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1.
Nat Commun ; 11(1): 4616, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934229

RESUMO

Para-nematic phases, induced by unwinding chiral helices, spontaneously relax to a chiral ground state through phase ordering dynamics that are of great interest and crucial for applications such as stimuli-responsive and biomimetic engineering. In this work, we characterize the cholesteric phase relaxation behaviors of ß-lactoglobulin amyloid fibrils and cellulose nanocrystals confined into cylindrical capillaries, uncovering two different equilibration pathways. The integration of experimental measurements and theoretical predictions reveals the starkly distinct underlying mechanism behind the relaxation dynamics of ß-lactoglobulin amyloid fibrils, characterized by slow equilibration achieved through consecutive sigmoidal-like steps, and of cellulose nanocrystals, characterized by fast equilibration obtained through smooth relaxation dynamics. Particularly, the specific relaxation behaviors are shown to emerge from the order parameter of the unwound cholesteric medium, which depends on chirality and elasticity. The experimental findings are supported by direct numerical simulations, allowing to establish hard-to-measure viscoelastic properties without applying magnetic or electric fields.


Assuntos
Lactoglobulinas/química , Cristais Líquidos/química , Amiloide/química , Coloides/química , Elasticidade , Cinética , Nanopartículas/química , Transição de Fase , Temperatura
2.
Proc Natl Acad Sci U S A ; 117(33): 20305-20315, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32737160

RESUMO

Posttranslational modifications (PTMs) of α-synuclein (α-syn), e.g., phosphorylation, play an important role in modulating α-syn pathology in Parkinson's disease (PD) and α-synucleinopathies. Accumulation of phosphorylated α-syn fibrils in Lewy bodies and Lewy neurites is the histological hallmark of these diseases. However, it is unclear how phosphorylation relates to α-syn pathology. Here, by combining chemical synthesis and bacterial expression, we obtained homogeneous α-syn fibrils with site-specific phosphorylation at Y39, which exhibits enhanced neuronal pathology in rat primary cortical neurons. We determined the cryo-electron microscopy (cryo-EM) structure of the pY39 α-syn fibril, which reveals a fold of α-syn with pY39 in the center of the fibril core forming an electrostatic interaction network with eight charged residues in the N-terminal region of α-syn. This structure composed of residues 1 to 100 represents the largest α-syn fibril core determined so far. This work provides structural understanding on the pathology of the pY39 α-syn fibril and highlights the importance of PTMs in defining the polymorphism and pathology of amyloid fibrils in neurodegenerative diseases.


Assuntos
Doença de Parkinson , alfa-Sinucleína/química , Amiloide/química , Amiloide/metabolismo , Animais , Células Cultivadas , Microscopia Crioeletrônica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Modelos Moleculares , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação , Conformação Proteica , Ratos , Ratos Sprague-Dawley , alfa-Sinucleína/síntese química , alfa-Sinucleína/metabolismo
3.
Nat Commun ; 11(1): 4090, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796831

RESUMO

hnRNPA2 is a human ribonucleoprotein (RNP) involved in RNA metabolism. It forms fibrils both under cellular stress and in mutated form in neurodegenerative conditions. Previous work established that the C-terminal low-complexity domain (LCD) of hnRNPA2 fibrillizes under stress, and missense mutations in this domain are found in the disease multisystem proteinopathy (MSP). However, little is known at the atomic level about the hnRNPA2 LCD structure that is involved in those processes and how disease mutations cause structural change. Here we present the cryo-electron microscopy (cryoEM) structure of the hnRNPA2 LCD fibril core and demonstrate its capability to form a reversible hydrogel in vitro containing amyloid-like fibrils. Whereas these fibrils, like pathogenic amyloid, are formed from protein chains stacked into ß-sheets by backbone hydrogen bonds, they display distinct structural differences: the chains are kinked, enabling non-covalent cross-linking of fibrils and disfavoring formation of pathogenic steric zippers. Both reversibility and energetic calculations suggest these fibrils are less stable than pathogenic amyloid. Moreover, the crystal structure of the disease-mutation-containing segment (D290V) of hnRNPA2 suggests that the replacement fundamentally alters the fibril structure to a more stable energetic state. These findings illuminate how molecular interactions promote protein fibril networks and how mutation can transform fibril structure from functional to a pathogenic form.


