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1.
Int J Mol Sci ; 22(19)2021 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-34639037

RESUMO

Amyloids are self-assembled protein aggregates that take cross-ß fibrillar morphology. Although some amyloid proteins are best known for their association with Alzheimer's and Parkinson's disease, many other amyloids are found across diverse organisms, from bacteria to humans, and they play vital functional roles. The rigidity, chemical stability, high aspect ratio, and sequence programmability of amyloid fibrils have made them attractive candidates for functional materials with applications in environmental sciences, material engineering, and translational medicines. This review focuses on recent advances in fabricating various types of macroscopic functional amyloid materials. We discuss different design strategies for the fabrication of amyloid hydrogels, high-strength materials, composite materials, responsive materials, extracellular matrix mimics, conductive materials, and catalytic materials.


Assuntos
Amiloide/química , Amiloide/metabolismo , Proteínas Amiloidogênicas/metabolismo , Aminoácidos/química , Amiloide/ultraestrutura , Proteínas Amiloidogênicas/química , Amiloidose/etiologia , Amiloidose/metabolismo , Amiloidose/patologia , Matriz Extracelular/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformação Proteica , Relação Estrutura-Atividade
3.
Sci Rep ; 11(1): 13785, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215782

RESUMO

The light chain (AL) amyloidosis is caused by the aggregation of light chain of antibodies into amyloid fibrils. There are plenty of computational resources available for the prediction of short aggregation-prone regions within proteins. However, it is still a challenging task to predict the amyloidogenic nature of the whole protein using sequence/structure information. In the case of antibody light chains, common architecture and known binding sites can provide vital information for the prediction of amyloidogenicity at physiological conditions. Here, in this work, we have compared classical sequence-based, aggregation-related features (such as hydrophobicity, presence of gatekeeper residues, disorderness, ß-propensity, etc.) calculated for the CDR, FR or VL regions of amyloidogenic and non-amyloidogenic antibody light chains and implemented the insights gained in a machine learning-based webserver called "VLAmY-Pred" ( https://web.iitm.ac.in/bioinfo2/vlamy-pred/ ). The model shows prediction accuracy of 79.7% (sensitivity: 78.7% and specificity: 79.9%) with a ROC value of 0.88 on a dataset of 1828 variable region sequences of the antibody light chains. This model will be helpful towards improved prognosis for patients that may likely suffer from diseases caused by light chain amyloidosis, understanding origins of aggregation in antibody-based biotherapeutics, large-scale in-silico analysis of antibody sequences generated by next generation sequencing, and finally towards rational engineering of aggregation resistant antibodies.


Assuntos
Amiloide/genética , Cadeias Leves de Imunoglobulina/genética , Amiloidose de Cadeia Leve de Imunoglobulina/genética , Agregação Patológica de Proteínas/genética , Sequência de Aminoácidos/genética , Amiloide/química , Amiloide/imunologia , Amiloide/ultraestrutura , Biologia Computacional , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/imunologia , Cadeias Leves de Imunoglobulina/ultraestrutura , Amiloidose de Cadeia Leve de Imunoglobulina/imunologia , Amiloidose de Cadeia Leve de Imunoglobulina/patologia , Modelos Moleculares , Agregação Patológica de Proteínas/patologia , Conformação Proteica
4.
Int J Mol Sci ; 22(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199513

RESUMO

Intrinsic disorder is a natural feature of polypeptide chains, resulting in the lack of a defined three-dimensional structure. Conformational changes in intrinsically disordered regions of a protein lead to unstable ß-sheet enriched intermediates, which are stabilized by intermolecular interactions with other ß-sheet enriched molecules, producing stable proteinaceous aggregates. Upon misfolding, several pathways may be undertaken depending on the composition of the amino acidic string and the surrounding environment, leading to different structures. Accumulating evidence is suggesting that the conformational state of a protein may initiate signalling pathways involved both in pathology and physiology. In this review, we will summarize the heterogeneity of structures that are produced from intrinsically disordered protein domains and highlight the routes that lead to the formation of physiological liquid droplets as well as pathogenic aggregates. The most common proteins found in aggregates in neurodegenerative diseases and their structural variability will be addressed. We will further evaluate the clinical relevance and future applications of the study of the structural heterogeneity of protein aggregates, which may aid the understanding of the phenotypic diversity observed in neurodegenerative disorders.


