RESUMO
BACKGROUND: Cholinesterase inhibitors (ChEIs) are prescribed for Alzheimer's disease (AD) and sometimes for mild cognitive impairment (MCI) without knowing underlying pathologies and its effect on cognition. We investigated the frequency of ChEI prescriptions in amyloid-negative MCI and their association with cognitive changes in the Alzheimer's Disease Neuroimaging Initiative (ADNI) cohort. METHODS: We included participants with amyloid positron emission tomography (PET)-negative MCI from the ADNI. We analyzed the associations of ChEI use with cognitive changes, brain volume, and cerebrospinal fluid (CSF) total tau (t-tau), hyperphosphorylated tau181 (p-tau181), and p-tau181/t-tau ratio. RESULTS: ChEIs were prescribed in 27.4% of amyloid PET-negative MCI and were associated with faster cognitive decline, reduced baseline hippocampal volume and entorhinal cortical thickness, and a longitudinal decrease in the frontal lobe cortical thickness. CONCLUSIONS: The association between ChEI use and accelerated cognitive decline may stem from underlying pathologies involving reduced hippocampal volume, entorhinal cortical thickness and faster frontal lobe atrophy. We suggest that ChEI use in amyloid PET-negative MCI patients might need further consideration, and studies investigating the causality between ChEI use and cognitive decline are warranted in the future.
Assuntos
Inibidores da Colinesterase , Disfunção Cognitiva , Tomografia por Emissão de Pósitrons , Humanos , Disfunção Cognitiva/diagnóstico por imagem , Masculino , Tomografia por Emissão de Pósitrons/métodos , Feminino , Idoso , Inibidores da Colinesterase/uso terapêutico , Proteínas tau/líquido cefalorraquidiano , Proteínas tau/metabolismo , Idoso de 80 Anos ou mais , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Imageamento por Ressonância Magnética , Pessoa de Meia-Idade , Amiloide/metabolismoRESUMO
C-reactive protein (CRP) binds to phosphocholine (PCh)-containing substances and subsequently activates the complement system to eliminate the ligand. The PCh-binding function of CRP has been conserved throughout evolution from arthropods to humans. Human CRP, in its structurally altered conformation at acidic pH, also binds to amyloid-ß (Aß) and prevents the formation of Aß fibrils. It is unknown whether the Aß-binding function of CRP has also been evolutionarily conserved. The aim of this study was to determine whether CRP isolated from American horseshoe crab Limulus polyphemus was also anti-amyloidogenic and whether this function required structural alteration of Limulus CRP (Li-CRP). Two CRP species Li-CRP-I and Li-CRP-II were purified from hemolymph by employing PCh-affinity chromatography and phosphoethanolamine-affinity chromatography, respectively. Both Li-CRP-I and Li-CRP-II bound to immobilized Aß at physiological pH. Unlike human CRP, Li-CRP did not require any changes in its overall structure to bind to Aß. Both Li-CRP-I and Li-CRP-II bound to Aß in the fluid phase also and prevented the fibrillation of Aß. Additionally, ion-exchange chromatography of purified Li-CRP indicated that a variety of Li-CRP molecules of different subunit compositions were present in Limulus hemolymph, raising the possibility that the presence of various Li-CRP species in hemolymph facilitates the recognition of a range of proteins with differing amyloidogenicity. We conclude that the binding of CRP to Aß is an ancient function of CRP. In invertebrates, the Aß-binding function of CRP can protect the host from toxicity caused by amyloidogenic and pathogenic proteins. In humans, the Aß-binding function of CRP can protect against inflammatory diseases in which the host proteins are ectopically deposited on either host cells or foreign cells in an inflammatory milieu since immobilized proteins may expose Aß-like structures after deposition at places where they are not supposed to be.
