The formation of amyloid-like fibrils of α-chymotrypsin in different aqueous organic solvents.

The formation of amyloid-like fibrils of α-chymotrypsin was studied in aqueous ethanol, methanol, tertbutanol, dimethylformamide and acetonitrile. Thioflavin T (ThT), Congo red (CR) and 1-anilino-8-naphthalenesulfonic acid (ANS) binding, turbidity, intrinsic fluorescence and far-UV circular dichroism measurements were employed to characterize the amyloid fibril formation. The greatest extent of fibril formation after incubation for 24 h at pH 7.0 and at 24 °C was in ethanol at 55%, in methanol and dimethylformamide (DMF) at 60-70% and in tert-butanol at 60-80%. The ANS binding and intrinsic fluorescence results showed that the hydrophobic residues are more solvent-exposed in the aggregated form of α-chymotrypsin. The ThT, CR binding and far-UV CD measurements indicated that the formation of the cross-ß structure of α-chymotrypsin depends on the polarity of the organic solvent. To determine the role of surface charges in the aggregation, chemically modified forms of α-chymotrypsin were prepared. The citraconylated and succinylated enzymes exhibited a higher and the enzyme forms modified with aliphatic aldehydes a lower propensity for aggregation. These results suggest the important role of surface charges in the aggregation of α-chymotrypsin.

Controlled in situ preparation of A beta(1-42) oligomers from the isopeptide "iso-A beta(1-42)", physicochemical and biological characterization.

Beta-amyloid (A beta) peptides play a crucial role in the pathology of the neurodegeneration in Alzheimer's disease (AD). Biological experiments (both in vitro and animal model studies of AD) require synthetic A beta peptides of standard quality, aggregation grade, neurotoxicity and water solubility. The synthesis of A beta peptides has been difficult, owing to their hydrophobic character, poor solubility and high tendency for aggregation. Recently an isopeptide precursor (iso-A beta(1-42)) was synthesized by Fmoc-chemistry and transformed at neutral pH to A beta(1-42) by O-->N acyl migration in a short period of time. We prepared the same precursor peptide using Boc-chemistry and studied the transformation to A beta(1-42) by acyl migration. The peptide conformation and aggregation processes were studied by several methods (circular dichroism, atomic force and transmission electron microscopy, dynamic light scattering). The biological activity of the synthetic A beta(1-42) was measured by ex vivo (long-term potentiation studies in rat hippocampal slices) and in vivo experiments (spatial learning of rats). It was proven that O-->N acyl migration of the precursor isopeptide results in a water soluble oligomeric mixture of neurotoxic A beta(1-42). These oligomers are formed in situ just before the biological experiments and their aggregation grade could be standardized.

Synthesis and studies on cell-penetrating peptides.

The ability of different synthetic cell penetrating peptides, as Antennapedia (wild and Phe(6) mutated penetratins), flock house virus, and integrin peptides to form complexes with a 25mer antisense oligonucleotide was compared and their conformation was determined by circular dichroism spectroscopy. The efficiency for oligonucleotide delivery into cells was measured using peptides labeled with a coumarin derivative showing blue fluorescence and the fluorescein-labeled antisense oligonucleotide showing green fluorescence. Fluorescence due to the excitation energy transfer confirmed the interaction of the antisense oligonucleotide and cell-penetrating peptides. The most efficient oligonucleotide delivery was found for penetratins. Comparison of the two types of penetratins shows that the wild-type penetratin proved to be more efficient than mutated penetratin. The paper also emphasizes that the attachment of a fluorescent label may have an effect on the conformation and flexibility of cell-penetrating peptides that must be taken into consideration when evaluating biological experiments.

Aggregation of Abeta(1-42) in the presence of short peptides: conformational studies.

