ABSTRACT
ß-amyloid (Aß) aggregates involved in Alzheimer's disease (AD) are resistant to proteases but could be destabilized by small peptides designed to target specific hydrophobic regions of Aß that take part in aggregate assembly. Since thrombin and AD are intricately connected, and elastase modulates thrombin activity, elastase-digested thrombin peptides were verified for intervention in the Aß-aggregation pathway. Intact or elastase-digested thrombin destabilized Aß fibril, as demonstrated by thioflavin T assay. Peptides were synthesized employing thrombin as a template, of which, a hexapeptide (T3) showed maximum destabilization at 1 µm. ExPASy peptide cutter software coupled with mass spectrometric analysis confirmed the generation of T3 peptide from elastase-digested thrombin. TEM micrographs revealed that 30-day incubation of preformed Aß fibrils or monomers with T3 resulted in destabilization or inhibition, respectively, leading mostly to particles of 1.74 ± 0.17 nm, which roughly corresponded to Aß monomer. Surface plasmon resonance employing CM5 chip coupled with Aß40 mouse monoclonal antibody showed a drop in response when T3 was incubated with Aß fibrils between 2 and 8 h. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide and confocal microscopy demonstrated the ability of T3 to rescue neuroblastoma cells from Aß oligomer-induced cytotoxic damage. Although no [Aß-T3] adduct could be detected by mass spectrometry, an initial interaction appeared to facilitate the process of destabilization/inhibition of aggregation. T3 was comparable to standard ß-sheet breaker peptides, LPFFD and KLVFF in terms of Aß aggregate destabilization. High hydrophobicity values coupled with recognition and breaking elements make T3 a potential candidate for future therapeutic applications.
Subject(s)
Alzheimer Disease/drug therapy , Amyloid beta-Peptides/genetics , Protein Aggregation, Pathological/drug therapy , Thrombin/pharmacology , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Amyloid/antagonists & inhibitors , Amyloid/genetics , Amyloid beta-Peptides/antagonists & inhibitors , Animals , Benzothiazoles/pharmacology , Humans , Hydrophobic and Hydrophilic Interactions/drug effects , Mice , Neuroprotection/drug effects , Peptide Hydrolases/genetics , Peptides/pharmacology , Protein Aggregation, Pathological/genetics , Protein Aggregation, Pathological/pathology , Thrombin/geneticsABSTRACT
BACKGROUNDS: Spontaneous deamidation and isoaspartate (IsoAsp) formation contributes to aging and reduced longevity in cells. A protein-l-isoaspartate (d-aspartate) O-methyltransferase (PCMT) is responsible for minimizing IsoAsp moieties in most organisms. METHODS: PCMT was purified in its native form from yeast Candida utilis. The role of the native PCMT in cell survival and protein repair was investigated by manipulating intracellular PCMT levels with Oxidized Adenosine (AdOx) and Lithium Chloride (LiCl). Proteomic Identification of possible cellular targets was carried out using 2-dimensional gel electrophoresis, followed by on-Blot methylation and mass spectrometric analysis. RESULTS: The 25.4 kDa native PCMT from C. utilis was found to have a Km of 3.5 µM for AdoMet and 33.36 µM for IsoAsp containing Delta Sleep Inducing Peptide (DSIP) at pH 7.0. Native PCMT comprises of 232 amino acids which is coded by a 698 bp long nucleotide sequence. Phylogenetic comparison revealed the PCMT to be related more closely with the prokaryotic homologs. Increase in PCMT levels in vivo correlated with increased cell survival under physiological stresses. PCMT expression was seen to be linked with increased intracellular reactive oxygen species (ROS) concentration. Proteomic identification of possible cellular substrates revealed that PCMT interacts with proteins mainly involved with cellular housekeeping. PCMT effected both functional and structural repair in aged proteins in vitro. GENERAL SIGNIFICANCE: Identification of PCMT in unicellular eukaryotes like C. utilis promises to make investigations into its control machinery easier owing to the familiarity and flexibility of the system.