Assuntos
Peptídeos beta-Amiloides , Amiloide , Proteína C-Reativa , Caranguejos Ferradura , Animais , Proteína C-Reativa/metabolismo , Proteína C-Reativa/química , Caranguejos Ferradura/metabolismo , Humanos , Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Ligação Proteica , Hemolinfa/metabolismo , Evolução Molecular , Fosforilcolina/metabolismoRESUMO
Tannic acid (TA)-derived carbon dots (TACDs) were synthesized for the first time via a solvothermal method using TA as one of the raw materials, which may effectively inhibit amyloid fibril aggregation and disaggregate mature fibril. The fluorescent property of TACDs were modulated by adjusting the ratio of TA to o-phenylenediamine (oPD), and TACDs fabricated with the precursor ratio as 1:1 showed the best fluorescent property. Circular dichroism spectra (CD) showed that the structure of ß-sheet decreased as the concentration of TACDs increased. The inhibition efficiency, as confirmed by thioflavin T (ThT) and transmission electron microscopy (TEM), is extraordinary at 98.16%, whereas disaggregation efficiency is noteworthy at 97.97%, and the disaggregated lysozyme fibrils did not reaggregate after 7 days. More critically, TACDs can also alleviate the cellular toxicity caused by Aß fibrils and improve cell viability. This work offers a new perspective on the design of scavengers for amyloid plaques.
Assuntos
Carbono , Agregados Proteicos , Taninos , Taninos/química , Taninos/farmacologia , Carbono/química , Humanos , Agregados Proteicos/efeitos dos fármacos , Muramidase/química , Muramidase/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Pontos Quânticos/química , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Amiloide/química , Amiloide/metabolismo , Fenilenodiaminas/química , Fenilenodiaminas/farmacologia , Animais , PolifenóisRESUMO
Age-related neurodegenerative disorders like Alzheimer's disease (AD) and Parkinson's disease (PD) are characterized by deposits of protein aggregates, or amyloid, in various regions of the brain. Historically, aggregation of a single protein was observed to be correlated with these different pathologies: tau in AD and α-synuclein (αS) in PD. However, there is increasing evidence that the pathologies of these two diseases overlap, and the individual proteins may even promote each other's aggregation. Both tau and αS are intrinsically disordered proteins (IDPs), lacking stable secondary and tertiary structure under physiological conditions. In this study we used a combination of biochemical and biophysical techniques to interrogate the interaction of tau with both soluble and fibrillar αS. Fluorescence correlation spectroscopy (FCS) was used to assess the interactions of specific domains of fluorescently labeled tau with full length and C-terminally truncated αS in both monomer and fibrillar forms. We found that full-length tau as well as individual tau domains interact with monomer αS weakly, but this interaction is much more pronounced with αS aggregates. αS aggregates also mildly slow the rate of tau aggregation, although not the final degree of aggregation. Our findings suggest that co-occurrence of tau and αS in disease are more likely to occur through monomer-fiber binding interactions, rather than monomer-monomer or co-aggregation.
Assuntos
alfa-Sinucleína , Proteínas tau , alfa-Sinucleína/metabolismo , alfa-Sinucleína/química , Proteínas tau/metabolismo , Proteínas tau/química , Humanos , Ligação Proteica , Agregados Proteicos , Amiloide/metabolismo , Amiloide/química , Espectrometria de Fluorescência , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Agregação Patológica de Proteínas/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologiaRESUMO
Self-assembly is a key process in living systems-from the microscopic biological level (e.g. assembly of proteins into fibrils within biomolecular condensates in a human cell) through to the macroscopic societal level (e.g. assembly of humans into common-interest communities across online social media platforms). The components in such systems (e.g. macromolecules, humans) are highly diverse, and so are the self-assembled structures that they form. However, there is no simple theory of how such structures assemble from a multi-species pool of components. Here we provide a very simple model which trades myriad chemical and human details for a transparent analysis, and yields results in good agreement with recent empirical data. It reveals a new inhibitory role for biomolecular condensates in the formation of dangerous amyloid fibrils, as well as a kinetic explanation of why so many diverse distrust movements are now emerging across social media. The nonlinear dependencies that we uncover suggest new real-world control strategies for such multi-species assembly.