Assuntos
Amiloide/química , Amiloide/metabolismo , Microscopia Crioeletrônica/métodos , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/ultraestrutura , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/ultraestrutura , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/química , Humanos , Hidrogéis/química , Proteínas de Ligação a RNA/química
4.
Nat Commun ; 11(1): 3781, 2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32728047

RESUMO

Nanomechanical mass spectrometry has proven to be well suited for the analysis of high mass species such as viruses. Still, the use of one-dimensional devices such as vibrating beams forces a trade-off between analysis time and mass resolution. Complex readout schemes are also required to simultaneously monitor multiple resonance modes, which degrades resolution. These issues restrict nanomechanical MS to specific species. We demonstrate here single-particle mass spectrometry with nano-optomechanical resonators fabricated with a Very Large Scale Integration process. The unique motion sensitivity of optomechanics allows designs that are impervious to particle position, stiffness or shape, opening the way to the analysis of large aspect ratio biological objects of great significance such as viruses with a tail or fibrils. Compared to top-down beam resonators with electrical read-out and state-of-the-art mass resolution, we show a three-fold improvement in capture area with no resolution degradation, despite the use of a single resonance mode.


Assuntos
Espectrometria de Massas/métodos , Nanotecnologia/métodos , Dispositivos Ópticos , Imagem Individual de Molécula/métodos , Amiloide/química , Desenho de Equipamento , Espectrometria de Massas/instrumentação , Nanopartículas/química , Nanotecnologia/instrumentação , Imagem Individual de Molécula/instrumentação , Vírus/química
5.
Proc Natl Acad Sci U S A ; 117(28): 16363-16372, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601205

RESUMO

The epididymal lumen contains a complex cystatin-rich nonpathological amyloid matrix with putative roles in sperm maturation and sperm protection. Given our growing understanding for the biological function of this and other functional amyloids, the problem still remains: how functional amyloids assemble including their initial transition to early oligomeric forms. To examine this, we developed a protocol for the purification of nondenatured mouse CRES, a component of the epididymal amyloid matrix, allowing us to examine its assembly to amyloid under conditions that may mimic those in vivo. Herein we use X-ray crystallography, solution-state NMR, and solid-state NMR to follow at the atomic level the assembly of the CRES amyloidogenic precursor as it progressed from monomeric folded protein to an advanced amyloid. We show the CRES monomer has a typical cystatin fold that assembles into highly branched amyloid matrices, comparable to those in vivo, by forming ß-sheet assemblies that our data suggest occur via two distinct mechanisms: a unique conformational switch of a highly flexible disulfide-anchored loop to a rigid ß-strand and by traditional cystatin domain swapping. Our results provide key insight into our understanding of functional amyloid assembly by revealing the earliest structural transitions from monomer to oligomer and by showing that some functional amyloid structures may be built by multiple and distinctive assembly mechanisms.


Assuntos
Amiloide/química , Proteínas Amiloidogênicas/química , Cistatinas/química , Amiloide/metabolismo , Amiloide/ultraestrutura , Proteínas Amiloidogênicas/metabolismo , Animais , Cristalografia por Raios X , Cistatinas/metabolismo , Epididimo/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Multimerização Proteica
6.
Nat Commun ; 11(1): 3281, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32612175

RESUMO

Amyloid fibrils result from the aggregation of host cell-encoded proteins, many giving rise to specific human illnesses such as Alzheimer's disease. Here we show that the major virulence factor of Rift Valley fever virus, the protein NSs, forms filamentous structures in the brain of mice and affects mortality. NSs assembles into nuclear and cytosolic disulfide bond-dependent fibrillary aggregates in infected cells. NSs structural arrangements exhibit characteristics typical for amyloids, such as an ultrastructure of 12 nm-width fibrils, a strong detergent resistance, and interactions with the amyloid-binding dye Thioflavin-S. The assembly dynamics of viral amyloid-like fibrils can be visualized in real-time. They form spontaneously and grow in an amyloid fashion within 5 hours. Together, our results demonstrate that viruses can encode amyloid-like fibril-forming proteins and have strong implications for future research on amyloid aggregation and toxicity in general.


