Fibrillar Assemblies Research Articles

Site-Specific Alkylation of the Islet Amyloid Polypeptide Accelerates Self-Assembly and Potentiates Perturbation of Lipid Membranes

The accumulation of insoluble amyloids in the pancreatic islets is a pathological hallmark of type II diabetes and correlates closely with the loss of β-cell mass. The predominant component of these amyloid deposits is the islet amyloid polypeptide (IAPP). The factors contributing to the conversion of IAPP from a monomeric bioactive peptide hormone into insoluble amyloid fibrils remain partially elusive. In this study, we investigated the effect of the oxidative non-enzymatic post-translational modification induced by the reactive metabolite 4-hydroxynonenal (HNE) on IAPP aggregation and cytotoxicity. Incubation of IAPP with exogenous HNE accelerated its self-assembly into β-sheet fibrils and led to the formation of a Michael adduct on the His-18 side chain. To model this covalent modification, the imidazole N(π) position of histidine was alkylated using a close analogue of HNE, the octyl chain. IAPP lipidated at His-18 showed a hastened random coil-to-β-sheet conformational conversion into fibrillar assemblies with a distinct morphology, a low level of binding to thioflavin T, and a high surface hydrophobicity. Introducing an octyl chain on His-18 enhanced the ability of the peptide to perturb synthetic lipid vesicles, to permeabilize the plasma membrane, and to induce the death of pancreatic β-cells. Alkylated IAPP triggered the self-assembly of unmodified IAPP by prompting primary nucleation and increased its capacity to perturb the plasma membrane, indicating that only a small proportion of the modified peptide is necessary to shift the balance toward the formation of proteotoxic species. This study underlines the importance of studying IAPP post-translational modifications induced by oxidative metabolites in the context of pancreatic amyloids.

Phosphorylation and O-GlcNAcylation of the PHF-1 Epitope of Tau Protein Induce Local Conformational Changes of the C-Terminus and Modulate Tau Self-Assembly Into Fibrillar Aggregates.

Phosphorylation of the neuronal microtubule-associated Tau protein plays a critical role in the aggregation process leading to the formation of insoluble intraneuronal fibrils within Alzheimer’s disease (AD) brains. In recent years, other posttranslational modifications (PTMs) have been highlighted in the regulation of Tau (dys)functions. Among these PTMs, the O-β-linked N-acetylglucosaminylation (O-GlcNAcylation) modulates Tau phosphorylation and aggregation. We here focus on the role of the PHF-1 phospho-epitope of Tau C-terminal domain that is hyperphosphorylated in AD (at pS396/pS404) and encompasses S400 as the major O-GlcNAc site of Tau while two additional O-GlcNAc sites were found in the extreme C-terminus at S412 and S413. Using high resolution NMR spectroscopy, we showed that the O-GlcNAc glycosylation reduces phosphorylation of PHF-1 epitope by GSK3β alone or after priming by CDK2/cyclin A. Furthermore, investigations of the impact of PTMs on local conformation performed in small peptides highlight the role of S404 phosphorylation in inducing helical propensity in the region downstream pS404 that is exacerbated by other phosphorylations of PHF-1 epitope at S396 and S400, or O-GlcNAcylation of S400. Finally, the role of phosphorylation and O-GlcNAcylation of PHF-1 epitope was probed in in-vitro fibrillization assays in which O-GlcNAcylation slows down the rate of fibrillar assembly while GSK3β phosphorylation stimulates aggregation counteracting the effect of glycosylation.

The ultrastructure of infectious L-type bovine spongiform encephalopathy prions constrains molecular models.

