Amyloidogenic Aggregation Research Articles

English

Introduction Conformational disorders such as Alzheimer’s, Parkinson’s, familial amyloidotic polyneuropathy and spongiform encephalopaties are a consequence of protein misfolding and aggregation predominantly in the form of amyloid ϐibrils. These pathologies represent a major health problem, which most probably will overwhelm the health systems of developed countries in the near future. Signiϐicant progress has been made recently to understanding the underlying mechanism of protein misfolding and aggregation. The current picture of protein aggregation is a phenomenon resulting from protein conformational ϐluctuations leading to misfolded intermediates prone to form non-native interactions with other intermediates, resulting in amyloid ϐibril formation. Fortunately just a small group of proteins are associated with human conformational disorders. The primary causes that lead this group of proteins to misfolding and aggregation are point mutations, protein over-expression and failure of protein quality-control system. Beside amyloid formation, there are other types of aggregation available to a misfold-disease-related polypeptide chain in the proteinfree energy landscape. Among them, native-like aggregation is becoming a widely studied topic of research. This aggregation type, simultaneously straightforward and ubiquitous, seems to be involved concurrently in the pathway of amyloid ϐibril formation and disruption. In this review, the pathways of misfold and aggregation of a protein are accessed along with the primary causes that turn a native soluble protein into amyloid ϐibrils or native-like aggregate. In addition, an insight into the biophysical and biochemical aspects fundamental to amyloid ϐibril formation and nativelike aggregation is provided. Finally some clues are presented about what makes a protein follow an amyloidogenic or native-like aggregation pathway. Conclusion More laboratory data should be gathered about the structure, stability, dynamics and aggregation kinetics, in order to get a clearer picture of the biophysical mechanisms underlying native-like aggregation.

Inhibition of TTR Aggregation-Induced Cell Death – A New Role for Serum Amyloid P Component

BackgroundSerum amyloid P component (SAP) is a glycoprotein that is universally found associated with different types of amyloid deposits. It has been suggested that it stabilizes amyloid fibrils and therefore protects them from proteolytic degradation.Methodology/Principal FindingsIn this paper, we show that SAP binds not only to mature amyloid fibrils but also to early aggregates of amyloidogenic mutants of the plasma protein transthyretin (TTR). It does not inhibit fibril formation of TTR mutants, which spontaneously form amyloid in vitro at physiological pH. We found that SAP prevents cell death induced by mutant TTR, while several other molecules that are also known to decorate amyloid fibrils do not have such effect. Using a Drosophila model for TTR-associated amyloidosis, we found a new role for SAP as a protective factor in inhibition of TTR-induced toxicity. Overexpression of mutated TTR leads to a neurological phenotype with changes in wing posture. SAP-transgenic flies were crossed with mutated TTR-expressing flies and the results clearly confirmed a protective effect of SAP on TTR-induced phenotype, with an almost complete reduction in abnormal wing posture. Furthermore, we found in vivo that binding of SAP to mutated TTR counteracts the otherwise detrimental effects of aggregation of amyloidogenic TTR on retinal structure.Conclusions/SignificanceTogether, these two approaches firmly establish the protective effect of SAP on TTR-induced cell death and degenerative phenotypes, and suggest a novel role for SAP through which the toxicity of early amyloidogenic aggregates is attenuated.

The Local Mechanical Properties of Lipid Bilayers are Altered by Amyloid-Forming Proteins

A vast number of protein misfolding diseases are characterized by the formation of nanoscale protein aggregates generally termed amyloids. Such diseases include Alzheimer's disease (AD), Huntington's disease (HD), and type 2 diabetes. While the precise mechanism by which amyloidogenic aggregates are toxic remains unclear, various amyloid-forming proteins interact strongly with lipid membranes. We investigated how mechanical properties of model total brain lipid extract bilayers are altered when exposed to different amyloid-forming proteins (As, huntingtin, synthetic polyQ peptide, and amylin) utilizing in situ tapping mode atomic force microscopy (AFM) and scanning probe acceleration microscopy (SPAM). The advantage of the SPAM technique is that provides nanoscale spatially resolved maps of tip/sample tapping forces, which are directly correlated to mechanical properties of the surface. As a result, mechanical changes of lipid membranes can be mapped and correlated with changes in surface topography associated with protein aggregation. using this technique, we demonstrate that lipid bilayer structure is disrupted by amyloid-forming proteins. Disrupted regions of the bilayer were associated with decreased compression modulus and reduced adhesion to the AFM probe. Both of these observed mechanical changes are consistent with a decrease in the packing efficiency of the lipids within the bilayer. The interpretation of the mechanical changes in the lipid bilayers as measured by the SPAM technique were validated via numerical simulations of the tip/surface force interaction under a variety of conditions. These changes in bilayer mechanical properties associated with exposure to amyloid forming proteins may represent a common mechanism leading to membrane dysfunction in protein misfolding diseases.

