Rationally designed dehydroalanine (ΔAla)‐containing peptides inhibit amyloid‐β (Aβ) peptide aggregation

Among the pathological hallmarks of Alzheimer's disease (AD) is the deposition of amyloid‐β (Aβ) peptides, primarily Aβ (1–40) and Aβ (1–42), in the brain as senile plaques. A large body of evidence suggests that cognitive decline and dementia in AD patients arise from the formation of various aggregated forms of Aβ, including oligomers, protofibrils and fibrils. Hence, there is increasing interest in designing molecular agents that can impede the aggregation process and that can lead to the development of therapeutically viable compounds. Here, we demonstrate the ability of the specifically designed α,β‐dehydroalanine (ΔAla)‐containing peptides P1 (K‐L‐V‐F‐ΔA‐I‐ΔA) and P2 (K‐F‐ΔA‐ΔA‐ΔA‐F) to inhibit Aβ (1–42) aggregation. The mechanism of interaction of the two peptides with Aβ (1–42) seemed to be different and distinct. Overall, the data reveal a novel application of ΔAla‐containing peptides as tools to disrupt Aβ aggregation that may lead to the development of anti‐amyloid therapies not only for AD but also for many other protein misfolding diseases. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 456–465, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Synthesis of Aβ[1‐42] and its derivatives with improved efficiency

It has been proved that the principal component of senile plaques is aggregates of β‐amyloid peptide (Aβ) in cases of one of the most common forms of age‐related neurodegenerative disorders, Alzheimer's disease (AD). Although the synthetic methods for the synthesis of Aβ peptides have been developed since their first syntheses, Aβ[1‐42] is still problematic to prepare. The highly hydrophobic composition of Aβ[1‐42] results in aggregation between resin‐bound peptide chains or intrachain aggregation which leads to a decrease in the rates of deprotection and repetitive incomplete coupling reactions during 9‐flurenylmethoxycarbonyl (Fmoc) synthesis. In order to avoid aggregation and/or disrupt internal aggregation during stepwise Fmoc solid phase synthesis and to improve the quality of crude products, several attempts have been made. Since highly pure Aβ peptides in large quantities are used in biological experiments, we wanted to develop a method for a rational synthesis of human Aβ[1‐42] with high purity and adequate yield. This paper reports a convenient methodology with a novel solvent system for the synthesis of Aβ[1‐42], its N‐terminally truncated derivatives Aβ[4‐42] and Aβ[5‐42], and Aβ[1‐42] labeled with 7‐amino‐4‐methyl‐3‐coumarinylacetic acid (AMCA) at the N‐terminus using Fmoc strategy. The use of 10% anisole in Dimethylformamide/Dichloromethane (DMF/DCM) can substantially improve the purity and yield of crude Aβ[1‐42] and has been shown to be an optimal coupling condition for the synthesis of Aβ[1‐42]. Anisole is a cheap and simple aid in the synthesis of ‘difficult sequences’ where other solvents are less successful in the prevention of aggregation during the synthesis. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd.     

Inhibition of Aβ42 aggregation using peptides selected from combinatorial libraries

Increasing evidence suggests that the aggregation of the small peptide Aβ42 plays an important role in the development of Alzheimer's disease. Inhibiting the initial aggregation of Aβ42 may be an effective treatment for preventing, or slowing, the onset of the disease. Using an in vivo screen based on the enzyme EGFP, we have searched through two combinatorially diverse peptide libraries to identify peptides capable of inhibiting Aβ42 aggregation. From this initial screen, three candidate peptides were selected and characterized. ThT studies indicated that the selected peptides were capable of inhibiting amyloid aggregation. Additional ThT studies showed that one of the selected peptides was capable of disaggregating preformed Aβ42 fibers. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Interactions of amyloid Aβ(1–42) peptide with self‐assembled peptide nanospheres

