Uncovering the pathological functions of Ser404 phosphorylation by semisynthesis of a phosphorylated TDP-43 prion-like domain.

Herein, we have successfully semi-synthesized a TDP-43 prion-like domain with Ser404 phosphorylation. We have demonstrated that Ser404 phosphorylation could accelerate the amyloid aggregation of the TDP-43 prion-like domain and aggravate its cytotoxicity.

Surface Functionalization of Gold Nanorods Improves Nanostructure Assemblies on Amyloid Fibril Scaffolds.

A hybrid scaffold containing gold nanorods and lysozyme amyloid fibrils has been fabricated, and the effect of surface modification on improving nanostructure assembly on the biological template has been investigated. The nanohybrid system was characterized by monitoring surface plasmon resonance bands, dynamic light scattering spectroscopy, Thioflavin-T assay, and transmission electron microscopy. Surface of gold nanorods (GNRs) was modified with polystyrene sulfonate (PSS), and possible difference in assembly of the pristine and modified nanostructures was compared upon interaction with amyloid fibrils. Analysis of transmission electron microscopy showed that changing the surface charge of GNRs with biocompatible polymer improved electrostatic interactions between the nanostructures and amyloid fibril templates. Analysis of cell viability assays also showed that surface functionalization of GNRs remarkably improved biocompatibility of the nanoscaffold. Results of this study encourage utilization of modification strategies to fabricate a new generation of nanoscaffolds with fruitful applications in regenerative medicine.

Ubiquitination and SUMOylation of Amyloid and Amyloid-like Proteins in Health and Disease.

Post-translational modifications (PTMs) play important roles in altering the structure and function of proteins. In this article, we focus on ubiquitination and SUMOylation of amyloidogenic proteins. We discuss the functional contributions of PTMs on proteins involved in amyloid-related diseases as well as the aberrant PTM signatures of the disease agents. In addition, we extend our discussion to the nascent field of functional amyloids, a subclass of amyloids that perform physiological functions. Here, we present examples from mammals and yeast to gain insight into physiological regulation of amyloid-like proteins.

Analysis of Prairie Vole Amylin Reveals the Importance of the N-Terminus and Residue 22 in Amyloidogenicity and Cytotoxicity.

Amyloid formation by amylin contributes to ß-cell dysfunction in type 2 diabetes. The features that control the amyloidogenicity and toxicity of amylin are not understood. Not all species form islet amyloid, and its presence or absence correlates with the in vitro behavior of the polypeptide. Rats do not develop type 2 diabetes or islet amyloid, and rat amylin is non-amyloidogenic, except at very high concentrations. This has led to the notion that rodent amylins are non-amyloidogenic. Prairie vole amylin has an unusual sequence compared to those of human and rat amylin, including nonconservative Lys-1 to Glu and Asn-22 to Gly substitutions. The first reduces the net charge on the peptide, while the second disrupts a potential network of side chain hydrogen bonds in the amyloid fiber, a so-called Asn ladder. The prairie vole polypeptide forms amyloid more slowly than human amylin and is considerably less cytotoxic. An Asn-22 to Gly substitution in human amylin significantly reduces toxicity, increasing the effective concentration of amylin required to reach 50% toxicity by >7-fold, but has modest effects on the time to form amyloid. A Lys-1 to Glu replacement has a weaker effect but does reduce toxicity relative to that of human amylin, without having a significant impact on the time to form amyloid. The effect of the Lys-1 to Glu substitution on amyloid kinetics is more significant in Tris buffer than in phosphate-buffered saline. This work demonstrates that the N-terminus of amylin plays a role in modulating toxicity and highlights the key role of position 22.

A new approach for the separation, characterization and testing of potential prionoid protein aggregates through hollow-fiber flow field-flow fractionation and multi-angle light scattering.