Bovine spongiform encephalopathy (BSE) is a prion disease of cattle that is caused by the misfolding of the cellular prion protein (PrPC) into an infectious conformation (PrPSc). PrPC is a predominantly α-helical membrane protein that misfolds into a β-sheet rich, infectious state, which has a high propensity to self-assemble into amyloid fibrils. Three strains of BSE prions can cause prion disease in cattle, including classical BSE (C-type) and two atypical strains, named L-type and H-type BSE. To date, there is no detailed information available about the structure of any of the infectious BSE prion strains. In this study, we purified L-type BSE prions from transgenic mouse brains and investigated their biochemical and ultrastructural characteristics using electron microscopy, image processing, and immunogold labeling techniques. By using phosphotungstate anions (PTA) to precipitate PrPSc combined with sucrose gradient centrifugation, a high yield of proteinase K-resistant BSE amyloid fibrils was obtained. A morphological examination using electron microscopy, two-dimensional class averages, and three-dimensional reconstructions revealed two structural classes of L-type BSE amyloid fibrils; fibrils that consisted of two protofilaments with a central gap and an average width of 22.5 nm and one-protofilament fibrils that were 10.6 nm wide. The one-protofilament fibrils were found to be more abundant compared to the thicker two-protofilament fibrils. Both fibrillar assemblies were successfully decorated with monoclonal antibodies against N- and C-terminal epitopes of PrP using immunogold-labeling techniques, confirming the presence of polypeptides that span residues 100-110 to 227-237. The fact that the one-protofilament fibrils contain both N- and C-terminal PrP epitopes constrains molecular models for the structure of the infectious conformer in favour of a compact four-rung β-solenoid fold.

Suppression of α-synuclein propagation after intrastriatal injection in FABP3 null mice

Accumulation and aggregation of α-synuclein (αSyn) trigger neuronal loss in the substantia nigra pars compacta (SNpc), which in turn causes motor symptoms in Parkinson’s disease. We previously demonstrated that fatty acid-binding protein 3 (FABP3), an intracellular fatty acid carrier protein, enhances αSyn neurotoxicity in SNpc and motor impairments after intranigral injection of αSyn fibrils. However, the temporal profile of αSyn fibril spread and their toxicity remains unclear. In the present study, we investigated the temporal profile of αSyn fibril spread and its toxicity, which induces intracellular fibril formation. Monomeric and fibrillar aSyn assemblies were labeled with ATTO550 to distinguish the exogenous form from the endogenous species and injected into bilateral striatum in Fabp3+/+ (wild type) and Fabp3-/- mice. Accumulation of both monomeric and fibrillar exogenous αSyn in the SNpc was drastically decreased in Fabp3-/- mice compared to that in the Fabp3+/+ counterparts. Deletion of Fabp3 also prevented exogenous αSyn fibril-induced seeding of the endogenous αSyn into aggregates containing phosphorylated and filamentous forms in the SNpc. Consistent with these results, loss of dopaminergic neurons and subsequent impaired motor behavior were attenuated in Fabp3-/- mice. These results highlight the crucial role of FABP3 in pathogenic αSyn accumulation and its seeding ability. Taken together, FABP3 could be a potential therapeutic target against αSyn propagation in synucleinopathies.

Fibronectin Extra Domains tune cellular responses and confer topographically distinct features to fibril networks.

Cellular fibronectin (FN; also known as FN1) variants harboring one or two alternatively spliced so-called extra domains (EDB and EDA) play a central bioregulatory role during development, repair processes and fibrosis. Yet, how the extra domains impact fibrillar assembly and function of the molecule remains unclear. Leveraging a unique biological toolset and image analysis pipeline for direct comparison of the variants, we demonstrate that the presence of one or both extra domains impacts FN assembly, function and physical properties of the matrix. When presented to FN-null fibroblasts, extra domain-containing variants differentially regulate pH homeostasis, survival and TGF-β signaling by tuning the magnitude of cellular responses, rather than triggering independent molecular switches. Numerical analyses of fiber topologies highlight significant differences in variant-specific structural features and provide a first step for the development of a generative model of FN networks to unravel assembly mechanisms and investigate the physical and functional versatility of extracellular matrix landscapes.This article has an associated First Person interview with the first author of the paper.

Multi-target inhibition ability of neohesperidin dictates its neuroprotective activity: Implication in Alzheimer's disease therapeutics

The polygenic nature of Alzheimer's disease (AD) and cross-talk between several signaling cascades make it harder to decode the disease pathogenesis. β-secretase (BACE1) works upstream in the amyloidogenic processing of amyloid precursor protein (APP) to generate Aβ that rapidly aggregates to form fibrils, the most abundant component of plaques observed in AD brains. Here, we report dual inhibition of BACE1 and Aβ aggregation by neohesperidin, a flavonoid glycoconjugate, using multi-spectroscopic approaches, force microscopy, molecular modeling, and validated the potency in SH-SY5Y neuroblastoma cell lines. Steady-state and time-resolved fluorescence reveal that neohesperidin binds close to the catalytic aspartate dyad. This binding conformationally restricts the protein in closed form which possibly precludes APP recognition and thereby inhibits BACE1 activity. Neohesperidin also dose-dependently inhibits the amyloid fibril formation, as evident from ANS, ThT assay, and AFM. Neohesperidin ameliorates aggregated Aβ25–35 induced ROS generation and mitochondrial dysfunction in the SH-SY5Y cell line. As a result, the amyloid induced apoptosis is significantly prohibited and normal neuronal morphology is rescued. These findings suggest neohesperidin as an inhibitor of the pathogenic conversion of Aβ to fibrillar amyloid assembly. Neohesperidin thus emerges as a non-toxic multi-potent scaffold for the development of AD therapeutics.