Intraneuronal β‐amyloid and its interactions with proteins and subcellular organelles

Amyloidogenic aggregation and misfolding of proteins are linked to neurodegeneration. The mechanism of neurodegeneration in Alzheimer's disease, which gives rise to severe neuronal death and memory loss, is not yet fully understood. The amyloid hypothesis remains the most accepted theory for the pathomechanism of the disease. It was suggested that β-amyloid accumulation may play a key role in initiating the neurodegenerative processes. The recent intracellular β-amyloid (iAβ) hypothesis emphasizes the primary role of iAβ to initiate the disease by interaction with cytoplasmic proteins and cell organelles, thereby triggering apoptosis. Sophisticated methods (proteomics, protein microarray, and super resolution microscopy) have been used for studying iAβ interactions with proteins and membraneous structures. The present review summarizes the studies on the origin of iAβ and the base of its neurotoxicity: interactions with cytosolic proteins and several cell organelles such as endoplasmic reticulum, endosomes, lysosomes, ribosomes, mitochondria, and the microtubular system.

Early Amyloidogenic Oligomerization Studied through Fluorescence Lifetime Correlation Spectroscopy

Amyloidogenic protein aggregation is a persistent biomedical problem. Despite active research in disease-related aggregation, the need for multidisciplinary approaches to the problem is evident. Recent advances in single-molecule fluorescence spectroscopy are valuable for examining heterogenic biomolecular systems. In this work, we have explored the initial stages of amyloidogenic aggregation by employing fluorescence lifetime correlation spectroscopy (FLCS), an advanced modification of conventional fluorescence correlation spectroscopy (FCS) that utilizes time-resolved information. FLCS provides size distributions and kinetics for the oligomer growth of the SH3 domain of α-spectrin, whose N47A mutant forms amyloid fibrils at pH 3.2 and 37 °C in the presence of salt. The combination of FCS with additional fluorescence lifetime information provides an exciting approach to focus on the initial aggregation stages, allowing a better understanding of the fibrillization process, by providing multidimensional information, valuable in combination with other conventional methodologies.

Techniques for Monitoring Protein Misfolding and Aggregation in Vitro and in Living Cells.

Protein misfolding and aggregation have been considered important in understanding many neurodegenerative diseases and recombinant biopharmaceutical production. Therefore, various traditional and modern techniques have been utilized to monitor protein aggregation in vitro and in living cells. Fibril formation, morphology and secondary structure content of amyloidogenic proteins in vitro have been monitored by molecular probes, TEM/AFM, and CD/FTIR analyses, respectively. Protein aggregation in living cells has been qualitatively or quantitatively monitored by numerous molecular folding reporters based on either fluorescent protein or enzyme. Aggregation of a target protein is directly correlated to the changes in fluorescence or enzyme activity of the folding reporter fused to the target protein, which allows non-invasive monitoring aggregation of the target protein in living cells. Advances in the techniques used to monitor protein aggregation in vitro and in living cells have greatly facilitated the understanding of the molecular mechanism of amyloidogenic protein aggregation associated with neurodegenerative diseases, optimizing culture conditions to reduce aggregation of biopharmaceuticals expressed in living cells, and screening of small molecule libraries in the search for protein aggregation inhibitors.