Assuntos
Doenças Neurodegenerativas/genética , Agregados Proteicos/genética , Agregação Patológica de Proteínas/genética , Conformação Proteica em Folha beta , Amiloide/genética , Amiloide/ultraestrutura , Humanos , Proteínas Intrinsicamente Desordenadas , Doenças Neurodegenerativas/patologia , alfa-Sinucleína/genética , alfa-Sinucleína/ultraestrutura , Proteínas tau/genética , Proteínas tau/ultraestrutura
5.
J Mol Biol ; 433(20): 167124, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34224749

RESUMO

The prediction of highly ordered three-dimensional structures of amyloid protein fibrils from the amino acid sequences of their monomeric self-assembly precursors constitutes a challenging and unresolved aspect of the classical protein folding problem. Because of the polymorphic nature of amyloid assembly whereby polypeptide chains of identical amino acid sequences under identical conditions are capable of self-assembly into a spectrum of different fibril structures, the prediction of amyloid structures from an amino acid sequence requires a detailed and holistic understanding of its assembly free energy landscape. The full extent of the structure space accessible to the cross-ß molecular architecture of amyloid must also be resolved. Here, we review the current understanding of the diversity and the individuality of amyloid structures, and how the polymorphic landscape of amyloid links to biology and disease phenotypes. We present a comprehensive review of structural models of amyloid fibrils derived by cryo-EM, ssNMR and AFM to date, and discuss the challenges ahead for resolving the structural basis and the biological consequences of polymorphic amyloid assemblies.


Assuntos
Amiloide/química , Amiloide/metabolismo , Amiloide/ultraestrutura , Amiloidose/metabolismo , Animais , Humanos , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína
6.
Nat Commun ; 12(1): 4231, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244499

RESUMO

Pathological aggregation of the protein tau into insoluble aggregates is a hallmark of neurodegenerative diseases. The emergence of disease-specific tau aggregate structures termed tau strains, however, remains elusive. Here we show that full-length tau protein can be aggregated in the absence of co-factors into seeding-competent amyloid fibrils that sequester RNA. Using a combination of solid-state NMR spectroscopy and biochemical experiments we demonstrate that the co-factor-free amyloid fibrils of tau have a rigid core that is similar in size and location to the rigid core of tau fibrils purified from the brain of patients with corticobasal degeneration. In addition, we demonstrate that the N-terminal 30 residues of tau are immobilized during fibril formation, in agreement with the presence of an N-terminal epitope that is specifically detected by antibodies in pathological tau. Experiments in vitro and in biosensor cells further established that co-factor-free tau fibrils efficiently seed tau aggregation, while binding studies with different RNAs show that the co-factor-free tau fibrils strongly sequester RNA. Taken together the study provides a critical advance to reveal the molecular factors that guide aggregation towards disease-specific tau strains.


Assuntos
Amiloide/metabolismo , Agregação Patológica de Proteínas/patologia , RNA/metabolismo , Proteínas tau/metabolismo , Amiloide/ultraestrutura , Técnicas Biossensoriais , Humanos , Ressonância Magnética Nuclear Biomolecular , RNA/ultraestrutura , RNA Fúngico/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Proteínas tau/isolamento & purificação , Proteínas tau/ultraestrutura
7.
J Biol Chem ; 297(1): 100858, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34097874

RESUMO

Protein aggregation in the outermost layers of the cornea, which can lead to cloudy vision and in severe cases blindness, is linked to mutations in the extracellular matrix protein transforming growth factor-ß-induced protein (TGFBIp). Among the most frequent pathogenic mutations are R124H and R555W, both associated with granular corneal dystrophy (GCD) characterized by the early-onset formation of amorphous aggregates. The molecular mechanisms of protein aggregation in GCD are largely unknown. In this study, we determined the crystal structures of R124H, R555W, and the lattice corneal dystrophy-associated A546T. Although there were no changes in the monomeric TGFBIp structure of any mutant that would explain their propensity to aggregate, R124H and R555W demonstrated a new dimer interface in the crystal packing, which is not present in wildtype TGFBIp or A546T. This interface, as seen in both the R124H and R555W structures, involves residue 124 of the first TGFBIp molecule and 555 in the second. The interface is not permitted by the Arg124 and Arg555 residues of wildtype TGFBIp and may play a central role in the aggregation exhibited by R124H and R555W in vivo. Using cross-linking mass spectrometry and in-line size exclusion chromatography-small-angle X-ray scattering, we characterized a dimer formed by wildtype and mutant TGFBIps in solution. Dimerization in solution also involves interactions between the N- and C-terminal domains of two TGFBIp molecules but was not identical to the crystal packing dimerization. TGFBIp-targeted interventions that disrupt the R124H/R555W crystal packing dimer interface might offer new therapeutic opportunities to treat patients with GCD.