Assuntos
Amiloide , Condensados Biomoleculares , Humanos , Amiloide/química , Amiloide/metabolismo , Condensados Biomoleculares/metabolismo , Condensados Biomoleculares/química , Cinética , Mídias SociaisRESUMO
Amyloid PET plays a crucial role in the early diagnosis of Alzheimer's disease and the determination of the feasibility of disease-modifying therapies. It offers several advantages, including high sensitivity and specificity, minimal invasiveness, and the ability to provide spatial evaluation, all of which contribute to the optimization of dementia care. However, proper use and interpretation of the results require a thorough understanding of their limitations. Although careful consideration is necessary when using scans on asymptomatic individuals, clinical applications could broaden if preemptive treatments and high-precision individual risk assessments for the preclinical stage are developed.
Assuntos
Doença de Alzheimer , Tomografia por Emissão de Pósitrons , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/metabolismo , Doença de Alzheimer/diagnóstico , Humanos , Amiloide/metabolismoRESUMO
Systemic amyloidosis involves the deposition of misfolded proteins in organs/tissues, leading to progressive organ dysfunction and failure. Congo red is the gold-standard chemical stain for visualizing amyloid deposits in tissue, showing birefringence under polarization microscopy. However, Congo red staining is tedious and costly to perform, and prone to false diagnoses due to variations in amyloid amount, staining quality and manual examination of tissue under a polarization microscope. We report virtual birefringence imaging and virtual Congo red staining of label-free human tissue to show that a single neural network can transform autofluorescence images of label-free tissue into brightfield and polarized microscopy images, matching their histochemically stained versions. Blind testing with quantitative metrics and pathologist evaluations on cardiac tissue showed that our virtually stained polarization and brightfield images highlight amyloid patterns in a consistent manner, mitigating challenges due to variations in chemical staining quality and manual imaging processes in the clinical workflow.
Assuntos
Amiloide , Aprendizado Profundo , Microscopia de Fluorescência , Coloração e Rotulagem , Humanos , Birrefringência , Amiloide/metabolismo , Microscopia de Fluorescência/métodos , Coloração e Rotulagem/métodos , Vermelho Congo , Microscopia de Polarização/métodos , Amiloidose/patologia , Amiloidose/metabolismo , Amiloidose/diagnóstico por imagem , Imagem Óptica/métodos , Placa Amiloide/patologia , Placa Amiloide/metabolismo , Placa Amiloide/diagnóstico por imagem , Miocárdio/patologia , Miocárdio/metabolismoRESUMO
Alkaptonuria (AKU) is a rare autosomal recessive metabolic disorder caused by mutations in the homogentisate 1,2-dioxygenase (HGD) gene, leading to the accumulation of homogentisic acid (HGA), causing severe inflammatory conditions. Recently, the presence of serum amyloid A (SAA) has been reported in AKU tissues, classifying AKU as novel secondary amyloidosis; AA amyloidosis is characterized by the extracellular tissue deposition of fibrils composed of fragments of SAA. AA amyloidosis may complicate several chronic inflammatory conditions, like rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, chronic infections, neoplasms, etc. Treatments of AA amyloidosis relieve inflammatory disorders by reducing SAA concentrations; however, no definitive therapy is currently available. SAA regulation is a crucial step to improve AA secondary amyloidosis treatments. Here, applying a comprehensive in vitro and in silico approach, we provided evidence that HGA is a disruptor modulator of SAA, able to enhance its polymerization, fibril formation, and aggregation upon SAA/SAP colocalization. In silico studies deeply dissected the SAA misfolding molecular pathway and SAA/HGA binding, suggesting novel molecular insights about it. Our results could represent an important starting point for identifying novel therapeutic strategies in AKU and AA secondary amyloidosis-related diseases.
