The increasing realization of the prevalence of liquid–liquid phase separation (LLPS) across multiple length scales of biological constructs, from intracellular membraneless organelles to extracellular load-bearing tissues, has raised intriguing questions about intermolecular interactions regulating LLPS at the atomic level. Squid-beak derived histidine (His)- and tyrosine (Tyr)-rich peptides (HBpeps) have recently emerged as suitable short model peptides to precisely assess the roles of peptide motifs and single residues on the phase behavior and material properties of microdroplets obtained by LLPS. In this study, by systematically introducing single mutations in an HBpep, we have identified specific sticker residues that attract peptide chains together. We find that His and Tyr residues located near the sequence termini drive phase separation, forming interaction nodes that stabilize microdroplets. Combining quantum chemistry simulations with NMR studies, we predict atomic-level bond geometries and uncover inter-residue supramolecular interactions governing LLPS. These results are subsequently used to propose possible topological arrangements of the peptide chains, which upon expansion can help explain the three-dimensional network of microdroplets. The stability of the proposed topologies carried out through all-atom molecular dynamics simulations predicts chain topologies that are more likely to stabilize the microdroplets. Overall, this study provides useful guidelines for the de novo design of peptide coacervates with tunable phase behavior and material properties. In addition, the analysis of nanoscale topologies may pave the way to understand how client molecules can be trapped within microdroplets, with direct implications for the encapsulation and controlled release of therapeutics for drug delivery applications.

Graphical abstract

Studying pathogenic effects of amyloids requires homogeneous amyloidogenic peptide samples. Recombinant production of these peptides is challenging due to their susceptibility to aggregation and chemical modifications. Thus, chemical synthesis is primarily used to produce amyloidogenic peptides suitable for high‐resolution structural studies. Here, we exploited the shielded environment of protein condensates formed via liquid–liquid phase separation (LLPS) as a protective mechanism against premature aggregation. We designed a fusion protein tag undergoing LLPS in Escherichia coli and linked it to highly amyloidogenic peptides, including β amyloids. We find that the fusion proteins form membraneless organelles during overexpression and remain fluidic‐like. We also developed a facile purification method of functional Aβ peptides free of chromatography steps. The strategy exploiting LLPS can be applied to other amyloidogenic, hydrophobic, and repetitive peptides that are otherwise difficult to produce.

Anticholinergic drugs can be used as a treatment for many diseases. However, anticholinergic drugs are also known for their cognition-related side effects. Recently, there has been an increasing number of reports indicating a positive association between exposure to anticholinergic drugs and Alzheimer's disease (AD). Our novel study provides evidence of interactions between two representative anticholinergic drugs [Chlorpheniramine (CPM), a common antihistamine, and Trazodone (TRD), an antidepressant] with neuroprotective amyloid-beta (Ab) chaperone, lipocalin-type prostaglandin D synthase (L-PGDS) and the amyloid beta-peptide (1-40). Here, we demonstrate that CPM and TRD bind to L-PGDS with high affinity where chlorpheniramine exhibited higher inhibitory effects on L-PGDS as compared to Trazodone. We also show that the interactions between the drug molecules and Ab(1-40) peptides result in a higher fibrillar content of Ab(1-40) fibrils with altered fibril morphology. These altered fibrils possess higher cytotoxicity compared to Ab(1-40) fibrils formed in the absence of the drugs. Overall, our data suggest a mechanistic link between exposure to anticholinergic drugs and increased risk of Alzheimer's disease via inhibition of the neuroprotective chaperone L-PGDS and direct modification of Ab amyloid morphology and cytotoxicity.

With a significant proportion of the global population growing older (>60 years), the low success rates of current diagnoses for early neurodegeneration signs are disappointing. Early detection of Alzheimer's disease (AD) can improve acclimatization and quality of life for patients in their later years. Endogenous proteins, such as the most abundant secreted protein in cerebrospinal fluid, lipocalin‐type prostaglandin d synthase (L‐PGDS), can bind the early toxic oligomers of amyloid β (Aβ) peptides implicated in AD and prevent their aggregation. Herein, the utility of L‐PGDS for detection of amyloids is demonstrated. L‐PGDS is conjugated with different iron‐oxide magnetic nanoparticles for contrast‐enhanced visualization using magnetic resonance imaging (MRI). These conjugates inhibit amyloid aggregation in vitro and improve viability in neuronal cells incubated with amyloid fibrils, showing a potential neuroprotective function. L‐PGDS‐ferritin conjugates, when administered intraventricularly, localize to AD‐associated amyloid‐rich regions in mice brain imaged using MRI and histological stains. As a proof‐of‐concept, it is demonstrated that L‐PGDS conjugates could reach the brain regions through non‐invasive intranasal administration. These conjugates are developed as the first entirely protein‐based nanoprobes for early detection of brain amyloids. The results of this study open a wider avenue for study of endogenous proteins as potential theranostics for AD.