RESUMO
Cerebral (Aß) plaque and (pTau) tangle deposition are hallmarks of Alzheimer's disease (AD), yet are insufficient to confer complete AD-like neurodegeneration experimentally. Factors acting upstream of Aß/pTau in AD remain unknown, but their identification could enable earlier diagnosis and more effective treatments. T cell abnormalities are emerging AD hallmarks, and CD8 T cells were recently found to mediate neurodegeneration downstream of tangle deposition in hereditary neurodegeneration models. The precise impact of T cells downstream of Aß/pTau, however, appears to vary depending on the animal model. Our prior work suggested that antigen-specific memory CD8 T ("hiT") cells act upstream of Aß/pTau after brain injury. Here, we examine whether hiT cells influence sporadic AD-like pathophysiology upstream of Aß/pTau. Examining neuropathology, gene expression, and behavior in our hiT mouse model we show that CD8 T cells induce plaque and tangle-like deposition, modulate AD-related genes, and ultimately result in progressive neurodegeneration with both gross and fine features of sporadic human AD. T cells required Perforin to initiate this pathophysiology, and IFNγ for most gene expression changes and progression to more widespread neurodegenerative disease. Analogous antigen-specific memory CD8 T cells were significantly elevated in the brains of human AD patients, and their loss from blood corresponded to sporadic AD and related cognitive decline better than plasma pTau-217, a promising AD biomarker candidate. We identify an age-related factor acting upstream of Aß/pTau to initiate AD-like pathophysiology, the mechanisms promoting its pathogenicity, and its relevance to human sporadic AD.
Assuntos
Doença de Alzheimer , Linfócitos T CD8-Positivos , Modelos Animais de Doenças , Doença de Alzheimer/imunologia , Doença de Alzheimer/patologia , Doença de Alzheimer/genética , Animais , Linfócitos T CD8-Positivos/imunologia , Camundongos , Humanos , Placa Amiloide/patologia , Placa Amiloide/imunologia , Peptídeos beta-Amiloides/metabolismo , Camundongos Transgênicos , Encéfalo/patologia , Encéfalo/imunologia , Masculino , Interferon gama/metabolismo , Interferon gama/imunologia , Envelhecimento/imunologia , Memória Imunológica , Células T de Memória/imunologia , Perforina/metabolismo , Perforina/genética , FemininoRESUMO
Amyloid aggregation is a key feature of Alzheimer's disease (AD) and a primary target for past and present therapeutic efforts. Recent research is making it increasingly clear that the heterogeneity of amyloid deposits, extending past the commonly targeted amyloid-ß (Aß), must be considered for successful therapy. We recently demonstrated that amyloid-α (Aα or p3), a C-terminal peptidic fragment of Aß, aggregates rapidly to form amyloids and can expedite the aggregation of Aß through seeding. Here, we advance the understanding of Aα biophysics and biology in several important ways. We report the first cryogenic electron microscopy (cryo-EM) structure of an Aα amyloid fibril, proving unambiguously that the peptide is fibrillogenic. We demonstrate that Aα induces Aß to form amyloid aggregates that are less toxic than pure Aß aggregates and use nuclear magnetic resonance spectroscopy (NMR) to provide insights into specific interactions between Aα and Aß in solution. This is the first evidence that Aα can coassemble with Aß and alter its biological effects at relatively low concentrations. Based on the above, we urge researchers in the field to re-examine the significance of Aα in AD.
Assuntos
Doença de Alzheimer , Precursor de Proteína beta-Amiloide , Humanos , Amiloide/química , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/químicaRESUMO
The rippled ß-sheet was theorized by Pauling and Corey in 1953 as a structural motif in which mirror image peptide strands assemble into hydrogen-bonded periodic arrays with strictly alternating chirality. Structural characterization of the rippled ß-sheet was limited to biophysical methods until 2022 when atomic resolution structures of the motif were first obtained. The crystal structural foundation is restricted to four model tripeptides composed exclusively of aromatic residues. Here, we report five new rippled sheet crystal structures derived from amyloid ß and amylin, the aggregating toxic peptides of Alzheimer's disease and type II diabetes, respectively. Despite the variation in peptide sequence composition, all five structures form antiparallel rippled ß-sheets that extend, like a fibril, along the entire length of the crystalline needle. The long-range packing of the crystals, however, varies. In three of the crystals, the sheets pack face-to-face and exclude water, giving rise to cross-ß architectures grossly resembling the steric zipper motif of amyloid fibrils but differing in fundamental details. In the other two crystals, the solvent is encapsulated between the sheets, yielding fibril architectures capable of host-guest chemistry. Our study demonstrates that the formation of rippled ß-sheets from aggregating racemic peptide mixtures in three-dimensional (3D) assemblies is a general phenomenon and provides a structural basis for targeting intrinsically disordered proteins.
