Misfolded protein oligomers: mechanisms of formation, cytotoxic effects, and pharmacological approaches against protein misfolding diseases.

The conversion of native peptides and proteins into amyloid aggregates is a hallmark of over 50 human disorders, including Alzheimer's and Parkinson's diseases. Increasing evidence implicates misfolded protein oligomers produced during the amyloid formation process as the primary cytotoxic agents in many of these devastating conditions. In this review, we analyze the processes by which oligomers are formed, their structures, physicochemical properties, population dynamics, and the mechanisms of their cytotoxicity. We then focus on drug discovery strategies that target the formation of oligomers and their ability to disrupt cell physiology and trigger degenerative processes.

Secondary Processes Dominate the Quiescent, Spontaneous Aggregation of α-Synuclein at Physiological pH with Sodium Salts.

The accurate recapitulation in an in vitro assay of the aggregation process of α-synuclein in Parkinson's disease has been a significant challenge. As α-synuclein does not aggregate spontaneously in most currently used in vitro assays, primary nucleation is triggered by the presence of surfaces such as lipid membranes or interfaces created by shaking, to achieve aggregation on accessible time scales. In addition, secondary nucleation is typically only observed by lowering the pH below 5.8. Here we investigated assay conditions that enables spontaneous primary nucleation and secondary nucleation at pH 7.4. Using 400 mM sodium phosphate, we observed quiescent spontaneous aggregation of α-synuclein and established that this aggregation is dominated by secondary processes. Furthermore, the presence of potassium ions enhanced the reproducibility of quiescent α-synuclein aggregation. This work provides a framework for the study of spontaneous α-synuclein aggregation at physiological pH.

EGCG inactivates a pore-forming toxin by promoting its oligomerization and decreasing its solvent-exposed hydrophobicity.

Natural proteinaceous pore-forming agents can bind and permeabilize cell membranes, leading to ion dyshomeostasis and cell death. In the search for antidotes that can protect cells from peptide toxins, we discovered that the polyphenol epigallocatechin gallate (EGCG) interacts directly with melittin from honeybee venom, resulting in the elimination of its binding to the cell membrane and toxicity by markedly lowering the extent of its solvent-exposed hydrophobicity and promoting its oligomerization into larger species. These physicochemical parameters have also been shown to play a key role in the binding to cells of misfolded protein oligomers in a host of neurodegenerative diseases, where oligomer-membrane binding and associated toxicity have been shown to correlate negatively with oligomer size and positively with solvent-exposed hydrophobicity. For melittin, which is not an amyloid-forming protein and has a very distinct mechanism of toxicity compared to misfolded oligomers, we find that the size-hydrophobicity-toxicity relationship also rationalizes the pharmacological attenuation of melittin toxicity by EGCG. These results highlight the importance of the physicochemical properties of pore forming agents in mediating their interactions with cell membranes and suggest a possible therapeutic approach based on compounds with a similar mechanism of action as EGCG.

A Brain-Permeable Aminosterol Regulates Cell Membranes to Mitigate the Toxicity of Diverse Pore-Forming Agents.

The molecular composition of the plasma membrane plays a key role in mediating the susceptibility of cells to perturbations induced by toxic molecules. The pharmacological regulation of the properties of the cell membrane has therefore the potential to enhance cellular resilience to a wide variety of chemical and biological compounds. In this study, we investigate the ability of claramine, a blood-brain barrier permeable small molecule in the aminosterol class, to neutralize the toxicity of acute biological threat agents, including melittin from honeybee venom and α-hemolysin from Staphylococcus aureus. Our results show that claramine neutralizes the toxicity of these pore-forming agents by preventing their interactions with cell membranes without perturbing their structures in a detectable manner. We thus demonstrate that the exogenous administration of an aminosterol can tune the properties of lipid membranes and protect cells from diverse biotoxins, including not just misfolded protein oligomers as previously shown but also biological protein-based toxins. Our results indicate that the investigation of regulators of the physicochemical properties of cell membranes offers novel opportunities to develop countermeasures against an extensive set of cytotoxic effects associated with cell membrane disruption.

A Structural Ensemble of a Tau-Microtubule Complex Reveals Regulatory Tau Phosphorylation and Acetylation Mechanisms.

Tau is a microtubule-associated protein that regulates the stability of microtubules. We use metainference cryoelectron microscopy, an integrative structural biology approach, to determine an ensemble of conformations representing the structure and dynamics of a tau-microtubule complex comprising the entire microtubule-binding region of tau (residues 202-395). We thus identify the ground state of the complex and a series of excited states of lower populations. A comparison of the interactions in these different states reveals positions along the tau sequence that are important to determine the overall stability of the tau-microtubule complex. This analysis leads to the identification of positions where phosphorylation and acetylation events have destabilizing effects, which we validate by using site-specific post-translationally modified tau variants obtained by chemical mutagenesis. Taken together, these results illustrate how the simultaneous determination of ground and excited states of macromolecular complexes reveals functional and regulatory mechanisms.

Nonhuman IAPP Variants Inhibit Human IAPP Aggregation.

AIM: To identify naturally occurring variants of IAPP capable of inhibiting the aggregation of human IAPP and protecting living cells from the toxic effects of human IAPP. BACKGROUND: The loss of insulin-producing ß-cells and the overall progression of type 2 diabetes appears to be linked to the formation of toxic human IAPP (hIAPP, Islet Amyloid Polypeptide, amylin) amyloid in the pancreas. Inhibiting the initial aggregation of hIAPP has the potential to slow, if not stop entirely, the loss of ß-cells and halt the progression of the disease. OBJECTIVE: To identify and characterize naturally occurring variants of IAPP capable of inhibiting human IAPP aggregation. METHODS: Synthetic human IAPP was incubated with synthetic IAPP variants identified from natural sources under conditions known to promote amyloid-based aggregation. To identify IAPP variants capable of inhibiting human IAPP aggregation, Thioflavin T-binding fluorescence, atomic force microscopy, and cell-rescue assays were performed. RESULTS: While most IAPP variants showed little to no ability to inhibit human IAPP aggregation, several variants showed some ability to inhibit aggregation, with two variants showing substantial inhibitory potential. CONCLUSION: Several naturally occurring IAPP variants capable of inhibiting human IAPP aggregation were identified and characterized.

Amyloidogenicity of naturally occurring full-length animal IAPP variants.

The aggregation of the 37-amino acid polypeptide human islet amyloid polypeptide (hIAPP), as either insoluble amyloid or as small oligomers, appears to play a direct role in the death of human pancreatic ß-islet cells in type 2 diabetes. hIAPP is considered to be one of the most amyloidogenic proteins known. The quick aggregation of hIAPP leads to the formation of toxic species, such as oligomers and fibers, that damage mammalian cells (both human and rat pancreatic cells). Whether this toxicity is necessary for the progression of type 2 diabetes or merely a side effect of the disease remains unclear. If hIAPP aggregation into toxic amyloid is on-path for developing type 2 diabetes in humans, islet amyloid polypeptide (IAPP) aggregation would likely need to play a similar role within other organisms known to develop the disease. In this work, we compared the aggregation potential and cellular toxicity of full-length IAPP from several diabetic and nondiabetic organisms whose aggregation propensities had not yet been determined for full-length IAPP.