Protein misfolding and aggregation are the common mechanisms in a variety of aggregation-dependent diseases. The compromised proteins often assemble into toxic, accumulating amyloid-like structures of various lengths and their toxicity can also be transferred both in vivo and in vitro a prion-like behavior. The characterization of protein interactions, degradation and conformational dynamics in biological systems still represents an analytical challenge in the prion-like protein comprehension. In our work, we investigated the nature of a transferable cytotoxic agent, presumably a misfolded protein, through the coupling of a multi-detector, non-destructive separation platform based on hollow-fiber flow field-flow fractionation with imaging and downstream in vitro tests. After purification with ion exchange chromatography, the transferable cytotoxic agentwas analyzed with Atomic Force Microscopy and statistical analysis, showing that the concentration of protein dimers and low n-oligomer forms was higher in the cytotoxic sample than in the control preparation. To assess whether the presence of these species was the actual toxic and/or self-propagating factor, we employed HF5 fractionation, with UV and Multi-Angle Light Scattering detection, to define proteins molar mass distribution and abundance, and fractionate the sample into size-homogeneous fractions. These fractions were then tested individually in vitro to investigate the direct correlation with cytotoxicity. Only the later-eluted fraction, which contains high-molar mass aggregates, proved to be toxic onto cell cultures. Moreover, it was observed that the selective transfer of toxicity also occurs for one lower-mass fraction, suggesting that two different mechanisms, acute and later induced toxicity, are in place. These results strongly encourage the efficacy of this platform to enable the identification of protein toxicants.

Protein misfolding, aggregation and mechanism of amyloid cytotoxicity: An overview and therapeutic strategies to inhibit aggregation.

Protein and peptides are converted from their soluble forms into highly ordered fibrillar aggregates under various conditions inside the cell. Such transitions confer diverse neurodegenerative diseases including Alzheimer's disease, Huntington's disease Prion's disease, Parkinson's disease, polyQ and share abnormal folding of potentially cytotoxic protein species linked with degeneration and death of precise neuronal populations. Presently, major advances are made to understand and get detailed insight into the structural basis and mechanism of amyloid formation, cytotoxicity and therapeutic approaches to combat them. Here we highlight classifies and summarizes the detailed overview of protein misfolding and aggregation at their molecular level including the factors that promote protein aggregation under in vivo and in vitro conditions. In addition, we describe the recent technologies that aid the characterization of amyloid aggregates along with several models that might be responsible for amyloid induced cytotoxicity to cells. Overview on the inhibition of amyloidosis by targeting different small molecules (both natural and synthetic origin) have been also discussed, that provides important approaches to identify novel targets and develop specific therapeutic strategies to combat protein aggregation related neurodegenerative diseases.

Amyloid-like Behavior of Site-Specifically Citrullinated Myelin Oligodendrocyte Protein (MOG) Peptide Fragments inside EBV-Infected B-Cells Influences Their Cytotoxicity and Autoimmunogenicity.

Multiple sclerosis (MS) is an autoimmune disorder manifested via chronic inflammation, demyelination, and neurodegeneration inside the central nervous system. The progressive phase of MS is characterized by neurodegeneration, but unlike classical neurodegenerative diseases, amyloid-like aggregation of self-proteins has not been documented. There is evidence that citrullination protects an immunodominant peptide of human myelin oligodendrocyte glycoprotein (MOG34-56) against destructive processing in Epstein-Barr virus-infected B-lymphocytes (EBV-BLCs) in marmosets and causes exacerbation of ongoing MS-like encephalopathies in mice. Here we collected evidence that citrullination of MOG can also lead to amyloid-like behavior shifting the disease pathogenesis toward neurodegeneration. We observed that an immunodominant MOG peptide, MOG35-55, displays amyloid-like behavior upon site-specific citrullination at positions 41, 46, and/or 52. These amyloid aggregates are shown to be toxic to the EBV-BLCs and to dendritic cells at concentrations favored for antigen presentation, suggesting a role of amyloid-like aggregation in the pathogenesis of progressive MS.

Cysteine as a potential anti-amyloidogenic agent with protective ability against amyloid induced cytotoxicity.

