In vitro Aggregation Ability of Five Commercially Available Aβ42 Peptides

Background: As the most basic material, synthetic human Amyloid-β (1-42) (Aβ42) pep- tides from different manufacturers have been widely used. Their aggregation ability is vital to the reliability, repeatability and comparability of studies on Aβ42 physiology and pathology. However, it has not been evaluated and compared. Objectives: To analyze the consistency of the aggregation ability of 5 commercially available Aβ42 peptides. Methods: 5 Aβ42 peptides represented as A, B, C, D and E were pretreated by HFIP. The pretreated Aβ42 peptides were dissolved in Thioflavin T (ThT) solution, and their aggregation kinetics was monitored for 30 h with the aggregation kinetics test. Meanwhile, the pretreated peptides were ag- gregated in phosphate buffered saline. After aggregated for 12 h, they were detected by methods of ThT fluorescence, far-UV circular dichroism (CD), SDS-PAGE, western blot, and transmission electron microscopy (TEM), respectively. After aggregation for 8 h and 12 h, their cytotoxicity to SH-SY5Y cells was further evaluated using Cell Counting Kit-8. Results: For aggregation kinetics, peptides A, C and E remained low level curves, while peptides B and D presented typical sigmoidal kinetics curves. In CD measurement, the aggregates of pep- tides B and D showed relatively high negative CD peaks with the height of -8.09 mdeg and -14.37 mdeg, while the height of peptides A, C and E was -1.04, -3.55, and -3.88. In ThT assay, relative fluorescence intensity of the aggregates of peptides B and D were 7.79 and 8.82, higher than 1.19, 1.71, and 2.70 of peptide A, C and E, respectively. In SDS-PAGE, all aggregates contained monomers and eleven polymers. Moreover, peptide B-E presented a trapezoidal distribution from dimers to trimers, and peptide A aggregated to dimers. By western blot, the bands of monomers re- mained in all aggregates. Furthermore, peptides B and D aggregated to dimers and trimers, pep- tides A and C only aggregated to dimers, and peptide E showed a strong band of trimers. By TEM, protofibrils were observed only in peptide B, while substantial spherical aggregates were formed in other peptides. Additionally, peptides B, D and E exhibited higher cytotoxicity after being aggregat- ed for 8 h, whereas peptides A, B and D presented relatively high cytotoxicity after 12-hour aggre- gation. Conclusion: Commercially available Aβ42 peptides showed obvious differences in aggregation abil- ity, which should arouse enough attention in the field of basic study related to Aβ42. The aggrega- tion ability evaluation with the various assay methods has some discrepancies, and it is highly ur- gent to establish a reasonable and uniform measurement strategy.

The β-amylase7 gene in Zea mays encodes a protein with structural and catalytic properties similar to Arabidopsis BAM2

Starch accumulates in the plastids of green plant tissue during the day to provide carbon for metabolism at night. Starch hydrolysis is catalyzed by members of the β-amylase (BAM) family, which in Arabidopsis thaliana (At), includes nine structurally and functionally diverse members. One of these enzymes, AtBAM2, is a plastid-localized enzyme that is unique among characterized β-amylases since it is tetrameric and exhibits sigmoidal kinetics. Sequence alignments show that the BAM domains of AtBAM7, a catalytically inactive, nuclear-localized transcription factor with an N-terminal DNA binding domain, and AtBAM2 are more closely related to each other than they are to any other AtBAM. Since BAM2 is found in more ancient lineages, it was hypothesized that BAM7 evolved from BAM2. However, analysis of the genomes of 48 flowering plants revealed 12 species that appear to have a BAM7 gene but lack a BAM2 gene. Upon closer inspection, these BAM7 proteins have a greater percent identity to AtBAM2 than to AtBAM7, and they share all of the AtBAM2 functional residues that BAM7 proteins normally lack. We hypothesize that these genes may encode a BAM2-like protein although they are currently annotated as BAM7-like genes. To test this hypothesis, we designed a cDNA of the short form of corn BAM7 (ZmBAM7-S) for expression in E. coli. Small Angle X-Ray Scattering data indicate that ZmBAM7-S has a tetrameric solution structure more similar to that of AtBAM2 than AtBAM1. In addition, partially purified ZmBAM7-S is catalytically active and exhibits sigmoidal kinetics. Together these data suggest that some BAM7 genes may encode a functional BAM2. Exploring and understanding β-amylase gene structure could have impacts on the current annotation of genes.