CD and infrared spectroscopic studies were performed on (i) the inhibitory effects of equimolar quantities of LPFFD-OH and LPYFD-NH(2) on the time-dependent aggregation of amyloid beta-protein (Abeta) (1-42) and (ii) the beta-sheet-breaker effects of two-fold molar excess of the pentapeptides on aggregated Abeta(1-42) aged 1 week. The data obtained from the time-dependent studies demonstrated that LPFFD-OH did not significantly influence, whereas LPYFD-NH(2) exerted some inhibitory effect on the aggregation of Abeta(1-42). When added to a solution of Abeta(1-42) aged 1 week, LPFFD-OH accelerated, while LPYFD-NH(2) delayed, but did not prevent further fibrillogenesis. The difference in the effects of these two pentapeptides on the aggregational profile of Abeta(1-42) is probably due to the difference in their conformational preferences: LPFFD-OH adopts a beta-turn and extended structures, while LPYFD-NH(2) adopts a prevailing beta-turn conformation.

N-terminal acylation of the SV40 nuclear localization signal peptide enhances its oligonucleotide binding and membrane translocation efficiencies.

Octanoyl and palmitoyl groups were coupled to the N-terminus of an analog of the SV40 nuclear localization signal peptide, SV126-133(Ser128), to study the effect of the fatty acid chain length on the complex formation with a single-stranded antisense oligodeoxynucleotide (ODN) and on the cellular uptake of the complex. The strongest binding affinity was observed for the palmitoylated peptide, indicating the better accessibility of the positively charged lysyl and arginyl side-chains to the phosphate groups due to the turn structures stabilized by the palmitoyl group. On increase of the peptide to ODN molar ratio (rM), gradual unstacking of the bases was observed, the maximal rate being reached at rM=10. At rM>10 restacking of the nucleotide bases was detected and the ODN was completely encapsulated in a liposome-like structure made up of palmitoylated peptides. Cell translocation experiments revealed a highly efficient cell transport of the ODN by palmitoylated SV40 peptide at rM>10.

Endomorphin-2, an endogenous tetrapeptide, protects against Abeta1-42 in vitro and in vivo.

The underlying cause of Alzheimer's disease (AD) is thought to be the beta-amyloid aggregates formed mainly by Abeta1-42 peptide. Protective pentapeptides [e.g., Leu-Pro-Phe-Phe-Asp (LPFFD)] have been shown to prevent neuronal toxicity of Abeta1-42 by arresting and reversing fibril formation. Here we report that an endogenous tetrapeptide, endomorphin-2 (End-2, amino acid sequence: YPFF), defends against Abeta1-42 induced neuromodulatory effects at the cellular level. Although End-2 does not interfere with the kinetics of Abeta fibrillogenesis according to transmission electron microscopic studies and quasielastic light scattering measurements, it binds to Abeta1-42 during aggregation, as revealed by tritium-labeled End-2 binding assay and circular dichroism measurements. The tetrapeptide attenuates the inhibitory effect on cellular redox activity of Abeta1-42 in a dose-dependent manner, as measured by 3-(4,5-dimethylthiazolyl-2)-2,-5-diphenyltetrazolium bromide (MTT) assay. In vitro and in vivo electrophysiological experiments show that End-2 also protects against the field excitatory postsynaptic potential attenuating and the NMDA-evoked response-enhancing effect of Abeta1-42. Studies using [D-Ala (2), N-Me-Phe (4), Gly (5)-ol]-enkephalin (DAMGO), a mu-opioid receptor agonist, show that the protective effects of the tetrapeptide are not mu-receptor modulated. The endogenous tetrapeptide End-2 may serve as a lead compound for the drug development in the treatment of AD.

The effects of Al(III) speciation on the activity of trypsin.

The effects of the different forms of Al(III) on the catalytic activity of the serine protease trypsin were studied. Enzyme activity was measured by BAEE assay in the presence of AlCl(3), Al(III):lactic acid 1:3, Al(III):maltol 1:3 or Al(III):nitrilotriacetic acid (NTA) 1:1 at a nominal Al(III) concentration of 0.01 M, and the ligand alone at pH 7.4 at 25 degrees C. Maltol and NTA caused approximately 30% inhibition, while that for the corresponding Al(III) complex was less than half of this. Al(III) in the form of the chloride or in three equivalents of lactic acid did not influence the activity of the enzyme, probably because most of the Al(III) was precipitated as Al(OH)(3). No direct interaction could be detected between the enzyme and the Al(III) complexes, either by ultrafiltration or by CD spectroscopy. These results strongly suggest that there is no direct involvement of Al(III) in the enzymatic reactions of trypsin.