Assuntos
Alcaptonúria , Ácido Homogentísico , Proteína Amiloide A Sérica , Alcaptonúria/metabolismo , Alcaptonúria/patologia , Proteína Amiloide A Sérica/metabolismo , Proteína Amiloide A Sérica/genética , Humanos , Ácido Homogentísico/metabolismo , Agregados Proteicos , Amiloidose/metabolismo , Amiloidose/patologia , Amiloide/metabolismo , Modelos Biológicos , Homogentisato 1,2-Dioxigenase/metabolismo , Homogentisato 1,2-Dioxigenase/genéticaRESUMO
Functional amyloids, beneficial to the organism producing them, are found throughout life, from bacteria to humans. While disease-related amyloids form by uncontrolled aggregation, the fibrillation of functional amyloid is regulated by complex cellular machinery and optimized sequences, including so-called gatekeeper residues such as Asp. However, the molecular basis for this regulation remains unclear. Here we investigate how the introduction of additional gatekeeper residues affects fibril formation and stability in the functional amyloid CsgA from E. coli. Step-wise introduction of additional Asp gatekeepers gradually eliminated fibrillation unless preformed fibrils were added, illustrating that gatekeepers mainly affect nucleus formation. Once formed, the mutant CsgA fibrils were just as stable as wild-type CsgA. HSQC NMR spectra confirmed that CsgA is intrinsically disordered, and that the introduction of gatekeeper residues does not alter this ensemble. NMR-based Dark-state Exchange Saturation Transfer (DEST) experiments on the different CsgA variants, however, show a decrease in transient interactions between monomeric states and the fibrils, highlighting a critical role for these interactions in the fibrillation process. We conclude that gatekeeper residues affect fibrillation kinetics without compromising structural integrity, making them useful and selective modulators of fibril properties.
Assuntos
Amiloide , Proteínas de Escherichia coli , Escherichia coli , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Amiloide/química , Amiloide/metabolismo , Amiloide/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/química , Estabilidade Proteica , Ressonância Magnética Nuclear Biomolecular , MutaçãoRESUMO
BACKGROUND AND OBJECTIVES: Alzheimer disease (AD) is associated with a 2 to 3-fold increased risk of developing late-onset focal epilepsy, yet it remains unclear how development of focal epilepsy in AD is related to AD pathology. The objective of this study was to examine spatial relationships between the epileptogenic zone and tau deposition, amyloid deposition, and brain atrophy in individuals with AD who developed late-onset, otherwise unexplained focal epilepsy. We hypothesized that if network hyperexcitability is mechanistically linked to AD pathology, then there would be increased tau and amyloid deposition within the epileptogenic hemisphere. METHODS: In this cross-sectional study, we performed tau and amyloid PET imaging, brain MRI, and overnight scalp EEG in individuals with early clinical stages of AD who developed late-onset, otherwise unexplained focal epilepsy (AD-Ep). Participants were referred from epilepsy and memory disorders clinics at our institutions. We determined epilepsy localization based on EEG findings and seizure semiology. We quantified tau deposition, amyloid deposition, and atrophy across brain regions and calculated asymmetry indices for these measures. We compared findings in AD-Ep with those in a control AD group without epilepsy (AD-NoEp). RESULTS: The AD-Ep group included 8 individuals with a mean age of 69.5 ± 4.2 years at PET imaging. The AD-NoEp group included 14 individuals with a mean age of 71.7 ± 9.8 years at PET imaging. In AD-Ep, we found a highly asymmetric pattern of tau deposition, with significantly greater tau in the epileptogenic hemisphere. Amyloid deposition and cortical atrophy were also greater in the epileptogenic hemisphere, although the magnitudes of asymmetry were reduced compared with tau. Compared with AD-NoEp, the AD-Ep group had significantly greater tau asymmetry and trends toward greater asymmetry of amyloid and atrophy. AD-Ep also had significantly greater amyloid burden bilaterally and trends toward greater tau burden within the epileptogenic hemisphere, compared with AD-NoEp. DISCUSSION: Our results reveal a spatial association between the epileptogenic focus and tau deposition, amyloid deposition, and neurodegeneration in early clinical stages of AD. Within the limitations of a cross-sectional study with small sample sizes, these findings contribute to our understanding of the clinicopathologic heterogeneity of AD, demonstrating an association between focal epilepsy and lateralized pathology in AD.
