Superoxide dismutase-1 alters the rate of prion protein aggregation and resulting fibril conformation.

The assembly of amyloidogenic proteins into highly-structured fibrillar aggregates is related to the onset and progression of several amyloidoses, including neurodegenerative Alzheimer's or Parkinson's diseases. Despite years of research and a general understanding of the process of such aggregate formation, there are currently still very few drugs and treatment modalities available. One of the factors that is relatively insufficiently understood is the cross-interaction between different amyloid-forming proteins. In recent years, it has been shown that several of these proteins or their aggregates can alter each other's fibrillization properties, however, there are still many unknowns in the amyloid interactome. In this work, we examine the interaction between amyloid disease-related prion protein and superoxide dismutase-1. We show that not only does superoxide dismutase-1 increase the lag time of prion protein fibril formation, but it also changes the conformation of the resulting aggregates.

Filarial thioredoxin reductase exerts anti-inflammatory effects upon lipopolysaccharide induced inflammation in macrophages.

Lymphatic filariasis and its associated health hazards have taken enormous tolls especially in the tropical and sub-tropical countries round the globe. Our present work contemplates the immunomodulatory role of filarial Thioredoxin reductase (TrxR) for the survival of the parasite inside the human host. For this, the protein TrxR was purified from the filarial parasite Setaria cervi and further substantiated through specific anti-TrxR antibody raised in mice. Both commercially available anti-TrxR antibody and laboratory raised antibody produced a single band with a molecular mass of ~80 kDa on western blot. The protein is optimally active at pH 7.0 and at temperature 37 °C. This protein contains both alpha helix and beta pleated sheet with selenocysteine at its active site. The Km was found to be 2.75 ± 0.49 mM. TrxR was found to downregulate lipopolysaccharide (LPS)-induced inflammation in macrophages due to inhibition of TLR4-NF-κB pathway. The result was further supported by the downregulation of inflammasome pathway and activation of alternatively activated macrophages upon TrxR treatment. Hence this study projects insights into the importance of filarial TrxR in host-parasite interface as well as it illustrates novel therapeutic strategy towards anti-filarial drug development.

Arginine induces protein self-assembly into nanofibers for triggering osteogenic differentiation of stem cells.

Although silk proteins are considered promising in building a scaffold for tissue engineering, one of the silk proteins, Bombyx mori silk sericin (BS), has limited processability in producing nanofibrous scaffolds because its surface charge anisotropy promotes gelation instead. To overcome this daunting challenge, we developed a mild and simple procedure for assembling BS into nanofibers and nanofibrous scaffolds. Briefly, arginine was added to the aqueous BS solution to reduce the negative charge of BS, thereby inducing BS to self-assemble into nanofibers in the solution. Circular dichroism (CD) and Fourier transform infrared (FT-IR) spectra showed that arginine promoted the formation of ß-sheet conformation in BS and increased its thermal stability. Furthermore, the arginine-induced BS nanofiber solution could be casted into scaffolds made of abundant network-like nanofibrous structures. The BS scaffolds promoted cell adhesion and growth and stimulated osteogenic differentiation of the bone marrow mesenchymal stem cells (BMSCs) in the absence of differentiation inducers in culture media. Our study presents a new strategy for assembling proteins into osteogenic nanofibrous scaffolds for inducing stem cell differentiation in regenerative medicine.

ßB2 W151R mutant is prone to degradation, aggregation and exposes the hydrophobic side chains in the fourth Greek Key motif.

Studies have established that congenital cataract is the major cause of blindness in children across the globe. The ß-crystallin protein family is the richest and most soluble structural protein in the lens. Their solubility and stability are essential in maintaining lens transparency. In this study, we identified a novel ßB2 mutation W151R in a rare progressive cortical congenital cataract family and explored its pathogenesis using purified protein and mutant related cataract-cell models. Due to its low solubility and poor structural stability, the ßB2 W151R mutation was prone to aggregation. Moreover, the W151R mutation enhanced the exposure of the hydrophobic side chains in the fourth Greek Key motif, which were readily degraded by trypsin. However, upon the administration of lanosterol, the negative effect of the W151R mutation was reversed. Therefore, lanosterol is a potential therapeutic option for cataracts.