Mechanistic Inferences From Analysis of Measurements of Protein Phase Transitions in Live Cells

The combination of phase separation and disorder-to-order transitions can give rise to ordered, semi-crystalline fibrillar assemblies that underlie prion phenomena namely, the non-Mendelian transfer of information across cells. Recently, a method known as Distributed Amphifluoric Förster Resonance Energy Transfer (DAmFRET) was developed to study the convolution of phase separation and disorder-to-order transitions in live cells. In this assay, a protein of interest is expressed to a broad range of concentrations and the acquisition of local density and order, measured by changes in FRET, is used to map phase transitions for different proteins. The high-throughput nature of this assay affords the promise of uncovering sequence-to-phase behavior relationships in live cells. Here, we report the development of a supervised method to obtain automated and accurate classifications of phase transitions quantified using the DAmFRET assay. Systems that we classify as undergoing two-state discontinuous transitions are consistent with prion-like behaviors, although the converse is not always true. We uncover well-established and surprising new sequence features that contribute to two-state phase behavior of prion-like domains. Additionally, our method enables quantitative, comparative assessments of sequence-specific driving forces for phase transitions in live cells. Finally, we demonstrate that a modest augmentation of DAmFRET measurements, specifically time-dependent protein expression profiles, can allow one to apply classical nucleation theory to extract sequence-specific lower bounds on the probability of nucleating ordered assemblies. Taken together, our approaches lead to a useful analysis pipeline that enables the extraction of mechanistic inferences regarding phase transitions in live cells.

Seeding Propensity and Characteristics of Pathogenic αSyn Assemblies in Formalin-Fixed Human Tissue from the Enteric Nervous System, Olfactory Bulb, and Brainstem in Cases Staged for Parkinson's Disease.

We investigated α-synuclein’s (αSyn) seeding activity in tissue from the brain and enteric nervous system. Specifically, we assessed the seeding propensity of pathogenic αSyn in formalin-fixed tissue from the gastric cardia and five brain regions of 29 individuals (12 Parkinson’s disease, 8 incidental Lewy body disease, 9 controls) using a protein misfolding cyclic amplification assay. The structural characteristics of the resultant αSyn assemblies were determined by limited proteolysis and transmission electron microscopy. We show that fixed tissue from Parkinson’s disease (PD) and incidental Lewy body disease (ILBD) seeds the aggregation of monomeric αSyn into fibrillar assemblies. Significant variations in the characteristics of fibrillar assemblies derived from different regions even within the same individual were observed. This finding suggests that fixation stabilizes seeds with an otherwise limited seeding propensity, that yield assemblies with different intrinsic structures (i.e., strains). The lag phase preceding fibril assembly for patients ≥80 was significantly shorter than in other age groups, suggesting the existence of increased numbers of seeds or a higher seeding potential of pathogenic αSyn with time. Seeding activity did not diminish in late-stage disease. No statistically significant difference in the seeding efficiency of specific regions was found, nor was there a relationship between seeding efficiency and the load of pathogenic αSyn in a particular region at a given neuropathological stage.

Computational Reverse-Engineering Analysis of Scattering Experiments (CREASE) on Amphiphilic Block Polymer Solutions: Cylindrical and Fibrillar Assembly

