Segregation between an ornamental and a disease driver gene provides insights into pigment cell regulation.

Genetic interactions are adaptive within a species. Hybridization can disrupt such species-specific genetic interactions and creates novel interactions that alter the hybrid progeny overall fitness. Hybrid incompatibility, which refers to degenerative genetic interactions that decrease the overall hybrid survival, is one of the results from combining two diverged genomes in hybrids. The discovery of spontaneous lethal tumorigenesis and underlying genetic interactions in select hybrids between diverged Xiphophorus species showed that lethal pathological process can result from degenerative genetic interactions. Such genetic interactions leading to lethal phenotype are thought to shield gene flow between diverged species. However, hybrids between certain Xiphophorus species do not develop such tumors. Here we report the identification of a locus residing in the genome of one Xiphophorus species that represses an oncogene from a different species. Our finding provides insights into normal and pathological pigment cell development, regulation and molecular mechanism in hybrid incompatibility. Significance: The Dobzhansky-Muller model states epistatic interactions occurred between genes in diverged species underlies hybrid incompatibility. There are a few vertebrate interspecies hybrid cases that support the Dobzhansky-Muller model. This study reports a fish hybrid system where incompatible genetic interactions are involved in neuronal regulation of pigment cell biology, and also identified a novel point of regulation for pigment cells.

Modulating Nanodroplet Formation En Route to Fibrillization of Amyloid Peptides with Designed Flanking Sequences.

Soluble oligomers populating early amyloid aggregation can be regarded as nanodroplets of liquid-liquid phase separation (LLPS). Amyloid peptides typically contain hydrophobic aggregation-prone regions connected by hydrophilic linkers and flanking sequences, and such a sequence hydropathy pattern drives the formation of supramolecular structures in the nanodroplets and modulates subsequent fibrillization. Here, we studied LLPS and fibrillization of coarse-grained amyloid peptides with increasing flanking sequences. Nanodroplets assumed lamellar, cylindrical micellar, and spherical micellar structures with increasing peptide hydrophilic/hydrophobic ratios, and such morphologies governed subsequent fibrillization processes. Adding glycine-serine repeats as flanking sequences to Aß16-22, the amyloidogenic core of amyloid-ß, our computational predictions of morphological transitions were corroborated experimentally. The uncovered inter-relationships between the peptide sequence pattern, oligomer/nanodroplet morphology, and fibrillization pathway, kinetics, and structure may contribute to our understanding of pathogenic amyloidosis in aging, facilitate future efforts ameliorating amyloidosis through peptide engineering, and aid in the design of novel amyloid-based functional nanobiomaterials and nanocomposites.

Inhibition of Amyloid Aggregation and Toxicity with Janus Iron Oxide Nanoparticles.

Amyloid aggregation is a ubiquitous form of protein misfolding underlying the pathologies of Alzheimer's disease (AD), Parkinson's disease (PD) and type 2 diabetes (T2D), three primary forms of human amyloid diseases. While much has been learned about the origin, diagnosis and management of these neurological and metabolic disorders, no cure is currently available due in part to the dynamic and heterogeneous nature of the toxic oligomers induced by amyloid aggregation. Here we synthesized beta casein-coated iron oxide nanoparticles (ßCas IONPs) via a BPA-P(OEGA-b-DBM) block copolymer linker. Using a thioflavin T kinetic assay, transmission electron microscopy, Fourier transform infrared spectroscopy, discrete molecular dynamics simulations and cell viability assays, we examined the Janus characteristics and the inhibition potential of ßCas IONPs against the aggregation of amyloid beta (Aß), alpha synuclein (αS) and human islet amyloid polypeptide (IAPP) which are implicated in the pathologies of AD, PD and T2D. Incubation of zebrafish embryos with the amyloid proteins largely inhibited hatching and elicited reactive oxygen species, which were effectively rescued by the inhibitor. Furthermore, Aß-induced damage to mouse brain was mitigated in vivo with the inhibitor. This study revealed the potential of Janus nanoparticles as a new nanomedicine against a diverse range of amyloid diseases.

Amyloid Aggregation under the Lens of Liquid-Liquid Phase Separation.