In this work we have probed the interactions of the amyloid Aβ(1–42) peptide with self‐assembled nanospheres. The nanospheres were formed by self‐assembly of a newly developed bolaamphiphile bis(N‐alpha‐amido‐methionine)‐1,8 octane dicarboxylate under aqueous conditions. It was found that the interactions of the Aβ(1–42) peptide with the nanospheres were concentration as well as pH dependent and the peptide largely adopts a random coil structure upon interacting with the nanospheres. Further, upon incorporation with the nanospheres, we observed a relative diminution in the aggregation of Aβ(1–42) at low concentrations of Aβ(1–42). The interactions between the nanospheres and the Aβ(1–42) peptide were investigated by atomic force microscopy, transmission electron microscopy, circular dichroism, FTIR and fluorescence spectroscopy, and the degree of fibrillation in the presence and absence of nanospheres was monitored by the Thioflavine T assay. We believe that the outcome from this work will help further elucidate the binding properties of Aβ peptide as well as designing nanostructures as templates for further investigating the nucleation and fibrillation process of Aβ‐like peptides. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

A synthetic peptide corresponding to a region of the human pericentriolar material 1 (PCM‐1) protein binds β‐amyloid (Aβ1‐42) oligomers

We have recently reported that a ~19‐kDa polypeptide, rPK‐4, is a protein kinase Cs inhibitor that is 89% homologous to the 1171–1323 amino acid region of the 228‐kDa human pericentriolar material‐1 (PCM‐1) protein (Chakravarthy et al. 2012). We have now discovered that rPK‐4 binds oligomeric amyloid‐β peptide (Aβ)_1‐42 with high affinity. Most importantly, a PCM‐1‐selective antibody co‐precipitated Aβ and amyloid β precursor protein (AβPP) from cerebral cortices and hippocampi from AD (Alzheimer's disease) transgenic mice that produce human AβPP and Aβ_1‐42, suggesting that PCM‐1 may interact with amyloid precursor protein/Aβ in vivo. We have identified rPK‐4′s Aβ‐binding domain using a set of overlapping synthetic peptides. We have found with ELISA, dot‐blot, and polyacrylamide gel electrophoresis techniques that a ~ 5 kDa synthetic peptide, amyloid binding peptide (ABP)‐p4‐5 binds Aβ_1‐42 at nM levels. Most importantly, ABP‐p4‐5, like rPK‐4, appears to preferentially bind Aβ_1‐42 oligomers, believed to be the toxic AD‐drivers. As expected from these observations, ABP‐p4‐5 prevented Aβ_1‐42 from killing human SH‐SY5Y neuroblastoma cells via apoptosis. These findings indicate that ABP‐p4‐5 is a possible candidate therapeutic for AD.     

Wild‐type,Flemish, and Dutch amyloid‐β exhibit different cytotoxicities depending on Aβ40 to Aβ42 interaction time and concentration ratio

Addition of amyloid β (Aβ) peptide Aβ40 to Aβ42 can delay Aβ42 aggregation, but consequent cytotoxicity has been reported to be enhanced or diminished. In the present study, we found that cytotoxicity was enhanced when human neuroblastoma SH‐SY5Y cells were incubated in a mixture of wt Aβ42 and Aβ40wt at a ratio of 1 : 10–20 (0.1 : 1–2 μM) for 24–36 h, whereas the enhancement was detected in cells incubated for longer times (48–60 h) with the less amyloidogenic Flemish Aβ40 variant or in cells incubated for as short as 12 h with the more amyloidogenic Dutch variant. Reductions in cytotoxicity by Aβ40 were most prominently observed in the Flemish and wt Aβ40/Aβ42 mixture at ratio 1 : 20 incubated for a short time (~12 h). The most cytotoxic Aβ40/Aβ42 mixtures were enriched in Aβ protofibril‐like structures, implying a strong correlation between cytotoxicity and this structure, the formation of which was dependent on amyloidogenic properties and incubation time. The consequences of the interactions were probably because of the different amyloidogenic properties of the Aβ40 variants, rather than to those of Aβ42, because aggregation rates of Aβ40 variants were highly dependent on sequence, whereas those of Aβ42 variants were not. These studies highlight a potential role for Aβ40 in cytotoxicity and provide novel mechanistic insights into the pathogenesis of each familial Alzheimer's disease‐associated Aβ40 variant. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Retracted: S14G‐humanin inhibits Aβ1–42 fibril formation,disaggregates preformed fibrils,and protects against Aβ‐induced cytotoxicity in vitro