Assuntos
Amiloide/metabolismo , Proteínas Amiloidogênicas/metabolismo , Febre do Vale de Rift/metabolismo , Vírus da Febre do Vale do Rift/metabolismo , Proteínas não Estruturais Virais/metabolismo , Amiloide/química , Amiloide/ultraestrutura , Proteínas Amiloidogênicas/química , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Núcleo Celular/virologia , Chlorocebus aethiops , Células HeLa , Humanos , Camundongos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Agregação Patológica de Proteínas/metabolismo , Febre do Vale de Rift/virologia , Vírus da Febre do Vale do Rift/patogenicidade , Células Vero , Proteínas não Estruturais Virais/química , Virulência , Fatores de Virulência
7.
Nat Commun ; 11(1): 3314, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620861

RESUMO

The amyloid conformation can be adopted by a variety of sequences, but the precise boundaries of amyloid sequence space are still unclear. The currently charted amyloid sequence space is strongly biased towards hydrophobic, beta-sheet prone sequences that form the core of globular proteins and by Q/N/Y rich yeast prions. Here, we took advantage of the increasing amount of high-resolution structural information on amyloid cores currently available in the protein databank to implement a machine learning approach, named Cordax (https://cordax.switchlab.org), that explores amyloid sequence beyond its current boundaries. Clustering by t-Distributed Stochastic Neighbour Embedding (t-SNE) shows how our approach resulted in an expansion away from hydrophobic amyloid sequences towards clusters of lower aliphatic content and higher charge, or regions of helical and disordered propensities. These clusters uncouple amyloid propensity from solubility representing sequence flavours compatible with surface-exposed patches in globular proteins, functional amyloids or sequences associated to liquid-liquid phase transitions.


Assuntos
Algoritmos , Amiloide/química , Proteínas Amiloidogênicas/química , Modelos Químicos , Peptídeos/química , Amiloide/metabolismo , Proteínas Amiloidogênicas/metabolismo , Amiloidose/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Aprendizado de Máquina , Peptídeos/metabolismo , Conformação Proteica , Engenharia de Proteínas/métodos , Solubilidade
8.
Proc Natl Acad Sci U S A ; 117(22): 12087-12094, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32414930

RESUMO

The spontaneous assembly of proteins into amyloid fibrils is a phenomenon central to many increasingly common and currently incurable human disorders, including Alzheimer's and Parkinson's diseases. Oligomeric species form transiently during this process and not only act as essential intermediates in the assembly of new filaments but also represent major pathogenic agents in these diseases. While amyloid fibrils possess a common, defining set of physicochemical features, oligomers, by contrast, appear much more diverse, and their commonalities and differences have hitherto remained largely unexplored. Here, we use the framework of chemical kinetics to investigate their dynamical properties. By fitting experimental data for several unrelated amyloidogenic systems to newly derived mechanistic models, we find that oligomers present with a remarkably wide range of kinetic and thermodynamic stabilities but that they possess two properties that are generic: they are overwhelmingly nonfibrillar, and they predominantly dissociate back to monomers rather than maturing into fibrillar species. These discoveries change our understanding of the relationship between amyloid oligomers and amyloid fibrils and have important implications for the nature of their cellular toxicity.


Assuntos
Amiloide/química , Proteínas Amiloidogênicas/química , Cinética , Doença de Alzheimer , Peptídeos beta-Amiloides/química , Amiloidose , Modelos Teóricos , Agregados Proteicos , Termodinâmica
9.
PLoS Comput Biol ; 16(5): e1007767, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32365068

RESUMO

Many proteins have the potential to aggregate into amyloid fibrils, protein polymers associated with a wide range of human disorders such as Alzheimer's and Parkinson's disease. The thermodynamic stability of amyloid fibrils, in contrast to that of folded proteins, is not well understood: the balance between entropic and enthalpic terms, including the chain entropy and the hydrophobic effect, are poorly characterised. Using a combination of theory, in vitro experiments, simulations of a coarse-grained protein model and meta-data analysis, we delineate the enthalpic and entropic contributions that dominate amyloid fibril elongation. Our prediction of a characteristic temperature-dependent enthalpic signature is confirmed by the performed calorimetric experiments and a meta-analysis over published data. From these results we are able to define the necessary conditions to observe cold denaturation of amyloid fibrils. Overall, we show that amyloid fibril elongation is associated with a negative heat capacity, the magnitude of which correlates closely with the hydrophobic surface area that is buried upon fibril formation, highlighting the importance of hydrophobicity for fibril stability.


