We demonstrate PolyAla synthesis using 5% piperazine + 2% DBU, which significantly reduces deletion products arising due to incomplete Fmoc-deprotection and can be used for deletion-free assembly of aggregation prone difficult peptides.

Intra-neuronal misfolding of monomeric tau protein to toxic β-sheet rich neurofibrillary tangles is a hallmark of Alzheimer’s disease (AD). Tau pathology correlates not only with progressive dementia but also with microglia-mediated inflammation in AD. Amyloid-beta (Aβ), another pathogenic peptide involved in AD, has been shown to activate NLRP3 inflammasome (NOD-like receptor family, pyrin domain containing 3), triggering the secretion of proinflammatory interleukin-1β (IL1β) and interleukin-18 (IL18). However, the effect of tau protein on microglia concerning inflammasome activation, microglial polarization, and autophagy is poorly understood. In this study, human microglial cells (HMC3) were stimulated with the unaggregated and aggregated forms of the tau-derived PHF6 peptide (VQIVYK). Modulation of NLRP3 inflammasome was examined by qRT-PCR, immunocytochemistry, and Western blot. We demonstrate that fibrillar aggregates of VQIVYK upregulated the NLRP3 expression at both mRNA and protein levels in a dose- and time-dependent manner, leading to increased expression of IL1β and IL18 in HMC3 cells. Aggregated PHF6-peptide also activated other related inflammation and microglial polarization markers. Furthermore, we also report a time-dependent effect of the aggregated PHF6 on BECN1 (Beclin-1) expression and autophagy. Overall, the PHF6 model system-based study may help to better understand the complex interconnections between Alzheimer’s PHF6 peptide aggregation and microglial inflammation, polarization, and autophagy.

Designing programmable biomaterials that could act as extracellular matrices and permit functionalization is a current need for tissue engineering advancement.

Tau is an intrinsically disordered protein responsible for maintaining the structure and stability of axonal microtubules. However, in certain disease conditions including Alzheimer’s disease, tau protein may undergo biochemical and structural changes to form intracellular aggregates. Since tau is a proline- and arginine-rich eukaryotic protein, heterologous expression in Escherichia coli often results in poor yield and has been a major technical challenge. In the current work, we have improved the expressed yield of tau by overcoming codon bias problem and established a simplified protocol for efficient extraction. The reported method has two distinct features: (i) enhanced tau expression (upto eightfold) by supplementing deficient tRNAs that aid in rapid translation and (ii) direct boiling of expressed E. coli cells to extract tau with no separate cell lysis step. We further demonstrate that tau extracted by the direct boiling method is similar to tau purified by size-exclusion chromatography exhibiting similar structural and biophysical characteristics including aggregation propensity. Since morphologies and in vitro toxicity of fibrillar tau aggregates were also similar, tau extracted by the one-step direct boiling method can be used for tau aggregation assays without any additional purification.