The aggregation of soluble amyloid‐beta (Aβ) peptide into oligomers/fibrils is one of the key pathological features in Alzheimer's disease (AD). The Aβ aggregates are considered to play a pivotal role in the pathogenesis of AD. Therefore, inhibiting Aβ aggregation and destabilizing preformed Aβ fibrils would be an attractive therapeutic target for prevention and treatment of AD. S14G‐humanin (HNG), a synthetic derivative of Humanin (HN), has been shown to be a strong neuroprotective agent against various AD‐related insults. Recent studies have shown that HNG can significantly improve cognitive deficits and reduce insoluble Aβ levels as well as amyloid plaque burden without affecting amyloid precursor protein processing and Aβ production in transgenic AD models. However, the potential mechanisms by which HNG reduces Aβ‐related pathology in vivo remain obscure. In the present study, we found that HNG could significantly inhibit monomeric Aβ1–42 aggregation into fibrils and destabilize preformed Aβ1–42 fibrils in a concentration‐dependent manner by Thioflavin T fluorescence assay. In transmission electron microscope study, we observed that HNG was effective in inhibiting Aβ1–42 fibril formation and disrupting preformed Aβ1–42 fibrils, exhibiting various types of amorphous aggregates without identifiable Aβ fibrils. Furthermore, HNG‐treated monomeric or fibrillar Aβ1–42 was found to significantly reduce Aβ1–42‐mediated cytotoxic effects on PC12 cells in a dose‐dependent manner by MTT assay. Collectively, our results demonstrate for the first time that HNG not only inhibits Aβ1–42 fibril formation but also disaggregates preformed Aβ1–42 fibrils, which provides the novel evidence that HNG may have anti‐Aβ aggregation and fibrillogenesis, and fibril‐destabilizing properties. Together with previous studies, we concluded that HNG may have promising therapeutic potential as a multitarget agent for the prevention and/or treatment of AD. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Alzheimer's Aβ42 and Aβ40 peptides form interlaced amyloid fibrils

Deposition of amyloid β (Aβ) in the brain is a pathological hallmark of Alzheimer's disease. There are two major isoforms of Aβ: the 42‐residue Aβ42 and the 40‐residue Aβ40. The only difference between Aβ42 and Aβ40 is that Aβ42 has two extra residues at the C‐terminus. The amyloid plaques in Alzheimer's brains consist of mostly Aβ42 and some plaques contain only Aβ42, even though Aβ40 concentration is several‐fold more than Aβ42. Using electron paramagnetic resonance, we studied the formation of amyloid fibrils using a mixture of Aβ42 and Aβ40 in vitro. We show that Aβ42 and Aβ40 form mixed fibrils in an interlaced manner, although Aβ40 is not as efficient as Aβ42 in terms of being incorporated into Aβ42 fibrils. Our results suggest that both Aβ42 and Aβ40 would be present in amyloid plaques if in vivo aggregation of Aβ were similar to the in vitro process. Therefore, there must be some mechanisms that lead to the preferential deposition of Aβ42 at the extracellular space. Identifying such mechanisms may open new avenues for therapeutic interventions to treat Alzheimer's disease.     

Molecular characterization of the β‐amyloid(4‐10) epitope of plaque specific Aβ antibodies by affinity‐mass spectrometry using alanine site mutation