Assuntos
Amiloide/química , Amiloide/fisiologia , Amiloide/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/fisiologia , Proteínas Amiloidogênicas/química , Proteínas Amiloidogênicas/fisiologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Teóricos , Simulação de Dinâmica Molecular , Desnaturação Proteica , Dobramento de Proteína , Temperatura , Termodinâmica
10.
Nat Commun ; 11(1): 2643, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32457390

RESUMO

Amyloid aggregation of α-synuclein (α-syn) is closely associated with Parkinson's disease (PD) and other synucleinopathies. Several single amino-acid mutations (e.g. E46K) of α-syn have been identified causative to the early onset of familial PD. Here, we report the cryo-EM structure of an α-syn fibril formed by N-terminally acetylated E46K mutant α-syn (Ac-E46K). The fibril structure represents a distinct fold of α-syn, which demonstrates that the E46K mutation breaks the electrostatic interactions in the wild type (WT) α-syn fibril and thus triggers the rearrangement of the overall structure. Furthermore, we show that the Ac-E46K fibril is less resistant to harsh conditions and protease cleavage, and more prone to be fragmented with an enhanced seeding capability than that of the WT fibril. Our work provides a structural view to the severe pathology of the PD familial mutation E46K of α-syn and highlights the importance of electrostatic interactions in defining the fibril polymorphs.


Assuntos
Proteínas Mutantes/química , Proteínas Mutantes/genética , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/genética , Acetilação , Sequência de Aminoácidos , Substituição de Aminoácidos , Amiloide/química , Amiloide/genética , Amiloide/ultraestrutura , Microscopia Crioeletrônica , Humanos , Microscopia de Força Atômica , Modelos Moleculares , Proteínas Mutantes/ultraestrutura , Mutação de Sentido Incorreto , Conformação Proteica , Estabilidade Proteica , Eletricidade Estática , alfa-Sinucleína/ultraestrutura
11.
PLoS Comput Biol ; 16(5): e1007647, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32453794

RESUMO

The use of yeast systems to study the propagation of prions and amyloids has emerged as a crucial aspect of the global endeavor to understand those mechanisms. Yeast prion systems are intrinsically multi-scale: the molecular chemical processes are indeed coupled to the cellular processes of cell growth and division to influence phenotypical traits, observable at the scale of colonies. We introduce a novel modeling framework to tackle this difficulty using impulsive differential equations. We apply this approach to the [PSI+] yeast prion, which is associated with the misconformation and aggregation of Sup35. We build a model that reproduces and unifies previously conflicting experimental observations on [PSI+] and thus sheds light onto characteristics of the intracellular molecular processes driving aggregate replication. In particular our model uncovers a kinetic barrier for aggregate replication at low densities, meaning the change between prion or prion-free phenotype is a bi-stable transition. This result is based on the study of prion curing experiments, as well as the phenomenon of colony sectoring, a phenotype which is often ignored in experimental assays and has never been modeled. Furthermore, our results provide further insight into the effect of guanidine hydrochloride (GdnHCl) on Sup35 aggregates. To qualitatively reproduce the GdnHCl curing experiment, aggregate replication must not be completely inhibited, which suggests the existence of a mechanism different than Hsp104-mediated fragmentation. Those results are promising for further development of the [PSI+] model, but also for extending the use of this novel framework to other yeast prion or amyloid systems.


