RESUMO
This study assessed the effect of konjac glucomannan (KGM) on the aggregation of soy protein isolate (SPI) and its gel-related structure and properties. Raman results showed that KGM promoted the rearrangement of SPI to form more ß-sheets, contributing to the formation of an ordered structure. Atomic force microscopy, confocal laser scanning microscopy, and small-angle X-ray scattering results indicated that KGM reduced the size of SPI particles, narrowed their size distribution, and loosened the large aggregates formed by the stacking of SPI particles, improving the uniformity of gel system. As the hydrogen bonding between the KGM and SPI molecules enhanced, a well-developed network structure was obtained, further reducing the immobilized water's content (T22) and increasing the water-holding capacity (WHC) of SPI gel. Furthermore, this gel structure showed improved gel hardness and resistance to both small and large deformations. These findings facilitate the design and production of SPI-based gels with desired performance.
Assuntos
Géis , Mananas , Proteínas de Soja , Proteínas de Soja/química , Mananas/química , Géis/química , Tamanho da Partícula , Agregados ProteicosRESUMO
The mechanisms of trypsin hydrolysis time on the structure of soy protein hydrolysate fibril aggregates (SPHFAs) and the stability of SPHFAs-high internal phase Pickering emulsions (HIPPEs) were investigated. SPHFAs were prepared using soy protein hydrolysate (SPH) with different trypsin hydrolysis time (0 min-120 min) to stabilize SPHFAs-HIPPEs. The results showed that moderate trypsin hydrolysis (30 min, hydrolysis degree of 2.31 %) induced SPH unfolding and increased the surface hydrophobicity of SPH, thereby promoting the formation of flexible SPHFAs with maximal thioflavin T intensity and ζ-potential. Moreover, moderate trypsin hydrolysis improved the viscoelasticity of SPHFAs-HIPPEs, and SPHFAs-HIPPEs remained stable after storage at 25 °C for 80 d and heating at 100 °C for 1 h. Excessive trypsin hydrolysis (> 30 min) decreased the stability of SPHFAs-HIPPEs. In conclusion, moderate trypsin hydrolysis promoted the formation of flexible SPHFAs with high surface charge by inducing SPH unfolding, thereby promoting the stability of SPHFAs-HIPPEs.
Assuntos
Emulsões , Interações Hidrofóbicas e Hidrofílicas , Hidrolisados de Proteína , Proteínas de Soja , Tripsina , Tripsina/química , Hidrólise , Emulsões/química , Proteínas de Soja/química , Hidrolisados de Proteína/química , Agregados ProteicosRESUMO
Titin is a multidomain protein of striated and smooth muscles of vertebrates. The protein consists of repeating immunoglobulin-like (Ig) and fibronectin-like (FnIII) domains, which are ß-sandwiches with a predominant ß-structure, and also contains disordered regions. In this work, the methods of atomic force microscopy (AFM), X-ray diffraction, and Fourier transform infrared spectroscopy were used to study the morphology and structure of aggregates of rabbit skeletal muscle titin obtained in two different solutions: 0.15 M glycine-KOH, pH 7.0 and 200 mM KCl, 10 mM imidazole, pH 7.0. According to AFM data, skeletal muscle titin formed amorphous aggregates of different morphologies in the above two solutions. Amorphous aggregates of titin formed in a solution containing glycine consisted of much larger particles than aggregates of this protein formed in a solution containing KCl. The "KCl-aggregates" according to AFM data had the form of a "sponge"-like structure, while amorphous "glycine-aggregates" of titin formed "branching" structures. Spectrofluorometry revealed the ability of "glycine-aggregates" of titin to bind to the dye thioflavin T (TT), and X-ray diffraction revealed the presence of one of the elements of the amyloid cross ß-structure, a reflection of ~4.6 Å, in these aggregates. These data indicate that "glycine-aggregates" of titin are amyloid or amyloid-like. No similar structural features were found in "KCl-aggregates" of titin; they also did not show the ability to bind to thioflavin T, indicating the non-amyloid nature of these titin aggregates. Fourier transform infrared spectroscopy revealed differences in the secondary structure of the two types of titin aggregates. The data we obtained demonstrate the features of structural changes during the formation of intermolecular bonds between molecules of the giant titin protein during its aggregation. The data expand the understanding of the process of amyloid protein aggregation.
