Jin-Tang Du

Copper binding properties of a tau peptide associated with Alzheimer's disease studied by CD, NMR, and MALDI-TOF MS

We have previously reported the copper binding properties of R3 peptide (residues 318-335: VTSKCGSLGNIHHKPGGG, according to the longest tau protein) derived from the third repeat microtubule-binding domain of water-soluble tau protein. In this work, we have investigated copper binding properties of R2 peptide (residues 287-304: VQSKCGSKDNIKHVPGGG) derived from the second repeat region of tau protein. Similar to R3 peptide, R2 peptide also plays an important role in the formation of neurofibrillary tangles (NFTs) which is one of the two main biological characteristics of Alzheimers disease (AD). Based on the copper binding properties of R2 peptide, the possible influences of the binding on the formation of NFTs were investigated. Results from circular dichroism (CD) spectra, nuclear magnetic resonance (NMR) spectroscopy, and matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) suggest that the binding is pH-dependent and stoichiometry-determined. In addition, these results also reveal that R2 peptide adopts a monomeric alpha-helical structure in aqueous solutions at physiological pH after the addition of 1 mol equiv. of Cu2+. Since alpha-helix structure is responsible for the formation of paired helical filaments (PHFs) which aggregate into NFTs, it is hypothesized that Cu2+ induces R2 peptide to self-assemble into a PHFs-like structure. Hence, it is postulated that Cu2+ plays an important role in the aggregation of R2 peptide and tau protein and that copper binding to R2 peptide may be another possible involvement in AD.

Copper (II) modulates in vitro aggregation of a tau peptide.

Copper (II) has been implicated in the pathology of Alzheimers disease (AD) for the impaired homeostatic mechanism found in the brains of AD patients. Here we studied the binding properties of Cu(II) with the first microtubule-binding repeat, encompassing residues 256-273 of the human tau441 sequence. Additionally, the effect of Cu(II) on the assembly of this repeat was also investigated. Our results indicate that Cu(II) can bind to this repeat with His(268) involved and has an inhibiting effect on the in vitro aggregation of this repeat. This work provides new insight into the role of Cu(II) in Alzheimers disease.

O-GlcNAcylation modulates the self-aggregation ability of the fourth microtubule-binding repeat of tau

In Alzheimers disease (AD), tau protein is abnormally hyperphosphorylated and aggregated into paired helical filaments (PHFs). It was discovered recently that tau is also O-GlcNAcylated in human brains. And O-GlcNAcylation may regulate phosphorylation of tau in a site-specific manner. In this work, we focused on the fourth microtubule-binding repeat (R4) of tau, which has an O-GlcNAcylation site-Ser356. The aggregation behavior of this repeat and its O-GlcNAcylated form was investigated by turbidity, precipitation assay and electron microscopy. In addition, conformations of these two peptides were analyzed with circular dichroism (CD). Our results revealed that O-GlcNAcylation at Ser356 could greatly slow down the aggregation speed of R4 peptide. This modulation of O-GlcNAcylation on tau aggregation implies a new perspective of tau pathology.

Phosphorylation modulates the local conformation and self‐aggregation ability of a peptide from the fourth tau microtubule‐binding repeat

Phosphorylation of tau protein modulates both its physiological role and its aggregation into paired helical fragments, as observed in Alzheimers diseased neurons. It is of fundamental importance to study paired helical fragment formation and its modulation by phosphorylation. This study focused on the fourth microtubule‐binding repeat of tau, encompassing an abnormal phosphorylation site, Ser356. The aggregation propensities of this repeat peptide and its corresponding phosphorylated form were investigated using turbidity, thioflavin T fluorescence and electron microscopy. There is evidence for a conformational change in the fourth microtubule‐binding repeat of tau peptide upon phosphorylation, as well as changes in aggregation activity. Although both tau peptides have the ability to aggregate, this is weaker in the phosphorylated peptide. This study reveals that both tau peptides are capable of self‐aggregation and that phosphorylation at Ser356 can modulate this process.

Synthesis and conformational properties of phosphopeptides related to the human tau protein

In the brains of Alzheimers disease patients, the tau protein dissociates from the axonal microtubule and abnormally aggregates to form a paired helical filament (PHF). One of the priorities in Alzheimer research is to determine the effects of abnormal phosphorylation on the local structure. A series of peptides corresponding to isolated regions of tau protein have been successfully synthesized using Fmoc-based chemistry and their conformations were determined by 1H NMR spectroscopy and circular dichroism (CD) spectroscopy. Immunodominant peptides corresponding to tau-(256-273), tau-(350-367) and two phosphorylated derivatives in which a single Ser was phosphorylated at positions 262 and 356, respectively, were the main focus of the study. A direct alteration of the local structure after phosphorylation constitutes a new strategy through which control of biological activity can be enforced. In our study on Ser262 in R1 peptide and Ser356 in R4 peptide, phosphorylation modifies both the negative charge and the local conformation nearby the phosphorylation sites. Together, these structural changes indicate that phosphorylation may act as a conformational switch in the binding domain of tau protein to alter specificity and affinity of binding to microtubule, particularly in response to the abnormal phosphorylation events associated with Alzheimers disease.

Concise preparation of N α -Fmoc- N ɛ -(Boc, methyl)-lysine and its application in the synthesis of site-specifically lysine monomethylated peptide

Summary.A concise preparation of Nα-Fmoc-Nɛ-(Boc, methyl)-lysine and its application in the synthesis of site-specifically lysine monomethylated peptide is described. Nα-Fmoc-Nɛ-(Boc, methyl)-lysine is obtained, via consecutive reductive benzylation and reductive methylation in a one-pot reaction, followed by debenzylation through catalytic hydrogenolysis and Boc protection in another one-pot reaction. A peptide containing monomethylated lysine is successfully synthesized by incorporating Nα-Fmoc-Nɛ-(Boc, methyl)-lysine as a building block via solid-phase peptide synthesis.