Novel Design of Neuropeptide-Based Drugs with ß-Sheet Breaking Potential in Amyloid-Beta Cascade: Molecular and Structural Deciphers.

Our work discusses the investigation of 75 peptide-based drugs with the potential ability to break the ß-sheet structures of amyloid-beta peptides from senile plaques. Hence, this study offers a unique insight into the design of neuropeptide-based drugs with ß-sheet breaker potential in the amyloid-beta cascade for Alzheimer's disease (AD). We started with five peptides (15QKLVFF20, 16KLVFF20, 17LVFF20, 16KLVF19 and 15QKLV18), to which 14 different organic acids were attached at the N-terminal. It was necessary to evaluate the physiochemical features of these sequences due to the biological correlation with our proposal. Hence, the preliminary analysis of different pharmacological features provided the necessary data to select the peptides with the best biocompatibility for administration purposes. Our approaches demonstrated that the peptides 17LVFF20, NA-17LVFF20, 16KLVF19 and NA-16KLVF19 (NA-nicotinic acid) have the ability to interfere with fibril formation and hence improve the neuro and cognitive functions. Moreover, the peptide conjugate NA-16KLVF19 possesses attractive pharmacological properties, demonstrated by in silico and in vitro studies. Tandem mass spectrometry showed no fragmentation for the spectra of 16KLVF19. Such important results suggest that under the action of protease, the peptide cleavage does not occur at all. Additionally, circular dichroism confirmed docking simulations and showed that NA-16KLVF19 may improve the ß-sheet breaker mechanism, and thus the entanglement process of amyloid-beta peptides can be more effective.

Aluminium Binding to Modified Amyloid-ß Peptides: Implications for Alzheimer's Disease.

Aluminium (Al) is clearly neurotoxic and considerable evidence exists that Al may play a role in the aetiology or pathogenesis of Alzheimer's disease (AD). Nevertheless, the link between AD pathology and Al is still open to debate. Therefore, we investigated here the interaction of aluminium ions with two Aß peptide fragments and their analogues. First, we synthesised by the Fmoc/tBu solid-phase peptide synthesis (SPPS) strategy using an automated peptide synthesiser two new peptides starting from the Aß(1-16) native peptide fragment. For this purpose, the three histidine residues (H6, H13, and H14) of the Aß(1-16) peptide were replaced by three alanine and three serine residues to form the modified peptides Aß(1-16)A36,13,14 and Aß(1-16)S36,13,14 (primary structures: H-1DAEFRADSGYEVAAQK16-NH2 and H-1DAEFRSDSGYEVSSQK16-NH2). In addition, the Aß(9-16) peptide fragment (H-9GYEVHHQK16-NH2) and its glycine analogues, namely Aß(9-16)G110, (H-9GGEVHHQK16-NH2), Aß(9-16)G213,14 (H-9GYEVGGQK16-NH2), and Aß(9-16)G310,13,14 (H-9GGEVGGQK16-NH2), were manually synthesised in order to study Al binding to more specific amino acid residues. Both the peptides and the corresponding complexes with aluminium were comparatively investigated by mass spectrometry (MS), circular dichroism spectroscopy (CD), atomic force microscopy (AFM), scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FT-IR). Al-peptide molecular ions and Al-fragment ions were unambiguously identified in the MS and MS/MS spectra. AFM images showed dramatic changes in the film morphology of peptides upon Al binding. Our findings from the investigation of N-terminal 1-16 and even 9-16 normal and modified sequences of Aß peptides suggest that they have the capability to be involved in aluminium ion binding associated with AD.

Mass spectrometric characterization of the zein protein composition in maize flour extracts upon protein separation by SDS-PAGE and 2D gel electrophoresis.