Assuntos
Conectina , Microscopia de Força Atômica , Músculo Esquelético , Agregados Proteicos , Conectina/química , Conectina/metabolismo , Conectina/genética , Coelhos , Animais , Músculo Esquelético/metabolismo , Músculo Esquelético/química , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios X , BenzotiazóisRESUMO
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
BACKGROUND: GALNTs (UDP-GalNAc; polypeptide N-acetylgalactosaminyltransferases) initiate mucin-type O-GalNAc glycosylation by adding N-GalNAc to protein serine/threonine residues. Abnormalities in O-GalNAc glycosylation are involved in various disorders such as Parkinson's disease (PD), a neurodegenerative disorder. GALNT9 is potentially downregulated in PD patients. METHODS: To determine whether GALNT9 enrichment ameliorates cytotoxicity related to PD-like variations, a pcDNA3.1-GALNT9 plasmid was constructed and transfected into SH-SY5Y cells to establish a GALNT9-overexpressing cell model. RESULTS: Downregulation of GALNT9 and O-GalNAc glycosylation was confirmed in our animal and cellular models of PD-like variations. GALNT9 supplementation greatly attenuated cytotoxicity induced by MPP+ (1-Methyl-4-phenylpyridinium iodide) since it led to increased levels of tyrosine hydroxylase and dopamine, reduced rates of apoptosis, and significantly ameliorated MPP+-induced mitochondrial dysfunction by alleviating abnormal levels of mitochondrial membrane potential and reactive oxygen species. A long-lasting mPTP (mitochondrial permeability transition pores) opening and calcium efflux resulted in significantly lower activity in the cytochrome C-associated apoptotic pathway and mitophagy process, signifying that GALNT9 supplementation maintained neuronal cell health under MPP+ exposure. Additionally, it was found that glycans linked to proteins influenced the formation of protein aggregates containing α-synuclein, and GALNT9 supplement dramatically reduced such insoluble protein aggregations under MPP+ treatment. Glial GALNT9 predominantly appears under pathological conditions like PD-like variations. CONCLUSIONS: GALNT9 enrichment improved cell survival, and glial GALNT9 potentially represents a pathogenic index for PD patients. This study provides insights into the development of therapeutic strategies for the treatment of PD.
Assuntos
1-Metil-4-fenilpiridínio , Mitocôndrias , N-Acetilgalactosaminiltransferases , Polipeptídeo N-Acetilgalactosaminiltransferase , alfa-Sinucleína , N-Acetilgalactosaminiltransferases/metabolismo , N-Acetilgalactosaminiltransferases/genética , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Animais , 1-Metil-4-fenilpiridínio/toxicidade , 1-Metil-4-fenilpiridínio/farmacologia , Agregados Proteicos , Doença de Parkinson/metabolismo , Doença de Parkinson/genética , Linhagem Celular Tumoral , Camundongos , Apoptose/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Glicosilação , Potencial da Membrana Mitocondrial/efeitos dos fármacos , MasculinoRESUMO
Importin ß-superfamily nuclear import receptors (NIRs) mitigate mislocalization and aggregation of RNA-binding proteins (RBPs), like FUS and TDP-43, which are implicated in neurodegenerative diseases. NIRs potently disaggregate RBPs by recognizing their nuclear localization signal (NLS). However, disease-causing mutations in NLS compromise NIR binding and activity. Here, we define features that characterize the anti-aggregation activity of NIR and NLS. We find that high binding affinity between NIR and NLS, and optimal NLS location relative to the aggregating domain plays a role in determining NIR disaggregation activity. A designed FUS chimera (FUSIBB), carrying the importin ß binding (IBB) domain, is solubilized by importin ß in vitro, translocated to the nucleus in cultured cells, and downregulates the expression of endogenous FUS. In this study, we posit that guiding the mutual recognition of NLSs and NIRs will aid the development of therapeutics, illustrated by the highly soluble FUSIBB replacing the aggregation-prone endogenous FUS.