Assuntos
Peptídeos beta-Amiloides , Diabetes Mellitus Tipo 2 , Humanos , Peptídeos beta-Amiloides/química , Conformação Proteica em Folha beta , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Modelos Moleculares , Amiloide/químicaRESUMO
Evidence linking amyloid beta (Aß) cellular uptake and toxicity has burgeoned, and mechanisms underlying this association are subjects of active research. Two major, interconnected questions are whether Aß uptake is aggregation-dependent and whether it is sequence-specific. We recently reported that the neuronal uptake of Aß depends significantly on peptide chirality, suggesting that the process is predominantly receptor-mediated. Over the past decade, the cellular prion protein (PrPC) has emerged as an important mediator of Aß-induced toxicity and of neuronal Aß internalization. Here, we report that the soluble, nonfibrillizing Aß (1-30) peptide recapitulates full-length Aß stereoselective cellular uptake, allowing us to decouple aggregation from cellular, receptor-mediated internalization. Moreover, we found that Aß (1-30) uptake is also dependent on PrPC expression. NMR-based molecular-level characterization identified the docking site on PrPC that underlies the stereoselective binding of Aß (1-30). Our findings therefore identify a specific sequence within Aß that is responsible for the recognition of the peptide by PrPC, as well as PrPC-dependent cellular uptake. Further uptake stereodifferentiation in PrPC-free cells points toward additional receptor-mediated interactions as likely contributors for Aß cellular internalization. Taken together, our results highlight the potential of targeting cellular surface receptors to inhibit Aß cellular uptake as an alternative route for future therapeutic development for Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteínas PrPC/metabolismo , Células HEK293 , HumanosRESUMO
Spurred in part by the failure of recent therapeutics targeting amyloid ß plaques in Alzheimer's Disease (AD), attention is increasingly turning to the oligomeric forms of this peptide that form early in the aggregation process. However, while numerous amyloid ß fibril structures have been characterized, primarily by NMR spectroscopy and cryo-EM, obtaining structural information on the low molecular weight forms of amyloid ß that presumably precede and/or seed fibril formation has proved challenging. These transient forms are heterogeneous, and depend heavily on experimental conditions such as buffer, temperature, concentration, and degree of quiescence during measurement. Here, we present the concept for a new approach to delineating structural features of early-stage low molecular weight amyloid ß oligomers, using a solvent accessibility assay in conjunction with simultaneous fluorescence measurements.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Humanos , Peptídeos beta-Amiloides/química , Peso Molecular , Amiloide/química , Espectroscopia de Ressonância Magnética , Fragmentos de Peptídeos/químicaRESUMO
The study of intrinsically disordered and amyloidogenic proteins poses a major challenge to researchers due to the propensity of the system to aggregate and to form amyloid fibrils and deposits. This intrinsic nature limits the way amyloids can be studied and increases the level of complexity of the techniques needed to study the system of interest. Recent reports suggest that cellular recognition and internalization of pre-fibrillary species of amyloidogenic peptides and proteins may initiate some of its toxic actions. Therefore, developing novels tools to facilitate the understanding and determination of the interactions between intrinsically disordered proteins and the cellular membrane is becoming increasingly valuable. Here, we present and propose an approach for the study of the interactions of intrinsically disordered proteins with the cellular surface based on the use of enantiomeric fragment pairs (EFPs). By following a stepwise methodology in which the amyloidogenic peptide or protein is fragmented into specific segments, we show how this approach can be exploited to differentiate between different types of cellular uptake, to determine the degree of receptor-mediated cellular internalization of intrinsically disordered peptides and proteins, and to pinpoint the specific regions within the amino acid sequence responsible for the cellular recognition. Adopting this approach overcomes aggregation-related challenges and offers a particularly well-suited platform for the elucidation of receptor-intermediated recognition, uptake, and toxicity.