Assuntos
Proteínas Priônicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Amiloide/química , Simulação por Computador , Guanidina/farmacologia , Proteínas de Choque Térmico/metabolismo , Cinética , Modelos Biológicos , Modelos Teóricos , Fatores de Terminação de Peptídeos/metabolismo , Fenótipo , Proteínas de Saccharomyces cerevisiae/metabolismo
12.
Nat Struct Mol Biol ; 27(5): 417-423, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32284600

RESUMO

Self-templating assemblies of the human prion protein are clinically associated with transmissible spongiform encephalopathies. Here we present the cryo-EM structure of a denaturant- and protease-resistant fibril formed in vitro spontaneously by a 9.7-kDa unglycosylated fragment of the human prion protein. This human prion fibril contains two protofilaments intertwined with screw symmetry and linked by a tightly packed hydrophobic interface. Each protofilament consists of an extended beta arch formed by residues 106 to 145 of the prion protein, a hydrophobic and highly fibrillogenic disease-associated segment. Such structures of prion polymorphs serve as blueprints on which to evaluate the potential impact of sequence variants on prion disease.


Assuntos
Príons/química , Príons/metabolismo , Amiloide/química , Animais , Microscopia Crioeletrônica , Cristalografia por Raios X , Humanos , Interações Hidrofóbicas e Hidrofílicas , Mamíferos , Modelos Moleculares , Fragmentos de Peptídeos/química , Peptídeo Hidrolases/metabolismo , Doenças Priônicas/etiologia , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
13.
Phys Rev Lett ; 124(11): 118102, 2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-32242730

RESUMO

The fine interplay between the simultaneous stretching and confinement of amyloid fibrils is probed by combining a microcapillary setup with atomic force microscopy. Single-molecule statistics reveal how the stretching of fibrils changed from force to confinement dominated at different length scales. System order, however, is solely ruled by confinement. Coarse-grained simulations support the results and display the potential to tailor system properties by tuning the two effects. These findings may further help shed light on in vivo amyloid fibril growth and transport in highly confined environments such as blood vessels.


Assuntos
Amiloide/química , Modelos Químicos , Amiloide/metabolismo , Simulação por Computador , Microscopia de Força Atômica/métodos
14.
Proc Natl Acad Sci U S A ; 117(19): 10322-10328, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32345723

RESUMO

Atomistic description of protein fibril formation has been elusive due to the complexity and long time scales of the conformational search. Here, we develop a multiscale approach combining numerous atomistic simulations in explicit solvent to construct Markov State Models (MSMs) of fibril growth. The search for the in-register fully bound fibril state is modeled as a random walk on a rugged two-dimensional energy landscape defined by ß-sheet alignment and hydrogen-bonding states, whereas transitions involving states without hydrogen bonds are derived from kinetic clustering. The reversible association/dissociation of an incoming peptide and overall growth kinetics are then computed from MSM simulations. This approach is applied to derive a parameter-free, comprehensive description of fibril elongation of Aß16-22 and how it is modulated by phenylalanine-to-cyclohexylalanine (CHA) mutations. The trajectories show an aggregation mechanism in which the peptide spends most of its time trapped in misregistered ß-sheet states connected by weakly bound states twith short lifetimes. Our results recapitulate the experimental observation that mutants CHA19 and CHA1920 accelerate fibril elongation but have a relatively minor effect on the critical concentration for fibril growth. Importantly, the kinetic consequences of mutations arise from cumulative effects of perturbing the network of productive and nonproductive pathways of fibril growth. This is consistent with the expectation that nonfunctional states will not have evolved efficient folding pathways and, therefore, will require a random search of configuration space. This study highlights the importance of describing the complete energy landscape when studying the elongation mechanism and kinetics of protein fibrils.


Assuntos
Peptídeos beta-Amiloides/química , Amiloide/química , Mutação , Fragmentos de Peptídeos/química , Fenilalanina/análogos & derivados , Fenilalanina/genética , Peptídeos beta-Amiloides/genética , Simulação por Computador , Humanos , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Fragmentos de Peptídeos/genética , Estrutura Secundária de Proteína , Termodinâmica
15.
Science ; 367(6483): 1230-1234, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32165583

RESUMO

How long-lived memories withstand molecular turnover is a fundamental question. Aggregates of a prion-like RNA-binding protein, cytoplasmic polyadenylation element-binding (CPEB) protein, is a putative substrate of long-lasting memories. We isolated aggregated Drosophila CPEB, Orb2, from adult heads and determined its activity and atomic structure, at 2.6-angstrom resolution, using cryo-electron microscopy. Orb2 formed ~75-nanometer-long threefold-symmetric amyloid filaments. Filament formation transformed Orb2 from a translation repressor to an activator and "seed" for further translationally active aggregation. The 31-amino acid protofilament core adopted a cross-ß unit with a single hydrophilic hairpin stabilized through interdigitated glutamine packing. Unlike the hydrophobic core of pathogenic amyloids, the hydrophilic core of Orb2 filaments suggests how some neuronal amyloids could be a stable yet regulatable substrate of memory.


