RESUMO
Several aspects of telomere biology are regulated by the telomeric repeat-containing RNA TERRA. While TERRA expression is conserved through evolution, species-specific mechanisms regulate its biogenesis and function. Here we report on the expression of TERRA in Caenorhabditis elegans. We show that C. elegans TERRA is regulated by the telomere-binding proteins POT-1 and POT-2 which repress TERRA in a telomere-specific manner. C. elegans TERRA transcripts are heterogeneous in length and form discrete nuclear foci, as detected by RNA FISH, in both postmitotic and germline cells; a fraction of TERRA foci localizes to telomeres. Interestingly, in germ cells, TERRA is expressed in all stages of meiotic prophase I, and it increases during pachytene, a stage in meiosis when homologous recombination is ongoing. We used the MS2-GFP system to study the spatiotemporal dynamics of single-telomere TERRA molecules. Single particle tracking revealed different types of motilities, suggesting complex dynamics of TERRA transcripts. Finally, we unveiled distinctive features of C. elegans TERRA, which is regulated by telomere shortening in a telomere-specific manner, and it is upregulated in the telomerase-deficient trt-1; pot-2 double mutant prior to activation of the alternative lengthening mechanism ALT. Interestingly, in these worms TERRA displays distinct dynamics with a higher fraction of fast-moving particles.
Assuntos
Proteínas de Caenorhabditis elegans , RNA Longo não Codificante , Telomerase , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/genética , Meiose , RNA Longo não Codificante/genética , Telomerase/genética , Telômero/genética , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Depression, either bipolar or unipolar, is a highly prevalent and disabling condition. Even though several treatment options exist for depressed patients, a significant portion of individuals receiving conventional pharmacotherapy fails to achieve and sustain remission. For this reason, there is a strong need for effective alternatives to pharmacotherapy. In this respect, non-invasive brain stimulation (NIBS), including transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (tDCS), have been increasingly investigated in the last two decade as promising treatment strategies for major depression and treatment-resistant depression (TRD). Indeed, due to their safety and tolerability and to the growing evidence on their efficacy, NIBS has been included in international treatment guidelines, having become part of the standard clinical practice. Even though several clinical trials involving NIBS in patients with major depression and TRD have been conducted, literature in specific areas is still marked by some inconsistencies, due to small sample-sizes, lack of multicentre-studies and to the difficulty in comparing different treatment modalities and stimulation protocols. In light of the above, we sought to provide a brief, updated compendium of the latest innovative acquisition for the use of NIBS in the treatment of depression, either unipolar or bipolar, as well as TRD with a specific focus on innovative set-up, devices, target areas, and parameters that may affect the outcome.
Assuntos
Transtorno Bipolar , Transtorno Depressivo Maior , Estimulação Transcraniana por Corrente Contínua , Humanos , Transtorno Bipolar/terapia , Estimulação Transcraniana por Corrente Contínua/métodos , Estimulação Magnética Transcraniana/métodos , Transtorno Depressivo Maior/terapia , Encéfalo/fisiologiaRESUMO
In recent years, biomaterials from abundant and renewable sources have shown potential in medicine and materials science alike. In this study, we combine theoretical modeling, molecular dynamics simulations, and several experimental techniques to understand the regeneration of cellulose/silk-, chitin/silk-, and chitosan/silk-based biocomposites after dissolution in ionic liquid and regeneration in water. We propose a novel theoretical model that correlates the composite's microscopic structure to its bulk properties. We rely on modeling non-cross-linked biopolymers that present layer-like structures such as ß-sheets and we successfully predict structural, thermal, and mechanical properties of a mixture of these biomolecules. Our model and experiments show that the solubility of the pure substance in the chosen solvent can be used to modulate the amount of crystallinity of the biopolymer blend, as measured by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). Thermogravimetric analysis (TGA) shows that the decomposition temperature of the blended biocomposites compared to their pure counterparts is reduced in accordance with our theoretical predictions. The morphology of the material is further characterized through scanning electron microscopy (SEM) and shows differently exposed surface area depending on the blend. Finally, differential scanning calorimetry (DSC) is performed to characterize the residual water content in the material, essential for explaining the regeneration process in water. As a final test of the model, we compare our model's prediction of the Young's modulus with existing data in the literature. The model correctly reproduces experimental trends observed in the Young's modulus due to varying the concentration of silk in the biopolymer blend.