Highly homogenous α zein protein was isolated from maize kernels in an environment-friendly process using 95% ethanol as solvent. Due to the polyploidy and genetic polymorphism of the plant source, the application of high resolution separation methods in conjunction with precise analytical methods, such as MALDI-TOF-MS, is required to accurately estimate homogeneity of products that contain natural zein protein. The α zein protein product revealed two main bands in SDS-PAGE analysis, one at 25 kDa and other at 20 kDa apparent molecular mass. Yet, high resolution 2DE revealed approximately five protein spot groups in each row, the first at ca. 25 kDa and the second at ca. 20 kDa. Peptide mass fingerprinting data of the proteins in the two dominant SDS-PAGE bands matched to 30 amino acid sequence entries out of 102 non-redundant data base entries. MALDI-TOF-MS peptide mapping of the proteins from all spots indicated the presence of only α zein proteins. The most prominent ion signals in the MALDI mass spectra of the protein mixture of the 25 kDa SDS gel band after in-gel digestion were found at m/z 1272.6 and m/z 2009.1, and the most prominent ion signals of the protein mixture of the 20 kDa band after in-gel digestion were recorded at m/z 1083.5 and m/z 1691.8. These ion signals have been found typical for α zein proteins and may serve as marker ion signals which upon chymotryptic digestion reliably indicate the presence of α zein protein in two hybrid corn products.

Stoichiometry of Heavy Metal Binding to Peptides Involved in Alzheimer's Disease: Mass Spectrometric Evidence.

Mass spectrometry is a powerful analytical technique becoming increasingly important in different biomedical research area. Mass spectrometric based methods were developed and applied to detect and identify multiple metal ion complexes of peptides and proteins with high sensitivity and high mass accuracy. Aggregation of amyloid-ß (Aß) peptides is one of the main pathological features of Alzheimer's disease (AD), and some metal ions seem to play a key role in AD pathogenesis. Consequently, mass spectrometry was used to investigate heavy metal binding to AD-related peptides. Therefore, the purpose of this chapter is to review the methodology and application of identifying coordination chemistry and binding properties of several metal ion-binding sites to synthetic ß-amyloid (Aß) and anti-amyloid model peptides. The selective metal-amyloid-ß peptide interaction studies using (a) Matrix-assisted laser desorption/ionization mass spectrometry (MALDI); (b) Electrospray ionization mass spectrometry (ESI-MS), and (c) Tandem mass spectrometry (MS/MSn) will be reported.

Letter: Mass spectrometric evidence for iron binding to the neuroprotective peptide NAP and its Cys5 mutant.

The NAP peptide (H(2)N-(1)NAPVSIPQ(8)-CONH(2)) is a truncated version of the activity-dependent neuroprotective protein. Its neuroprotective activities consist of the inhibition of Aß(25-35) and Aß(1-40) fibrillogenesis as well as protection against Aß-induced neurotoxicity and prevention of microtubule disruption associated with Alzheimer's disease. Therefore, we synthesized NAP and its mutant peptide with the sequence: H(2)N-(1)NAPVCIPQ(8)-COOH (NAPCOH), by replacing serine S(5) with cysteine C(5). Both native and mutant peptides were further used to study their interaction with iron ions. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry, Fourier transform infrared spectroscopy and also atomic force microscopy were used to probe Fe(3+) binding to both peptides. Contrary to the expected results, the investigated peptides underwent different oxidation processes, with resultant reduced Fe(2+) ions. These ions, and not the original Fe(3+) ions, were found to bind to each of non-oxidized peptides.

Study on the mechanism of ferrite-induced dinitrophenol photodegradation.

We report here the effectiveness of gas chromatography mass spectrometry techniques in establishing the ferrite-associated photocatalytic degradation mechanism of pesticide 2,4-dinitrophenol (2,6-DNP). Unlike the previously discussed DNP-degradation mechanisms that involve either oxidation or reduction reactions, ferrite-based ultraviolet (UV) photodegradation of DNP affords the nontoxic 6-hydroxy-3,5-dinitrohexa-2,4-dienal by an unusual water addition to the benzene core. We searched for and demonstrated the presence of an epoxide of DNP within the photodegradation process, which may unambiguously explain the novel photochemical mechanism. During the 15 min UV photoinduced process, DNP degradation efficiency on the zinc ferrite catalyst was calculated to be 82%, whereas the first-order kinetic rate constant k was as high as 3.4 x 10(-2)min(-1).