In this paper, we extend a recently developed computational reverse-engineering analysis for scattering experiments (CREASE) approach to analyze cylindrical, fibrillar, and elliptical cylindrical structures assembled in amphiphilic block copolymer solutions. With CREASE, one can deduce information about assembled structures characterized via small-angle scattering without having to rely on fits using conventional analytical scattering models that may be approximate or inapplicable for the system at hand. With scattering intensity profiles and information about the polymer solution (e.g., molecular weight, composition, and sequence) provided to CREASE as an input, the output from CREASE includes the shape, morphology, dimensions, and molecular packing in the assembled polymer structures within the solution. CREASE is comprised of two steps: the first step involves a genetic algorithm (GA) to determine the shape and dimensions of the domains in the assembled structure and the second step uses molecular simulations to reconstruct chain conformations and monomer-level arrangements within the assembled structure. This paper builds on our recent work that was focused on spherical assembled structures and extends it to nonspherical assembled structures like cylinders, fibrils, and cylinders with elliptical cross sections. We validate the GA step within CREASE by taking input scattering intensity profiles from a variety of assembled shapes with known shapes and dimensions and by producing outputs that match those known shapes and target dimensions. To demonstrate its use in real situations where microscopy may hint at the potential shapes without the user knowing the dimensions with certainty, we apply CREASE with different potential assembled shapes and compare the structural dimensions of the results.

Basic Structure, Physiology, and Biochemistry of Connective Tissues and Extracellular Matrix Collagens.

The physiology of connective tissues like tendons and ligaments is highly dependent upon the collagens and other such extracellular matrix molecules hierarchically organized within the tissues. By dry weight, connective tissues are mostly composed of fibrillar collagens. However, several other forms of collagens play essential roles in the regulation of fibrillar collagen organization and assembly, in the establishment of basement membrane networks that provide support for vasculature for connective tissues, and in the formation of extensive filamentous networks that allow for cell-extracellular matrix interactions as well as maintain connective tissue integrity. The structures and functions of these collagens are discussed in this chapter. Furthermore, collagen synthesis is a multi-step process that includes gene transcription, translation, post-translational modifications within the cell, triple helix formation, extracellular secretion, extracellular modifications, and then fibril assembly, fibril modifications, and fiber formation. Each step of collagen synthesis and fibril assembly is highly dependent upon the biochemical structure of the collagen molecules created and how they are modified in the cases of development and maturation. Likewise, when the biochemical structures of collagens or are compromised or these molecules are deficient in the tissues - in developmental diseases, degenerative conditions, or injuries - then the ultimate form and function of the connective tissues are impaired. In this chapter, we also review how biochemistry plays a role in each of the processes involved in collagen synthesis and assembly, and we describe differences seen by anatomical location and region within tendons. Moreover, we discuss how the structures of the molecules, fibrils, and fibers contribute to connective tissue physiology in health, and in pathology with injury and repair.

The differential solvent exposure of N-terminal residues provides “fingerprints” of alpha-synuclein fibrillar polymorphs

Synucleinopathies are neurodegenerative diseases characterized by the presence of intracellular deposits containing the protein alpha-synuclein (aSYN) within patients’ brains. It has been shown that aSYN can form structurally distinct fibrillar assemblies, also termed polymorphs. We previously showed that distinct aSYN polymorphs assembled in vitro, named fibrils, ribbons, and fibrils 91, differentially bind to and seed the aggregation of endogenous aSYN in neuronal cells, which suggests that distinct synucleinopathies may arise from aSYN polymorphs. In order to better understand the differential interactions of aSYN polymorphs with their partner proteins, we mapped aSYN polymorphs surfaces. We used limited proteolysis, hydrogen–deuterium exchange, and differential antibody accessibility to identify amino acids on their surfaces. We showed that the aSYN C-terminal region spanning residues 94 to 140 exhibited similarly high solvent accessibility in these three polymorphs. However, the N-terminal amino acid residues 1 to 38 of fibrils were exposed to the solvent, while only residues 1 to 18 within fibrils 91 were exposed, and no N-terminal residues within ribbons were solvent-exposed. It is likely that these differences in surface accessibility contribute to the differential binding of distinct aSYN polymorphs to partner proteins. We thus posit that the polypeptides exposed on the surface of distinct aSYN fibrillar polymorphs are comparable to fingerprints. Our findings have diagnostic and therapeutic potential, particularly in the prion-like propagation of fibrillar aSYN, as they can facilitate the design of ligands that specifically bind and distinguish between fibrillar polymorphs.