Assuntos
Celulose/química , Quitina/química , Quitosana/química , Líquidos Iônicos/química , Modelos Teóricos , Seda/química , Água/química , Animais , Materiais Biocompatíveis/química , Bombyx , Módulo de Elasticidade , RegeneraçãoRESUMO
The metabolism of living systems involves many enzymes that play key roles as catalysts and are essential to biological function. Searching ligands with the ability to modulate enzyme activities is central to diagnosis and therapeutics. Peptides represent a promising class of potential enzyme modulators due to the large chemical diversity, and well-established methods for library synthesis. Peptides and their derivatives are found to play critical roles in modulating enzymes and mediating cellular uptakes, which are increasingly valuable in therapeutics. We present a methodology that uses molecular dynamics (MD) and point-variant screening to identify short peptide motifs that are critical for inhibiting ß-galactosidase (ß-Gal). MD was used to simulate the conformations of peptides and to suggest short motifs that were most populated in simulated conformations. The function of the simulated motifs was further validated by the experimental point-variant screening as critical segments for inhibiting the enzyme. Based on the validated motifs, we eventually identified a 7-mer short peptide for inhibiting an enzyme with low µM IC50. The advantage of our methodology is the relatively simplified simulation that is informative enough to identify the critical sequence of a peptide inhibitor, with a precision comparable to truncation and alanine scanning experiments. Our combined experimental and computational approach does not rely on a detailed understanding of mechanistic and structural details. The MD simulation suggests the populated motifs that are consistent with the results of the experimental alanine and truncation scanning. This approach appears to be applicable to both natural and artificial peptides. With more discovered short motifs in the future, they could be exploited for modulating biocatalysis, and developing new medicine.
Assuntos
Peptídeos/química , Motivos de Aminoácidos , Sítios de Ligação , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Concentração Inibidora 50 , Simulação de Dinâmica Molecular , Peptídeos/farmacologia , Ligação Proteica , Estrutura Secundária de Proteína , beta-Galactosidase/antagonistas & inibidoresRESUMO
The microtubule-associated protein tau regulates the stability of microtubules within neurons in the central nervous system. In turn, microtubules are responsible for the remodeling of the cytoskeleton that ultimately leads to the formation or pruning of new connections among neurons. As a consequence, dysfunction of tau is associated with many forms of dementia as well as Alzheimer's disease. In the brain, tau activity is regulated by its phosphorylation state. Phosphorylation is a post-translational modification of proteins that adds a phosphate group to the side chain of an amino acid. Phosphorylation at key locations in the tau sequence leads to a higher or lower affinity for microtubules. In Alzheimer's disease, tau is present in an abnormal phosphorylation state. However, studying the effect of phosphorylation experimentally has been extremely challenging as there is no viable way of exactly selecting the location and the number of phosphorylated sites. For this reason, researchers have turned to pseudophosphorylation. In this technique, actual phosphorylation is mimicked by mutating the selected amino acid into glutamate or aspartate. Whether this methodology is equivalent to actual phosphorylation is still open to debate. In this study, we will show that phosphorylation and pseudophosphorylation are not exactly equivalent. Although for larger aggregates the two techniques lead to similar structures, the kinetics of the process may be altered. In addition, very little is known about the impact that this may have on the early stages of aggregation, such as nucleation and conformational rearrangement. In this study, we show that the two methods may produce a similar ensemble of conformations, even though the kinetic and chemical details that lead to it are quite different.
Assuntos
Agregados Proteicos , Agregação Patológica de Proteínas , Proteínas tau/química , Proteínas tau/metabolismo , Algoritmos , Humanos , Ligação de Hidrogênio , Fosforilação , Teoria QuânticaRESUMO
Self-aggregation of the microtubule-binding protein Tau reduces its functionality and is tightly associated with Tau-related diseases, termed tauopathies. Tau aggregation is also strongly associated with two nucleating six-residue segments, namely PHF6 (VQIVYK) and PHF6* (VQIINK). In this paper, using experiments and computational modeling, we study the self-assembly of individual and binary mixtures of Tau fragments containing PHF6* (R2/wt; (273)GKVQIINKKLDL(284)) and PHF6 (R3/wt; (306)VQIVYKPVDLSK(317)) and a mutant R2/ΔK280 associated with a neurodegenerative tauopathy. The initial stage of aggregation is probed by ion-mobility mass spectrometry, the kinetics of aggregation monitored with Thioflavin T assays, and the morphology of aggregates visualized by transmission electron microscopy. Insights into the structure of early aggregates and the factors stabilizing the aggregates are obtained from replica exchange molecular dynamics simulations. Our data suggest that R3/wt has a much stronger aggregation propensity than either R2/wt or R2/ΔK280. Heterodimers containing R3/wt are less stable than R3/wt homodimers but much more stable than homodimers of R2/wt and R2/ΔK280, suggesting a possible role of PHF6*-PHF6 interactions in initiating the aggregation of full-length Tau. Lastly, R2/ΔK280 binds more strongly to R3/wt than R2/wt, suggesting a possible mechanism for a pathological loss of normal Tau function.