Assuntos
Doença de Alzheimer , Atrofia , Encéfalo , Eletroencefalografia , Imageamento por Ressonância Magnética , Tomografia por Emissão de Pósitrons , Convulsões , Proteínas tau , Humanos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/patologia , Atrofia/patologia , Masculino , Feminino , Proteínas tau/metabolismo , Idoso , Estudos Transversais , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Encéfalo/metabolismo , Convulsões/diagnóstico por imagem , Convulsões/metabolismo , Convulsões/patologia , Pessoa de Meia-Idade , Idoso de 80 Anos ou mais , Amiloide/metabolismo , Epilepsias Parciais/diagnóstico por imagem , Epilepsias Parciais/metabolismo , Epilepsias Parciais/patologiaRESUMO
Tau is an intrinsically disordered protein involved in several neurodegenerative diseases where a common hallmark is the appearance of tau aggregates in the brain. One common approach to elucidate the mechanisms behind the aggregation of tau has been to recapitulate in vitro the self-assembly process in a fast and reproducible manner. While the seeding of tau aggregation is prompted by negatively charged cofactors, the obtained fibrils are morphologically distinct from those found in vivo. The Tau AD core fragment (TADC, tau 306-378) has emerged as a new model and potential solution for the cofactor-free in vitro aggregation of tau. Here, we use TADC to further study this process combining multiple amyloid-detecting fluorophores and fibril bioimaging. We confirmed by transmission electron microscopy that this fragment forms fibrils after quiescent incubation at 37 °C. We then employed a panel of eight amyloid-binding fluorophores to query the formed species by acquiring their emission spectra. The results obtained showed that nearly all dyes detect TADC self-assembled species. However, the successful monitoring of TADC aggregation kinetics was limited to three fluorophores (X-34, Bis-ANS, and pFTAA) which yielded sigmoidal curves but different aggregation half-times, hinting to different species being detected. Altogether, this study highlights the potential of using multiple extrinsic fluorescent probes, alone or in combination, as tools to further clarify mechanisms behind the aggregation of amyloidogenic proteins.
Assuntos
Doença de Alzheimer , Amiloide , Corantes Fluorescentes , Proteínas tau , Proteínas tau/metabolismo , Proteínas tau/química , Proteínas tau/ultraestrutura , Humanos , Amiloide/metabolismo , Amiloide/química , Corantes Fluorescentes/química , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Agregados Proteicos , Agregação Patológica de Proteínas/metabolismo , Cinética , Ligação ProteicaRESUMO
Alzheimer's disease (AD) is one of the most common causes of dementia, accounting for more than 60% of all cases. It is a neurodegenerative disease in which symptoms such as a decline in memory, thinking, learning, and organizing skills develop gradually over many years and eventually become more severe. To date, there is no effective treatment for the cause of Alzheimer's disease, and the existing pharmacological options primarily help manage symptoms. Treatment is mainly based on acetylcholinesterase (AChE) inhibitors such as donepezil, rivastigmine, and galantamine, which exhibit numerous adverse cardiovascular and gastrointestinal effects due to excessive stimulation of peripheral cholinergic activity involving muscarinic receptors. Therefore, in addition to the obvious drugs that act on the cause of the disease, new drugs based on AChE inhibition that show the fewest side effects are needed. One potential drug could be a new compound under study, tetrahydroacridine derivative (CHDA), which showed significant potential to inhibit the AChE enzyme in previous in vitro studies. The present study shows that while having very potent AChE inhibitory properties, CHDA is a compound with low toxicity to nerve cell culture and living organisms. In addition, it exhibits dissociative activity against amyloid ß fibrils, which is extremely important for applications in Alzheimer's disease therapy.
