Amyloid Fibrils Formed by Short Prion-Inspired Peptides Are Metalloenzymes

Altres ajuts: acords transformatius de la UABEnzymes typically fold into defined 3D protein structures exhibiting a high catalytic efficiency and selectivity. It has been proposed that the earliest enzymes may have arisen from the self-assembly of short peptides into supramolecular amyloid-like structures. Several artificial amyloids have been shown to display catalytic activity while offering advantages over natural enzymes in terms of modularity, flexibility, stability, and reusability. Hydrolases, especially esterases, are the most common artificial amyloid-like nanozymes with some reported to act as carbonic anhydrases (CA). Their hydrolytic activity is often dependent on the binding of metallic cofactors through a coordination triad composed of His residues in the β-strands, which mimic the arrangement found in natural metalloenzymes. Tyr residues contribute to the coordination of metal ions in the active center of metalloproteins; however, their use has been mostly neglected in the design of metal-containing amyloid-based nanozymes. We recently reported that four different polar prion-inspired heptapeptides spontaneously self-assembled into amyloid fibrils. Their sequences lack His but contain three alternate Tyr residues exposed to solvent. We combine experiments and simulations to demonstrate that the amyloid fibrils formed by these peptides can efficiently coordinate and retain different divalent metal cations, functioning as both metal scavengers and nanozymes. The metallized fibrils exhibit esterase and CA activities without the need for a histidine triad. These findings highlight the functional versatility of prion-inspired peptide assemblies and provide a new sequential context for the creation of artificial metalloenzymes. Furthermore, our data support amyloid-like structures acting as ancestral catalysts at the origin of life

Computational modeling of fluorescent markers for amyloid detection

Premi Extraordinari de Doctorat concedit pels programes de doctorat de la UAB per curs acadèmic 2016-2017La marca distintiva principal de la enfermedad de Alzheimer es la deposición en el cerebro de fibras que resultan de la agregación del amiloide-β, un péptido hidrofóbico de 40 o 42 aminoácidos. En los últimos años, se ha dedicado mucho esfuerzo al desarrollo de técnicas de fluorescencia para la detección de fibras amiloides in vivo. La peculiar arquitectura "cross-b" de las fibras genera canales, definidos por las cadenas laterales de los residuos hidrofóbicos, que pueden alojar moléculas pequeñas que pueden actuar como marcadores fluorescentes Dado que el objetivo es la detección de agregados amiloides, el interés está en detectar selectivamente la fluorescencia emitida por los marcadores enlazados con las fibras amiloides. Paraello, los marcadores deben sufrir importantes modificaciones en sus espectros de fluorescencia cuando éstos se enlacen con las fibras. A pesar de que se hayan propuesto muchas clases de marcadores en los últimos 10 años, todavía no se conocen profundamente los orígenes de estos cambios en los espectros de fluorescencia. En esta tesis se estudian tanto las propiedades fotofísicas de los marcadores fluorescentes como su interacción con las fibras amiloides mediante un conjunto de técnicas computacionales. Los resultados obtenidos indican que los cambios en los espectros de fluorescencia pueden atribuirse a una variedad de fenómenos. Por ejemplo, los marcadores pueden sufrir procesos de agregación-desagregación, es decir, en disolución, los agregados formados espontáneamente no emiten fluorescencia, mientras que en presencia de fibras se recupera una sola molécula, que sí puede emitir. Por otra parte, algunos de estos marcadores se pueden desactivar a través de un cruce intersistema, cuya eficiencia depende de la polaridad del medio. De esta manera, cuando la molécula se transfiere desde el medio biológico, que es esencialmente una disolución acuosa, al núcleo hidrofóbico de la fibra, se modifica la velocidad de cruce intersistema, y consecuentemente, el rendimiento cuántico de la fluorescencia. Por último, dado que estos marcadores fluorescentes contienen enlaces dobles, pueden existir intersecciones cónicas correspondientes a torsiones de 90 grados alrededor de estos enlaces que den lugar a una desactivación no radiativa. Estas torsiones se pueden producir libremente cuando la molécula está en disolución. Sin embargo, el impedimento estérico existente en el interior de la fibra hace que éstas sean más difíciles, si no imposibles, reduciendo la eficiencia de conversión interna y aumentando la fluorescencia. Estas observaciones permiten racionalizar el comportamiento de estos marcadores y guiar el diseño de la síntesis de nuevos marcadores con rendimiento óptimo.The main hallmark of Alzheimer's disease is the deposition in the brain of fibrils resulting from the aggregation of amyloid-β, a hydrophobic peptide composed of 40 or 42 aminoacids. In recent years, a lot of interest has been devoted to the development of fluorescence imaging techniques that allow the in vivo detection of amyloid fibrils. The peculiar cross-β architecture of amyloid fibrils yields channel-shaped binding pockets, defined by the side chains of hydrophobic residues, which can accommodate small molecules that can act as fluorescent markers. Since the aim is to detect amyloid aggregates, the interest relies in collecting selectively the fluorescence emitted by markers that are actually bound to amyloid fibrils, and not by free molecules. In this regard, fluorescent markers should undergo important variations of their fluorescence spectra upon binding to amyloid fibrils. Despite the fact that several classes of fluorescent markers have been proposed over the last 10 years, little is known about the origin of these spectral modifications. In this thesis, both the photophysical properties of fluorescent markers and their binding to amyloid fibrils were studied using a pool of computational techniques. A deep understanding of these phenomena is essential for the rational design of markers with tailored properties. Results indicate that a variety of properties may be responsible for these spectral modifications occurring upon binding to amyloid fibrils. For instance, the markers may undergo an aggregation-disaggregation process, which is accompanied by an important enhancement of fluorescence intensity. This can be explained considering that while the fluorescence of spontaneously formed aggregates of these markers is quenched in aqueous solution, in the presence of amyloid fibrils they disaggregate, yielding the single, emitting, marker molecule. Some of these markers may also undergo intersystem-crossing, whose efficiency has been demonstrated to be strongly affected by the polarity of the environment. In this way, when the molecule moves from the polar biological medium, which is essentially an aqueous solution, inside the hydrophobic core of amyloid fibrils, the rate of intersystem crossing changes, and with it the quantum yield of fluorescence. Last, since these fluorescent markers contain double bonds, conical intersections corresponding to torsions of 90 degrees around the double bonds can lead to non-radiative deactivation. While these torsions are relatively easy for the free molecule, they become hindered, if not impossible, when the marker is packed inside amyloid fibrils. This reduces the efficiency of internal conversion upon binding, which in turn enhances fluorescence. These findings allow the rationalization of the spectroscopical behavior of these markers and can guide the design of new markers with improved performances

