Human Plasma Protein Corona of Aß Amyloid and Its Impact on Islet Amyloid Polypeptide Cross-Seeding.

Alzheimer's disease (AD) is the most severe form of neurological disorder, characterized by the presence of extracellular amyloid-ß (Aß) plaques and intracellular tau tangles. For decades, therapeutic strategies against the pathological symptoms of AD have often relied on the delivery of monoclonal antibodies to target specifically Aß amyloid or oligomers, largely to no avail. Aß can be traced in the brain as well as in cerebrospinal fluid and the circulation, giving rise to abundant opportunities to interact with their environmental proteins. Using liquid chromatography tandem-mass spectrometry, here we identified for the first time the protein coronae of the two major amyloid forms of Aß-Aß1-42 and Aß1-40-exposed to human blood plasma. Out of the proteins identified in all groups, 58 proteins were unique to the Aß1-42 samples and 31 proteins unique to the Aß1-40 samples. Both fibrillar coronae consisted of proteins significant in complement activation, inflammation, and protein metabolic pathways involved in the pathology of AD. Structure-wise, the coronal proteins often possessed multidomains of high flexibility to maximize their association with the amyloid fibrils. The protein corona hindered recognition of Aß1-42 fibrils by their structurally specific antibodies and accelerated the aggregation but not the ß-cell toxicity of human islet amyloid polypeptide, the peptide associated with type 2 diabetes. This study highlights the importance of understanding the structural, functional, and pathological implications of the amyloid protein corona for the development of therapeutics against AD and a range of amyloid diseases.

Single-Molecular Heteroamyloidosis of Human Islet Amyloid Polypeptide.

Human amyloids and plaques uncovered post mortem are highly heterogeneous in structure and composition, yet literature concerning the heteroaggregation of amyloid proteins is extremely scarce. This knowledge deficiency is further exacerbated by the fact that peptide delivery is a major therapeutic strategy for targeting their full-length counterparts associated with the pathologies of a range of human diseases, including dementia and type 2 diabetes (T2D). Accordingly, here we examined the coaggregation of full-length human islet amyloid polypeptide (IAPP), a peptide associated with type 2 diabetes, with its primary and secondary amyloidogenic fragments 19-29 S20G and 8-20. Single-molecular aggregation dynamics was obtained by high-speed atomic force microscopy, augmented by transmission electron microscopy, X-ray diffraction, and super-resolution stimulated emission depletion microscopy. The coaggregation significantly prolonged the pause phase of fibril elongation, increasing its dwell time by 3-fold. Surprisingly, unidirectional elongation of mature fibrils, instead of protofilaments, was observed for the coaggregation, indicating a new form of tertiary protein aggregation unknown to existing theoretical models. Further in vivo zebrafish embryonic assay indicated improved survival and hatching, as well as decreased frequency and severity of developmental abnormalities for embryos treated with the heteroaggregates of IAPP with 19-29 S20G, but not with 8-20, compared to the control, indicating the therapeutic potential of 19-29 S20G against T2D.

Differential Roles of Plasma Protein Corona on Immune Cell Association and Cytokine Secretion of Oligomeric and Fibrillar Beta-Amyloid.

Alzheimer's disease (AD) is a primary neurological disease with no effective cure. A hallmark of AD is the presence of intracellular tangles and extracellular plaques derived from the aberrant aggregation of tau- and beta-amyloid (Aß). Aß presents in the brain as well as in cerebrospinal fluid and the circulation, and Aß toxicity has been attributed to amyloidosis and inflammation, among other causes. In this study, the effects of the plasma protein corona have been investigated with regard to the blood cell association and cytokine secretion of oligomeric (Aßo) and fibrillar Aß1-42(Aßf), two major forms of the peptide aggregates. Aßo displayed little change in membrane association in whole blood or washed blood (i.e., cells in the absence of plasma proteins) at 37 °C, while Aßf showed a clear preference for binding with all cell types sans plasma proteins. Immune cells exposed to Aßo, but not to Aßf, resulted in significant expression of cytokines IL-6 and TNF measured in real-time by a localized surface plasmon resonance sensor. These observations indicate greater immune cell association and cytokine stimulation of Aßo than Aßf and shed new light on the contrasting toxicities of Aßo and Aßf resulting from their differential capacities in acquiring a plasma protein corona. These results further implicate a close connection between Aß amyloidosis and immunopathology in AD.