Alzheimer disease is a neurodegenerative disease affecting an increasing number of patients worldwide. Current therapeutic strategies are directed to molecules capable to block the aggregation of the β‐amyloid(1‐42) (Aβ) peptide and its shorter naturally occurring peptide fragments into toxic oligomers and amyloid fibrils. Aβ‐specific antibodies have been recently developed as powerful antiaggregation tools. The identification and functional characterization of the epitope structures of Aβ antibodies contributes to the elucidation of their mechanism of action in the human organism. In previous studies, the Aβ(4‐10) peptide has been identified as an epitope for the polyclonal anti‐Aβ(1‐42) antibody that has been shown capable to reduce amyloid deposition in a transgenic Alzheimer disease mouse model. To determine the functional significance of the amino acid residues involved in binding to the antibody, we report here the effects of alanine single‐site mutations within the Aβ‐epitope sequence on the antigen‐antibody interaction. Specific identification of the essential affinity preserving mutant peptides was obtained by exposing a Sepharose‐immobilized antibody column to an equimolar mixture of mutant peptides, followed by analysis of bound peptides using high‐resolution MALDI‐Fourier transform‐Ion Cyclotron Resonance mass spectrometry. For the polyclonal antibody, affinity was preserved in the H6A, D7A, S8A, and G9A mutants but was lost in the F4, R5, and Y10 mutants, indicating these residues as essential amino acids for binding. Enzyme‐linked immunosorbent assays confirmed the binding differences of the mutant peptides to the polyclonal antibody. In contrast, the mass spectrometric analysis of the mutant Aβ(4‐10) peptides upon affinity binding to a monoclonal anti‐Aβ(1‐17) antibody showed complete loss of binding by Ala‐site mutation of any residue of the Aβ(4‐10) epitope. Surface plasmon resonance affinity determination of wild‐type Aβ(1‐17) to the monoclonal Aβ antibody provided a binding constant K_D in the low nanomolar range. These results provide valuable information in the elucidation of the binding mechanism and the development of Aβ‐specific antibodies with improved therapeutic efficacy.     

Terminal sidechain packing of a designed β‐hairpin influences conformation and stability

While end capping in α‐helices is well understood, the concept of capping a β‐hairpin is a relatively recent development; to date, favorable Coulombic interactions are the only example of sidechains at the termini influencing the overall stability of a β‐hairpin. While cross‐strand hydrophobic residues generally provide hairpin stabilization, particular when flanking the turn region, those remote from this location appear to provide little stabilization. While probing for an optimal residue at a hydrogen bond position near the terminus of a designed β‐hairpin a conservative, hydrophobic, V → I mutation was observed to not only result in a significant change in fold population but also effected major changes in the structuring shifts at numerous sites in the peptide. Mutational studies reveal that there is an interaction between the sidechain at this H‐bonded site and the sidechain at the C‐terminal non‐H‐bonded site of the hairpin. This interaction, which appears to be hydrophobic in character, requires a highly twisted hairpin structure. Modifications at the C‐terminal site, for example an E → A mutation (ΔΔG_U = 6 kJ/mol), have profound affects on fold structure and stability. The data suggests that this may be a case of hairpin end capping by the formation of a hydrophobic cluster. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 557–564, 2009. This article was originally published online as an accepted preprint. The “Published Online”date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Rational design of amyloid β peptide–binding proteins: Pseudo‐Aβ β‐sheet surface presented in green fluorescent protein binds tightly and preferentially to structured Aβ

Some neurodegenerative diseases such as Alzheimer disease (AD) and Parkinson disease are caused by protein misfolding. In AD, amyloid β‐peptide (Aβ) is thought to be a toxic agent by self‐assembling into a variety of aggregates involving soluble oligomeric intermediates and amyloid fibrils. Here, we have designed several green fluorescent protein (GFP) variants that contain pseudo‐Aβ β‐sheet surfaces and evaluated their abilities to bind to Aβ and inhibit Aβ oligomerization. Two GFP variants P13H and AP93Q bound tightly to Aβ, K_d = 260 nM and K_d = 420 nM, respectively. Moreover, P13H and AP93Q were capable of efficiently suppressing the generation of toxic Aβ oligomers as shown by a cell viability assay. By combining the P13H and AP93Q mutations, a super variant SFAB4 comprising four strands of Aβ‐derived sequences was designed and bound more tightly to Aβ (K_d = 100 nM) than those having only two pseudo‐Aβ strands. The SFAB4 protein preferentially recognized the soluble oligomeric intermediates of Aβ more than both unstructured monomer and mature amyloid fibrils. Thus, the design strategy for embedding pseudo‐Aβ β‐sheet structures onto a protein surface arranged in the β‐barrel structure is useful to construct molecules capable of binding tightly to Aβ and inhibiting its aggregation. This strategy may provide implication for the diagnostic and therapeutic development in the treatment of AD. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Conformational evaluation of labeled C3′‐O‐P‐13CH2‐O‐C4″ phosphonate internucleotide linkage,a phosphodiester isostere