Assuntos
Córnea/ultraestrutura , Distrofias Hereditárias da Córnea/genética , Proteínas da Matriz Extracelular/genética , Agregados Proteicos/genética , Fator de Crescimento Transformador beta/genética , Amiloide/genética , Amiloide/ultraestrutura , Córnea/metabolismo , Distrofias Hereditárias da Córnea/patologia , Cristalografia por Raios X , Proteínas da Matriz Extracelular/ultraestrutura , Humanos , Mutação de Sentido Incorreto/genética , Multimerização Proteica/genética
8.
J Biol Chem ; 297(1): 100860, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34102212

RESUMO

Formation of biomolecular condensates through liquid-liquid phase separation (LLPS) has been described for several pathogenic proteins linked to neurodegenerative diseases and is discussed as an early step in the formation of protein aggregates with neurotoxic properties. In prion diseases, neurodegeneration and formation of infectious prions is caused by aberrant folding of the cellular prion protein (PrPC). PrPC is characterized by a large intrinsically disordered N-terminal domain and a structured C-terminal globular domain. A significant fraction of mature PrPC is proteolytically processed in vivo into an entirely unstructured fragment, designated N1, and the corresponding C-terminal fragment C1 harboring the globular domain. Notably, N1 contains a polybasic motif that serves as a binding site for neurotoxic Aß oligomers. PrP can undergo LLPS; however, nothing is known how phase separation of PrP is triggered on a molecular scale. Here, we show that the intrinsically disordered N1 domain is necessary and sufficient for LLPS of PrP. Similar to full-length PrP, the N1 fragment formed highly dynamic liquid-like droplets. Remarkably, a slightly shorter unstructured fragment, designated N2, which lacks the Aß-binding domain and is generated under stress conditions, failed to form liquid-like droplets and instead formed amorphous assemblies of irregular structures. Through a mutational analysis, we identified three positively charged lysines in the postoctarepeat region as essential drivers of condensate formation, presumably largely via cation-π interactions. These findings provide insights into the molecular basis of LLPS of the mammalian prion protein and reveal a crucial role of the Aß-binding domain in this process.


Assuntos
Doenças Neurodegenerativas/genética , Doenças Priônicas/genética , Proteínas Priônicas/genética , Príons/genética , Amiloide/genética , Amiloide/ultraestrutura , Animais , Fenômenos Biofísicos , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/ultraestrutura , Extração Líquido-Líquido , Doenças Neurodegenerativas/patologia , Doenças Priônicas/patologia , Proteínas Priônicas/ultraestrutura , Domínios Proteicos/genética , Dobramento de Proteína
9.
J Biol Chem ; 297(1): 100804, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34044018

RESUMO

The functional amyloid Orb2 belongs to the cytoplasmic polyadenylation element binding (CPEB) protein family and plays an important role in long-term memory formation in Drosophila. The Orb2 domain structure combines RNA recognition motifs with low-complexity sequences similar to many RNA-binding proteins shown to form protein droplets via liquid-liquid phase separation (LLPS) in vivo and in vitro. This similarity suggests that Orb2 might also undergo LLPS. However, cellular Orb2 puncta have very little internal protein mobility, and Orb2 forms fibrils in Drosophila brains that are functionally active indicating that LLPS might not play a role for Orb2. In the present work, we reconcile these two views on Orb2 droplet formation. Using fluorescence microscopy, we show that soluble Orb2 can indeed phase separate into protein droplets. However, fluorescence recovery after photobleaching (FRAP) data shows that these droplets have either no or only an extremely short-lived liquid phase and appear maturated right after formation. Orb2 fragments that lack the C-terminal RNA-binding domain (RBD) form fibrils out of these droplets. Solid-state NMR shows that these fibrils have well-ordered static domains in addition to the Gln/His-rich fibril core. Further, we find that full-length Orb2B, which is by far the major component of Orb2 fibrils in vivo, does not transition into fibrils but remains in the droplet phase. Together, our data suggest that phase separation might play a role in initiating the formation of functional Orb2 fibrils.