Assuntos
Amiloide/química , Proteínas de Drosophila/química , Memória de Longo Prazo , Neurônios/metabolismo , Agregados Proteicos , Proteínas de Ligação a RNA/química , Fatores de Transcrição/química , Fatores de Poliadenilação e Clivagem de mRNA/química , Animais , Microscopia Crioeletrônica , Drosophila melanogaster , Glutamina/química , Interações Hidrofóbicas e Hidrofílicas , Conformação Proteica
16.
Subcell Biochem ; 94: 421-436, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32189310

RESUMO

As normal constituents of blood serum, the Serum Amyloid A (SAA) proteins are small (104 amino acids in humans) and remarkably well-conserved in mammalian evolution. They are synthesized prominently, but not exclusively, in the liver. Fragments of SAA can associate into insoluble fibrils (called "amyloid") characteristic of "secondary" amyloid disease in which they can interrupt normal physiology and lead to organ failure. SAA proteins comprise a family of molecules, two members of which (SAA1 and SAA2) are (along with C-reactive protein, CRP) the most prominent members of the acute phase response (APR) during which their serum levels rise dramatically after trauma, infection and other stimuli. Biologic function (s) of SAA are unresolved but features are consistent with a prominent role in primordial host defense (including the APR ). SAA proteins are lipophilic and contribute to high density lipoproteins (HDL) and cholesterol transport. SAA proteins interact with specific receptors and have been implicated in tissue remodeling through metalloproteinases, local tissue changes in atherosclerosis, cancer metastasis, lung inflammation, maternal-fetal health and intestinal physiology. Molecular details of some of these are emerging.


Assuntos
Proteína Amiloide A Sérica , Reação de Fase Aguda , Amiloide/química , Amiloide/metabolismo , Animais , Colesterol/metabolismo , Doença , Humanos , Lipoproteínas HDL/metabolismo , Fígado/metabolismo , Proteína Amiloide A Sérica/química , Proteína Amiloide A Sérica/metabolismo
17.
Nat Chem Biol ; 16(6): 653-659, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32152544

RESUMO

Defining the biologically active structures of proteins in their cellular environments remains challenging for proteins with multiple conformations and functions, where only a minor conformer might be associated with a given function. Here, we use deep mutational scanning to probe the structure and dynamics of α-synuclein, a protein known to adopt disordered, helical and amyloid conformations. We examined the effects of 2,600 single-residue substitutions on the ability of intracellularly expressed α-synuclein to slow the growth of yeast. Computational analysis of the data showed that the conformation responsible for this phenotype is a long, uninterrupted, amphiphilic helix with increasing dynamics toward the C terminus. Deep mutational scanning can therefore determine biologically active conformations in cellular environments, even for a highly dynamic multi-conformational protein.


Assuntos
Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutação , alfa-Sinucleína/química , alfa-Sinucleína/genética , Sequência de Aminoácidos , Amiloide/química , Biblioteca Genômica , Modelos Moleculares , Fenótipo , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Leveduras/metabolismo
18.
J Mol Biol ; 432(7): 1910-1925, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32169484

RESUMO

Liquid-liquid phase separation (LLPS) of proteins underlies the formation of membrane-less organelles. While it has been recognized for some time that these organelles are of key importance for normal cellular functions, a growing number of recent observations indicate that LLPS may also play a role in disease. In particular, numerous proteins that form toxic aggregates in neurodegenerative diseases, such as amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and Alzheimer's disease, were found to be highly prone to phase separation, suggesting that there might be a strong link between LLPS and the pathogenic process in these disorders. This review aims to assess the molecular basis of this link through exploration of the intermolecular interactions that underlie LLPS and aggregation and the underlying mechanisms facilitating maturation of liquid droplets into more stable assemblies, including so-called labile fibrils, hydrogels, and pathological amyloids. Recent insights into the structural basis of labile fibrils and potential mechanisms by which these relatively unstable structures could transition into more stable pathogenic amyloids are also discussed. Finally, this review explores how the environment of liquid droplets could modulate protein aggregation by altering kinetics of protein self-association, affecting folding of protein monomers, or changing aggregation pathways.