Self-Assembly of a Novel Pentapeptide into Hydrogelated Dendritic Architecture: Synthesis, Properties, Molecular Docking and Prospective Applications.

Currently, ultrashort oligopeptides consisting of fewer than eight amino acids represent a cutting-edge frontier in materials science, particularly in the realm of hydrogel formation. By employing solid-phase synthesis with the Fmoc/tBu approach, a novel pentapeptide, FEYNF-NH2, was designed, inspired by a previously studied sequence chosen from hen egg-white lysozyme (FESNF-NH2). Qualitative peptide analysis was based on reverse-phase high performance liquid chromatography (RP-HPLC), while further purification was accomplished using solid-phase extraction (SPE). Exact molecular ion confirmation was achieved by matrix-assisted laser desorption-ionization mass spectrometry (MALDI-ToF MS) using two different matrices (HCCA and DHB). Additionally, the molecular ion of interest was subjected to tandem mass spectrometry (MS/MS) employing collision-induced dissociation (CID) to confirm the synthesized peptide structure. A combination of research techniques, including Fourier-transform infrared spectroscopy (FTIR), fluorescence analysis, transmission electron microscopy, polarized light microscopy, and Congo red staining assay, were carefully employed to glean valuable insights into the self-assembly phenomena and gelation process of the modified FEYNF-NH2 peptide. Furthermore, molecular docking simulations were conducted to deepen our understanding of the mechanisms underlying the pentapeptide's supramolecular assembly formation and intermolecular interactions. Our study provides potential insights into amyloid research and proposes a novel peptide for advancements in materials science. In this regard, in silico studies were performed to explore the FEYNF peptide's ability to form polyplexes.

Letter: Mass spectrometric approach of high pH- and copper-induced glutathione oxidation.

The interaction between copper ions and gamma-L-glutamyl-L-cysteinyl-glycine [glutathione (GSH)] molecules may lead to the formation of the physiologically occurring Cu[I)-[GSH]2 and Cu(II)-GSSG complexes. Since glutathione depletion in neurons and aberrant copper metabolism have been implicated in several neurodegenerative disorders, we studied here the interaction of GSH with copper ions (Cu2+) by electrospray ionization ion trap mass spectrometry (ESI-IT-MS). Besides, we extended this approach to pH in excess of 10 by adding ethanolamine to the solution being investigated. As a result, the ESI-IT-MS spectra revealed novel aspects regarding the speciation of copper-glutathione complex.

A Diphenylalanine Based Pentapeptide with Fibrillating Self-Assembling Properties.

Peptides and their related compounds can self-assemble into diverse nanostructures of different shapes and sizes in response to various stimuli such as pH, temperature or ionic strength. Here we report the synthesis and characterization of a lysozyme derived pentapeptide and its ability to build well-defined fibrillar structures. Lysozyme FESNF peptide fragment was synthesized by solid phase peptide synthesis using the Fmoc/t-Bu strategy, purified by analytical high-performance liquid chromatography (HPLC) and its molecular weight was confirmed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Spectroscopic features of this pentapeptide were investigated by UV-visible spectroscopy and fluorimetry showing the pattern of marginal phenylalanine residues within the peptide sequence. Self-assembling properties were determined using atomic force microscopy (AFM), aggregation index and thioflavin T assay (ThT). FESNF generating fibrillar structures observed by AFM and aggregation propensity were primarily influenced by pH conditions. Moreover, the experimental data were confirmed by molecular dynamics simulation studies. The obtained fibrils will be used next to explore their potential to act as support material for medical and cosmetic application.

A computational study of metal ions interaction with amyloid-ß 1-42 peptide structure in hyperpyrexia: Implications for Alzheimer disease.