Assuntos
Amiloide/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Sequência de Aminoácidos , Amiloide/ultraestrutura , Animais , Benzotiazóis/metabolismo , Isótopos de Carbono , Proteínas de Drosophila/química , Drosophila melanogaster/ultraestrutura , Fluorescência , Concentração Osmolar , Domínios Proteicos , Fatores de Transcrição/química , Fatores de Poliadenilação e Clivagem de mRNA/química
10.
Int J Mol Sci ; 22(6)2021 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-33809196

RESUMO

Deposition of amyloid ß (Aß) fibrils in the brain is a key pathologic hallmark of Alzheimer's disease. A class of polyphenolic biflavonoids is known to have anti-amyloidogenic effects by inhibiting aggregation of Aß and promoting disaggregation of Aß fibrils. In the present study, we further sought to investigate the structural basis of the Aß disaggregating activity of biflavonoids and their interactions at the atomic level. A thioflavin T (ThT) fluorescence assay revealed that amentoflavone-type biflavonoids promote disaggregation of Aß fibrils with varying potency due to specific structural differences. The computational analysis herein provides the first atomistic details for the mechanism of Aß disaggregation by biflavonoids. Molecular docking analysis showed that biflavonoids preferentially bind to the aromatic-rich, partially ordered N-termini of Aß fibril via the π-π interactions. Moreover, docking scores correlate well with the ThT EC50 values. Molecular dynamic simulations revealed that biflavonoids decrease the content of ß-sheet in Aß fibril in a structure-dependent manner. Hydrogen bond analysis further supported that the substitution of hydroxyl groups capable of hydrogen bond formation at two positions on the biflavonoid scaffold leads to significantly disaggregation of Aß fibrils. Taken together, our data indicate that biflavonoids promote disaggregation of Aß fibrils due to their ability to disrupt the fibril structure, suggesting biflavonoids as a lead class of compounds to develop a therapeutic agent for Alzheimer's disease.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Benzotiazóis/farmacologia , Biflavonoides/farmacologia , Agregação Patológica de Proteínas/tratamento farmacológico , Doença de Alzheimer/patologia , Amiloide/antagonistas & inibidores , Amiloide/efeitos dos fármacos , Amiloide/ultraestrutura , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/ultraestrutura , Biflavonoides/química , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Humanos , Ligação de Hidrogênio/efeitos dos fármacos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/patologia
11.
PLoS Genet ; 17(3): e1009407, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33657088

RESUMO

Parkinson's disease is a neurodegenerative disorder associated with misfolding and aggregation of α-synuclein as a hallmark protein. Two yeast strain collections comprising conditional alleles of essential genes were screened for the ability of each allele to reduce or improve yeast growth upon α-synuclein expression. The resulting 98 novel modulators of α-synuclein toxicity clustered in several major categories including transcription, rRNA processing and ribosome biogenesis, RNA metabolism and protein degradation. Furthermore, expression of α-synuclein caused alterations in pre-rRNA transcript levels in yeast and in human cells. We identified the nucleolar DEAD-box helicase Dbp4 as a prominent modulator of α-synuclein toxicity. Downregulation of DBP4 rescued cells from α-synuclein toxicity, whereas overexpression led to a synthetic lethal phenotype. We discovered that α-synuclein interacts with Dbp4 or its human ortholog DDX10, sequesters the protein outside the nucleolus in yeast and in human cells, and stabilizes a fraction of α-synuclein oligomeric species. These findings provide a novel link between nucleolar processes and α-synuclein mediated toxicity with DDX10 emerging as a promising drug target.