Increasing experiments suggest that amyloid peptides can undergo liquid-liquid phase separation (LLPS) before the formation of amyloid fibrils. However, the exact role of LLPS in amyloid aggregation at the molecular level remains elusive. Here, we investigated the LLPS and amyloid fibrillization of a coarse-grained peptide, capable of capturing fundamental properties of amyloid aggregation over a wide range of concentrations in molecular dynamics simulations. On the basis of the Flory-Huggins theory of polymer solutions, we determined the binodal and spinodal concentrations of LLPS in the low-concentration regime, ϕBL and ϕSL, respectively. Only at concentrations above ϕBL, peptides formed metastable or stable oligomers corresponding to the high-density liquid phase (HDLP) in LLPS, out of which the nucleated conformational conversion to fibril seeds occurred. Below ϕSL, the HDLP was metastable and transient, and the subsequent fibrillization process followed the traditional nucleation and elongation mechanisms. Only above ϕSL, the HDLP became stable, and the initial fibril nucleation and growth were governed by the high local peptide concentrations. The predicted saturation of amyloid aggregation half-times with increasing peptide concentration to a constant, instead of the traditional power-law scaling to zero, was confirmed by simulations and by a thioflavin-T kinetic assay and the transmission electron microscopy of islet amyloid polypeptide (IAPP) aggregation. Our study provides a unified picture of amyloid aggregation for a wide range of concentrations within the framework of LLPS, which may help us better understand the etiology of amyloid diseases, where the amyloid protein concentration can vary by ∼9 orders of magnitude depending on the organ location and facilitate the engineering of novel amyloid-based functional materials.

Morphological Determinants of Carbon Nanomaterial-Induced Amyloid Peptide Self-Assembly.

Hybridizing carbon nanomaterials (CNMs) with amyloid fibrils-the ordered nanostructures self-assembled by amyloidogenic peptides-has found promising applications in bionanotechology. Understanding fundamental interactions of CNMs with amyloid peptides and uncovering the determinants of their self-assembly structures and dynamics are, therefore, pivotal for enriching and enabling this novel class of hybrid nanomaterials. Here, we applied atomistic molecular dynamics simulations to investigate the self-assembly of two amyloid peptides-the amyloidogenic core residues 16-22 of amyloid-ß (Aß16-22) and the non-amyloid-ß core of α-synuclein (NACore68-78)-on the surface of carbon nanotubes (CNT) with different sizes and chirality. Our computational results showed that with small radial CNTs, both types of peptides could form ß-sheets wrapping around the nanotube surface into a supercoiled morphology. The angle between ß-strands and nanotube axes in the supercoil structure depended mainly on the peptide sequence and CNT radius, but also weakly on the CNT chirality. Large radial CNTs and the extreme case of the flat graphene nanosheet, on the other hand, could nucleate amyloid fibrils perpendicular to the surface. Our results provided new insights of hybridizing CNMs with amyloid peptides and also offered a novel approach to manipulate the morphology of CNM-induced amyloid assembly by tuning the surface curvature, peptide sequence, and molecular ratio between peptides and available CNM surface area, which may be useful in engineering nanocomposites with high-order structures.

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.

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.

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.

Amyloid Self-Assembly of hIAPP8-20 via the Accumulation of Helical Oligomers, α-Helix to ß-Sheet Transition, and Formation of ß-Barrel Intermediates.

The self-assembly of human islet amyloid polypeptide (hIAPP) into ß-sheet-rich nanofibrils is associated with the pathogeny of type 2 diabetes. Soluble hIAPP is intrinsically disordered with N-terminal residues 8-17 as α-helices. To understand the contribution of the N-terminal helix to the aggregation of full-length hIAPP, here the oligomerization dynamics of the hIAPP fragment 8-20 (hIAPP8-20) are investigated with combined computational and experimental approaches. hIAPP8-20 forms cross-ß nanofibrils in silico from isolated helical monomers via the helical oligomers and α-helices to ß-sheets transition, as confirmed by transmission electron microscopy, atomic force microscopy, circular dichroism spectroscopy, Fourier transform infrared spectroscopy, and reversed-phase high performance liquid chromatography. The computational results also suggest that the critical nucleus of aggregation corresponds to hexamers, consistent with a recent mass-spectroscopy study of hIAPP8-20 aggregation. hIAPP8-20 oligomers smaller than hexamers are helical and unstable, while the α-to-ß transition starts from the hexamers. Converted ß-sheet-rich oligomers first form ß-barrel structures as intermediates before aggregating into cross-ß nanofibrils. This study uncovers a complete picture of hIAPP8-20 peptide oligomerization, aggregation nucleation via conformational conversion, formation of ß-barrel intermediates, and assembly of cross-ß protofibrils, thereby shedding light on the aggregation of full-length hIAPP, a hallmark of pancreatic beta-cell degeneration.

Nucleation of ß-rich oligomers and ß-barrels in the early aggregation of human islet amyloid polypeptide.