Given the current context of the SARS-CoV-19 pandemic, among the interfering risky factors with the Aß peptide aggregation in the brains of Alzheimer's disease (AD) patients can be hyperpyrexia and increased intracranial pressure (ICP). According to our hypothesis on the relationship between hyperpyrexia and cognitive decline in AD, two models of Aß peptides were used in this study: the structure of AD amyloid beta-peptide and near-atomic resolution fibril structures of the Aß peptide. Therefore, the binding templates were constructed for Aß peptide regions able to bind 9 different metal ions. The fragment transformation method was used for the structural comparison between Aß chains. Molecular dynamics simulation (MDS) was applied using the Nose-Poincare-Anderson equation to generate a theoretically correct NPT (isothermal-isobaric ensemble). The smallest dissimilarities were observed in the case of Cu+ binding potential followed by Co2+, both with similar variation. Structural changes have also occurred as a result of the dynamic simulation. All these changes suggest an aggravating factor in both hyperpyretic and AD conditions. Our findings suggest that elevated temperature and increased intracranial pressure rise the effect of peptide aggregation, by converting α-helix motif to ß-sheet and random coil conformation, which are related to the formation of senile plaques in AD brains.

Structural Characterization of a New Collagen Biomimetic Octapeptide with Nanoscale Self-Assembly Potential: Experimental and Theoretical Approaches.

Bioinspired peptides are attractive biomolecules which can improve our understanding of self-assembly processes for rational design of new peptide-based materials. Herein, a new amidated peptide FRSAPFIE (FRS), based on a sequence present in human collagen, was synthesized, characterized by mass spectrometry and subjected to self-assembling investigations. The optimal conditions for self-assembly were disclosed by dynamic light scattering at 32 °C and a peptide concentration of 0.51 %. In addition, AFM studies revealed ellipsoidal FRS shapes with an area between 0.8 and 3.1 µm2 . The ability of self-assembly was also proved using FAD dye as extrinsic fluorescence reporter. According to the theoretical analysis, the FRS peptide tends to form a bundle-type association, with a type of fibrillary tangles particle. Altogether, our findings address new challenges regarding the FRS peptide which can be used in further self-assembly studies to design biocompatible drug-delivery platforms.

Silver-induced conformational changes of polypeptides: a CD study.

The role of silver ions in various pathologies, as well as their effect on peptide conformation and properties are less understood. Consequently, we synthesized several peptides with various residues in their sequence to investigate silver-induced conformational changes at various pH values by Circular Dichroism spectroscopy. Uniquely, the glycine-based, histidine-containing peptide showed a severe change from a random coil and ß-turn conformation to large α-helices during silver binding. When comparing the effect of silver ions on the conformation of bradykinin a similar tendency was found. Besides, silver ions reduced the amyloid-ß peptide tendency to aggregation. Our results suggest a specific and protective role for silver ions in brain pathologies, which is related to their high affinity toward physiologically and pharmacologically active peptides. Fourier transform infrared spectroscopy studies as well as the mass spectrometric ones support our conclusions.

Interaction of inorganic mercury with CoA-SH and acyl-CoAs.

Sulfur containing biomolecules are involved in complexes with mercury. CoA is an important cofactor for many enzymes involved in metabolic processes. Fatty acyl-CoA-thioesters, substrates of mitochondrial ß-oxidation, are sulfur containing compounds and potential mercury ligands. The CoA-Hg(2+) complex can be easily assessed by UV-Vis spectroscopy or indirectly by antibacterial tests that reconfirmed the protective role of CoA on E. coli. The characteristics of these complexes were determined by means of FTIR spectroscopy. The reverse phase liquid chromatography combined with electrospray ionization tandem mass spectrometry was used for detection of the side-product that resulted through the cleavage of thioesters in the presence of mercury. An unexpected result was the detection of octathioic acid as a product. Our study shows that mitochondrial ß-oxidation can be affected by thioesters depletion assisted by Hg(2+). The GC-MS technique could be used to detect some possible mitochondrial injuries due to the heavy metal ions.

Zinc Binding to NAP-Type Neuroprotective Peptides: Nuclear Magnetic Resonance Studies and Molecular Modeling.