In Vivo Mitigation of Amyloidogenesis through Functional-Pathogenic Double-Protein Coronae.

Amyloid diseases are global epidemics with no cure available. Herein, we report a first demonstration of in vivo mitigation of amyloidogenesis using biomimetic nanotechnology. Specifically, the amyloid fragments (ba) of ß-lactoglobulin, a whey protein, were deposited onto the surfaces of carbon nanotubes (baCNT), which subsequently sequestered human islet amyloid polypeptide (IAPP) through functional-pathogenic double-protein coronae. Conformational changes at the ba-IAPP interface were studied by Fourier transform infrared, circular dichroism, and X-ray scattering spectroscopies. baCNT eliminated the toxic IAPP species from zebrafish embryos, as evidenced by the assays of embryonic development, cell morphology, hatching, and survival as well as suppression of oxidative stress. In addition to IAPP, baCNT also displayed high potency against the toxicity of amyloid-ß, thereby demonstrating the broad applicability of this biomimetic nanotechnology and the use of an embryonic zebrafish model for the high-throughput screening of a range of amyloidogenesis and their inhibitors in vivo.

Mitigating Human IAPP Amyloidogenesis In Vivo with Chiral Silica Nanoribbons.

Amyloid fibrils generally display chirality, a feature which has rarely been exploited in the development of therapeutics against amyloid diseases. This study reports, for the first time, the use of mesoscopic chiral silica nanoribbons against the in vivo amyloidogenesis of human islet amyloid polypeptide (IAPP), the peptide whose aggregation is implicated in type 2 diabetes. The thioflavin T assay and transmission electron microscopy show accelerated IAPP fibrillization through elimination of the nucleation phase and shortening of the elongation phase by the nanostructures. Coarse-grained simulations offer complementary molecular insights into the acceleration of amyloid aggregation through their nonspecific binding and directional seeding with the nanostructures. This accelerated IAPP fibrillization translates to reduced toxicity, especially for the right-handed silica nanoribbons, as revealed by cell viability, helium ion microscopy, as well as zebrafish embryo survival, developmental, and behavioral assays. This study has implicated the potential of employing chiral nanotechnologies against the mesoscopic enantioselectivity of amyloid proteins and their associated diseases.

Complex chiral colloids and surfaces via high-index off-cut silicon.

Silicon wafers are commonly etched in potassium hydroxide solutions to form highly symmetric surface structures. These arise when slow-etching {111} atomic planes are exposed on standard low-index surfaces. However, the ability of nonstandard high-index wafers to provide more complex structures by tilting the {111} planes has not been fully appreciated. We demonstrate the power of this approach by creating chiral surface structures and nanoparticles of a specific handedness from gold. When the nanoparticles are dispersed in liquids, gold colloids exhibiting record molar circular dichroism (>5 × 10(9) M(-1) cm(-1)) at red wavelengths are obtained. The nanoparticles also present chiral pockets for binding.

Probing the Aggregation and Immune Response of Human Islet Amyloid Polypeptides with Ligand-Stabilized Gold Nanoparticles.

The use of nanomaterials has recently become an emerging strategy against protein amyloidosis associated with a range of metabolic and brain diseases. To facilitate research in this area, here we first demonstrated the use of hyperspectral imaging (HSI) and COMSOL simulations for reporting the aggregation of human islet amyloid polypeptides (IAPPs), a hallmark of type 2 diabetes, as well as the physical interactions between the peptide and gold nanoparticles (AuNPs) grafted with citrate and poly(ethylene glycol) (PEG400 and PEG3000). We found a distinct anticorrelation between increased IAPP aggregation and decreased spectral red shifts incurred in the AuNP plasmonic resonance. Moreover, Jurkat cells exposed to IAPP and AuNPs were characterized by quantifying their cytokine secretions with a localized surface plasmon resonance (LSPR) immunoassay, where a peak response was registered for the most toxic IAPP oligomers and most suppressed by citrate-coated AuNPs. This study demonstrated the potential of using HSI and LSPR as two new platforms for the facile examination of protein aggregation and their induced immune response associated with amyloid diseases.