Modified internucleotide linkage featuring the C3′‐O‐P‐CH₂‐O‐C4″ phosphonate grouping as an isosteric alternative to the phosphodiester C3′‐O‐P‐O‐CH₂‐C4″ bond was studied in order to learn more on its stereochemical arrangement, which we showed earlier to be of prime importance for the properties of the respective oligonucleotide analogues. Two approaches were pursued: First, the attempt to prepare the model dinucleoside phosphonate with ¹³C‐labeled CH₂ group present in the modified internucleotide linkage that would allow for a more detailed evaluation of the linkage conformation by NMR spectroscopy. Second, the use of ab initio calculations along with molecular dynamics (MD) simulations in order to observe the most populated conformations and specify main structural elements governing the conformational preferences. To deal with the former aim, a novel synthesis of key labeled reagent (CH₃O)₂P(O)¹³CH₂OH for dimer preparation had to be elaborated using aqueous ¹³C‐formaldehyde. The results from both approaches were compared and found consistent. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 514–529, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Comparison of neurotoxicity of different aggregated forms of Aβ40, Aβ42 and Aβ43 in cell cultures

The abnormal deposition of amyloid‐β (Aβ) peptides in the brain is the main neuropathological hallmark of Alzheimer's disease (AD). Amyloid deposits are formed by a heterogeneous mixture of Aβ peptides, among which the most studied are Aβ40 and Aβ42. Aβ40 is abundantly produced in the human brain, but the level of Aβ42 is remarkably increased in the brain of AD patients. Aside from Aβ40 and Aβ42, recent data have raised the possibility that Aβ43 peptides may be instrumental in AD pathogenesis. Besides its length, whether the Aβ aggregated form accounts for the neurotoxicity is also particularly controversial. Aβ fibrils are generally considered as key pathogenic substances in AD pathogenesis. Nevertheless, recent data implicated soluble Aβ oligomers as the main cause of synaptic dysfunction and memory loss in AD. To further address this uncertainty, we analyzed the neurotoxicity of different Aβ species and Aβ forms at the cellular level. The results showed that Aβ42 could form oligomers significantly faster than Aβ40 and Aβ43 and Aβ42 oligomers showed the greatest level of neurotoxicity. Regardless of the length of Aβ peptides, Aβ oligomers induced significantly higher cytotoxicity compared with the other two Aβ forms. Surprisingly, the neurotoxicity of fibrils in PC12 cells was only marginally but not significantly stronger than monomers, contrary to previous reports. Altogether, our findings demonstrate the high pathogenicity of Aβ42 among the three Aβ species and support the idea that Aβ42 oligomers contribute to the pathological events leading to neurodegeneration in AD. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Mg2+ ions bind at the C‐terminal region of skeletal muscle α‐tropomyosin

Tropomyosin (Tm) is a dimeric coiled‐coil protein that polymerizes through head‐to‐tail interactions. These polymers bind along actin filaments and play an important role in the regulation of muscle contraction. Analysis of its primary structure shows that Tm is rich in acidic residues, which are clustered along the molecule and may form sites for divalent cation binding. In a previous study, we showed that the Mg²⁺‐induced increase in stability of the C‐terminal half of Tm is sensitive to mutations near the C‐terminus. In the present report, we study the interaction between Mg²⁺ and full‐length Tm and smaller fragments corresponding to the last 65 and 26 Tm residues. Although the smaller Tm peptide (Tm_{259‐284(W269)}) is flexible and to large extent unstructured, the larger Tm_{220‐284(W269)} fragment forms a coiled coil in solution whose stability increases significantly in the presence of Mg²⁺. NMR analysis shows that Mg²⁺ induces chemical shift perturbations in both Tm_{220‐284(W269)} and Tm_{259‐284(W269)} in the vicinity of His276, in which are located several negatively charged residues. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 583–590, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Single‐molecule pair studies of the interactions of the α‐GalNAc (Tn‐antigen) form of porcine submaxillary mucin with soybean agglutinin