Assuntos
RNA Helicases DEAD-box/metabolismo , Agregados Proteicos , Agregação Patológica de Proteínas/metabolismo , Multimerização Proteica , alfa-Sinucleína/metabolismo , Amiloide/metabolismo , Amiloide/ultraestrutura , Regulação da Expressão Gênica , Humanos , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Modelos Biológicos , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Ligação Proteica , Transporte Proteico , Leveduras/genética , Leveduras/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/genética
12.
Nat Commun ; 12(1): 1627, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33712586

RESUMO

RIPK3 amyloid complex plays crucial roles during TNF-induced necroptosis and in response to immune defense in both human and mouse. Here, we have structurally characterized mouse RIPK3 homogeneous self-assembly using solid-state NMR, revealing a well-ordered N-shaped amyloid core structure featured with 3 parallel in-register ß-sheets. This structure differs from previously published human RIPK1/RIPK3 hetero-amyloid complex structure, which adopted a serpentine fold. Functional studies indicate both RIPK1-RIPK3 binding and RIPK3 amyloid formation are essential but not sufficient for TNF-induced necroptosis. The structural integrity of RIPK3 fibril with three ß-strands is necessary for signaling. Molecular dynamics simulations with a mouse RIPK1/RIPK3 model indicate that the hetero-amyloid is less stable when adopting the RIPK3 fibril conformation, suggesting a structural transformation of RIPK3 from RIPK1-RIPK3 binding to RIPK3 amyloid formation. This structural transformation would provide the missing link connecting RIPK1-RIPK3 binding to RIPK3 homo-oligomer formation in the signal transduction.


Assuntos
Amiloide/metabolismo , Amiloide/ultraestrutura , Necroptose/fisiologia , Proteína Serina-Treonina Quinases de Interação com Receptores/química , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Benzotiazóis , Sobrevivência Celular , Drosophila , Herpesviridae , Humanos , Camundongos , Simulação de Dinâmica Molecular , Necroptose/genética , Conformação Proteica , Ratos , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Alinhamento de Sequência , Análise de Sequência de Proteína , Transdução de Sinais
13.
Molecules ; 26(4)2021 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-33673072

RESUMO

Oligomeric intermediates on the pathway of amyloid fibrillation are suspected as the main cytotoxins responsible for amyloid-related pathogenicity. As they appear to be a part of the lag phase of amyloid fibrillation when analyzed using standard methods such as Thioflavin T (ThT) fluorescence, a more sensitive method is needed for their detection. Here we apply Fourier transform infrared spectroscopy (FTIR) in attenuated total reflectance (ATR) mode for fast and cheap analysis of destabilized hen-egg-white lysozyme solution and detection of oligomer intermediates of amyloid fibrillation. Standard methods of protein aggregation analysis- Thioflavin T (ThT) fluorescence, atomic force microscopy (AFM), and 8-anilinonaphthalene-1-sulphonic acid (ANS) fluorescence were applied and compared to FTIR spectroscopy data. Results show the great potential of FTIR for both, qualitative and quantitative monitoring of oligomer formation based on the secondary structure changes. While oligomer intermediates do not induce significant changes in ThT fluorescence, their secondary structure changes were very prominent. Normalization of specific Amide I region peak intensities by using Amide II peak intensity as an internal standard provides an opportunity to use FTIR spectroscopy for both qualitative and quantitative analysis of biological samples and detection of potentially toxic oligomers, as well as for screening of efficiency of fibrillation procedures.


Assuntos
Amiloide/química , Proteínas Amiloidogênicas/química , Benzotiazóis/química , Muramidase/química , Amiloide/ultraestrutura , Proteínas Amiloidogênicas/ultraestrutura , Animais , Galinhas , Fluorescência , Microscopia de Força Atômica , Muramidase/ultraestrutura , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier
14.
Biophys Chem ; 270: 106530, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33545456

RESUMO

Bovine milk αS2-casein, an intrinsically disordered protein, readily forms amyloid fibrils in vitro and is implicated in the formation of amyloid fibril deposits in mammary tissue. Its two cysteine residues participate in the formation of either intra- or intermolecular disulphide bonds, generating monomer and dimer species. X-ray solution scattering measurements indicated that both forms of the protein adopt large, spherical oligomers at 20 °C. Upon incubation at 37 °C, the disulphide-linked dimer showed a significantly greater propensity to form amyloid fibrils than its monomeric counterpart. Thioflavin T fluorescence, circular dichroism and infrared spectra were consistent with one or both of the dimer isomers (in a parallel or antiparallel arrangement) being predisposed toward an ordered, amyloid-like structure. Limited proteolysis experiments indicated that the region from Ala81 to Lys113 is incorporated into the fibril core, implying that this region, which is predicted by several algorithms to be amyloidogenic, initiates fibril formation of αS2-casein. The partial conservation of the cysteine motif and the frequent occurrence of disulphide-linked dimers in mammalian milks despite the associated risk of mammary amyloidosis, suggest that the dimeric conformation of αS2-casein is a functional, yet amyloidogenic, structure.