Assuntos
Acetilcolinesterase , Peptídeos beta-Amiloides , Inibidores da Colinesterase , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/química , Peptídeos beta-Amiloides/metabolismo , Humanos , Acetilcolinesterase/metabolismo , Acetilcolinesterase/química , Acridinas/farmacologia , Acridinas/química , Animais , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Amiloide/metabolismoRESUMO
Reversible and irreversible amyloids are two diverging cases of protein (mis)folding associated with the cross-ß motif in the protein folding and aggregation energy landscape. Yet, the molecular origins responsible for the formation of reversible vs irreversible amyloids have remained unknown. Here we provide evidence at the atomic level of distinct folding motifs for irreversible and reversible amyloids derived from a single protein sequence: human lysozyme. We compare the 2.8 Å structure of irreversible amyloid fibrils determined by cryo-electron microscopy helical reconstructions with molecular insights gained by solid-state NMR spectroscopy on reversible amyloids. We observe a canonical cross-ß-sheet structure in irreversible amyloids, whereas in reversible amyloids, there is a less-ordered coexistence of ß-sheet and helical secondary structures that originate from a partially unfolded lysozyme, thus carrying a "memory" of the original folded protein precursor. We also report the structure of hen egg-white lysozyme irreversible amyloids at 3.2 Å resolution, revealing another canonical amyloid fold, and reaffirming that irreversible amyloids undergo a complete conversion of the native protein into the cross-ß structure. By combining atomic force microscopy, cryo-electron microscopy and solid-state NMR, we show that a full unfolding of the native protein precursor is a requirement for establishing irreversible amyloid fibrils.
Assuntos
Amiloide , Microscopia Crioeletrônica , Muramidase , Dobramento de Proteína , Muramidase/química , Muramidase/ultraestrutura , Muramidase/metabolismo , Amiloide/química , Amiloide/ultraestrutura , Amiloide/metabolismo , Humanos , Modelos Moleculares , Animais , Galinhas , Espectroscopia de Ressonância Magnética , Estrutura Secundária de ProteínaRESUMO
Medin amyloid, prevalent in the vessel walls of 97â¯% of individuals over 50, contributes to arterial stiffening and cerebrovascular dysfunction, yet our understanding of its aggregation mechanism remains limited. Dividing the full-length 50-amino-acid medin peptide into five 10-residue segments, we conducted individual investigations on each segment's self-assembly dynamics via microsecond-timescale atomistic discrete molecular dynamics (DMD) simulations. Our findings showed that medin1-10 and medin11-20 segments predominantly existed as isolated unstructured monomers, unable to form stable oligomers. Medin31-40 exhibited moderate aggregation, forming dynamic ß-sheet oligomers with frequent association and dissociation. Conversely, medin21-30 and medin41-50 segments demonstrated significant self-assembly capability, readily forming stable ß-sheet-rich oligomers. Residue pairwise contact frequency analysis highlighted the critical roles of residues 22-26 and 43-49 in driving the self-assembly of medin21-30 and medin41-50, acting as the ß-sheet core and facilitating ß-strand formation in other regions within medin monomers, expecting to extend to oligomers and fibrils. Regions containing residues 22-26 and 43-49, with substantial self-assembly abilities and assistance in ß-sheet formation, represent crucial targets for amyloid inhibitor drug design against aortic medial amyloidosis (AMA). In summary, our study not only offers deep insights into the mechanism of medin amyloid formation but also provides crucial theoretical and practical guidance for future treatments of AMA.