Mucins form a group of heavily O‐glycosylated biologically important glycoproteins that are involved in a variety of biological functions, including modulating immune response, inflammation, and adhesion. Mucins are also involved in cancer and metastasis and often express diagnostic cancer antigens. Recently, a modified porcine submaxillary mucin (Tn‐PSM) containing GalNAcα1‐O‐Ser/Thr residues was shown to bind to soybean agglutinin (SBA) with ～10⁶‐fold enhanced affinity relative to GalNAcα1‐O‐Ser, the pancarcinoma carbohydrate antigen. In this study, dynamic force spectroscopy is used to investigate molecular pairs of SBA and Tn‐PSM. A number of force jumps that demonstrate unbinding or rebinding events were observed up to a distance equal to 2.0 μm, consistent with the length of the mucin chain. The unbinding force increased from 103 to 402 pN with increasing force loading rate. The position of the activation barrier in the energy landscape of the interaction was 0.1 nm. The lifetime of the SBA–TnPSM complex in the absence of applied force was determined to be in the range 1.3–1.9 s. Kinetic parameters describing the rate of dissociation of other sugar lectin interactions are in the range 3.3 × 10^?3–2.5 × 10^?3 s. The long lifetime of the SBA‐TnPSM complex is compatible with a binding model in which lectin molecules “bind and jump” from α‐GalNAc residue to α‐GalNAc residue along the polypeptide chain of Tn‐PSM before dissociating. These findings have important implications for the molecular recognition properties of mucins. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 719–728, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Heating‐induced conformational change of a novel β‐(1→3)‐D‐glucan from Pleurotus geestanus

Recently, we isolated and purified a neutral polysaccharide (PGN) from edible fungus Pleurotus geestanus. Its structure was characterized by a range of physical–chemical methods, including high performance anion exchange chromatography, uronic acid, and protein analyses, size exclusion chromatography with ultraviolet, refractive index and light scattering detectors, and nuclear magnetic resonance. Our results revealed that PGN is a novel β‐(1→3)‐D ‐glucan with glucose attached to every other sugar residues at Position 6 in the backbone. It has a degree of branching of 1/2. Such structure is different from typical β‐(1→3)‐D ‐glucans schizophyllan and lentinan in which DB is 1/3 and 2/5, respectively. Rheological study showed a very interesting melting behavior of PGN in water solution: heating PGN in water leads to two transitions, in the range of 8–12.5°C and 25–60°C, respectively. The melting behavior and conformational changes were characterized by rheometry, micro‐differential scan calorimetry, atomic force microscopy, static and dynamic light scattering at different temperatures. The first heating‐induced transition corresponds to the disintegration of polymer bundles into small helical clusters, resembling the heating‐induced dissociation of SPG in water at 7°C; the second one might correspond to the dissociation of helical strands to individual chains. The ability of PGN to undergo a conformation/viscosity transition in water upon heating is very valuable to immobilize cells or enzymes or therapeutic DNA/RNA, which makes PGN a potentially useful biomaterial. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 121–131, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Engineered cystine knot peptides that bind αvβ3, αvβ5, and α5β1 integrins with low‐nanomolar affinity

There is a critical need for compounds that target cell surface integrin receptors for applications in cancer therapy and diagnosis. We used directed evolution to engineer the Ecballium elaterium trypsin inhibitor (EETI‐II), a knottin peptide from the squash family of protease inhibitors, as a new class of integrin‐binding agents. We generated yeast‐displayed libraries of EETI‐II by substituting its 6‐amino acid trypsin binding loop with 11‐amino acid loops containing the Arg‐Gly‐Asp integrin binding motif and randomized flanking residues. These libraries were screened in a high‐throughput manner by fluorescence‐activated cell sorting to identify mutants that bound to α_vβ₃ integrin. Select peptides were synthesized and were shown to compete for natural ligand binding to integrin receptors expressed on the surface of U87MG glioblastoma cells with half‐maximal inhibitory concentration values of 10–30 nM. Receptor specificity assays demonstrated that engineered knottin peptides bind to both α_vβ₃ and α_vβ₅ integrins with high affinity. Interestingly, we also discovered a peptide that binds with high affinity to α_vβ₃, α_vβ₅, and α₅β₁ integrins. This finding has important clinical implications because all three of these receptors can be coexpressed on tumors. In addition, we showed that engineered knottin peptides inhibit tumor cell adhesion to the extracellular matrix protein vitronectin, and in some cases fibronectin, depending on their integrin binding specificity. Collectively, these data validate EETI‐II as a scaffold for protein engineering, and highlight the development of unique integrin‐binding peptides with potential for translational applications in cancer. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