Protein aggregation and misfolding have been allied with numerous human disorders and thus inhibition of such occurrence has been center for intense research efforts against these diseases. Here, we investigated anti-fibrillation activity of cysteine and its effect on kinetics of stem bromelain amyloid fibril formation. We established the anti-fibrillation and anti aggregation activities of cysteine by using multiple approaches like turbidity measurements, dye binding assays (ThT and ANS) and structural changes were monitored by circular dichroism (CD) followed by electron microscopy. Our experimental study inferred that cysteine inhibits temperature induced fibrillation of protein in a concentration dependent way. In addition, MDA-MB-231 cell viability of pre-formed amyloid was increased in presence of cysteine as compared to the fibrils alone. Furthermore, dynamic light scattering studies of native, aggregated as well as incubated (amyloids in presence of cysteine) samples indicates that cysteine restores native like structures of stem bromelain. Isothermal titration calorimetric results revealed that hydrogen bonding between cysteine and stem bromelain plays a significant role during inhibition of stem bromelain aggregation. However, thiophilic interaction between thiol group of cysteine and aromatic amino acid residue of stem bromelain may also have noteworthy role in inhibition of amyloid formation.

The Anti-Amyloid-ß and Neuroprotective Properties of a Novel Tricyclic Pyrone Molecule.

There is an urgent unmet need for new therapeutics for Alzheimer's disease (AD), the most common cause of dementia in the elderly. Therapeutic approaches targeting amyloid-ß (Aß) and its downstream toxicities have become major strategies in AD drug development. We have taken a rational design approach and synthesized a class of tricyclic pyrone (TP) compounds that show anti-Aß and other neuroprotective actions. The in vivo efficacy of a lead TP named CP2 to ameliorate AD-like pathologies has been shown in mouse models. Here we report the selection and initial characterization of a new lead TP70, which exhibited an anti-Aß therapeutic index even higher than CP2. Moreover, TP70 was able to reduce oxidative stress, inhibit acyl-coenzyme A:cholesterol acyltransferase (ACAT), and upregulate the expression of ATP-binding cassette subfamily A, member 1 (ABCA1), actions considered neuroprotective in AD. TP70 further showed excellent pharmacokinetic properties, including brain penetration and oral availability. When administered to 5xFAD mice via intraperitoneal or oral route, TP70 enhanced the overall solubility and decreased the level of cerebral Aß, including both fibrillary and soluble Aß species. Interestingly, TP70 enhanced N-methyl-D-aspartate (NMDA) receptor-mediated excitatory post-synaptic potential (EPSP) in the hippocampal CA1 area, increased the magnitude of NMDA-dependent hippocampal long-term potentiation (LTP), a cellular model of learning and memory, and prevented the Aß oligomer-impaired LTP. Significantly, a single dose of TP70 administered to aged 5xFAD mice was effective in mitigating the impaired LTP induction, recorded at 24 h after administration. Our results support a potential of TP70 in clinical development for AD in view of its synergistic neuroprotective actions, ability to positively modulate NMDA receptor-mediated hippocampal plasticity, and favorable pharmacokinetic properties in rodents.

Amyloidogenicity and toxicity of the reverse and scrambled variants of amyloid-ß 1-42.

ß-amyloid 1-42 (Aß1-42) is a self-assembling peptide that goes through many conformational and morphological changes before forming the fibrils that are deposited in extracellular plaques characteristic of Alzheimer's disease. The link between Aß1-42 structure and toxicity is of major interest, in particular, the neurotoxic potential of oligomeric species. Many studies utilise reversed (Aß42-1) and scrambled (AßS) forms of amyloid-ß as control peptides. Here, using circular dichroism, thioflavin T fluorescence and transmission electron microscopy, we reveal that both control peptides self-assemble to form fibres within 24 h. However, oligomeric Aß reduces cell survival of hippocampal neurons, while Aß42-1 and Aßs have reduced effect on cellular health, which may arise from their ability to assemble rapidly to form protofibrils and fibrils.