Assuntos
Regulação para Baixo , Sinais de Localização Nuclear , Proteína FUS de Ligação a RNA , beta Carioferinas , Proteína FUS de Ligação a RNA/metabolismo , Proteína FUS de Ligação a RNA/genética , Humanos , beta Carioferinas/metabolismo , beta Carioferinas/genética , Núcleo Celular/metabolismo , Ligação Proteica , Células HEK293 , Agregados Proteicos , Células HeLa , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/genética , Transporte Ativo do Núcleo CelularRESUMO
The inner capsid protein of rotavirus, VP6, emerges as a promising candidate for next-generation vaccines against rotaviruses owing to its abundance in virion particles and high conservation. However, the formation of inclusion bodies during prokaryotic VP6 expression poses a significant hurdle to rotavirus research and applications. Here, we employed experimental and computational approaches to investigate inclusion body formation and aggregation-prone regions (APRs). Heterologous recombinant VP6 expression in Escherichia coli BL21(DE3) cells resulted in inclusion body formation, confirmed by transmission electron microscopy revealing amorphous aggregates. Thioflavin T assay demonstrated incubation temperature-dependent aggregation of VP6 inclusion bodies. Computational predictions of APRs in rotavirus A VP6 protein were performed using sequence-based tools (TANGO, AGGRESCAN, Zyggregator, Waltz, FoldAmyloid, ANuPP, Camsol intrinsic) and structure-based tools (SolubiS, CamSol structurally corrected, Aggrescan3D). A total of 24 consensus APRs were identified, with 21 of them being surface-exposed in VP6. All identified APRs display a predominance of hydrophobic amino acids, ranging from 33 to 100%. Computational identification of these APRs corroborates our experimental observation of VP6 inclusion body or aggregate formation. Characterization of VP6's aggregation propensity facilitates understanding of its behaviour during prokaryotic expression and opens avenues for protein engineering of soluble variants, advancing research on rotavirus VP6 in pathology, therapy, and diagnostics.
Assuntos
Antígenos Virais , Proteínas do Capsídeo , Escherichia coli , Corpos de Inclusão , Rotavirus , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Antígenos Virais/genética , Antígenos Virais/metabolismo , Corpos de Inclusão/metabolismo , Rotavirus/genética , Rotavirus/metabolismo , Agregados Proteicos , Simulação por Computador , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Alzheimer's disease (AD) and more than twenty other dementias, termed tauopathies, are pathologically defined by insoluble aggregates of the microtubule-associated protein tau (MAPT). Although tau aggregation correlates with AD symptomology, the specific tau species, i.e., monomers, soluble oligomers, and insoluble aggregates that induce neurotoxicity are incompletely understood. We developed a light-responsive tau protein (optoTAU) and used viscosity-sensitive AggFluor probes to investigate the consequence(s) of tau aggregation in human neurons and identify modifiers of tau aggregation in AD and other tauopathies. We determined that optoTAU reproduces biological and structural properties of tau aggregation observed in human brains and the pathophysiological transition in tau solubility in live cells. We also provide proof-of-concept for the utilization of optoTAU as a pharmacological platform to identify modifiers of tau aggregation. These findings have broad implications for the characterization of aggregation-prone proteins and investigation of the complex relationship between protein solubility, cellular function, and disease progression.
Assuntos
Neurônios , Agregação Patológica de Proteínas , Proteínas tau , Proteínas tau/metabolismo , Proteínas tau/química , Humanos , Neurônios/metabolismo , Agregação Patológica de Proteínas/metabolismo , Agregados Proteicos , Tauopatias/metabolismo , Tauopatias/patologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , SolubilidadeRESUMO
The aim of the present study was to evaluate the effects of ultrasound-assisted intermittent tumbling (UT) at 300 W, 20 kHz and 40 min on the conformation, intermolecular interactions and aggregation of myofibrillar proteins (MPs) and its induced gelation properties at various tumbling times (4 and 6 h). Raman results showed that all tumbling treatments led the helical structure of MPs to unfold. In comparison to the single intermittent tumbling treatment (ST), UT treatment exerted more pronounced effects on strengthening the intermolecular hydrogen bonds and facilitating the formation of an ordered ß-sheet structure. When the tumbling time was the same, UT treatment caused higher surface hydrophobicity, fluorescence intensity and disulfide bond content in the MPs, inducing the occurrence of hydrophobic interaction and disulfide cross-linking between MPs molecules, thus forming the MPs aggregates. Additionally, results from the solubility, particle size, atomic force microscopy and SDS-PAGE further indicated that, relative to the ST treatment, UT treatment was more potent in promoting the polymerization of myosin heavy chain. The MPs aggregates in the UT group were more uniform than those in the ST group. During the gelation process, the pre-formed MPs aggregates in the UT treatment increased the thermal stability of myosin, rendering it more resistant to heat-induced unfolding of the myosin rod region. Furthermore, they improved the protein tail-tail interaction, resulting in the formation of a well-structured gel network with higher gel strength and cooking yield compared to the ST treatment.