Assuntos
Amiloide/química , Transição de Fase , Agregação Patológica de Proteínas , Dobramento de Proteína , Amiloide/metabolismo , Animais , Humanos , Ligação Proteica , Domínios Proteicos
19.
Proc Natl Acad Sci U S A ; 117(12): 6866-6874, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32161130

RESUMO

Increasing evidence suggests that amyloid polymorphism gives rise to different strains of amyloids with distinct toxicities and pathology-spreading properties. Validating this hypothesis is challenging due to a lack of tools and methods that allow for the direct characterization of amyloid polymorphism in hydrated and complex biological samples. Here, we report on the development of 11-mercapto-1-undecanesulfonate-coated gold nanoparticles (NPs) that efficiently label the edges of synthetic, recombinant, and native amyloid fibrils derived from different amyloidogenic proteins. We demonstrate that these NPs represent powerful tools for assessing amyloid morphological polymorphism, using cryogenic transmission electron microscopy (cryo-EM). The NPs allowed for the visualization of morphological features that are not directly observed using standard imaging techniques, including transmission electron microscopy with use of the negative stain or cryo-EM imaging. The use of these NPs to label native paired helical filaments (PHFs) from the postmortem brain of a patient with Alzheimer's disease, as well as amyloid fibrils extracted from the heart tissue of a patient suffering from systemic amyloid light-chain amyloidosis, revealed a high degree of homogeneity across the fibrils derived from human tissue in comparison with fibrils aggregated in vitro. These findings are consistent with, and strongly support, the emerging view that the physiologic milieu is a key determinant of amyloid fibril strains. Together, these advances should not only facilitate the profiling and characterization of amyloids for structural studies by cryo-EM, but also pave the way to elucidate the structural basis of amyloid strains and toxicity, and possibly the correlation between the pathological and clinical heterogeneity of amyloid diseases.


Assuntos
Amiloide/genética , Amiloide/metabolismo , Encéfalo/metabolismo , Microscopia Crioeletrônica/métodos , Ouro/química , Nanopartículas Metálicas/química , Polimorfismo Genético , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Amiloide/química , Humanos , Amiloidose de Cadeia Leve de Imunoglobulina/genética , Amiloidose de Cadeia Leve de Imunoglobulina/metabolismo , Amiloidose de Cadeia Leve de Imunoglobulina/patologia , Emaranhados Neurofibrilares
20.
Chem Commun (Camb) ; 56(21): 3147-3150, 2020 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-32057047

RESUMO

Amyloid fibrils, implicated in health and diseases, commonly exhibit a periodic twist trait relevant to the structures and dynamics of the fibrils. However, the origins and modulations of fibril twist in complex in vivo environments are not yet fully understood. Here we highlight an important factor that causes twist variations in amyloid fibril structures-the presence of surrounding surfaces. Using cholesterol-containing lipid bilayers with varying cholesterol contents, we have demonstrated via atomic force microscopy that amyloid-ß peptide fibrils initiated on membranes increase their average pitch size of twisting periodicity as the cholesterol content increases. These surface-induced twist variations arise from the enhanced hydrophobic interactions between the fibril and the surface distorting the torsional elastic energy of the fibril twisting as supported by a theory of an elastic model. These findings not only provide an important insight into fibril polymorphism phenomena resulting from the surface effects but also suggest a novel solution to modulate filament twisting on the nanoscale for biomaterials applications involving nanoscale features.


Assuntos
Amiloide/química , Colesterol/química , Bicamadas Lipídicas/química , Microscopia de Força Atômica , Tamanho da Partícula , Conformação Proteica , Propriedades de Superfície
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