Assuntos
Proteínas Intrinsicamente Desordenadas , Sequência de Aminoácidos , Amiloide/química , Proteínas Amiloidogênicas , Proteínas Intrinsicamente Desordenadas/química , Peptídeos/químicaRESUMO
When peptides are mixed with their mirror images in an equimolar ratio, two-dimensional periodic structural folds can form, in which extended peptide strands are arrayed with alternating chirality. The resultant topography class, termed the rippled ß-sheet, was introduced as a theoretical concept by Pauling and Corey in 1953. Unlike other fundamental protein structural motifs identified around that time, including the α-helix and the pleated ß-sheet, it took several decades before conclusive experimental data supporting the proposed rippled ß-sheet motif were gained. Much of the key experimental evidence was provided over the course of the past decade through the concurrent efforts of our three laboratories. Studies that focused on developing new self-assembling hydrogel materials have shown that certain amphiphilic peptides form fibrils and hydrogel networks that are more rigid and have a higher thermodynamic stability when made from racemic peptide mixtures as opposed to pure enantiomers. Related interrogation of assemblies composed of mixtures of l- and d-amphiphilic peptides confirmed that the resulting fibrils were composed of alternating l/d peptides consistent with rippled ß-sheets. It was also demonstrated that mirror-image amyloid beta (Aß) could act as a molecular chaperone to promote oligomer-to-fibril conversion of the natural Aß enantiomer, which was found to reduce Aß neurotoxicity against different neuronal cell models. With a cross-disciplinary approach that combines experiment and theory, our three laboratories have demonstrated the unique biophysical, biochemical, and biological properties that arise upon mixing of peptide enantiomers, in consequence of rippled ß-sheet formation. In this Account, we give an overview of the early history of the rippled ß-sheet and provide a detailed structural description/definition of this motif relative to the pleated ß-sheet. We then summarize the key findings, obtained on three unique sets of aggregating mirror-image peptide pairs through independent efforts of our three laboratories, and use these results to delineate the landscape of the rippled ß-sheet structural motif to inspire future studies. Peptide sequence parameters that favor rippled ß-sheet assembly are described, along with the accompanying kinetic and thermodynamic properties, as well as the resulting emergent physical properties of the assemblies. The Account then concludes with a brief overview of some key unresolved challenges in this nascent field. There is much potential for future applications of this unique supramolecular motif in the realm of materials design and biomedical research. We hope this Account will stimulate much-needed discussion of this fascinating structural class to eventually produce a fully quantitative, rational framework for the molecular engineering of rippled ß-sheets in the future.
Assuntos
Peptídeos/química , Cinética , Modelos Moleculares , Estrutura Secundária de Proteína , TermodinâmicaRESUMO
Recent findings suggest that amyloid-ß (Aß) may not be the only peptidic culprit for the cognitive decline observed in patients with Alzheimer's disease. A C-terminal fragment of Aß, amyloid-α (Aα), also known as p3, has been shown to form amyloidogenic oligomers and fibrils more rapidly than Aß. However, the insolubility and aggregation propensity of this 24-26-residue peptide make it exceptionally difficult to produce, purify, and subsequently study. This paper reports a reproducible, multi-step method for the purification and pre-treatment of Aα and related analogues, yielding 95%-99% pure peptides. We anticipate that the methods described herein will permit previously inaccessible biophysical and biological experiments that may be critical to understanding the role of this too long overlooked peptide in AD disease pathology.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Amiloide , Peptídeos beta-Amiloides/química , Humanos , Fragmentos de Peptídeos/químicaRESUMO
Previous studies have shown that racemic mixtures of 40- and 42-residue amyloid-ß peptides (d,l-Aß40 and d,l-Aß42) form amyloid fibrils with accelerated kinetics and enhanced stability relative to their homochiral counterparts (l-Aß40 and l-Aß42), suggesting a "chiral inactivation" approach to abrogating the neurotoxicity of Aß oligomers (Aß-CI). Here we report a structural study of d,l-Aß40 fibrils, using electron microscopy, solid-state nuclear magnetic resonance (NMR), and density functional theory (DFT) calculations. Two- and three-dimensional solid-state NMR spectra indicate molecular conformations in d,l-Aß40 fibrils that resemble those in known l-Aß40 fibril structures. However, quantitative measurements of 13C-13C and 15N-13C distances in selectively labeled d,l-Aß40 fibril samples indicate a qualitatively different supramolecular structure. While cross-ß structures in mature l-Aß40 fibrils are comprised of in-register, parallel ß-sheets, our data indicate antiparallel ß-sheets in d,l-Aß40 fibrils, with alternation of d and l molecules along the fibril growth direction, i.e., antiparallel "rippled sheet" structures. The solid-state NMR data suggest the coexistence of d,l-Aß40 fibril polymorphs with three different registries of intermolecular hydrogen bonds within the antiparallel rippled sheets. DFT calculations support an energetic preference for antiparallel alignments of the ß-strand segments identified by solid-state NMR. These results provide insight into the structural basis for Aß-CI and establish the importance of rippled sheets in self-assembly of full-length, naturally occurring amyloidogenic peptides.