Design of peptide-PEG-Thiazole bound polypyrrole supramolecular assemblies for enhanced neuronal cell interactions

ABSTRACT Brain injuries, and neurodegenerative diseases can result in significant disability, lowered quality of life, and mortality. Current treatments including autologous nerve grafts are insufficient. This work presents the creation of new biomimetic supramolecular assemblies as a scaffold for potential applications in neural tissue regeneration. It is composed of polyethylene glycol amide core-conjugated with a thiazole-based building block and a peptide sequence (RVYNMGKGKGFCVPRPLVVYR). This peptide sequence is derived from laminin, a basal membrane protein, and a component of the extracellular matrix of neuronal cells. The product formed was allowed to self-assemble and bound to polypyrrole by layer-by-layer assembly to impart conductive properties (Lam-PEG-thiazole-PPy) resulting in the formation of fibrillar assemblies as indicated by electron microscopy studies. Rheological studies demonstrated that incorporation of PPy resulted in higher storage modulus indicative of higher ability to store deformation energy. Culture of rat olfactory-bulb derived neurons with the assemblies demonstrated that the Lam-PEG-thiazole-PPy assemblies promoted cell proliferation and were able to interact favorably with the cells. After 10 days of culture with the assemblies interconnected networks could be visualized. Furthermore, electrical stimulation resulted in a significant increase in axonal outgrowths. Thus, the assemblies presented herein exhibit potential for applications as a novel scaffold for neural tissue engineering.

Extracellular Vesicles-Encapsulated Yeast Prions and What They Can Tell Us about the Physical Nature of Propagons

The yeast Saccharomyces cerevisiae hosts an ensemble of protein-based heritable traits, most of which result from the conversion of structurally and functionally diverse cytoplasmic proteins into prion forms. Among these, [PSI+], [URE3] and [PIN+] are the most well-documented prions and arise from the assembly of Sup35p, Ure2p and Rnq1p, respectively, into insoluble fibrillar assemblies. Yeast prions propagate by molecular chaperone-mediated fragmentation of these aggregates, which generates small self-templating seeds, or propagons. The exact molecular nature of propagons and how they are faithfully transmitted from mother to daughter cells despite spatial protein quality control are not fully understood. In [PSI+] cells, Sup35p forms detergent-resistant assemblies detectable on agarose gels under semi-denaturant conditions and cytosolic fluorescent puncta when the protein is fused to green fluorescent protein (GFP); yet, these macroscopic manifestations of [PSI+] do not fully correlate with the infectivity measured during growth by the mean of protein infection assays. We also discovered that significant amounts of infectious Sup35p particles are exported via extracellular (EV) and periplasmic (PV) vesicles in a growth phase and glucose-dependent manner. In the present review, I discuss how these vesicles may be a source of actual propagons and a suitable vehicle for their transmission to the bud.

Naphthoquinone-Dopamine Hybrids Inhibit α-Synuclein Aggregation, Disrupt Preformed Fibrils, and Attenuate Aggregate-Induced Toxicity.

Accumulation and aggregation of the intrinsically disordered protein α-synuclein (α-Syn) into amyloid fibrils are hallmarks of a series of heterogeneous neurodegenerative disorders, known as synucleinopathies and most notably Parkinson's disease (PD). The crucial role of α-Syn aggregation in PD makes it an attractive target for the development of disease-modifying therapeutics that would inhibit α-Syn aggregation or disrupt its preformed fibrillar assemblies. To this end, we have designed and synthesized two naphthoquinone-dopamine-based hybrid small molecules, NQDA and Cl-NQDA, and demonstrated their ability to inhibit in vitro amyloid formation by α-Syn using ThT assay, CD, TEM, and Congo red birefringence. Moreover, these hybrid molecules efficiently disassembled preformed fibrils of α-Syn into nontoxic species, as evident from LUV leakage assay. NQDA and Cl-NQDA were found to have low cytotoxicity and they attenuated the toxicity induced by α-Syn towards SH-SY5Y neuroblastoma cells. NQDA was found to efficiently cross an in vitro human blood-brain barrier model. These naphthoquinone-dopamine based derivatives can be an attractive scaffold for therapeutic design towards PD.

FMRP-PKA Activity Negative Feedback Regulates RNA Binding-Dependent Fibrillation in Brain Learning and Memory Circuitry