Assuntos
Amiloide/química , Caseínas/química , Multimerização Proteica , Amiloide/ultraestrutura , Animais , Caseínas/ultraestrutura , Bovinos , Cisteína/análise , Dissulfetos/análise , Leite/química
15.
Int J Mol Sci ; 22(4)2021 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-33579016

RESUMO

Protein aggregation into amyloid fibrils is linked to multiple disorders. The understanding of how natively non-harmful proteins convert to these highly cytotoxic amyloid aggregates is still not sufficient, with new ideas and hypotheses being presented each year. Recently it has been shown that more than one type of protein aggregates may co-exist in the affected tissue of patients suffering from amyloid-related disorders, sparking the idea that amyloid aggregates formed by one protein may induce another protein's fibrillization. In this work, we examine the effect that lysozyme fibrils have on insulin amyloid aggregation. We show that not only do lysozyme fibrils affect insulin nucleation, but they also alter the mechanism of its aggregation.


Assuntos
Amiloide/metabolismo , Insulina/metabolismo , Muramidase/metabolismo , Agregação Patológica de Proteínas/metabolismo , Amiloide/ultraestrutura , Animais , Galinhas , Humanos , Agregados Proteicos , Proteínas Recombinantes/metabolismo
16.
Nat Commun ; 12(1): 875, 2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33558536

RESUMO

Systemic AL amyloidosis is a debilitating and potentially fatal disease that arises from the misfolding and fibrillation of immunoglobulin light chains (LCs). The disease is patient-specific with essentially each patient possessing a unique LC sequence. In this study, we present two ex vivo fibril structures of a λ3 LC. The fibrils were extracted from the explanted heart of a patient (FOR005) and consist of 115-residue fibril proteins, mainly from the LC variable domain. The fibril structures imply that a 180° rotation around the disulfide bond and a major unfolding step are necessary for fibrils to form. The two fibril structures show highly similar fibril protein folds, differing in only a 12-residue segment. Remarkably, the two structures do not represent separate fibril morphologies, as they can co-exist at different z-axial positions within the same fibril. Our data imply the presence of structural breaks at the interface of the two structural forms.


Assuntos
Amiloide/ultraestrutura , Microscopia Crioeletrônica , Amiloidose de Cadeia Leve de Imunoglobulina/metabolismo , Sequência de Aminoácidos , Feminino , Humanos , Cadeias Leves de Imunoglobulina/metabolismo , Pessoa de Meia-Idade , Mutação/genética , Agregados Proteicos , Conformação Proteica
17.
Nucleic Acids Res ; 49(5): 2403-2417, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33621982

RESUMO

TIA-1 is an RNA-binding protein that sequesters target RNA into stress granules under conditions of cellular stress. Promotion of stress granule formation by TIA-1 depends upon self-association of its prion-like domain that facilitates liquid-liquid phase separation and is thought to be enhanced via RNA binding. However, the mechanisms underlying the influence of RNA on TIA-1 self-association have not been previously demonstrated. Here we have investigated the self-associating properties of full-length TIA-1 in the presence of designed and native TIA-1 nucleic acid binding sites in vitro, monitoring phase separation, fibril formation and shape. We show that single stranded RNA and DNA induce liquid-liquid phase separation of TIA-1 in a multisite, sequence-specific manner and also efficiently promote formation of amyloid-like fibrils. Although RNA binding to a single site induces a small conformational change in TIA-1, this alone does not enhance phase separation of TIA-1. Tandem binding sites are required to enhance phase separation of TIA-1 and this is finely tuned by the protein:binding site stoichiometry rather than nucleic acid length. Native tandem TIA-1 binding sites within the 3' UTR of p53 mRNA also efficiently enhance phase separation of TIA-1 and thus may potentially act as potent nucleation sites for stress granule assembly.