Assuntos
Amiloide , Simulação de Dinâmica Molecular , Humanos , Amiloide/química , Amiloide/metabolismo , Aorta/metabolismo , Agregados Proteicos , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica em Folha beta , Antígenos de Superfície/metabolismo , Antígenos de Superfície/química , Sequência de Aminoácidos , Proteínas do LeiteRESUMO
The islet amyloid polypeptide (IAPP), also known as amylin, is a hormone playing key physiological roles. However, its aggregation and deposition in the pancreatic islets are associated with type 2 diabetes. While this peptide adopts mainly a random coil structure in solution, its secondary conformational conversion into α-helix represents a critical step for receptor activation and contributes to amyloid formation and associated cytotoxicity. Considering the large conformational landscape and high amyloidogenicity of the peptide, as well as the complexity of the self-assembly process, it is challenging to delineate the delicate interplay between helical folding, peptide aggregation, and receptor activation. In the present study, we probed the roles of helical folding on the function-toxicity duality of IAPP by restricting its conformational ensemble through side chain-to-side chain stapling via azide-alkyne cycloaddition. Intramolecular macrocyclization (i; i + 4) constrained IAPP into α-helix and inhibited its aggregation into amyloid fibrils. These helical derivatives slowed down the self-assembly of unmodified IAPP. Site-specific macrocyclization modulated the capacity of IAPP to perturb lipid bilayers and cell plasma membrane and reduced, or even fully inhibited, the cytotoxicity associated with aggregation. Furthermore, the α-helical IAPP analogs showed moderate to high potency toward cognate G protein-coupled receptors. Overall, these results indicate that macrocyclization represents a promising strategy to protect an amyloidogenic peptide hormone from aggregation and associated toxicity, while maintaining high receptor activity.
Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Humanos , Ciclização , Ligantes , Amiloide/química , Amiloide/metabolismo , Reação de CicloadiçãoRESUMO
Liquid-like protein condensates have recently attracted much attention due to their critical roles in biological phenomena. They typically show high fluidity and reversibility for exhibiting biological functions, while occasionally serving as sites for the formation of amyloid fibrils. To comprehend the properties of protein condensates that underlie biological function and pathogenesis, it is crucial to study them at the single-condensate level; however, this is currently challenging due to a lack of applicable methods. Here, we demonstrate that optical trapping is capable of inducing the formation of a single liquid-like condensate of α-synuclein in a spatiotemporally controlled manner. The irradiation of tightly focused near-infrared laser at an air/solution interface formed a condensate under conditions coexisting with polyethylene glycol. The fluorescent dye-labeled imaging showed that the optically induced condensate has a gradient of protein concentration from the center to the edge, suggesting that it is fabricated through optical pumping-up of the α-synuclein clusters and the expansion along the interface. Furthermore, Raman spectroscopy and thioflavin T fluorescence analysis revealed that continuous laser irradiation induces structural transition of protein molecules inside the condensate to ß-sheet rich structure, ultimately leading to the condensate deformation and furthermore, the formation of amyloid fibrils. These observations indicate that optical trapping is a powerful technique for examining the microscopic mechanisms of condensate appearance and growth, and furthermore, subsequent aging leading to amyloid fibril formation.
Assuntos
Amiloide , Pinças Ópticas , alfa-Sinucleína , alfa-Sinucleína/metabolismo , alfa-Sinucleína/química , Amiloide/química , Amiloide/metabolismo , Humanos , Análise Espectral Raman/métodosRESUMO
Amyloids are associated with over 50 human diseases and have inspired significant effort to identify small molecule remedies. Here, we present an in vivo platform that efficiently yields small molecule inhibitors of amyloid formation. We previously identified small molecules that kill the nematode C. elegans by forming membrane-piercing crystals in the pharynx cuticle, which is rich in amyloid-like material. We show here that many of these molecules are known amyloid-binders whose crystal-formation in the pharynx can be blocked by amyloid-binding dyes. We asked whether this phenomenon could be exploited to identify molecules that interfere with the ability of amyloids to seed higher-order structures. We therefore screened 2560 compounds and found 85 crystal suppressors, 47% of which inhibit amyloid formation. This hit rate far exceeds other screening methodologies. Hence, in vivo screens for suppressors of crystal formation in C. elegans can efficiently reveal small molecules with amyloid-inhibiting potential.