Aggregation of amyloid-ß peptides (Aß) is a hallmark of Alzheimer's disease (AD), which is affecting an increasing number of people. Hence, there is an urgent need to develop new pharmaceutical treatments which could be used to prevent the AD symptomatology. Activity-dependent neuroprotective protein (ADNP) was found to be deficient in AD, whereas NAP, an 8-amino-acid peptide (1NAPVSIPQ8) derived from ADNP, was shown to enhance cognitive function. The higher tendency of zinc ion to induce Aß aggregation and formation of amorphous aggregates is also well-known in the scientific literature. Although zinc binding to Aß peptides was extensively investigated, there is a shortage of knowledge regarding the relationship between NAP peptide and zinc ions. Therefore, here, we investigated the binding of zinc ions to the native NAP peptide and its analog obtained by replacing the serine residue in the NAP sequence with tyrosine (1NAPVYIPQ8) at various molar ratios and pH values by mass spectrometry (MS) and nuclear magnetic resonancespectroscopy (NMR). Matrix-assisted laser desorption/ionization time-of-flight (MALDI ToF) mass spectrometry confirmed the binding of zinc ions to NAP peptides, while the chemical shift of Asp1, observed in 1H-NMR spectra, provided direct evidence for the coordinating role of zinc in the N-terminal region. In addition, molecular modeling has also contributed largely to our understanding of Zn binding to NAP peptides.

Hydrogen cyanide and carboxyhemoglobin assessment in an open space fire-related fatality.

Hydrogen cyanide (HCN) can be a major contributory factor in death from fire-related inhalation injury. Although carbon monoxide (CO) is considered the lethal agent of smoke in fires, its liability as a cause of death is sometimes debatable. The purpose of this report is to present the case of an 80-year-old man with locomotor disabilities who died due to an open space fire of vegetation debris and household waste in his yard. We evaluated here the concentrations of HCN and carboxyhemoglobin (COHb) and their contribution to the mechanism of death. In addition, the risk factors and the contributing effect of the factors that compose the complex toxic environment that develops in fires were discussed. COHb was determined by spectrophotometry as recommended by Katsumata et al. in 1982. HCN was determined with ninhydrin in postmortem blood samples after removal with 20% phosphoric acid and capture in a potassium carbonate solution. A toxic concentration of 1.3 µg ml-1 HCN and a lethal COHb level of 73.7% were determined in the blood samples. Although death was mainly attributed to CO poisoning and extremely severe burns in this open space burning case, the additive effect of HCN in the mechanism of death was also highlighted. The results suggested the possibility that the man's clothing may have played an important role in the production of HCN in this open space fire, as well as other types of garbage that were burned.

Toxic Blood Hydrogen Cyanide Concentration as a Vital Sign of a Deceased Room Fire Victim-Case Report.

Carbon monoxide (CO) and hydrogen cyanide (HCN) are two common toxic products of combustion. HCN concentrations of fire victims are not routinely determined in most legal medicine services in Romania. We present the case of a room fire victim in which we evaluated the concentrations of HCN and carboxyhemoglobin (COHb), their contribution to the mechanism of death, and the possibility that HCN concentration can be interpreted as vital sign. COHb was determined by spectrophotometry. HCN was spectrophotometrically determined with ninhydrin in postmortem blood samples after its removal with 20% phosphoric acid and uptake into a solution of potassium carbonate. The presence of ethyl alcohol was determined by gas chromatography. The COHb concentration was 6.15%, while the blood HCN concentration was 1.043 µg × mL-1 and the total HCN was 1.904 µg × ml-1. A blood alcohol content of 4.36 g‰ and a urine alcohol content of 5.88 g‰ were also found. Although the fire produced a considerable amount of soot, and there were signs of inhalation of soot particles, the COHb level cannot be interpreted as a vital sign. Toxic concentrations of HCN and total HCN can be interpreted as a vital sign and indicates a contributive effect of HCN in the mechanism of death.

Electrospray ionization mass spectrometric approach of conformationally-induced metal binding to oligopeptides.