Assuntos
Proteínas tau/química , Proteínas tau/genética , Benzotiazóis , Dimerização , Humanos , Espectrometria de Massas , Microscopia Eletrônica de Transmissão , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Tiazóis/químicaRESUMO
Intrinsically disordered proteins (IDPs) are a unique class of proteins that have no stable native structure, a feature that allows them to adopt a wide variety of extended and compact conformations that facilitate a large number of vital physiological functions. One of the most well-known IDPs is the microtubule-associated tau protein, which regulates microtubule growth in the nervous system. However, dysfunctions in tau can lead to tau oligomerization, fibril formation, and neurodegenerative disease, including Alzheimer's disease. Using a combination of simulations and experiments, we explore the role of osmolytes in regulating the conformation and aggregation propensities of the R2/wt peptide, a fragment of tau containing the aggregating paired helical filament (PHF6*). We show that the osmolytes urea and trimethylamine N-oxide (TMAO) shift the population of IDP monomer structures, but that no new conformational ensembles emerge. Although urea halts aggregation, TMAO promotes the formation of compact oligomers (including helical oligomers) through a newly proposed mechanism of redistribution of water around the perimeter of the peptide. We put forth a "superposition of ensembles" hypothesis to rationalize the mechanism by which IDP structure and aggregation is regulated in the cell.
Assuntos
Simulação de Dinâmica Molecular , Peptídeos/química , Agregação Patológica de Proteínas , Proteínas tau/química , Humanos , Metilaminas/química , Estrutura Secundária de Proteína , Ureia/químicaRESUMO
The structural properties of water molecules surrounding TMAO molecules are studied using a newly developed atomistic force field for TMAO, combined with a multiscale coarse-graining (MS-CG) force field derived from the atomistic simulations. The all-atom force field is parametrized to work with the OPLS force field and with SPC, TIP3P, and TIP4P water models. The dual-resolution modeling enables a complete study of the dynamical and structural properties of the system, with the CG model providing important new physical insights into which interactions are critical in determining the structure of water around TMAO. TMAO is an osmolyte that stabilizes protein structures under conditions of chemical, thermal, and pressure denaturation. This molecule is excluded from the surface of proteins, and its effect on protein stability is mediated through TMAO-water interactions. We find that TMAO strongly binds two to three water molecules and, surprisingly, that methyl groups repel both the other methyl groups of TMAO and water molecules alike. The latter result is important because it shows that methyl groups are not interacting with each other through the expected hydrophobic effect (which would be attractive and not repulsive) and that the repulsion of water molecules forces a clathrate-like hydrogen bond network around them. We speculate that TMAO is excluded from the vicinity of the protein because the peculiar structure of water around TMAO prevents this molecule from coming in close contact with the protein.
Assuntos
Metilaminas/química , Água/química , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Pressão , Desnaturação Proteica , Proteínas/química , Proteínas/metabolismoRESUMO
The microtubule associated protein tau is essential for the development and maintenance of the nervous system. Tau dysfunction is associated with a class of diseases called tauopathies, in which tau is found in an aggregated form. This paper focuses on a small aggregating fragment of tau, (273)GKVQIINKKLDL(284), encompassing the (PHF6*) region that plays a central role in tau aggregation. Using a combination of simulations and experiments, we probe the self-assembly of this peptide, with an emphasis on characterizing the early steps of aggregation. Ion-mobility mass spectrometry experiments provide a size distribution of early oligomers, TEM studies provide a time course of aggregation, and enhanced sampling molecular dynamics simulations provide atomistically detailed structural information about this intrinsically disordered peptide. Our studies indicate that a point mutation, as well the addition of heparin, lead to a shift in the conformations populated by the earliest oligomers, affecting the kinetics of subsequent fibril formation as well as the morphology of the resulting aggregates. In particular, a mutant associated with a K280 deletion (a mutation that causes a heritable form of neurodegeneration/dementia in the context of full length tau) is seen to aggregate more readily than its wild-type counterpart. Simulations and experiment reveal that the ΔK280 mutant peptide adopts extended conformations to a greater extent than the wild-type peptide, facilitating aggregation through the pre-structuring of the peptide into a fibril-competent structure.