Reversible supramolecular assembly of the anti-microbial peptide plectasin into helical non-amyloid fibrils

Self-assembly and fibril formation play important roles in protein behavior. Amyloid fibrils formation is well-studied due to its role in neurodegenerative diseases and characterized by refolding of the protein into predominant β-sheet form. However, much less is known about the assembly of proteins into other types of supramolecular structures. Using cryo-electron microscopy at a resolution of 1.97 Angstroem, we show that a triple-mutant of the anti-microbial peptide plectasin assembles reversibly into helical non-amyloid fibrils. Plectasin contains a cysteine-stabilized alpha-helix/beta-sheets structure, which remains intact upon fibril formation. Two fibrils form a right-handed superstructure with each fibril consisting of double helical, left-handed structures. The fibril formation is reversible and follows sigmoidal kinetics with a pH-dependent equilibrium between soluble monomer and protein fibril. The anti-microbial activity does not appear compromised by fibril formation. This is the first high-resolution structure of this type of alpha/beta protein fibrils.

Exploring the occurrence of thioflavin-T-positive insulin amyloid aggregation intermediates

The aggregation of proteins is considered to be the main cause of several neurodegenerative diseases. Despite much progress in amyloid research, the process of fibrillization is still not fully understood, which is one of the main reasons why there are still very few effective treatments available. When the aggregation of insulin, a model amyloidogenic protein, is tracked using thioflavin-T (ThT), an amyloid specific dye, there is an anomalous occurrence of double-sigmoidal aggregation kinetics. Such an event is likely related to the formation of ThT-positive intermediates, which may affect the outcome of both aggregation kinetic data, as well as final fibril structure. In this work we explore insulin fibrillization under conditions, where both normal and double-sigmoidal kinetics are observed and show that, despite their dye-binding properties and random occurrence, the ThT-positive intermediates do not significantly alter the overall aggregation process.

A Peptidomimetic Fluorescent Probe to Detect the Trypsin β2 Subunit of the Human 20S Proteasome

This work describes the chemical synthesis, combinatorial selection, and enzymatic evaluation of peptidomimetic fluorescent substrates specific for the trypsin-like (β2) subunit of the 20S human proteasome. After deconvolution of a library comprising nearly 6000 compounds composed of peg substituted diaminopropionic acid DAPEG building blocks, the sequence ABZ–Dap(O2(Cbz))–Dap(GO1)–Dap(O2(Cbz))–Arg–ANB–NH2, where ABZ is 2-aminobenzoic acid, and ANB- 5 amino 2- nitro benzoic acid was selected. Its cleavage followed sigmoidal kinetics, characteristic for allosteric enzymes, with Km = 3.22 ± 0.02 μM, kcat = 245 s−1, and kcat/Km = 7.61 × 107 M−1 s−1. This process was practically halted when a selective inhibitor of the β2 subunit of the 20S human proteasome was supplemented to the reaction system. Titration of the substrate resulting in decreased amounts of proteasome 20S produced a linear signal up to 10−11 M. Using this substrate, we detected human proteasome 20S in human urine samples taken from the bladders of cancer patients. This observation could be useful for the noninvasive diagnosis of this severe disease.

Phenomenological Inferences on the Kinetics of a Mechanically Activated Knoevenagel Condensation: Understanding the “Snowball” Kinetic Effect in Ball Milling

We focus on understanding the kinetics of a mechanically activated Knoevenagel condensation conducted in a ball mill, that is characterized by sigmoidal kinetics and the formation of a rubber-like cohesive intermediate state coating the milling ball. The previously described experimental findings are explained using a phenomenological kinetic model. It is assumed that reactants transform into products already at the very first collision of the ball with the wall of the jar. The portion of reactants that are transformed into products during each oscillation is taken to be a fraction of the amount of material that is trapped between the ball and the wall of the jar. This quantity is greater when the reaction mixture transforms from its initial powder form to the rubber-like cohesive coating on the ball. Further, the amount of reactants processed in each collision varies proportionally with the total area of the layer coating the ball. The total area of this coating layer is predicted to vary with the third power of time, thus accounting for the observed dramatic increase of the reaction rate. Supporting experiments, performed using a polyvinyl acetate adhesive as a nonreactive but cohesive material, confirm that the coating around the ball grows with the third power of time.