Hidden Aggregation Hot-Spots on Human Apolipoprotein E: A Structural Study

Human apolipoprotein E (apoE) is a major component of lipoprotein particles, and under physiological conditions, is involved in plasma cholesterol transport. Human apolipoprotein E found in three isoforms (E2; E3; E4) is a member of a family of apolipoproteins that under pathological conditions are detected in extracellular amyloid depositions in several amyloidoses. Interestingly, the lipid-free apoE form has been shown to be co-localized with the amyloidogenic Aβ peptide in amyloid plaques in Alzheimer’s disease, whereas in particular, the apoE4 isoform is a crucial risk factor for late-onset Alzheimer’s disease. Evidence at the experimental level proves that apoE self-assembles into amyloid fibrilsin vitro, although the misfolding mechanism has not been clarified yet. Here, we explored the mechanistic insights of apoE misfolding by testing short apoE stretches predicted as amyloidogenic determinants by AMYLPRED, and we computationally investigated the dynamics of apoE and an apoE−Αβ complex. Our in vitro biophysical results prove that apoE peptide−analogues may act as the driving force needed to trigger apoE aggregation and are supported by the computational apoE outcome. Additional computational work concerning the apoE−Αβ complex also designates apoE amyloidogenic regions as important binding sites for oligomeric Αβ; taking an important step forward in the field of Alzheimer’s anti-aggregation drug development

Hsp70 chaperone blocks α-synuclein oligomer formation via a novel engagement mechanism.

Overexpression and aggregation of α-synuclein (ASyn) are linked to the onset and pathology of Parkinson's disease and related synucleinopathies. Elevated levels of the stress-induced chaperone Hsp70 protect against ASyn misfolding and ASyn-driven neurodegeneration in cell and animal models, yet there is minimal mechanistic understanding of this important protective pathway. It is generally assumed that Hsp70 binds to ASyn using its canonical and promiscuous substrate-binding cleft to limit aggregation. Here we report that this activity is due to a novel and unexpected mode of Hsp70 action, involving neither ATP nor the typical substrate-binding cleft. We use novel ASyn oligomerization assays to show that Hsp70 directly blocks ASyn oligomerization, an early event in ASyn misfolding. Using truncations, mutations, and inhibitors, we confirm that Hsp70 interacts with ASyn via an as yet unidentified, noncanonical interaction site in the C-terminal domain. Finally, we report a biological role for a similar mode of action in H4 neuroglioma cells. Together, these findings suggest that new chemical approaches will be required to target the Hsp70-ASyn interaction in synucleinopathies. Such approaches are likely to be more specific than targeting Hsp70's canonical action. Additionally, these results raise the question of whether other misfolded proteins might also engage Hsp70 via the same noncanonical mechanism

Bacterial Lectin FimH and Its Aggregation Hot-Spots: An Alternative Strategy against Uropathogenic Escherichia coli

Type I fimbriae are the main adhesive organelles of uropathogenic Escherichia coli (UPEC), consisting of four different subunits. Their component with the most important role in establishing bacterial infections is the FimH adhesin located at the fimbrial tip. This two-domain protein mediates adhesion to host epithelial cells through interaction with terminal mannoses on epithelial glycoproteins. Here, we propose that the amyloidogenic potential of FimH can be exploited for the development of therapeutic agents against Urinary Tract Infections (UTIs). Aggregation-prone regions (APRs) were identified via computational methods, and peptide-analogues corresponding to FimH lectin domain APRs were chemically synthesized and studied with the aid of both biophysical experimental techniques and molecular dynamic simulations. Our findings indicate that these peptide-analogues offer a promising set of antimicrobial candidate molecules since they can either interfere with the folding process of FimH or compete for the mannose-binding pocket

Arabidopsis thaliana Plant Natriuretic Peptide Active Domain Forms Amyloid-like Fibrils in a pH-Dependent Manner

Plant natriuretic peptides (PNPs) are hormones that have been extracted from many different species, with the Arabidopsis thaliana PNP (AtPNP-A) being the most studied among them. AtPNP-A is a signaling molecule that consists of 130 residues and is secreted into the apoplast, under conditions of biotic or abiotic stress. AtPNP-A has distant sequence homology with human ANP, a protein that forms amyloid fibrils in vivo. In this work, we investigated the amyloidogenic properties of a 34-residue-long peptide, located within the AtPNP-A sequence, in three different pH conditions, using transmission electron microscopy, X-ray fiber diffraction, ATR FT-IR spectroscopy, Congo red and Thioflavin T staining assays. We also utilize bioinformatics tools to study its association with known plant amyloidogenic proteins and other A. thaliana proteins. Our results reveal a new case of a pH-dependent amyloid forming peptide in A. thaliana, with a potential functional role