Fluorous l -Carbidopa Precursors : Highly Enantioselective Synthesis and Computational Prediction of Bioactivity

New fluorous enantiopure (S)-α-aminated β-keto esters were prepared through a highly enantioselective electrophilic α-amination step in the presence of europium triflate and (R,R)-phenyl-pybox. These compounds are precursors of fluorinated analogues of l-carbidopa, which is known to inhibit DOPA decarboxylase (DDC), a key protein in Parkinson's disease. Fluorination provides better stability for biological applications, which could possibly lead to DDC inhibitors better than l-carbidopa itself. Induced fit docking computational simulations performed on the new structures interacting with DDC highlight that for an efficient binding at the DDC site, at least one hydroxyl substituent must be present at the aromatic ring of the l-carbidopa analogues and show that the presence of fluorine can further fix the position of the ligand in the active site

Chemometric-assisted cocrystallization: Supervised pattern recognition for predicting the formation of new functional cocrystals

Owing to the antimicrobial and insecticide properties, the use of natural compounds like essential oils and their active components has proven to be an effective alternative to synthetic chemicals in different fields ranging from drug delivery to agriculture and from nutrition to food preservation. Their limited application due to the high volatility and scarce water solubility can be expanded by using crystal engineering approaches to tune some properties of the active molecule by combining it with a suitable partner molecule (coformer). However, the selection of coformers and the experimental effort required for discovering cocrystals are the bottleneck of cocrystal engineering. This study explores the use of chemometrics to aid the discovery of cocrystals of active ingredients suitable for various applications. Partial Least Squares–Discriminant Analysis is used to discern cocrystals from binary mixtures based on the molecular features of the coformers. For the first time, by including failed cocrystallization data and considering a variety of chemically diverse compounds, the proposed method resulted in a successful prediction rate of 85% for the test set in the model validation phase and of 74% for the external test set

pH-responsive self-assembly of amyloid fibrils for dual hydrolase-oxidase reactions

