes mellitus (T2DM). Aggregates of Amylin hormone, which is co-secreted with insulin from the pancreatic ?-cells, inhibit the activities of insulin and glucagon and cause T2DM. Importance of the conformationally restricted peptides for drug design against T2DM has been invigorated by recent FDA approval of Symlin, which is a large conformationally restricted peptide. However, Symlin still has some issues including solubility, oral bioavailability and cost of preparation. Herein, we introduced a novel strategy for conformationally restricted peptide design adopting a minimalistic approach for cost reduction. We have demonstrated efficient inhibition of amyloid formation of Amylin and its disruption by a novel class of conformationally restricted ?-sheet breaker hybrid peptidomimetics (BSBHps). We have inserted ?, ? and ? -aminobenzoic acid separately into an amyloidogenic peptide sequence, synthesized ?/?, ?/? and ?/? hybrid peptidomimetics, respectively. Interestingly, we observed the aggregation inhibitory efficacy of ?/? and ?/? BSBHps, but not of ?/? analogues. They also disrupt existing amyloids into non-toxic forms. Results may be useful for newer drug design against T2DM as well as other amyloidoses and understanding amyloidogenesis.
Project description:A circuit breaker is widely used to protect electric power system from fault currents or system errors; in particular, the opening mechanism in a circuit breaker is important to protect current overflow in the electric system. In this paper, multibody dynamic model of a circuit breaker including switch mechanism was developed including the electromagnetic actuator system. Since the opening mechanism operates sequentially, optimization of the switch mechanism was carried out to improve the current breaking time. In the optimization process, design parameters were selected from length and shape of each latch, which changes pivot points of bearings to shorten the breaking time. To validate optimization results, computational results were compared to physical tests with a high speed camera. Opening time of the optimized mechanism was decreased by 2.3 ms, which was proved by experiments. Switch mechanism design process can be improved including contact-latch system by using this process.
Project description:It has been proposed that singing evolved to facilitate social cohesion. However, it remains unclear whether bonding arises out of properties intrinsic to singing or whether any social engagement can have a similar effect. Furthermore, previous research has used one-off singing sessions without exploring the emergence of social bonding over time. In this semi-naturalistic study, we followed newly formed singing and non-singing (crafts or creative writing) adult education classes over seven months. Participants rated their closeness to their group and their affect, and were given a proxy measure of endorphin release, before and after their class, at three timepoints (months 1, 3 and 7). We show that although singers and non-singers felt equally connected by timepoint 3, singers experienced much faster bonding: singers demonstrated a significantly greater increase in closeness at timepoint 1, but the more gradual increase shown by non-singers caught up over time. This represents the first evidence for an 'ice-breaker effect' of singing in promoting fast cohesion between unfamiliar individuals, which bypasses the need for personal knowledge of group members gained through prolonged interaction. We argue that singing may have evolved to quickly bond large human groups of relative strangers, potentially through encouraging willingness to coordinate by enhancing positive affect.
Project description:Scorpion venoms are complex polypeptide mixtures, the ion channel blockers and antimicrobial peptides being the best studied components. The coagulopathic properties of scorpion venoms are poorly studied and the data about substances exhibiting these properties are very limited. During research on the Heterometrus laoticus scorpion venom, we have isolated low-molecular compounds with anticoagulant activity. Determination of their structure has shown that one of them is adenosine, and two others are dipeptides LeuTrp and IleTrp. The anticoagulant properties of adenosine, an inhibitor of platelet aggregation, are well known, but its presence in scorpion venom is shown for the first time. The dipeptides did not influence the coagulation time in standard plasma coagulation tests. However, similarly to adenosine, both peptides strongly prolonged the bleeding time from mouse tail and in vitro clot formation in whole blood. The dipeptides inhibited the secondary phase in platelet aggregation induced by ADP, and IleTrp decreased an initial rate of platelet aggregation induced by collagen. This suggests that their anticoagulant effects may be realized through the deterioration of platelet function. The ability of short peptides from venom to slow down blood coagulation and their presence in scorpion venom are established for the first time. Further studies are needed to elucidate the precise molecular mechanism of dipeptide anticoagulant activity.
Project description:MHC class I molecules bind only those peptides with high affinity that conform to stringent length and sequence requirements. We have now investigated which peptides can aid the in vitro folding of class I molecules, and we find that the dipeptide glycyl-leucine efficiently supports the folding of HLA-A*02:01 and H-2K(b) into a peptide-receptive conformation that rapidly binds high-affinity peptides. Treatment of cells with glycyl-leucine induces accumulation of peptide-receptive H-2K(b) and HLA-A*02:01 at the surface of cells. Other dipeptides with a hydrophobic second amino acid show similar enhancement effects. Our data suggest that the dipeptides bind into the F pocket like the C-terminal amino acids of a high-affinity peptide.