Amphiphilic surface chemistry of fullerenols is necessary for inhibiting the amyloid aggregation of alpha-synuclein NACore.

Featuring small sizes, caged structures, low cytotoxicity and the capability to cross biological barriers, fullerene hydroxy derivatives named fullerenols have been explored as nanomedicinal candidates for amyloid inhibition. Understanding the surface chemistry effect of hydroxylation extents and the corresponding amyloid inhibition mechanisms is necessary for enabling applications of fullerenols and also future designs of nanomedicines in mitigating amyloid aggregation. Here, we investigated effects of C60(OH)n with n = 0-40 on the aggregation of NACore (the amyloidogenic core region of the non-amyloid-ß component in α-synuclein), the amyloidogenic core of α-synuclein, by computational simulations, transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, thioflavin-T (ThT) fluorescence kinetics and viability assays. Computationally, NACore assembled into cross-ß aggregates via intermediates including ß-barrels, which are postulated as toxic oligomers of amyloid aggregation. Hydrophobic C60 preferred to self-assemble, and NACore bound to the surface of C60 nano-clusters formed ß-sheet rich aggregates - i.e., having little inhibition effect. Amphiphilic C60(OH)n with n = 4-20 displayed significant inhibition effects on NACore aggregation, where hydrogen bonding between hydroxyls and peptide backbones interrupted the formation of ß-sheets between peptides adsorbed onto the surfaces of fullerenols or fullerenol nano-assemblies due to hydrophobic interactions. Thus, both cross-ß aggregates and ß-barrel intermediates were significantly suppressed. With hydroxyls increased to 40, fullerenols became highly hydrophilic with reduced peptide binding and thus an inhibition effect on amyloid aggregation. ThT, FTIR and TEM characterization of C60(OH)n with n = 0, 24, & 40 confirmed the computational predictions. Our results and others underscore the importance of amphiphilic surface chemistry and the capability of polar groups in forming hydrogen bonds with peptide backbones to render amyloid inhibition, offering a new insight for de-novo design of anti-amyloid inhibitors.

Inhibition of amyloid beta toxicity in zebrafish with a chaperone-gold nanoparticle dual strategy.

Alzheimer's disease (AD) is the most prevalent form of neurodegenerative disorders, yet no major breakthroughs have been made in AD human trials and the disease remains a paramount challenge and a stigma in medicine. Here we eliminate the toxicity of amyloid beta (Aß) in a facile, high-throughput zebrafish (Danio rerio) model using casein coated-gold nanoparticles (ßCas AuNPs). ßCas AuNPs in systemic circulation translocate across the blood brain barrier of zebrafish larvae and sequester intracerebral Aß42 and its elicited toxicity in a nonspecific, chaperone-like manner. This is evidenced by behavioral pathology, reactive oxygen species and neuronal dysfunction biomarkers assays, complemented by brain histology and inductively coupled plasma-mass spectroscopy. We further demonstrate the capacity of ßCas AuNPs in recovering the mobility and cognitive function of adult zebrafish exposed to Aß. This potent, safe-to-use, and easy-to-apply nanomedicine may find broad use for eradicating toxic amyloid proteins implicated in a range of human diseases.

Physical and Toxicological Profiles of Human IAPP Amyloids and Plaques.

Although much has been learned about the fibrillization kinetics, structure and toxicity of amyloid proteins, the properties of amyloid fibrils beyond the saturation phase are often perceived as chemically and biologically inert, despite evidence suggesting otherwise. To fill this knowledge gap, we examined the physical and biological characteristics of human islet amyloid polypeptide (IAPP) fibrils that were aged up to two months. Not only did aging decrease the toxicity of IAPP fibrils, but the fibrils also sequestered fresh IAPP and suppressed their toxicity in an embryonic zebrafish model. The mechanical properties of IAPP fibrils in different aging stages were probed by atomic force microscopy and sonication, which displayed comparable stiffness but age-dependent fragmentation, followed by self-assembly of such fragments into the largest lamellar amyloid structures reported to date. The dynamic structural and toxicity profiles of amyloid fibrils and plaques suggest that they play active, long-term roles in cell degeneration and may be a therapeutic target for amyloid diseases.