The amyloid interactome: Exploring protein aggregation

<div><p>Protein-protein interactions are the quintessence of physiological activities, but also participate in pathological conditions. Amyloid formation, an abnormal protein-protein interaction process, is a widespread phenomenon in divergent proteins and peptides, resulting in a variety of aggregation disorders. The complexity of the mechanisms underlying amyloid formation/amyloidogenicity is a matter of great scientific interest, since their revelation will provide important insight on principles governing protein misfolding, self-assembly and aggregation. The implication of more than one protein in the progression of different aggregation disorders, together with the cited synergistic occurrence between amyloidogenic proteins, highlights the necessity for a more universal approach, during the study of these proteins. In an attempt to address this pivotal need we constructed and analyzed the human amyloid interactome, a protein-protein interaction network of amyloidogenic proteins and their experimentally verified interactors. This network assembled known interconnections between well-characterized amyloidogenic proteins and proteins related to amyloid fibril formation. The consecutive extended computational analysis revealed significant topological characteristics and unraveled the functional roles of all constituent elements. This study introduces a detailed protein map of amyloidogenicity that will aid immensely towards separate intervention strategies, specifically targeting sub-networks of significant nodes, in an attempt to design possible novel therapeutics for aggregation disorders.</p></div

Clustering analysis of the amyloid interactome.

<p>The 11 clusters with 3 or more nodes of the amyloid interaction network, derived utilizing the MCL algorithm [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173163#pone.0173163.ref097" target="_blank">97</a>]. Cytoscape [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173163#pone.0173163.ref051" target="_blank">51</a>] was used as a visualization tool. The visual legend summarizes the shortcuts of node colour and node shape (See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173163#pone.0173163.g001" target="_blank">Fig 1</a>). The highly connected subnetwork of the first cluster within the amyloid interactome reveals the strong affinity between 7 amyloidogenic proteins (cluster 1—red nodes) and the integral representation of the proteins presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173163#pone.0173163.t002" target="_blank">Table 2</a> (cluster 1—yellow nodes) (Interactive cluster subnetworks available at <a href="http://83.212.109.111/amyloid_interactome" target="_blank">http://83.212.109.111/amyloid_interactome</a>).</p

Subnetworks of molecular chaperones participating in the amyloid interactome.

<p>3 important subnetworks were isolated from the entire amyloid interactome: (A) Subnetwork of Hsp90 co-chaperone Cdc37, Hsc70-interacting protein, Hsp 90-alpha, Hsc71 and their first neighbors, (B) Subnetwork of Serum albumin and Hsc70-interacting protein and their first neighbors and (C) Subnetwork of Clusterin, Large proline-rich protein BAG6 and their first neighbors. The aforementioned proteins, having chaperone or co-chaperone activity, were found to play a pivotal role in the integrity of the interactome (See section Network Analysis Based on Graph Theory). A highly selective and direct correlation of Serum albumin and 6 amyloidogenic proteins was observed (B), whereas indirect interactions between Serum albumin and 2 amyloidogenic proteins were recorded (A). Hsc70-interacting protein is a significant element of the interactome, since it conciliates interactions between Apolipoproteins and ACys or ATTR (A,B). Clusterin synergistically with Large proline-rich protein BAG6 interferes with APrp and Aβ2M (C). The finding that more than one chaperones mediate the interconnection between different amyloidogenic proteins deserves further investigation.</p

The amyloid interactome.

<p>Interaction data for the creation of this network were gathered from the publicly available database IntAct [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173163#pone.0173163.ref045" target="_blank">45</a>] and Cytoscape [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173163#pone.0173163.ref051" target="_blank">51</a>] was used as a visualization tool (Interactive network available at <a href="http://83.212.109.111/amyloid_interactome" target="_blank">http://83.212.109.111/amyloid_interactome</a>). The network consists of 353 nodes and 1178 edges. Proteins are depicted as nodes and interactions as edges. Red-coloured nodes represent known <i>in vivo</i> amyloidogenic proteins, whereas yellow-coloured nodes represent <i>in vitro</i> amyloid-forming proteins or proteins related to amyloid fibril formation (see also Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173163#pone.0173163.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173163#pone.0173163.t002" target="_blank">2</a>). Green-coloured nodes are proteins, listed as other interaction partners. Hubs and bottlenecks are depicted as triangles (▲) and squares (■), respectively. Protein-nodes, which are both hubs and bottlenecks are shown as diamonds (◆). Important molecular chaperones are highlighted with a blue outline.</p

The dataset of 13 proteins related to amyloid fibril formation.

<p>The dataset of 13 proteins related to amyloid fibril formation.</p