SUMMARYFragile X mental retardation protein (FMRP) promotes cyclic AMP (cAMP) signaling. Using an in vivo protein kinase A activity sensor (PKA-SPARK), we find that Drosophila FMRP (dFMRP) and human FMRP (hFMRP) enhance PKA activity in a central brain learning and memory center. Increasing neuronal PKA activity suppresses FMRP in Kenyon cells, demonstrating an FMRP-PKA negative feedback loop. A patient-derived R140Q FMRP point mutation mislocalizes PKA-SPARK activity, whereas deletion of the RNA-binding argi-nine-glycine-glycine (RGG) box (hFMRP-ΔRGG) produces fibrillar PKA-SPARK assemblies colocalizing with ribonucleoprotein (RNP) and aggregation (thioflavin T) markers, demonstrating fibrillar partitioning of cytosolic protein aggregates. hFMRP-ΔRGG reduces dFMRP levels, indicating RGG-independent regulation. Short-term hFMRP-ΔRGG induction produces activated PKA-SPARK puncta, whereas long induction drives fibrillar assembly. Elevated temperature disassociates hFMRP-ΔRGG aggregates and blocks activated PKA-SPARK localization. These results suggest that FMRP regulates compartmentalized signaling via complex assembly, directing PKA activity localization, with FMRP RGG box RNA binding restricting separation via low-complexity interactions.

Process, Outcomes and Possible Elimination of Aggregation with Special Reference to Heme Proteins; Likely Remediations of Proteinopathies.

Protein folding is a natural phenomenon through which a linear polypeptide possessing necessary information attains three-dimension functionally active conformation. This is a complex and multistep process and therefore, the presence of several intermediary structures could be speculated as a result of protein folding. In in vivo, this folding process is governed by the assistance of other proteins called molecular chaperones and heat shock proteins. Due to the mechanism of protein folding, these intermediary structures remain major challenge for modern biology. Mutation in gene encoding amino acid can cause adverse environmental conditions which may result in misfolding of the linear polypeptide followed by the formation of aggregates and amyloidosis. Aggregation contributes to the pathophysiology of several maladies including diabetes mellitus, Huntington's and Alzheimer's disease. The propensity of native structure to form aggregated and fibrillar assemblies is a hallmark of amyloidosis. During aggregation of a protein, transition from α helix to β sheet is observed, and mainly β sheeted structure is visualised in a mature fibril. Heme proteins are very crucial for major life activities like transport of oxygen and carbon dioxide, synthesis of ATP, role in electron transport chain, and detoxification of free radicals formed during biochemical reactions. Any structural variation in the heme proteins may lead to a fatal response. Hence characterization of the folding intermediates becomes crucial. The characterization has been deciphered with the help of strong denaturants like acetonitrile and TFE. Moreover, possible role of elimination of these aggregates and prevention of protein denaturation is also discussed. Current review deals with the basic process and mechanism of the protein folding in general and the ultimate outcomes of the protein misfolding. Since Native conformation of heme proteins is essential for some vital activities as listed above, we have discussed possible prevention of denaturation and aggregation of heme proteins such as Hb, cyt c, catalase & peroxidase.

Revisiting the Amyloid Cascade Hypothesis: From Anti-Aβ Therapeutics to Auspicious New Ways for Alzheimer's Disease.

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder related to age, characterized by the cerebral deposition of fibrils, which are made from the amyloid-β (Aβ), a peptide of 40–42 amino acids. The conversion of Aβ into neurotoxic oligomeric, fibrillar, and protofibrillar assemblies is supposed to be the main pathological event in AD. After Aβ accumulation, the clinical symptoms fall out predominantly due to the deficient brain clearance of the peptide. For several years, researchers have attempted to decline the Aβ monomer, oligomer, and aggregate levels, as well as plaques, employing agents that facilitate the reduction of Aβ and antagonize Aβ aggregation, or raise Aβ clearance from brain. Unluckily, broad clinical trials with mild to moderate AD participants have shown that these approaches were unsuccessful. Several clinical trials are running involving patients whose disease is at an early stage, but the preliminary outcomes are not clinically impressive. Many studies have been conducted against oligomers of Aβ which are the utmost neurotoxic molecular species. Trials with monoclonal antibodies directed against Aβ oligomers have exhibited exciting findings. Nevertheless, Aβ oligomers maintain equivalent states in both monomeric and aggregation forms; so, previously administered drugs that precisely decrease Aβ monomer or Aβ plaques ought to have displayed valuable clinical benefits. In this article, Aβ-based therapeutic strategies are discussed and several promising new ways to fight against AD are appraised.