Assuntos
RNA/metabolismo , Antígeno-1 Intracelular de Células T/química , Regiões 3' não Traduzidas , Amiloide/ultraestrutura , Sítios de Ligação , DNA/química , DNA/metabolismo , Humanos , Modelos Moleculares , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Conformação Proteica , RNA/química , Antígeno-1 Intracelular de Células T/metabolismo , Antígeno-1 Intracelular de Células T/ultraestrutura , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
18.
J Biol Chem ; 296: 100334, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33508322

RESUMO

Systemic light chain (AL) amyloidosis is a fatal protein misfolding disease in which excessive secretion, misfolding, and subsequent aggregation of free antibody light chains eventually lead to deposition of amyloid plaques in various organs. Patient-specific mutations in the antibody VL domain are closely linked to the disease, but the molecular mechanisms by which certain mutations induce misfolding and amyloid aggregation of antibody domains are still poorly understood. Here, we compare a patient VL domain with its nonamyloidogenic germline counterpart and show that, out of the five mutations present, two of them strongly destabilize the protein and induce amyloid fibril formation. Surprisingly, the decisive, disease-causing mutations are located in the highly variable complementarity determining regions (CDRs) but exhibit a strong impact on the dynamics of conserved core regions of the patient VL domain. This effect seems to be based on a deviation from the canonical CDR structures of CDR2 and CDR3 induced by the substitutions. The amyloid-driving mutations are not necessarily involved in propagating fibril formation by providing specific side chain interactions within the fibril structure. Rather, they destabilize the VL domain in a specific way, increasing the dynamics of framework regions, which can then change their conformation to form the fibril core. These findings reveal unexpected influences of CDR-framework interactions on antibody architecture, stability, and amyloid propensity.


Assuntos
Amiloide/ultraestrutura , Regiões Determinantes de Complementaridade/genética , Amiloidose de Cadeia Leve de Imunoglobulina/genética , Placa Amiloide/genética , Sequência de Aminoácidos/genética , Amiloide/genética , Amiloide/imunologia , Proteínas Amiloidogênicas/genética , Proteínas Amiloidogênicas/imunologia , Proteínas Amiloidogênicas/ultraestrutura , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/ultraestrutura , Humanos , Amiloidose de Cadeia Leve de Imunoglobulina/imunologia , Amiloidose de Cadeia Leve de Imunoglobulina/metabolismo , Mutação/genética , Placa Amiloide/imunologia , Placa Amiloide/patologia , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/imunologia , Agregação Patológica de Proteínas/patologia , Conformação Proteica , Dobramento de Proteína
19.
Proc Natl Acad Sci U S A ; 118(4)2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33431654

RESUMO

Amyloid-ß (Aß) fibrils exhibit self-propagating, molecular-level polymorphisms that may contribute to variations in clinical and pathological characteristics of Alzheimer's disease (AD). We report the molecular structure of a specific fibril polymorph, formed by 40-residue Aß peptides (Aß40), that is derived from cortical tissue of an AD patient by seeded fibril growth. The structure is determined from cryogenic electron microscopy (cryoEM) images, supplemented by mass-per-length (MPL) measurements and solid-state NMR (ssNMR) data. Previous ssNMR studies with multiple AD patients had identified this polymorph as the most prevalent brain-derived Aß40 fibril polymorph from typical AD patients. The structure, which has 2.8-Å resolution according to standard criteria, differs qualitatively from all previously described Aß fibril structures, both in its molecular conformations and its organization of cross-ß subunits. Unique features include twofold screw symmetry about the fibril growth axis, despite an MPL value that indicates three Aß40 molecules per 4.8-Å ß-sheet spacing, a four-layered architecture, and fully extended conformations for molecules in the central two cross-ß layers. The cryoEM density, ssNMR data, and MPL data are consistent with ß-hairpin conformations for molecules in the outer cross-ß layers. Knowledge of this brain-derived fibril structure may contribute to the development of structure-specific amyloid imaging agents and aggregation inhibitors with greater diagnostic and therapeutic utility.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/ultraestrutura , Amiloide/ultraestrutura , Córtex Cerebral/química , Fragmentos de Peptídeos/ultraestrutura , Doença de Alzheimer/patologia , Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Sítios de Ligação , Córtex Cerebral/patologia , Microscopia Crioeletrônica , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Termodinâmica
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