Specific Disruption of Transthyretin(105–115) Fibrilization Using “Stabilizing” Inhibitors of Transthyretin Amyloidogenesis

Transthyretin (TTR) is a cerebrospinal fluid and serum protein that undergoes ordered aggregation (amyloidogenesis) in familial amyloidotic polyneuropathy (FAP) and senile systemic amyloidosis (SSA). It is now widely accepted that dissociation of the native TTR tetramer is a precondition for amyloidogenesis; thus, molecules that stabilize the tetramer have received much attention as potential TTR amyloidosis inhibitors. Many of these inhibitors bind to the thyroxine (T(4)) binding pocket and interact specifically with a section of the TTR sequence, corresponding to residues 105-115, that is implicated in amyloidogenic propensity. In this work, we study the effects of "stabilizing" inhibitors on ordered aggregation of TTR(105-115) peptide. We show that molecules known to bind full-length TTR at the T(4) site are potent, specific inhibitors of ordered aggregation, while molecules that do not interact with TTR exhibit milder, nonspecific disruption through a "hyperbundling" effect. Our results suggest that, in addition to annealing the native tetramer, "stabilizing" inhibitors may also directly disrupt amyloidogenic aggregation of TTR monomers through specific interactions with the exposed TTR(105-115) sequence.

Driving Forces and Structural Determinants of Steric Zipper Peptide Oligomer Formation Elucidated by Atomistic Simulations

Understanding the structural and energetic requirements of non-fibrillar oligomer formation harbors the potential to decipher an important yet still elusive part of amyloidogenic peptide and protein aggregation. Low-molecular-weight oligomers are described to be transient and polymorphic intermediates in the nucleated self-assembly process to highly ordered amyloid fibers and were additionally found to exhibit a profound cytotoxicity. However, detailed structural information on the oligomeric species involved in the nucleation cannot be readily inferred from experiments.Here, we study the spontaneous assembly of steric zipper peptides from the tau protein, insulin and α-synuclein with atomistic molecular dynamics simulations on the microsecond timescale. Detailed analysis of the forces driving the oligomerization reveals a common two-step process akin to a general condensation-ordering mechanism and thus provides a rational understanding of the molecular basis of peptide self-assembly. Our results suggest that the initial formation of partially ordered peptide oligomers is governed by the solvation free energy, whereas the dynamical ordering and emergence of β-sheets are mainly driven by optimized inter-peptide interactions in the collapsed state.A novel mapping technique based on collective coordinates is employed to highlight similarities and differences in the conformational ensemble of small oligomer structures. Elucidating the dynamical and polymorphic β-sheet oligomer conformations at atomistic detail furthermore suggests complementary sheet packing characteristics similar to steric zipper structures, but with a larger heterogeneity in the strand alignment pattern and sheet-to-sheet arrangements compared to the cross-β motif found in the fibrillar or crystalline states.

AFM Imaging and Single-Molecule Force Spectroscopy of Transthyretin Amyloid Intermediates

Aggregation of Transthyretin (TTR) in the form of amyloid fibrils is associated with systemic and neurological disorders. As part of the amyloid fibrilogenesis process, several oligomeric and protofibrillar intermediate structures have been identified and found to possess greater cytotoxicity than the mature amyloid fibrils. The structural dynamics and the molecular mechanisms behind the cytotoxicity of these intermediates are largely unknown.In the present work we explored the structure, dynamics and mechanics of TTR amyloid intermediates with a combination of AFM imaging and single-molecule force spectroscopy. We show that annular oligomers display a tendency of association into a linear array. AFM imaging and force measurements indicated that annular oligomers are constituted by 8 TTR dimers that are largely unfolded when compared with native TTR. Associated annular oligomers undergo a collapsing structural transition before the appearance of amyloid protofibrils. The emerging TTR protofibrils display morphological features predicted by the double-helical model of the amyloid protofilament. Upon evoking their disassembly, protofibrils dissociated into annular oligomers. Thus, annual oligomeric species represent an important structural intermediate along both the assembly and disassembly pathways.In this study we have dissected several structural transitions associated with the formation of TTR amyloid protofibrils. Because amyloidogenic aggregation is a potentially universal feature of proteins, the transitions reported here might be also relevant for other amyloidogenic disorders as well.

Amyloid-like Aggregates Sequester Numerous Metastable Proteins with Essential Cellular Functions

Protein aggregation is linked with neurodegeneration and numerous other diseases by mechanisms that are not well understood. Here, we have analyzed the gain-of-function toxicity of artificial β sheet proteins that were designed to form amyloid-like fibrils. Using quantitative proteomics, we found that the toxicity of these proteins in human cells correlates with the capacity of their aggregates to promote aberrant protein interactions and to deregulate the cytosolic stress response. The endogenous proteins that are sequestered by the aggregates share distinct physicochemical properties: They are relatively large in size and significantly enriched in predicted unstructured regions, features that are strongly linked with multifunctionality. Many of the interacting proteins occupy essential hub positions in cellular protein networks, with key roles in chromatin organization, transcription, translation, maintenance of cell architecture and protein quality control. We suggest that amyloidogenic aggregation targets a metastable subproteome, thereby causing multifactorial toxicity and, eventually, the collapse of essential cellular functions.