Assuntos
Peptídeos beta-Amiloides/química , Amiloide/química , Teoria da Densidade Funcional , Ressonância Magnética Nuclear Biomolecular , Humanos , Microscopia EletrônicaRESUMO
The rippled cross-ß sheet, a topography, in which mirror-image peptides are arranged with alternating chirality into a periodic two-dimensional network, is burgeoning as a new design principle for materials and biomedical applications. Experiments by the Schneider, Nilsson, and Raskatov labs have independently shown diverse racemic mixtures of aggregation-prone peptide of different sizes to favor the rippled over the pleated topography. Yet, systematic ab initio studies are lacking, and the field is yet to develop rules that would enable the design of new rippled cross-ß frameworks from first principles. Here, DFT calculations were performed on a set of model systems, designed to begin understanding the impact that bulky, hydrophobic sidechains have upon the formation of pleated and rippled cross-ß frameworks. It is hoped that this study will help stimulate the development of a predictive, general framework to enable rational design of rippled cross-ß sheets in the future.
Assuntos
Teoria da Densidade Funcional , Peptídeos/química , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Peptídeos/metabolismo , Conformação Proteica em Folha beta , TermodinâmicaRESUMO
We recently introduced amyloid ß chiral inactivation (Aß-CI) as a molecular approach that uses mirror-image peptides to chaperone the natural Aß stereoisomer into a less toxic state. The oligomer-to-fibril conversion mechanism remains the subject of active research. Perhaps the most striking feature of Aß-CI is the virtual obliteration of the incubation/induction phase that is so characteristic of Aß fibril formation kinetics. This qualitative change is indicative of the distinct mechanistic pathway Aß-CI operates through. The current working model of Aß-CI invokes the formation of "rippled" cross-ß sheets, in which alternating l- and d-peptide strands form periodic networks. However, the assumption of rippled cross-ß sheets does not per se explain the dramatic changes in reaction kinetics upon mixing of Aß enantiomers. Herein, it is shown by DFT computational methods that the individual peptide strands in rippled cross-ß networks are less conformationally strained than their pleated counterparts. This means that the adoption of fibril-seeding conformations is more probable for rippled cross-ß. Conformational selection is thus suggested as the mechanistic rationale for the acceleration of fibril formation upon Aß-CI.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Teoria da Densidade Funcional , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Conformação Proteica , EstereoisomerismoRESUMO
The difficulty of synthesizing and purifying the amyloid ß (Aß) peptide, combined with its high aggregation propensity and low solubility under physiological conditions, leads to a wide variety of experimental results from kinetic assays to biological activity. Thus, it becomes challenging to reproduce outcomes, and this limits our ability to rely on reported results as the foundation for new research. This article examines variability of the Aß peptide from different sources, comparing purity, and oligomer and fibril formation propensity side by side. The results highlight the importance of performing rigorous controls so that meaningful biophysical, biochemical, and neurobiological results can be obtained to improve our understanding on Aß.
Assuntos
Peptídeos beta-Amiloides/biossíntese , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Agregados ProteicosRESUMO
Methionine (Met) oxidation is an important biological redox node, with hundreds if not thousands of protein targets. The process yields methionine oxide (MetO). It renders the sulfur chiral, producing two distinct, diastereomerically related products. Despite the biological significance of Met oxidation, a reliable protocol to separate the resultant MetO diastereomers is currently lacking. This hampers our ability to make peptides and proteins that contain stereochemically defined MetO to then study their structural and functional properties. We have developed a facile method that uses supercritical CO2 chromatography and allows obtaining both diastereomers in purities exceeding 99 %. 1 Hâ NMR spectra were correlated with X-ray structural information. The stereochemical interconversion barrier at sulfur was calculated as 45.2â kcal mol-1 , highlighting the remarkable stereochemical stability of MetO sulfur chirality. Our protocol should open the road to synthesis and study of a wide variety of stereochemically defined MetO-containing proteins and peptides.