Assuntos
Géis , Miofibrilas , Reologia , Géis/química , Miofibrilas/química , Ondas Ultrassônicas , Proteínas Musculares/química , Conformação Proteica , Interações Hidrofóbicas e Hidrofílicas , Animais , Agregados ProteicosRESUMO
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
Atomic force microscopy (AFM) imaging enables the visualization of protein molecules with high resolution, providing insights into their shape, size, and surface topography. Here, we use AFM to study the aggregation process of protein S100A9 in physiological conditions, in the presence of calcium at a molar ratio 4Ca2+:S100A9. We find that S100A9 readily assembles into a worm-like fibril, with a period dimension along the fibril axis of 11.5 nm. The fibril's chain length extends up to 136 periods after an incubation time of 144 h. At room temperature, the fibril's bending stiffness was found to be 2.95×10-28 Nm2, indicating that the fibrils are relatively flexible. Additionally, the values obtained for the Young's modulus (Ex=6.96×105 Pa and Ey=3.37×105 Pa) are four orders of magnitude lower than those typically reported for canonical amyloid fibrils. Our findings suggest that, under the investigated conditions, a distinct aggregation mechanism may be in place in the presence of calcium. Therefore, the findings reported here could have implications for the field of biomedicine, particularly with regard to Alzheimer's disease.
Assuntos
Amiloide , Cálcio , Calgranulina B , Microscopia de Força Atômica , Microscopia de Força Atômica/métodos , Amiloide/química , Amiloide/ultraestrutura , Calgranulina B/química , Calgranulina B/metabolismo , Cálcio/metabolismo , Cálcio/química , Módulo de Elasticidade , Humanos , Agregados ProteicosRESUMO
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
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
Supramolecular medicine refers to the formulation of therapeutic and diagnostic agents through supramolecular techniques, amid treating, diagnosing, and preventing disease. Recently, there has been growing interest in developing metal nanoparticles (MNPs)-amyloid hybrid materials, which have the potential to revolutionize medical applications. Furthermore, the development of MNPs-amyloid hydrogel/scaffold supramolecules represents a promising new direction in amyloid nanotechnology, with potential applications in tissue engineering and biomedicine. This review first provides a brief introduction to the formation process of protein amyloid aggregates and their unique nanostructures. Subsequently, we focused on recent investigations into the use of MNPs-amyloid hybrid materials in tissue engineering and biomedicine. We anticipate that MNPs-amyloid supramolecular materials will pave the way for new functional materials in medical science, particularly in the field of tissue engineering.
Assuntos
Proteínas Amiloidogênicas , Sistemas de Liberação de Medicamentos , Nanopartículas Metálicas , Engenharia Tecidual , Engenharia Tecidual/métodos , Nanopartículas Metálicas/química , Sistemas de Liberação de Medicamentos/métodos , Agregados Proteicos , Alicerces Teciduais/química , Proteínas Amiloidogênicas/química , Dobramento de Proteína , Conformação Proteica , HumanosRESUMO
Dysregulation in protein homeostasis results in accumulation of protein aggregates, which are sequestered into dedicated insoluble compartments so-called inclusion bodies or aggresomes, where they are scavenged through different mechanisms to reduce proteotoxicity. The protein aggregates can be selectively scavenged by macroautophagy/autophagy called aggrephagy, which is mediated by the autophagic receptor SQSTM1. In this study, we have identified PLK2 as an important regulator of SQSTM1-mediated aggregation of polyubiquitinated proteins. PLK2 is upregulated following proteasome inhibition, and then associates with and phosphorylates SQSTM1 at S349. The phosphorylation of SQSTM1 S349 strengthens its binding to KEAP1, which is required for formation of large SQSTM1 aggregates/bodies upon proteasome inhibition. Our findings suggest that PLK2-mediated phosphorylation of SQSTM1 S349 represents a critical regulatory mechanism in SQSTM1-mediated aggregation of polyubiquitinated proteins.