Altres ajuts: ICREA Academia 2015There is an increasing interest in synthetic systems that can execute bioinspired chemical reactions without requiring the complex structures that characterize enzymes in their components. The hierarchical self-assembly of peptides provides a means to create catalytic microenvironments. Ideally, as it occurs in enzymes, the catalytic activity of peptide nanostructures should be reversibly regulated. In a typical enzyme mimetic design, the peptide's self-assembling and catalytic activities are segregated into different regions of the sequence. Here, we aimed to design minimal peptides in which the self-assembly and function were all encoded in the same amino acids. Moreover, we wanted to endow the resulting one-component nanomaterial with divergent, chemically unrelated, catalytic activities, a property not observed in natural enzymes. We show that short peptides consisting only of histidine and tyrosine residues, arranged in a binary pattern, form biocompatible amyloid-like fibrils and hydrogels combining hydrolytic and electrocatalytic activities. The nanofibers' mesoscopic properties are controlled by pH, the transition between assembled active β-sheet fibrils, and disassembled inactive random coil species occurring in a physiologically relevant pH range. The structure of one of such amyloid-like fibrils, as derived from molecular dynamic simulations, provides insights on how they attain this combination of structural and catalytic properties

The catalytic role of glutathione transferases in heterologous anthocyanin biosynthesis

Anthocyanins are ubiquitous plant pigments used in a variety of technological applications. Yet, after over a century of research, the penultimate biosynthetic step to anthocyanidins attributed to the action of leucoanthocyanidin dioxygenase has never been efficiently reconstituted outside plants, preventing the construction of heterologous cell factories. Through biochemical and structural analysis, here we show that anthocyanin-related glutathione transferases, currently implicated only in anthocyanin transport, catalyse an essential dehydration of the leucoanthocyanidin dioxygenase product, flavan-3,3,4-triol, to generate cyanidin. Building on this knowledge, introduction of anthocyanin-related glutathione transferases into a heterologous biosynthetic pathway in baker's yeast results in >35-fold increased anthocyanin production. In addition to unravelling the long-elusive anthocyanin biosynthesis, our findings pave the way for the colourants' heterologous microbial production and could impact the breeding of industrial and ornamental plants

Anatocismo nei piani di ammortamento standardizzati tradizionali

Il rapporto, integrando Matematica e Diritto, esamina se i piani di ammortamento "graduale" a tasso certo e costante nel tempo configurino la produzione di interessi su interessi, violando la norma civilistica sull'anatocismo. La conclusione è negativa

Molecular Recognition of Glycan-Bearing Glycomacromolecules Presented at Membrane Surfaces by Lectins: An NMR View

Lectin–glycan interactions are at the heart of a multitude of biological events. Glycans are usually presented in a multivalent manner on the cell surface as part of the so-called glycocalyx, where they interact with other entities. This multivalent presentation allows us to overcome the typical low affinities found for individual glycan–lectin interactions. Indeed, the presentation of glycans may drastically impact their binding by lectins, highly affecting the corresponding binding affinity and even selectivity. In this context, we herein present the study of the interaction of a variety of homo- and heteromultivalent lactose-functionalized glycomacromolecules and their lipid conjugates with two human galectins. We have employed as ligands the glycomacromolecules, as well as liposomes decorated with those structures, to evaluate their interactions in a cell-mimicking environment. Key details of the interaction have been unravelled by NMR experiments, both from the ligand and receptor perspectives, complemented by cryo-electron microscopy methods and molecular dynamics simulations.M.H. and L.H. thank the DFG for support through the ViroCarb research consortium (HA5950/5-2) and the CeMSA@HHU (Center for Molecular and Structural Analytics @ Heinrich-Heine University) for recording the mass spectrometric and the NMR-spectroscopic data for the structural conformation of the glycomacromolecules and their lipid conjugates. The CIC bioGUNE EM platform is also thanked for infrastructural support during cryo-EM data collection. The group in Spain thank the European Research Council (RECGLYCANMR, Advanced grant no. 788143), MCIN/AEI/10.13039/501100011033 for grants PDI2021-1237810B-C21, PID2021-126130OB-I00, CEX2021-001136-S, and CIBERES, an initiative of Instituto de Salud Carlos III (ISCIII), Madrid, Spain, for generous funding