Trapping of palindromic ligands within native transthyretin prevents amyloid formation

Transthyretin (TTR) amyloidosis is a fatal disease for which new therapeutic approaches are urgently needed. We have designed two palindromic ligands, 2,2'-(4,4'-(heptane-1,7-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (mds84) and 2,2'-(4,4'-(undecane-1,11-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (4ajm15), that are rapidly bound by native wild-type TTR in whole serum and even more avidly by amyloidogenic TTR variants. One to one stoichiometry, demonstrable in solution and by MS, was confirmed by X-ray crystallographic analysis showing simultaneous occupation of both T4 binding sites in each tetrameric TTR molecule by the pair of ligand head groups. Ligand binding by native TTR was irreversible under physiological conditions, and it stabilized the tetrameric assembly and inhibited amyloidogenic aggregation more potently than other known ligands. These superstabilizers are orally bioavailable and exhibit low inhibitory activity against cyclooxygenase (COX). They offer a promising platform for development of drugs to treat and prevent TTR amyloidosis.

Rational Manipulation of Amyloidogenesis Using an Atomic Level Map of Peptide−Fibril Interactions

Amyloidogenic aggregation has been the subject of intense research over the past few decades, but the mechanisms underlying the early stages of amyloidogenesis remain elusive. Here we demonstrate for the first time manipulation of amyloidogenesis based on an atomic level map of peptide-fibril interactions in early- and late-stage ordered aggregation. Several point mutants with specific amyloidogenic properties are introduced, including one that "stalls" early in the aggregation process, forming early-stage fibrillar aggregates, but not mature fibrils.

Role of Small Oligomers on the Amyloidogenic Aggregation Free-Energy Landscape

We combine atomic-force-microscopy particle-size-distribution measurements with earlier measurements on 1-anilino-8-naphthalene sulfonate, thioflavin T, and dynamic light scattering to develop a quantitative kinetic model for the aggregation of β-lactoglobulin into amyloid. We directly compare our simulations to the population distributions provided by dynamic light scattering and atomic force microscopy. We combine species in the simulation according to structural type for comparison with fluorescence fingerprint results. The kinetic model of amyloidogenesis leads to an aggregation free-energy landscape. We define the roles of and propose a classification scheme for different oligomeric species based on their location in the aggregation free-energy landscape. We relate the different types of oligomers to the amyloid cascade hypothesis and the toxic oligomer hypothesis for amyloid-related diseases. We discuss existing kinetic mechanisms in terms of the different types of oligomers. We provide a possible resolution to the toxic oligomer–amyloid coincidence.

Blood-based biomarkers of microvascular pathology in Alzheimer’s disease

Sporadic Alzheimer’s disease (AD) is a genetically complex and chronically progressive neurodegenerative disorder with molecular mechanisms and neuropathologies centering around the amyloidogenic pathway, hyperphosphorylation and aggregation of tau protein, and neurofibrillary degeneration. While cerebrovascular changes have not been traditionally considered to be a central part of AD pathology, a growing body of evidence demonstrates that they may, in fact, be a characteristic feature of the AD brain as well. In particular, microvascular abnormalities within the brain have been associated with pathological AD hallmarks and may precede neurodegeneration. In vivo assessment of microvascular pathology provides a promising approach to develop useful biological markers for early detection and pathological characterization of AD. This review focuses on established blood-based biological marker candidates of microvascular pathology in AD. These candidates include plasma concentration of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) that are increased in AD. Measures of endothelial vasodilatory function including endothelin (ET-1), adrenomedullin (ADM), and atrial natriuretic peptide (ANP), as well as sphingolipids are significantly altered in mild AD or during the predementia stage of mild cognitive impairment (MCI), suggesting sensitivity of these biomarkers for early detection and diagnosis. In conclusion, the emerging clinical diagnostic evidence for the value of blood-based microvascular biomarkers in AD is promising, however, still requires validation in phase II and III diagnostic trials. Moreover, it is still unclear whether the described protein dysbalances are early or downstream pathological events and how the detected systemic microvascular alterations relate to cerebrovascular and neuronal pathologies in the AD brain.