Viewing Extrinsic Proteotoxic Stress Through the Lens of Amyloid Cardiomyopathy.

Proteotoxicity refers to toxic stress caused by misfolded proteins of extrinsic or intrinsic origin and plays an integral role in the pathogenesis of cardiovascular diseases. Herein, we provide an overview of the current understanding of mechanisms underlying proteotoxicity and its contribution in the pathogenesis of amyloid cardiomyopathy.

Time-resolved studies define the nature of toxic IAPP intermediates, providing insight for anti-amyloidosis therapeutics.

Islet amyloidosis by IAPP contributes to pancreatic ß-cell death in diabetes, but the nature of toxic IAPP species remains elusive. Using concurrent time-resolved biophysical and biological measurements, we define the toxic species produced during IAPP amyloid formation and link their properties to induction of rat INS-1 ß-cell and murine islet toxicity. These globally flexible, low order oligomers upregulate pro-inflammatory markers and induce reactive oxygen species. They do not bind 1-anilnonaphthalene-8-sulphonic acid and lack extensive ß-sheet structure. Aromatic interactions modulate, but are not required for toxicity. Not all IAPP oligomers are toxic; toxicity depends on their partially structured conformational states. Some anti-amyloid agents paradoxically prolong cytotoxicity by prolonging the lifetime of the toxic species. The data highlight the distinguishing properties of toxic IAPP oligomers and the common features that they share with toxic species reported for other amyloidogenic polypeptides, providing information for rational drug design to treat IAPP induced ß-cell death.

pH Induced Conformational Transitions in the Transforming Growth Factor ß-Induced Protein (TGFßIp) Associated Corneal Dystrophy Mutants.

Most stromal corneal dystrophies are associated with aggregation and deposition of the mutated transforming growth factor-ß induced protein (TGFßIp). The 4(th)_FAS1 domain of TGFßIp harbors ~80% of the mutations that forms amyloidogenic and non-amyloidogenic aggregates. To understand the mechanism of aggregation and the differences between the amyloidogenic and non-amyloidogenic phenotypes, we expressed the 4(th)_FAS1 domains of TGFßIp carrying the mutations R555W (non-amyloidogenic) and H572R (amyloidogenic) along with the wild-type (WT). R555W was more susceptible to acidic pH compared to H572R and displayed varying chemical stabilities with decreasing pH. Thermal denaturation studies at acidic pH showed that while WT did not undergo any conformational transition, the mutants exhibited a clear pH-dependent irreversible conversion from αß conformation to ß-sheet oligomers. The ß-oligomers of both mutants were stable at physiological temperature and pH. Electron microscopy and dynamic light scattering studies showed that ß-oligomers of H572R were larger compared to R555W. The ß-oligomers of both mutants were cytotoxic to primary human corneal stromal fibroblast (pHCSF) cells. The ß-oligomers of both mutants exhibit variations in their morphologies, sizes, thermal and chemical stabilities, aggregation patterns and cytotoxicities.

Citrus bergamia Juice Extract Attenuates ß-Amyloid-Induced Pro-Inflammatory Activation of THP-1 Cells Through MAPK and AP-1 Pathways.

Flavonoids have been shown to be effective in protecting against age-related cognitive and motor decline in both in vitro and in vivo models. Recently, a flavonoid-rich extract of Citrus bergamia juice (BJe) has been shown to display anti-oxidant and anti-inflammatory properties against LPS-induced activation of human THP-1 monocytes. In the light of these observations, we wondered whether BJe may be beneficial against neuroinflammatory processes, such as those observed in Alzheimer's disease. To this aim we used THP-1 monocytes to investigate the mechanisms underlying the beneficial potential of BJe against amyloid-beta1-42 (Aß1-42) -mediated inflammation. Exposure of THP-1 cells to Aß1-42 significantly induced the expression and secretion of IL-6 and IL-1ß in THP-1 cells and increased the phosphorylation of ERK 1/2 as well as p46 and p54 members of JNK family. Moreover, Aß1-42 raises AP-1 DNA binding activity in THP-1-treated cells. Interestingly, all these effects were reduced in the presence of BJe. Our data indicate that BJe may effectively counteract the pro-inflammatory activation of monocytes/microglial cells exposed to amyloid fibrils, suggesting a promising role as a natural drug against neuroinflammatory processes.