Y12 nitration of human calcitonin (hCT): A promising strategy to produce non-aggregation bioactive hCT.

Irreversible aggregation can extremely limit the bioavailability and therapeutic activity of peptide-based drugs. There is therefore an urgent demand of effective strategy to control peptide aggregation. Recently, we found that tyrosine nitration at certain sites of peptide can effectively inhibit its aggregation. This minor modification may be an ideal strategy to the rational design of peptide-based drugs with low aggregation propensity yet without loss of bioactivity. Human calcitonin (hCT) is such a peptide hormone known for its hypocalcaemic effect but has limited pharmaceutical potential due to a high tendency to aggregate. In this study, by using multiple techniques including Fluorescence, TEM, Nu-PAGE and CD, we demonstrated that Y12 nitration of hCT would significantly inhibit its self-assembles, and we also found that this modification would not only reduce the cytotoxicity induced by peptide aggregation, but also had little effect on its potency. This finding may provide a novel strategy for clinically application of hCT instead of sCT.

Peptides based on the reactive center loop of Manduca sexta serpin-3 block its protease inhibitory function.

One innate immune response in insects is the proteolytic activation of hemolymph prophenoloxidase (proPO), regulated by protease inhibitors called serpins. In the inhibition reaction of serpins, a protease cleaves a peptide bond in a solvent-exposed reactive center loop (RCL) of the serpin, and the serpin undergoes a conformational change, incorporating the amino-terminal segment of the RCL into serpin ß-sheet A as a new strand. This results in an irreversible inhibitory complex of the serpin with the protease. We synthesized four peptides with sequences from the hinge region in the RCL of Manduca sexta serpin-3 and found they were able to block serpin-3 inhibitory activity, resulting in suppression of inhibitory protease-serpin complex formation. An RCL-derived peptide with the sequence Ser-Val-Ala-Phe-Ser (SVAFS) displayed robust blocking activity against serpin-3. Addition of acetyl-SVAFS-amide to hemolymph led to unregulated proPO activation. Serpin-3 associated with Ac-SVAFS-COO- had an altered circular dichroism spectrum and enhanced thermal resistance to change in secondary structure, indicating that these two molecules formed a binary complex, most likely by insertion of the peptide into ß-sheet A. The interference of RCL-derived peptides with serpin activity may lead to new possibilities of "silencing" arthropod serpins with unknown functions for investigation of their physiological roles.

Increasing the Assembly Efficacy of Peptidic ß-Sheets for a Highly-Sensitive HIV Detection.

Our recent publication illustrates the critical role of phenylalanine-mediated aromatic-aromatic interactions in determining the assembly of peptidic ß-sheets. However, the effect of phenylalanine number on regulating the assembly efficacy of peptidic ß-sheets remains poorly understood. We herein evaluate the assembly efficacy of ß-sheets of a series of oligopeptides which contain 0, 1, 2, or 3 phenylalanine in their molecular backbones. In our assembly system, two phenylalanine (2F) is the minimum number for driving the assembly of ß-sheets of oligopeptides. Oligopeptides with three phenylalanine (3F) show significantly increased assembly efficacy of ß-sheets compared to that with 2F. These results suggest a positive correlation between the phenylalanine number and assembly efficacy of ß-sheets. By improving the assembly efficacy of ß-sheets, we further develop a highly sensitive HIV analytical system in which the specific binding of ß-sheets with Congo Red induces enhanced fluorescence. For HIV p24 detection, the 3F-based analytical system (0.61 pg/mL) shows a significantly lower limit of detection (LOD) than the 2F-based analytical system (2.44 pg/mL), both of which are more sensitive than commercial ELISA (5 pg/mL) used in the clinic. This work not only illustrates the effect of phenylalanine number on regulating the assembly efficacy of ß-sheets but also provides a guideline for the construction of a highly sensitive analytical system of disease diagnosis.

Formation of α-synuclein aggregates in aqueous ethylammonium nitrate solutions.