Gold Nanoparticles and Microwave Irradiation Inhibit Beta-Amyloid Amyloidogenesis

Peptide-Gold nanoparticles selectively attached to β-amyloid protein (Aβ) amyloidogenic aggregates were irradiated with microwave. This treatment produces dramatic effects on the Aβ aggregates, inhibiting both the amyloidogenesis and the restoration of the amyloidogenic potential. This novel approach offers a new strategy to inhibit, locally and remotely, the amyloidogenic process, which could have application in Alzheimer’s disease therapy. We have studied the irradiation effect on the amyloidogenic process in the presence of conjugates peptide-nanoparticle by transmission electronic microscopy observations and by Thioflavine T assays to quantify the amount of fibrils in suspension. The amyloidogenic aggregates rather than the amyloid fibrils seem to be better targets for the treatment of the disease. Our results could contribute to the development of a new therapeutic strategy to inhibit the amyloidogenic process in Alzheimer’s disease.

Monitoring Copopulated Conformational States During Protein Folding Events Using Electrospray Ionization-Ion Mobility Spectrometry-Mass Spectrometry

The precise mechanism of protein folding remains elusive and there is a deficiency of biophysical techniques that are capable of monitoring the individual behavior of copopulated protein conformers during the folding process. Herein, an ion mobility spectrometry (IMS) device integrated with electrospray ionization mass spectrometry (ESI-MS) has been used to successfully separate and analyze protein conformers differing in cross section and/or charge state. In an initial test, an ensemble of folded and partially folded conformers of the protein cytochrome c was separated. A detailed study undertaken on the amyloidogenic protein β2-microglobulin (β2m), which forms fibrils by protein unfolding followed by self-aggregation and is responsible for the disease dialysis-related amyloidosis, has generated important insights into its folding landscape. Initially, a systematic titration of β2m over the pH range 2 to 7 using ESI-IMS-MS allowed individual conformers to be monitored and quantified throughout the acid denaturation process. Furthermore, a comparison of wild-type β2m with single and double amino acid variants with a range of folding stabilities and propensities for amyloid fibril formation has provided illuminating evidence of the role of different conformers in protein stability and amyloidogenic aggregation. The ESI-IMS-MS data presented here not only demonstrate an important and informative further dimension to ESI-MS, but also illustrate the potential of the ESI-IMS-MS technique for unravelling protein folding enigmas in general and studying protein misfolding diseases in particular.

Topological Determinants of Protein Domain Swapping

Protein domain swapping has been repeatedly observed in a variety of proteins and is believed to result from destabilization due to mutations or changes in environment. Based on results from our studies and others, we propose that structures of the domain-swapped proteins are mainly determined by their native topologies. We performed molecular dynamics simulations of seven different proteins, known to undergo domain swapping experimentally, under mildly denaturing conditions and found in all cases that the domain-swapped structures can be recapitulated by using protein topology in a simple protein model. Our studies further indicated that, in many cases, domain swapping occurs at positions around which the protein tends to unfold prior to complete unfolding. This, in turn, enabled prediction of protein structural elements that are responsible for domain swapping. In particular, two distinct domain-swapped dimer conformations of the focal adhesion targeting domain of focal adhesion kinase were predicted computationally and were supported experimentally by data obtained from NMR analyses.

Dopamine promotes α‐synuclein aggregation into SDS‐resistant soluble oligomers via a distinct folding pathway

Dopamine (DA) and alpha-synuclein (alpha-SN) are two key molecules associated with Parkinson's disease (PD). We have identified a novel action of DA in the initial phase of alpha-SN aggregation and demonstrate that DA induces alpha-SN to form soluble, SDS-resistant oligomers. The DA:alpha-SN oligomeric species are not amyloidogenic as they do not react with thioflavin T and lack the typical amyloid fibril structures as visualized with electron microscopy. Circular dichroism studies indicate that in the presence of lipid membranes DA interacts with alpha-SN, causing an alteration to the structure of the protein. Furthermore, DA inhibited the formation of iron-induced alpha-SN amyloidogenic aggregates, suggesting that DA acts as a dominant modulator of alpha-SN aggregation. These observations support the paradigm emerging for other neurodegenerative diseases that the toxic species is represented by a soluble oligomer and not the insoluble fibril.