Hemodynamic effects of combined focal cerebral ischemia and amyloid protein toxicity in a rat model: a functional CT study.

BACKGROUND/OBJECTIVE: Clinical evidence indicates that cerebral ischemia (CI) and a pathological factor of Alzheimer's disease, the ß-amyloid (Aß) protein, can increase the rate of cognitive impairment in the ageing population. Using the CT Perfusion (CTP) functional imaging, we sought to investigate the interaction between CI and the Aß protein on cerebral hemodynamics. METHODS: A previously established rat model of CI and Aß was used for the CTP study. Iodinated contrast was given intravenously, while serial CT images of sixteen axial slices were acquired. Cerebral blood flow (CBF) and blood volume (CBV) parametric maps were co-registered to a rat brain atlas and regions of interest were drawn on the maps. Microvascular alteration was investigated with histopathology. RESULTS: CTP results revealed that ipsilateral striatum of Aß+CI and CI groups showed significantly lower CBF and CBV than control at the acute phase. Striatal CBF and CBV increased significantly at week 1 in the CI and Aß+CI groups, but not in the Aß alone or control group. Histopathology showed that average density of dilated microvessels in the ipsilateral striatum in CI and Aß+CI groups was significantly higher than control at week 1, indicating this could be associated with hyperperfusion and hypervolemia observed from CTP results. CONCLUSION: These results demonstrate that CTP can quantitatively measure the hemodynamic disturbance on CBF and CBV functional maps in a rat model of CI interacting with Aß.

Therapeutic approaches against common structural features of toxic oligomers shared by multiple amyloidogenic proteins.

Impaired proteostasis is one of the main features of all amyloid diseases, which are associated with the formation of insoluble aggregates from amyloidogenic proteins. The aggregation process can be caused by overproduction or poor clearance of these proteins. However, numerous reports suggest that amyloid oligomers are the most toxic species, rather than insoluble fibrillar material, in Alzheimer's, Parkinson's, and Prion diseases, among others. Although the exact protein that aggregates varies between amyloid disorders, they all share common structural features that can be used as therapeutic targets. In this review, we focus on therapeutic approaches against shared features of toxic oligomeric structures and future directions.

Disrupting self-assembly and toxicity of amyloidogenic protein oligomers by "molecular tweezers" - from the test tube to animal models.

Despite decades of research, therapy for diseases caused by abnormal protein folding and aggregation (amyloidoses) is limited to treatment of symptoms and provides only temporary and moderate relief to sufferers. The failure in developing successful disease-modifying drugs for amyloidoses stems from the nature of the targets for such drugs - primarily oligomers of amyloidogenic proteins, which are distinct from traditional targets, such as enzymes or receptors. The oligomers are metastable, do not have well-defined structures, and exist in dynamically changing mixtures. Therefore, inhibiting the formation and toxicity of these oligomers likely will require out-of-the-box thinking and novel strategies. We review here the development of a strategy based on targeting the combination of hydrophobic and electrostatic interactions that are key to the assembly and toxicity of amyloidogenic proteins using lysine (K)-specific "molecular tweezers" (MTs). Our discussion includes a survey of the literature demonstrating the important role of K residues in the assembly and toxicity of amyloidogenic proteins and the development of a lead MT derivative called CLR01, from an inhibitor of protein aggregation in vitro to a drug candidate showing effective amelioration of disease symptoms in animal models of Alzheimer's and Parkinson's diseases.

The Hsp70/90 cochaperone, Sti1, suppresses proteotoxicity by regulating spatial quality control of amyloid-like proteins.