RESUMO
Amyloidogenic peptides and proteins aggregate into fibrillary structures that are usually deposited in tissues and organs and are often involved in the development of diseases. In contrast to native structured proteins, amyloids do not follow a defined energy landscape toward the fibrillary state and often generate a vast population of aggregation intermediates that are transient and exceedingly difficult to study. Here, we employ chiral editing as a tool to study the aggregation mechanism of the Amyloid ß (Aß) 42 peptide, whose aggregation intermediates are thought to be one of the main driving forces in Alzheimer's disease (AD). Through the design of a focused chiral mutant library (FCML) of 16 chiral Aß42 variants, we identified several point D-substitutions that allowed us to modulate the aggregation propensity and the biological activity of the peptide. Surprisingly, the reduced propensity toward aggregation and the stabilization of oligomeric intermediates did not always correlate with an increase in toxicity. In the present study, we show how chiral editing can be a powerful tool to trap and stabilize Aß42 conformers that might otherwise be too transient and dynamic to study, and we identify sites within the Aß42 sequence that could be potential targets for therapeutic intervention.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/genética , Humanos , Fragmentos de Peptídeos , Eletricidade EstáticaRESUMO
Alzheimer's amyloid beta can perform a wide variety of actions that are highly concentration dependent. This viewpoint aims to provide a framework for basic considerations on what might be considered brain-relevant concentrations of the peptide. Some implications for the therapeutic implementation of the recently emerged oligomer-to-fibril strategy are discussed.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Animais , Fenômenos Químicos , Humanos , Camundongos , Multimerização Proteica , RatosRESUMO
Amyloid ß 42 (Aß42) is an inherently disordered peptide, whose toxic actions are believed to play important roles in the etiology of Alzheimer's disease. Four fibril structures of the peptide that display broadly similar characteristics were recently published, but a systematic comparison of these structures is lacking. In this paper, a topological framework was created to enable such understanding and produced new insights into subtle structural elements that underlie the overall structural diversity. A DFT-based analysis illuminated some of the energetic differences that arise as a consequence.
Assuntos
Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Teoria da Densidade Funcional , Humanos , Modelos Químicos , Conformação ProteicaRESUMO
Amyloidâ ß is an inherently disordered peptide that can form diverse neurotoxic aggregates, and its 42-amino-acid isoform is believed to be the agent responsible for Alzheimer's disease (AD). Cellular uptake of the peptide is a pivotal step for it to be able to exert many of its toxic actions. The cellular uptake process is complex, and numerous competing internalization pathways have been proposed. To date, it remains unclear which of the uptake mechanisms are particularly important for the overall process, and improvement of this understanding is needed, so that better molecular AD therapeutics can be designed. Chirality can be used as a unique tool to study this process, because some of the proposed mechanisms are expected to proceed in stereoselective fashion, whereas others are not. To shed light on this important issue, we synthesized fluorescently labeled enantiomers of amyloidâ ß and quantified their cellular uptake, finding that uptake occurs in stereoselective fashion, with a typical preference for the l stereoisomer of ≈5:1. This suggests that the process is predominantly receptor-mediated, with likely minor contributions of non-stereoselective mechanisms.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , EstereoisomerismoRESUMO
The thermal (3 + 2) dipolar azide-alkyne cycloaddition, proceeding without copper or strained alkynes, is an underutilized ligation with potential applications in materials, bioorganic, and synthetic chemistry. Herein, we investigate the effects of alkyne substitution on the rate of this reaction, both experimentally and computationally. Electron-withdrawing groups accelerate the reaction, providing a range of relative rates from 1.0 to 2100 between the slowest and fastest alkynes studied. Unexpectedly, aryl groups conjugated to the alkyne significantly retard the reaction rate. In contrast, a sulfonyl, ester-substituted alkyne is reactive enough that it couples with an azide at room temperature in a few hours. This reactivity scale should provide a guide to those who wish to use this ligation under mild conditions.
RESUMO
Mixing of enantiomers of chiral molecules can have remarkable effects on the properties of their higher order assemblies. This, in turn, may have profound impact on both structural and functional outcomes, and has been noted across a wide range of contexts. This account presents key examples from organic, organometallic and bioorganic molecular sciences and showcases that, in disciplines as distinct as asymmetric catalysis and racemic protein crystallography, there is a fundamental way in which enantiomers differ from racemates.
RESUMO
Amyloid beta-protein 42 plays an important role in the onset and progression of Alzheimer's disease. Familial mutations have identified the glutamate residue 22 as a hotspot with regard to peptide neurotoxicity. We introduce an approach to study the influence of systematic sidechain modification at this residue, employing chirality as a structural probe. Circular dichroism experiments reveal that charge-preserving alterations of the amino acid sidechain attenuate the characteristic random coil to ß-sheet transition associated with the wildtype peptide. Removal of the negative charge from residue 22, a trait observed with all known familial mutations at this residue, gives rise to a peptide with limited random coil propensity and high ß-sheet characteristics. Our approach can be extended to other residues of Aß, as well as further amyloidogenic peptides.