Assuntos
Deleção de Sequência , Tauopatias/genética , Tauopatias/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismo , Sequência de Aminoácidos , Heparina/metabolismo , Humanos , Simulação de Dinâmica Molecular , Mutação Puntual , Conformação Proteica , Multimerização Proteica , Proteínas tau/química , Proteínas tau/ultraestruturaRESUMO
The amyloid-ß(25-35) peptide plays a key role in the etiology of Alzheimer's disease due to its extreme toxicity even in the absence of aging. Because of its high tendency to aggregate and its low solubility in water, the structure of this peptide is still unknown. In this work, we sought to understand the early stages of aggregation of the amyloid-ß(25-35) peptide by conducting simulations of oligomers ranging from monomers to tetramers. Our simulations show that although the monomer preferentially adopts a ß-hairpin conformation, larger aggregates have extended structures, and a clear transition from compact ß-hairpin conformations to extended ß-strand structures occurs between dimers and trimers. Even though ß-hairpins are not present in the final architecture of the fibril, our simulations indicate that they play a critical role in fibril growth. Our simulations also show that ß-sheet structures are stabilized when a ß-hairpin is present at the edge of the sheet. The binding of the hairpin to the sheet leads to a subsequent destabilization of the hairpin, with part of the hairpin backbone dangling in solution. This free section of the peptide can then recruit an extra monomer from solution, leading to further sheet extension. Our simulations indicate that the peptide must possess sufficient conformational flexibility to switch between a hairpin and an extended conformation in order for ß-sheet extension to occur, and offer a rationalization for the experimental observation that overstabilizing a hairpin conformation in the monomeric state (for example, through chemical cross-linking) significantly hampers the fibrillization process.
Assuntos
Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Multimerização Proteica , Modelos Moleculares , Estrutura Secundária de ProteínaRESUMO
Many problems of interest in modern science originate from the complex network of interactions of different molecular structures, each possessing its own typical length and time scale of relevance. In such materials, nontrivial properties emerge from the different length and time scales involved that could not be predicted from the properties of each individual subunit taken alone. A solution to the formidable theoretical and computational issues raised by these systems involves coarse-graining, a procedure in which multiple atoms are grouped into a few interaction sites. The coarse-grained approach aims at constructing an effective Hamiltonian from available information about the system and then using this Hamiltonian to investigate the behavior of the system on the length and time scales of interest. In this paper, we aim at determining how far we can coarse-grain a system using only the commonly used pairwise, spherically symmetric potentials, as well as assessing the impact of poor initial sampling on the quality of the resulting coarse-grained model. Coarse-graining is performed following the multiscale coarse-graining (MS-CG) methodology, and we use as a model system the N-methylacetamide (NMA) molecule, a simple representation of a peptide bond, which can adopt two conformations, cis and trans. Our simulations reveal that as the coarse-graining becomes more aggressive multibody effects start to emerge and that the initial sampling of conformations can adversely bias the model in the case of heavy coarse-graining.
RESUMO
A general mean field theory is presented for the construction of equilibrium coarse-grained models. Inverse methods that reconstruct microscopic models from low resolution experimental data can be derived as particular implementations of this theory. The theory also applies to the opposite problem of reduction, where relevant information is extracted from available equilibrium ensemble data. Additionally, a complementary approach is presented and problems of representability in coarse-grained modeling analyzed using information theoretic arguments. These problems are central to the construction of coarse-grained representations of complex systems, and commonly used coarse-graining methods and variational principles for coarse-graining are derived as particular cases of the general theory.
RESUMO
Many methodologies have been proposed to build reliable and computationally fast coarse-grained potentials. Typically, these force fields rely on the assumption that the relevant properties of the system under examination can be reproduced using a pairwise decomposition of the effective coarse-grained forces. In this work it is shown that an extension of the multiscale coarse-graining technique can be employed to parameterize a certain class of two-body and three-body force fields from atomistic configurations. The use of explicit three-body potentials greatly improves the results over the more commonly used two-body approximation. The method proposed here is applied to develop accurate one-site coarse-grained water models.