Assuntos
Complexo de Endopeptidases do Proteassoma , Agregados Proteicos , Proteínas Serina-Treonina Quinases , Proteína Sequestossoma-1 , Proteína Sequestossoma-1/metabolismo , Fosforilação , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Ubiquitinadas/metabolismo , Autofagia/fisiologia , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Células HEK293 , Ubiquitinação , Ligação ProteicaRESUMO
We developed a coarse-grained model for the protic ionic liquid, triethylammonium mesylate ([TEA]+[Ms]-), to characterize its inhibitory effects on amyloid aggregation using the K16LVFFAE22 fragment of the amyloid-ß (Aß16-22) as a model amyloidogenic peptide. In agreement with previous experiments, coarse-grained molecular dynamics simulations showed that increasing concentrations of [TEA]+[Ms]- in aqueous media led to increasingly small Aß16-22 aggregates with low beta-sheet contents. The cause of [TEA]+[Ms]-'s inhibition of peptide aggregation was found to be a result of two interrelated effects. At a local scale, the enrichment of interactions between [TEA]+ cations and hydrophobic phenylalanine side chains weakened the hydrophobic cores of amyloid aggregates, resulting in poorly ordered structures. At a global level, peptides tended to localize at the interfaces of IL-rich nanostructures with water. At high IL concentrations, when the IL-water interface was large or fragmented, Aß16-22 peptides were dispersed in the simulation cell, sometimes sequestered at unaggregated monomeric states. Together, these phenomena underlie [TEA]+[Ms]-'s inhibition of amyloid aggregation. This work addresses the critical lack of knowledge on the mechanisms of protein-ionic liquid interactions and may have broader implications for industrial applications.
Assuntos
Peptídeos beta-Amiloides , Interações Hidrofóbicas e Hidrofílicas , Líquidos Iônicos , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/antagonistas & inibidores , Líquidos Iônicos/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Agregados Proteicos/efeitos dos fármacos , Nanoestruturas/químicaRESUMO
Neurodegenerative diseases are the leading cause of human disability and immensely reduce patients' life span and quality. The diseases are characterized by the functional loss of neuronal cells and share several common pathogenic mechanisms involving the malfunction, structural distortion, or aggregation of multiple key regulatory proteins. Cellular phase separation is the formation of biomolecular condensates that regulate numerous biological processes, including neuronal development and synaptic signaling transduction. Aberrant phase separation may cause protein aggregation that is a general phenomenon in the neuronal cells of patients suffering neurodegenerative diseases. In this review, we summarize the pathological causes of common neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease, among others. We discuss the regulation of key amyloidogenic proteins with an emphasis of their aberrant phase separation and aggregation. We also introduce the approaches as potential therapeutic strategies to ameliorate neurodegenerative diseases through intervening protein aggregation. Overall, this review consolidates the research findings of phase separation and aggregation caused by misfolded proteins in a context of neurodegenerative diseases.
Assuntos
Doenças Neurodegenerativas , Agregação Patológica de Proteínas , Humanos , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Agregação Patológica de Proteínas/metabolismo , Animais , Proteínas Amiloidogênicas/metabolismo , Proteínas Amiloidogênicas/química , Agregados Proteicos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Separação de FasesRESUMO
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
Aggrescan4D (A4D) is an advanced computational tool designed for predicting protein aggregation, leveraging structural information and the influence of pH. Building upon its predecessor, Aggrescan3D (A3D), A4D has undergone numerous enhancements aimed at assisting the improvement of protein solubility. This manuscript reviews A4D's updated functionalities and explains the fundamental principles behind its pH-dependent calculations. Additionally, it presents an antibody case study to evaluate its performance in comparison with other structure-based predictors. Notably, A4D integrates advanced protein engineering protocols with pH-dependent calculations, enhancing its utility in advising solubility-enhancing mutations. A4D considers the impact of structural flexibility on aggregation propensities, and includes a large set of precalculated predictions. These capabilities should help to open new avenues for both understanding and managing protein aggregation. A4D is accessible through a dedicated web server at https://biocomp.chem.uw.edu.pl/a4d/.