Assuntos
Amiloide , Caenorhabditis elegans , Caenorhabditis elegans/metabolismo , Animais , Amiloide/metabolismo , Amiloide/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/química , Faringe/metabolismo , Faringe/efeitos dos fármacos , Humanos , Agregados Proteicos/efeitos dos fármacos , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos/métodosRESUMO
The inherent stochasticity associated with the hierarchical self-assembly of either native-like or partially-unfolded protein monomers leads to the formation of transient, morphologically-diverse prefibrillar species resulting in structurally-distinct polymorphic protein aggregates. High-resolution structural characterization of mature aggregates has revealed heterogeneous supramolecular packing of protofibrils within amyloid polymorphs. However, little is known about whether initial monomeric protein conformers engender polymorphism at the onset of aggregation. Here, we show that intrinsic conformational preference in aggregation-competent monomeric ovalbumin, an archetypal serpin, dictates fibrillar polymorphism by modulating aggregation pathways. Using fluorescence, FT-IR, and vibrational Raman spectroscopy coupled with dynamic light scattering and electron microscopy, we demonstrate that conformationally-diverse amyloidogenic monomers, formed via an interplay of electrostatic and hydrophobic interactions before the commencement of aggregation, play a crucial role in promoting amyloid polymorphism. Moreover, the monomeric conformational fingerprints, accrued at the onset of aggregation, persist and propagate during the formation of polymorphic amyloids. Our results delineate essential conformational characteristics of the monomeric protein preceding aggregation, which will have broad implications in the mechanistic understanding of amyloid strain diversity observed in disease-related proteins.
Assuntos
Amiloide , Conformação Proteica , Amiloide/química , Amiloide/metabolismo , Análise Espectral Raman , Ovalbumina/química , Ovalbumina/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Infravermelho com Transformada de Fourier , Agregados Proteicos , Eletricidade EstáticaRESUMO
Alzheimer's disease (AD) is a prevalent neurodegenerative disorder, impacting millions of individuals worldwide. Among its defining characteristics is the accumulation of senile plaques within the brain's gray matter, formed through the self-assembly of misfolded proteins contributing to the progressive symptoms of AD. This study investigates a polymorphic Aß fibril under static and oscillating electric fields using molecular dynamics simulation. Specifically, we utilized a polymorphic fibrillar complex composed of two intertwined pentamer-strands of the Aß1-40 peptide with the Osaka mutation (E22Δ), known for its toxicity and stable structure. Our findings demonstrate that a 0.3 and 0.4 V/nm electric field combined with a 0.20 GHz frequency effectively disrupts the polymorphic conformation of Aß fibrils. Furthermore, we elucidate the molecular mechanisms underlying this disruption, providing insights into the potential therapeutic use of oscillating electric fields for AD. This research offers valuable insights into novel therapeutic approaches for combating AD pathology.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Eletricidade , Simulação de Dinâmica Molecular , Mutação , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/química , Humanos , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Simulação por Computador , Amiloide/química , Amiloide/metabolismoRESUMO
The pathological process of prion diseases implicates that the normal physiological cellular prion protein (PrPC) converts into misfolded abnormal scrapie prion (PrPSc) through post-translational modifications that increase ß-sheet conformation. We recently demonstrated that HuPrP(90-231) thermal unfolding is partially irreversible and characterized by an intermediate state (ß-PrPI), which has been revealed to be involved in the initial stages of PrPC fibrillation, with a seeding activity comparable to that of human infectious prions. In this study, we report the thermal unfolding characterization, in cell-mimicking conditions, of the truncated (HuPrP(90-231)) and full-length (HuPrP(23-231)) human prion protein by means of CD and NMR spectroscopy, revealing that HuPrP(90-231) thermal unfolding is characterized by two successive transitions, as in buffer solution. The amyloidogenic propensity of HuPrP(90-231) under crowded conditions has also been investigated. Our findings show that although the prion intermediate, structurally very similar to ß-PrPI, forms at a lower temperature compared to when it is dissolved in buffer solution, in cell-mimicking conditions, the formation of prion fibrils requires a longer incubation time, outlining how molecular crowding influences both the equilibrium states of PrP and its kinetic pathways of folding and aggregation.