The effect of adding ethylammonium nitrate (EAN), which is an ionic liquid (IL), on the aggregate formation of α-synuclein (α-Syn) in aqueous solution has been investigated. FTIR and Raman spectroscopy were used to investigate changes in the secondary structure of α-Syn and in the states of water molecules and EAN. The results presented here show that the addition of EAN to α-Syn causes the formation of an intermolecular ß-sheet structure in the following manner: native disordered state → polyproline II (PPII)-helix → intermolecular ß-sheet (α-Syn amyloid-like aggregates: α-SynA). Although cations and anions of EAN play roles in masking the charged side chains and PPII-helix-forming ability involved in the formation of α-SynA, water molecules are not directly related to its formation. We conclude that EAN-induced α-Syn amyloid-like aggregates form at hydrophobic associations in the middle of the molecules after masking the charged side chains at the N- and C-terminals of α-Syn.

Beta-sheet-specific interactions with heat shock proteins define a mechanism of delayed tumor cell death in response to HAMLET.

As chaperones, heat shock proteins (HSPs) protect host cells against misfolded proteins that constitute a by-product of protein synthesis. Certain HSPs are also expressed on the surface of tumor cells, possibly to scavenge extracellular unfolded protein ligands and prevent them from becoming cytotoxic. HAMLET-a complex of partially unfolded alpha-lactalbumin and oleic acid-is relying on its N-terminal alpha-helical domain to perturb tumor cell membranes, and the cells die as a consequence of this interaction. Here we show that in parallel, cell surface HSPs bind the beta-sheet domain of alpha-lactalbumin and activate a temporarily protective loop, involving vesicular uptake and lysosomal accumulation. Later, HAMLET destroys lysosomal membrane integrity, and HAMLET release kills the remaining tumor cells. HSPs were identified as HAMLET targets in a proteomic screen and Hsp70-specific antibodies or shRNAs inhibited HAMLET uptake by tumor cells, which showed increased Hsp70 surface expression compared to differentiated cells. The results suggest that HAMLET engages tumor cells by two parallel recognition mechanisms, defined by alpha-helical- or beta-sheet domains of alpha-lactalbumin and resulting in an immediate death response, or a delay due to transient accumulation of the complex in the lysosomes. This dual response pattern was conserved among tumor cells but not seen in normal, differentiated cells. By two different mechanisms, HAMLET thus achieves a remarkably efficient elimination of tumor cells.

Supramolecular Nanofibers with Superior Bioactivity to Insulin-Like Growth Factor-I.

Bioactive peptides derived from proteins generally need to be folded into secondary structures to activate downstream signaling pathways. However, synthetic peptides typically form random-coils, thus losing their bioactivities. Here, we show that by introducing a self-assembling peptide motif and using different preparation pathways, a peptide from insulin-like growth factor-I (IGF-1) can be folded into an α-helix and ß-sheet. The ß-sheet one exhibits a low dissociation constant to the IGF-1 receptor (IGF-1R, 11.5 nM), which is only about 3 times higher than that of IGF-1 (4.3 nM). However, the α-helical one and the peptide without self-assembling motif show weak affinities to IGF-1R ( KD = 179.1 and 321.6 nM, respectively). At 10 nM, the ß-sheet one efficiently activates the IGF-1 downstream pathway, significantly enhancing HUVEC proliferation and preventing cell apoptosis. The ß-sheet peptide shows superior performance to IGF-1 in vivo, and it improves ischemic hind-limb salvage by significantly reducing muscle degradation and enhancing limb vascularization. Our study provides a useful strategy to constrain peptides into different conformations, which may lead to the development of supramolecular nanomaterials mimicking biofunctional proteins.

An In Vitro elucidation of the antiaggregatory potential of Diosminover thermally induced unfolding of hen egg white lysozyme; A preventive quest for lysozyme amyloidosis.