Electrospray ionization mass spectrometry was used to measure the binding of copper and nickel ions to the newly synthesized model peptides H(2)N-AAAAHAAAAHAAAAHAAAA-COOH (P19-H5) and H(2)N-AAAHAAAHAAAHAAAAAAA-COOH (P19-H4). The affinity of histidine-containing peptides toward heavy metal ions proved to be related to the position of each histidine residue in the peptide sequence. In contrast to P19-H5, P19-H4 peptide bound no nickel or copper ions in the gas phase, whereas its spectra showed an intense fragmentation. The role of spacing residues (Ala repeats) in selecting the various conformations was also investigated. Finally, the circular dichroism and Fourier transform infrared spectra indicated that these isomer peptides have quite different conformations. A close relationship between the conformation of alanine-based peptides and their affinity toward metal ions may result in different patterns of metal ion-peptide systems.

Direct evidence for binding of aluminum to NAP anti-amyloid peptide and its analogs.

NAP (NAPVSIPQ) is a small peptide derived from the activity-dependent neuroprotective protein (ADNP), which provides neuroprotection against amyloid-ß peptide toxicity associated with Alzheimer disease. Several metal ions are able to promote the formation of amyloid-ß peptide oligomers and protofibrils in human brain tissue. Although the relationship between metal ions and amyloid-ß peptide peptides is extensively investigated, that with the NAP peptide is less understood. Nevertheless, our previous research revealed unexpected iron binding to NAP peptide and its analogs. However, a link between aluminum ions, Alzheimer disease and amyloid-ß peptide or NAP peptides still remains controversial. Therefore, we have investigated the possible binding of aluminum ions to NAP peptide and its four analogs. Indeed, MALDI-ToF mass spectrometry (MS), including MS/MS study, and Fourier transform infrared (FT-IR) spectroscopy revealed an unexpected pattern of aluminum ion binding to both NAP peptide and its analogs. Our results have been discussed with respect to NAP protection against Alzheimer disease-related neurotoxicity.

Application of chemical and biological tests for estimation of current state of a tailing dump and surrounding soil from the region of Tarnita, Suceava, Romania.

This paper presents the results from a study on the current state of tailing dump, surrounding soil and water in the region of Tarnita-Suceava, Romania. A number of chemical analyses and germination tests were applied in an attempt to estimate the ability of soil to maintain the plants growing, the bioavailability, and heavy metals uptake. Total heavy metals, exchangeable metals, acidity, and carbon and nitrogen content were determined. A modified sequential extraction method was used to determine geochemical phase distribution of heavy metals. The most abundant heavy metals in the studied samples were Cu, Zn, and Pb. Elevated concentrations of As were also found. The results from sequential extraction revealed that up to 51% of copper was retained by amorphous and crystalline iron oxides in soil. Higher content of lead was noticed in amorphous iron oxide fraction. The heavy metal concentration in river water during dry season varied from 0.13 mg/L (Fe) to 4.2 mg/L (Zn) and was below the maximum contamination level for drinking water. The soil toxicity and heavy metal bioavailability of tailing dump material and surrounding soils were studied by germination tests. The germinated plantlets on the studied soils were found to accumulate elevated concentrations of heavy metals thus indicating the bioavailability of soil contaminants. Soil decontamination by distilled water or magnesium nitrate solution was found to be efficient enough to improve the capability of the studied soils to support the germination process.

An electrospray ionization mass spectrometric study of iron binding to amyloid-beta peptides.

Iron and other metal ions appear to play an important role in protein aggregation and are therefore likely to provide a link between protein aggregation and oxidative damage. This work reports on iron binding to amyloid- beta peptide (Abeta1-40), which affords a very specific electrospray ionization mass spectrometric (MS) spectrum. Both MS and MS/MS study confirmed that amyloid-beta peptide displays a high affinity toward iron(III) ions, producing multi charged molecular ions and peptide aggregates. Finally, the circular dichroism spectra indicate an unexpected modification of Abeta1-40 peptide conformation upon iron binding.