Graphene quantum dots rescue protein dysregulation of pancreatic ß-cells exposed to human islet amyloid polypeptide.

The amyloid aggregation of peptides and proteins is a hallmark of neurological disorders and type 2 diabetes. Human islet amyloid polypeptide (IAPP), co-secreted with insulin by pancreatic ß-cells, plays dual roles in both glycemic control and the pathology of type 2 diabetes. While IAPP can activate the NLRP3 inflammasome and modulate cellular autophagy, apoptosis and extracellular matrix metabolism, no data is available concerning intracellular protein expression upon exposure to the polypeptide. More surprisingly, how intracellular protein expression is modulated by nanoparticle inhibitors of protein aggregation remains entirely unknown. In this study, we first examined the changing proteomes of ßTC6, a pancreatic ß-cell line, upon exposure to monomeric, oligomeric and fibrillar IAPP, and detailed cellular protein expression rescued by graphene quantum dots (GQDs), an IAPP inhibitor. We found that 29 proteins were significantly dysregulated by the IAPP species, while majority of these proteins were nucleotide-binding proteins. Collectively, our liquid chromatography tandem-mass spectrometry, fluorescence quenching, helium ion microscopy, cytotoxicity and discreet molecular dynamics simulations data revealed a remarkable capacity of GQDs in regulating aberrant protein expression through H-bonding and hydrophobic interactions, pointing to nanomedicine as a new frontier against human amyloid diseases.

Serum albumin impedes the amyloid aggregation and hemolysis of human islet amyloid polypeptide and alpha synuclein.

Protein aggregation is a ubiquitous phenomenon underpinning the origins of a range of human diseases. The amyloid aggregation of human islet amyloid polypeptide (IAPP) and alpha synuclein (αS), specifically, is a hallmark of type 2 diabetes (T2D) and Parkinson's disease impacting millions of people worldwide. Although IAPP and αS are strongly associated with pancreatic ß-cell islets and presynaptic terminals, they have also been found in blood circulation and the gut. While extensive biophysical and biochemical studies have been focused on IAPP and αS interacting with cell membranes or model lipid vesicles, the roles of plasma proteins on the amyloidosis and membrane association of these two major types of amyloid proteins have rarely been examined. Using a thioflavin T kinetic assay, transmission electron microscopy and a hemolysis assay here we show that human serum albumin, the most abundant protein in the plasma, impeded the fibrillization and mitigated membrane damage of both IAPP and αS. This study offers a new insight on the native inhibition of amyloidosis.

Profiling the Serum Protein Corona of Fibrillar Human Islet Amyloid Polypeptide.

Amyloids may be regarded as native nanomaterials that form in the presence of complex protein mixtures. By drawing an analogy with the physicochemical properties of nanoparticles in biological fluids, we hypothesized that amyloids should form a protein corona in vivo that would imbue the underlying amyloid with a modified biological identity. To explore this hypothesis, we characterized the protein corona of human islet amyloid polypeptide (IAPP) fibrils in fetal bovine serum using two complementary methodologies developed herein: quartz crystal microbalance and "centrifugal capture", coupled with nanoliquid chromatography tandem mass spectroscopy. Clear evidence for a significant protein corona was obtained. No trends were identified for amyloid corona proteins based on their physicochemical properties, whereas strong binding with IAPP fibrils occurred for linear proteins or multidomain proteins with structural plasticity. Proteomic analysis identified amyloid-enriched proteins that are known to play significant roles in mediating cellular machinery and processing, potentially leading to pathological outcomes and therapeutic targets.

Nanoparticle-proteome in vitro and in vivo.

The protein corona is a concept central to a range of disciplines exploiting the bio-nano interface. As the literature continues to expand in this field, it is essential to condense and contextualize the in vitro and in vivo proteome databases accumulated over the past decade: a goal which this review intends to achieve for the benefit of nanomedicine and nanobiotechnology. The parameters used for our review are the physicochemical characteristics of the nanoparticles, their surface ligands, the biological matrix from which a corona was formed, methods employed, plus the top-ten enriched corona proteins. In addition, the protein coronal networks and their implications in vivo are highlighted for selected studies.