Protein misfolding diseases are associated with human pathologies. These neurodegenerative diseases remain challenging task for researchers because of their adverse effect on vital organs system. Lysozyme amyloidosis is also associated with multi-organ dysfunction. Hence elucidation of its folding pathway is of great importance, for which hen egg white lysozyme (HEWL) being homological to its human counterpart was taken into consideration. Here in this study we have investigated the effect of diosmin (DSN), a flavonoid over thermally aggregated HEWL. Decrease in ANS, ThT and Rayleigh scattering fluorescence intensity suggests the transition between ß to α conformations. Further decrease in absorbance at 360 nm and of congo red with slight blue shift also indicated the disappearance of ß sheeted structure under the under the influence of increasing concentration of DSN. These results were also supported by circular dichroism in which gradual appearance α helical structure was observed. Finally visualization under transmission electron microscopy (TEM) authenticated the maximum structural alteration in the previously formed aggregates of HEWL at 250 µM DSN. Molecular docking followed by 100 ns MD simulations help to understand the interaction mechanism of HEWL with DSN. Results suggest DSN could be a useful in the treatment of amyloid related disorders.

Nanodisc-Forming Scaffold Protein Promoted Retardation of Amyloid-Beta Aggregation.

Peptidic nanodiscs are useful membrane mimetic tools for structural and functional studies of membrane proteins, and membrane interacting peptides including amyloids. Here, we demonstrate anti-amyloidogenic activities of a nanodisc-forming 18-residue peptide (denoted as 4F), both in lipid-bound and lipid-free states by using Alzheimer's amyloid-beta (Aß40) peptide as an example. Fluorescence-based amyloid fibrillation kinetic assays showed a significant delay in Aß40 amyloid aggregation by the 4F peptide. In addition, 4F-encased lipid nanodiscs, at an optimal concentration of 4F (>20 µM) and nanodisc size (<10 nm), significantly affect amyloid fibrillation. A comparison of experimental results obtained from nanodiscs with that obtained from liposomes revealed a substantial inhibitory efficacy of 4F-lipid nanodiscs against Aß40 aggregation and were also found to be suitable to trap Aß40 intermediates. A combination of atomistic molecular dynamics simulations with NMR and circular dichroism experimental results exhibited a substantial change in Aß40 conformation upon 4F binding through electrostatic and π-π interactions. Specifically, the 4F peptide was found to interfere with the central ß-sheet-forming residues of Aß40 through substantial hydrogen, π-π, and π-alkyl interactions. Fluorescence experiments and coarse-grained molecular dynamics simulations showed the formation of a ternary complex, where Aß40 binds to the proximity of peptidic belt and membrane surface that deaccelerate amyloid fibrillation. Electron microscopy images revealed short and thick amyloid fibers of Aß40 formed in the presence of 4F or 4F-lipid nanodsics. These findings could aid in the development of amyloid inhibitors as well as in stabilizing Aß40 intermediates for high-resolution structural and neurobiological studies.

Quantitative assessment of paraoxon adsorption to amphiphilic ß-sheet peptides presenting the catalytic triad of esterases.

Organophosphate compounds that are used as pesticides affect the nervous system by binding irreversibly to the active site of the enzyme acetylcholine esterase (AChE) and disrupting neuro-signaling nerve cells. In this study we characterized adsorption of paraoxon to a set of designed peptides that present different arrangements of the three amino acids of the AChE catalytic site: histidine, glutamic-acid and serine. The peptides set included two ß-strands with no net charge and three ß-hairpins that differ in their net charge. Circular dichroism, Thioflavin T assays and TEM images provided only qualitative insights on paraoxon binding to the different peptides. Paraoxon binding to the different peptides was measured with dialysis membrane tubes filled with the peptide solutions and suspended in a reservoir of paraoxon solution. Among all the tested peptides, the single strand peptide, denoted ssESH exhibited at 100 µM in random conformation prefibrillar state, the maximum paraoxon adsorption, with a binding mol ratio of one paraoxon per two peptides and an estimated equilibrium binding constant 5 ∗ 104 M-1. The three ß-hairpin peptides demonstrated that a net negative charge is unfavorable for paraoxon adsorption. Surface enhanced Raman spectroscopy measurements with ssESH enabled the detection of nanomolar adsorbed concentrations of paraoxon.