Conformational diseases are associated with the conversion of normal proteins into aggregation-prone toxic conformers with structures similar to that of ß-amyloid. Spatial distribution of amyloid-like proteins into intracellular quality control centers can be beneficial, but cellular mechanisms for protective aggregation remain unclear. We used a high-copy suppressor screen in yeast to identify roles for the Hsp70 system in spatial organization of toxic polyglutamine-expanded Huntingtin (Huntingtin with 103Q glutamine stretch [Htt103Q]) into benign assemblies. Under toxic conditions, Htt103Q accumulates in unassembled states and speckled cytosolic foci. Subtle modulation of Sti1 activity reciprocally affects Htt toxicity and the packaging of Htt103Q into foci. Loss of Sti1 exacerbates Htt toxicity and hinders foci formation, whereas elevation of Sti1 suppresses Htt toxicity while organizing small Htt103Q foci into larger assemblies. Sti1 also suppresses cytotoxicity of the glutamine-rich yeast prion [RNQ+] while reorganizing speckled Rnq1-monomeric red fluorescent protein into distinct foci. Sti1-inducible foci are perinuclear and contain proteins that are bound by the amyloid indicator dye thioflavin-T. Sti1 is an Hsp70 cochaperone that regulates the spatial organization of amyloid-like proteins in the cytosol and thereby buffers proteotoxicity caused by amyloid-like proteins.

Inhibiting toxic aggregation of amyloidogenic proteins: a therapeutic strategy for protein misfolding diseases.

BACKGROUND: The deposition of self-assembled amyloidogenic proteins is associated with multiple diseases, including Alzheimer's disease, Parkinson's disease and type 2 diabetes mellitus. The toxic misfolding and self-assembling of amyloidogenic proteins are believed to underlie protein misfolding diseases. Novel drug candidates targeting self-assembled amyloidogenic proteins represent a potential therapeutic approach for protein misfolding diseases. SCOPE OF REVIEW: In this perspective review, we provide an overview of the recent progress in identifying inhibitors that block the aggregation of amyloidogenic proteins and the clinical applications thereof. MAJOR CONCLUSIONS: Compounds such as polyphenols, certain short peptides, and monomer- or oligomer-specific antibodies, can interfere with the self-assembly of amyloidogenic proteins, prevent the formation of oligomers, amyloid fibrils and the consequent cytotoxicity. GENERAL SIGNIFICANCE: Some inhibitors have been tested in clinical trials for treating protein misfolding diseases. Inhibitors that target the aggregation of amyloidogenic proteins bring new hope to therapy for protein misfolding diseases.

Binding with nucleic acids or glycosaminoglycans converts soluble protein oligomers to amyloid.

Ample evidence suggests that almost all polypeptides can either adopt a native structure (folded or intrinsically disordered) or form misfolded amyloid fibrils. Soluble protein oligomers exist as an intermediate between these two states, and their cytotoxicity has been implicated in the pathology of multiple human diseases. However, the mechanism by which soluble protein oligomers develop into insoluble amyloid fibrils is not clear, and investigation of this important issue is hindered by the unavailability of stable protein oligomers. Here, we have obtained stabilized protein oligomers generated from common native proteins. These oligomers exert strong cytotoxicity and display a common conformational structure shared with known protein oligomers. They are soluble and remain stable in solution. Intriguingly, the stabilized protein oligomers interact preferentially with both nucleic acids and glycosaminoglycans (GAG), which facilitates their rapid conversion into insoluble amyloid. Concomitantly, binding with nucleic acids or GAG strongly diminished the cytotoxicity of the protein oligomers. EGCG, a small molecule that was previously shown to directly bind to protein oligomers, effectively inhibits the conversion to amyloid. These results indicate that stabilized oligomers of common proteins display characteristics similar to those of disease-associated protein oligomers and represent immediate precursors of less toxic amyloid fibrils. Amyloid conversion is potently expedited by certain physiological factors, such as nucleic acids and GAGs. These findings concur with reports of cofactor involvement with disease-associated amyloid and shed light on potential means to interfere with the pathogenic properties of misfolded proteins.