NMR structure of rALF‐Pm3, an anti‐lipopolysaccharide factor from shrimp: Model of the possible lipid A‐binding site

The anti‐lipopolysaccharide factor ALF‐Pm3 is a 98‐residue protein identified in hemocytes from the black tiger shrimp Penaeus monodon. It was expressed in Pichia pastoris from the constitutive glyceraldehyde‐3‐phosphate dehydrogenase promoter as a folded and ¹⁵N uniformly labeled rALF‐Pm3 protein. Its 3D structure was established by NMR and consists of three α‐helices packed against a four‐stranded β‐sheet. The C³⁴? C⁵⁵ disulfide bond was shown to be essential for the structure stability. By using surface plasmon resonance, we demonstrated that rALF‐Pm3 binds to LPS, lipid A and to OM®‐174, a soluble analogue of lipid A. Biophysical studies of rALF‐Pm3/LPS and rALF‐Pm3/OM®‐174 complexes indicated rather high molecular sized aggregates, which prevented us to experimentally determine by NMR the binding mode of these lipids to rALF‐Pm3. However, on the basis of striking structural similarities to the FhuA/LPS complex, we designed an original model of the possible lipid A‐binding site of ALF‐Pm3. Such a binding site, located on the ALF‐Pm3 β‐sheet and involving seven charged residues, is well conserved in ALF‐L from Limulus polyphemus and in ALF‐T from Tachypleus tridentatus. In addition, our model is in agreement with experiments showing that β‐hairpin synthetic peptides corresponding to ALF‐L β‐sheet bind to LPS. Delineating lipid A‐binding site of ALFs will help go further in the de novo design of new antibacterial or LPS‐neutralizing drugs. © 2008 Wiley Periodicals, Inc. Biopolymers 91: 207–220, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Differential physiologic responses of α7 nicotinic acetylcholine receptors to β‐amyloid1–40 and β‐amyloid1–42

The β‐amyloid peptides (Aβ), Aβ_1–40 and Aβ_1–42, have been implicated in Alzheimer's disease (AD) pathology. Although Aβ_1–42 is generally considered to be the pathological peptide in AD, both Aβ_1–40 and Aβ_1–42 have been used in a variety of experimental models without discrimination. Here we show that monomeric or oligomeric forms of the two Aβ peptides, when interact with the neuronal cation channel, α7 nicotinic acetylcholine receptors (α7nAChR), would result in distinct physiologic responses as measured by acetylcholine release and calcium influx experiments. While Aβ_1–42 effectively attenuated these α7nAChR‐dependent physiology to an extent that was apparently irreversible, Aβ_1–40 showed a lower inhibitory activity that could be restored upon washings with physiologic buffers or treatment with α7nAChR antagonists. Our data suggest a clear pharmacological distinction between Aβ_1–40 and Aβ_1–42. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 25–30, 2003     

Aromatic‐interaction‐mediated inhibition of β‐amyloid assembly structures and cytotoxicity

Abnormal aggregation of β‐amyloid (Aβ) peptide plays an important role in the onset and progress of Alzheimer's disease (AD); hence, targeting Aβ aggregation is considered as an effective therapeutic strategy. Here, we studied the aromatic‐interaction‐mediated inhibitory effect of oligomeric polypeptides (K8Y8, K4Y8, K8W8) on Aβ42 fibrillization process. The polypeptides containing lysine as well as representative aromatic amino acids of tryptophan or tyrosine were found to greatly suppress the aggregation as evaluated by thioflavin T assay. Circular dichroism spectra showed that the β‐sheet formation of Aβ42 peptides decreased with the polypeptide additives. Molecular docking studies revealed that the oligomeric polypeptides could preferentially bind to Aβ42 through π–π stacking between aromatic amino acids and Phe19, together with hydrogen bonding. The cell viability assay confirmed that the toxicity of Aβ42 to SH‐SY5Y cells was markedly reduced in the presence of polypeptides. This study could be beneficial for developing peptide‐based inhibitory agents for amyloidoses. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.