The self-assembly of human islet amyloid polypeptide (hIAPP) into ß-sheet rich amyloid aggregates is associated with pancreatic ß-cell death in type 2 diabetes (T2D). Prior experimental studies of hIAPP aggregation reported the early accumulation of α-helical intermediates before the rapid conversion into ß-sheet rich amyloid fibrils, as also corroborated by our experimental characterizations with transmission electron microscopy and Fourier transform infrared spectroscopy. Although increasing evidence suggests that small oligomers populating early hIAPP aggregation play crucial roles in cytotoxicity, structures of these oligomer intermediates and their conformational conversions remain unknown, hindering our understanding of T2D disease mechanism and therapeutic design targeting these early aggregation species. We further applied large-scale discrete molecule dynamics simulations to investigate the oligomerization of full-length hIAPP, employing multiple molecular systems of increasing number of peptides. We found that the oligomerization process was dynamic, involving frequent inter-oligomeric exchanges. On average, oligomers had more α-helices than ß-sheets, consistent with ensemble-based experimental measurements. However, in ~4-6% independent simulations, ß-rich oligomers expected as the fibrillization intermediates were observed, especially in the pentamer and hexamer simulations. These ß-rich oligomers could adopt ß-barrel conformations, recently postulated to be the toxic oligomer species but only observed computationally in the aggregates of short amyloid protein fragments. Free-energy analysis revealed high energies of these ß-rich oligomers, supporting the nucleated conformational changes of oligomers in amyloid aggregation. ß-barrel oligomers of full-length hIAPP with well-defined three-dimensional structures may play an important pathological role in T2D etiology and may be a therapeutic target for the disease.

ß-barrel Oligomers as Common Intermediates of Peptides Self-Assembling into Cross-ß Aggregates.

Oligomers populated during the early amyloid aggregation process are more toxic than mature fibrils, but pinpointing the exact toxic species among highly dynamic and heterogeneous aggregation intermediates remains a major challenge. ß-barrel oligomers, structurally-determined recently for a slow-aggregating peptide derived from αB crystallin, are attractive candidates for exerting amyloid toxicity due to their well-defined structures as therapeutic targets and compatibility to the "amyloid-pore" hypothesis of toxicity. To assess whether ß-barrel oligomers are common intermediates to amyloid peptides - a necessary step toward associating ß-barrel oligomers with general amyloid cytotoxicity, we computationally studied the oligomerization and fibrillization dynamics of seven well-studied fragments of amyloidogenic proteins with different experimentally-determined aggregation morphologies and cytotoxicity. In our molecular dynamics simulations, ß-barrel oligomers were only observed in five peptides self-assembling into the characteristic cross-ß aggregates, but not the other two that formed polymorphic ß-rich aggregates as reported experimentally. Interestingly, the latter two peptides were previously found nontoxic. Hence, the observed correlation between ß-barrel oligomers formation and cytotoxicity supports the hypothesis of ß-barrel oligomers as the common toxic intermediates of amyloid aggregation.

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.

Lysophosphatidylcholine modulates the aggregation of human islet amyloid polypeptide.

Amyloid aggregation of human islet amyloid polypeptide (IAPP) is a hallmark of type 2 diabetes (T2D), a metabolic disease and a global epidemic. Although IAPP is synthesized in pancreatic ß-cells, its fibrils and plaques are found in the extracellular space indicating a causative transmembrane process. Numerous biophysical studies have revealed that cell membranes as well as model lipid vesicles promote the aggregation of amyloid-ß (associated with Alzheimer's), α-synuclein (associated with Parkinson's) and IAPP, through electrostatic and hydrophobic interactions between the proteins/peptides and lipid membranes. Using a thioflavin T kinetic assay, transmission electron microscopy, circular dichroism spectroscopy, discrete molecular dynamics simulations as well as free energy calculations here we show that micellar lysophosphatidylcholine (LPC), the most abundant lysophospholipid in the blood, inhibited the amyloid aggregation of IAPP through nonspecific interactions while elevating the α-helical peptide secondary structure. This surprising finding suggests a native protective mechanism against IAPP aggregation in vivo.

Effects of Protein Corona on IAPP Amyloid Aggregation, Fibril Remodelling, and Cytotoxicity.

Aggregation of islet amyloid polypeptide (IAPP), a peptide hormone co-synthesized and co-stored with insulin in pancreatic cells and also co-secreted to the circulation, is associated with beta-cell death in type-2 diabetes (T2D). In T2D patients IAPP is found aggregating in the extracellular space of the islets of Langerhans. Although the physiological environments of these intra- and extra-cellular compartments and vascular systems significantly differ, the presence of proteins is ubiquitous but the effects of protein binding on IAPP aggregation are largely unknown. Here we examined the binding of freshly-dissolved IAPP as well as pre-formed fibrils with two homologous proteins, namely cationic lysozyme (Lys) and anionic alpha-lactalbumin (aLac), both of which can be found in the circulation. Biophysical characterizations and a cell viability assay revealed distinct effects of Lys and aLac on IAPP amyloid aggregation, fibril remodelling and cytotoxicity, pointing to the role of protein "corona" in conferring the biological impact of amyloidogenic peptides. Systematic molecular dynamics simulations further provided molecular and structural details for the observed differential effects of proteins on IAPP amyloidosis. This study facilitates our understanding of the fate and transformation of IAPP in vivo, which are expected to have consequential bearings on IAPP glycemic control and T2D pathology.