6 research outputs found
Positional Effects of Phosphorylation on the Stability and Morphology of Tau-Related Amyloid Fibrils
Hyperphosphorylated forms of tau protein are the main
component
of paired helical filaments (PHFs) of neurofibrillary tangles in the
brain of Alzheimer’s disease patients. To understand the effect
of phosphorylation on the fibrillation of tau, we utilized tau-derived
phosphorylated peptides. The V<sub>306</sub>QIVYK<sub>311</sub> sequence
(PHF6) in the microtubule-binding domain is known to play a key role
in the fibrillation of tau, and the short peptide corresponding to
the PHF6 sequence forms amyloid-type fibrils similar to those generated
by full-length tau. We focused on the amino acid residue located at
the N-terminus of the PHF6 sequence, serine or lysine in the native
isoform of tau, and synthesized the PHF6 derivative peptides with
serine or lysine at the N-terminus of PHF6. Peptides phosphorylated
at serine and/or tyrosine were synthesized to mimic the possible phosphorylation
at these positions. The critical concentrations of the fibrillation
of peptides were determined to quantitatively assess fibril stability.
The peptide with the net charge of near zero tended to form stable
fibrils. Interestingly, the peptide phosphorylated at the N-terminal
serine residue exhibited remarkably low fibrillation propensity as
compared to the peptide possessing the same net charge. Transmission
electron microscopy measurements of the fibrils visualized the paired
helical or straight fibers and segregated masses of the fibers or
heterogeneous rodlike fibers depending on the phosphorylation status.
Further analyses of the fibrils by the X-ray fiber diffraction method
and Fourier transform infrared spectroscopic measurements indicated
that all the peptides shared a common cross-β structure. In
addition, the phosphoserine-containing peptides showed the characteristics
of β-sandwiches that could interact with both faces of the β-sheet.
On the basis of these observations, possible protofilament models
with four β-sheets were constructed to consider the positional
effects of the serine and/or tyrosine phosphorylations. The electrostatic
intersheet interaction between phosphate groups and the amino group
of lysine enhanced the lateral association between β-sheets
to compensate for the excess charge. In addition to the previously
postulated net charge of the peptide, the position of the charged
residue plays a critical role in the amyloid fibrillation of tau
Non-Enzymatic DNA Cleavage Reaction Induced by 5-Ethynyluracil in Methylamine Aqueous Solution and Application to DNA Concatenation
<div><p>DNA can be concatenated by hybridization of DNA fragments with protruding single-stranded termini. DNA cleavage occurring at a nucleotide containing a DNA base analogue is a useful method to obtain DNA with designed protruding termini. Here, we report a novel non-enzymatic DNA cleavage reaction for DNA concatenation. We found that DNA is cleaved at a nucleotide containing 5-ethynyluracil in a methylamine aqueous solution to generate 5′-phosphorylated DNA fragment as a cleavage product. We demonstrated that the reaction can be applied to DNA concatenation of PCR-amplified DNA fragments. This novel non-enzymatic DNA cleavage reaction is a simple practical approach for DNA concatenation.</p></div
Construction of plasmid from two PCR-amplified DNA fragments.
<p>(A) Scheme of plasmid construction. (B) Primer sequences used for PCR. The two sequences underlined in red and blue are complementary to each other. (C–G) Pictures of agarose gel electrophoresis. (C) PCR-amplified DNA fragments 1.5 (lane 2) and 2.2 kbp (lane 3). (D) 1.5 and 2.2 kbp DNA fragments before (lane 2,3) and after DNA cleavage at 25°C for 48 h (lane 4,5), 37°C for 10 h (lane 6,7), and 70°C for 0.5 h (lane 8,9). MeNH<sub>2</sub> was removed from the samples by speed-vac before electrophoresis. (E) Hybridized 1.5 and 2.2 kbp DNA fragments derived from those without cleavage reaction (lane 2) and cleaved at 25°C for 48 h (lane 3), 37°C for 10 h (lane 4), and 70°C for 0.5 h (lane 5). (F,G) Intact purified plasmids (F) and EcoRV-digested plasmids (G) derived from the DNA fragments cleaved at 25°C for 48 h (lane 2,3), 37°C for 10 h (lane 4–6), and 70°C for 0.5 h (lane 7–9). (H) Sequencing results of primer-derived regions of the plasmids. Underlined letters correspond to EU in the primers.</p
Chemical structures of thymine (T) and 5-ethynyluracil (EU).
<p>Chemical structures of thymine (T) and 5-ethynyluracil (EU).</p
Chemical formula of the DNA cleavage reaction.
<p>R is expected to be an abasic sugar derivative.</p
Degradation of DNA oligonucleotides containing 5-ethynyluracil.
<p>(A), (B) HPLC charts of T<sub>6</sub>(EU)T<sub>6</sub> before (gray) and after (black) the reaction in 14% NH<sub>3</sub>aq (A) or 20% MeNH<sub>2</sub>aq (B) at 70°C for 2 hours. (C), (D) (EU)T<sub>2</sub>AT<sub>2</sub>GT<sub>2</sub> (C) and T<sub>2</sub>AT<sub>2</sub>GT<sub>2</sub>(EU)T (D) before (gray) and after (black) the reaction in 20% MeNH<sub>2</sub>aq at 70°C for 2 hours.</p