Peptide Fibrillar Assemblies Exhibit Membranolytic Effects and Antimetastatic Activity on Lung Cancer Cells

Cancer metastasis is a central oncology concern that worsens patient conditions and increases mortality in a short period of time. During metastatic events, mitochondria undergo specific physiological alterations that have emerged as notable therapeutic targets to counter cancer progression. In this study, we use drug-free, cationic peptide fibrillar assemblies (PFAs) formed by poly(L-Lysine)-block-poly(L-Threonine) (Lys-b-Thr) to target mitochondria. These PFAs interact with cellular and mitochondrial membranes via electrostatic interactions, resulting in membranolysis. Charge repulsion and hydrogen-bonding interactions exerted by Lys and Thr segments dictate the packing of the peptides and enable the PFAs to display enhanced membranolytic activity toward cancer cells. Cytochrome c (cyt c), endonuclease G, and apoptosis-inducing factor were released from mitochondria after treatment of lung cancer cells, subsequently inducing caspase-dependent and caspase-independent apoptotic pathways. A metastatic xenograft mouse model was used to show how the PFAs significantly suppressed lung metastasis and inhibited tumor growth, while avoiding significant body weight loss and mortality. Antimetastatic activities of PFAs are also demonstrated by in vitro inhibition of lung cancer cell migration and clonogenesis. Our results imply that the cationic PFAs achieved the intended and targeted mitochondrial damage, providing an efficient antimetastatic therapy.

Self- and Cross-Seeding on α-Synuclein Fibril Growth Kinetics and Structure Observed by High-Speed Atomic Force Microscopy.

Fibril formation is an obligatory process in amyloid diseases and is characterized by nucleation and elongation phases that result in the formation of long filaments with cross-β sheet structure. The kinetics of this process, as well as that of secondary nucleation, is controlled by a variety of factors, including nucleus (seed) structure, monomer conformation, and biochemical milieu. Some fibrillar amyloid assemblies act as prions, replicating themselves from protein monomers templated by existing prion seeds. Prion strains, which are characterized by distinct physicochemical and pathologic properties, may also form due to perturbation of the templating process within the susceptible organism. Understanding the types and effects of perturbations occurring during the development and progression of Parkinson's disease is an area requiring more study. Here, we used high-speed atomic force microscopy to determine the kinetics and structural dynamics of α-synuclein fibril elongation initiated by self-seeding or cross-seeding of wild-type (WT) or mutant α-synuclein with WT or mutant α-synuclein seeds. We found that cross-seeding modulated not only elongation rates but also the structures of the growing fibrils. Some fibrils produced in this manner had structures distinct from their "parent" seeds. In other cases, cross-seeding was not observed at all. These findings suggest that α-synuclein sequence variants can produce different types of strains by self- or cross-seeding. Perpetuation of specific strains then would depend on the relative rates of fibril growth and the relative stabilities of the fibrils formed by each strain.

Peroxidasin is essential for endothelial cell survival and growth signaling by sulfilimine crosslink-dependent matrix assembly.

Peroxidasin (PXDN) has been reported to crosslink the C-terminal non-collagenous domains of collagen IV (Col IV) by forming covalent sulfilimine bond. Here, we explored the physiological role of PXDN and its mechanism of action in endothelial cell survival and growth. Silencing of PXDN using siRNAs decreased cell proliferation without increase of the number of detached cells and decreased cell viability under serum-starved condition with increased fragmented nuclei and caspase 3/7 activity. Conditioned medium (CM) containing wild-type PXDN restored the proliferation of PXDN-depleted cells, but CM containing mutant PXDN with deletion of either N-terminal extracellular matrix (ECM) motifs or peroxidase domain failed to restore PXDN function. Accordingly, anti-PXDN antibody [raised against IgC2 (3-4) subdomain within ECM motifs] and peroxidase inhibitor phloroglucinol prevented the rescue of the PXDN-depleted cells by PXDN-containing CM. PXDN depletion resulted in loss of sulfilimine crosslinks, and decreased dense fibrillar network assembly of not only Col IV, but also fibronectin and laminin like in Col IV knockdown. Exogenous PXDN-containing CM restored ECM assembly as well as proliferation of PXDN-depleted cells. Accordingly, purified recombinant PXDN protein restored the proliferation and ECM assembly, and prevented cell death of the PXDN-depleted cells. PXDN depletion also showed reduced growth factors-induced phosphorylation of FAK and ERK1/2. In addition, siPXDN-transfected cell-derived matrix failed to provide full ECM-mediated activation of FAK and ERK1/2. These results indicate that both the ECM motifs and peroxidase activity are essential for the cellular function of PXDN and that PXDN is crucial for ECM assembly for survival and growth signaling.