The activity of protein kinases is heavily dependent on the phosphorylation state of the protein. Kinase phosphorylation states have been prepared through biological or enzymatic means for biochemical evaluation, but the use of protein chemical modification as an investigative tool has not been addressed. By chemically reacting a genetically encoded cysteine, phosphocysteine was installed via dehydroalanine as a reactive intermediate. The installed phosphocysteine was intended as a surrogate to the naturally occurring phosphothreonine or phosphoserine of a phosphorylated protein kinase. Two model protein kinases were investigated on: MEK1 and p38α. The development of suitable protein variants and suitable reaction conditions on these two proteins is discussed in turn and in detail, resulting in p38α-pCys180 and MEK1-pCys222. Designed to be mimics of the naturally occurring p38α-pThr180 and MEK1-pSer222, these two chemically modified proteins were studied for their biological function. The core biological studies entailed the determination of enzymatic activity of both modified proteins, and included the necessary controls against their active counterparts. In addition, the studies on p38α-pCys180 also included a more detailed quantification of enzymatic activity, and the behaviour of this modified protein against known inhibitors of p38α was also investigated. Both modified proteins were shown to be enzymatically active and behave similarly to corresponding active species. The adaptation of mass spectrometry methods to handle the majority of project's analytical requirements, from monitoring chemical transformations to following enzyme kinetics was instrumental in making these studies feasible. The details of these technical developments are interwoven into the scientific discussion. Also included in this thesis is an introduction to the mechanism and function of protein kinases, and on the protein chemistry methods employed. The work is concluded with a projection of implications that this protein chemical modification technique has on kinase biomedical research.

Tesis por compendio / [ES] Debido a la creciente importancia de la dieta en el manejo de la salud, sigue habiendo un gran interés en desentrañar como se procesan los alimentos en el sistema digestivo humano. La estructura de los alimentos puede influir significativamente en su procesamiento, afectando al rendimiento durante la alimentación y la digestión. Específicamente, la digestión de alimentos a base de carbohidratos requiere una mayor comprensión debido a su contribución a los niveles de glucosa en sangre. El conocimiento de la cinética de digestión del almidón contribuirá a diseñar alimentos a medida para controlar los niveles de glucosa posprandial. El objetivo de esta tesis doctoral fue adquirir una mejor comprensión del impacto de la microestructura en la digestión del almidón y cómo las enzimas digestivas podrían ser moduladas por compuestos fenólicos. Con ese propósito, se evaluó el papel de la estructura del pan en la masticación in vivo y la digestión in vitro. Posteriormente, se produjeron geles de almidón de diferentes fuentes y se digirieron en un sistema de digestión oro-gastro-intestinal in vitro para analizar el impacto de la microestructura del gel. Después de los estudios de microestructura en geles de almidón y pan, se exploraron diferentes ácidos fenólicos o extractos polifenólicos de algas como inhibidores de enzimas digestivas de almidón, y se evaluó la participación de la microestructura del gel de almidón en la digestión enzimática. La masticación y la textura del bolo de panes tostados de trigo se vio afectada por su diferente estructura, a pesar de que no se observaron diferencias en la percepción sensorial. El proceso de panificación también ofreció la posibilidad de modificar la estructura del pan. De hecho, la variación de la forma de la masa dio lugar a panes con diferentes propiedades estructurales y texturales de la miga. La digestión de los panes con diferente estructura de miga confirmó que se disgregaban de manera diferente, produciendo variaciones en la posterior digestibilidad del almidón. Una vez que se estableció la importancia de la microestructura de la miga en la digestión del almidón, se cambió el enfoque para enlazar la microestructura de los geles de almidón con su digestión in vitro. Los geles obtenidos con almidones de distintas fuentes botánicas mostraron diferente digestibilidad, lo que se relacionó con su microestructura, pero también con su contenido de amilosa. Considerando la acción de las enzimas digestivas (α-amilasa y α-glucosidasa) sobre la hidrólisis del almidón, se estudiaron diferentes compuestos fenólicos para comprender las interacciones entre los compuestos fenólicos y las enzimas o sustratos. La forma más eficaz de inhibir las enzimas era incubarlas con ácidos fenólicos. Se necesitó una mayor concentración del inhibidor cuando los compuestos fenólicos interactuaban previamente con el sustrato, debido a su retención dentro del gel de almidón. La estructura química de los ácidos fenólicos controlaba la inhibición de la enzima. Asimismo, los extractos fenólicos complejos, como los extraídos de las algas A. nodosum, podrían utilizarse para inhibir las enzimas digestivas, mostrando mayor efecto inhibidor cuando fueron previamente incubados con la enzima, debido a la existencia de complejos carbohidrato-polifenoles con sus diferentes capacidades inhibitorias. Además, los ácidos fenólicos afectaron las propiedades de pegado y, por lo tanto, a la estructura y textura de geles de almidón. Sin embargo, esos cambios no fueron suficientes para controlar la hidrólisis enzimática del almidón, que estaba relacionada con la estructura química de los ácidos fenólicos y sus propiedades. En general, la microestructura de la miga o del gel puede limitar la accesibilidad de las enzimas digestivas, lo que reduciría la hidrólisis del almidón. Además, la inclusión de ácidos fenólicos en alimentos a base de almidón podría ser la alternativa para reducir el grado de digestión del almidón al inhibir estas enzimas. / [CA] A causa de la creixent importància de la dieta en el maneig de la salut, segueix sent de gran interès desentranyar com es processen els aliments en el sistema digestiu humà. L'estructura dels aliments pot influir significativament en el processament dels aliments, afectant al seu rendiment durant l'alimentació i la digestió. Específicament, la digestió d'aliments a base de carbohidrats requereix una major comprensió a conseqüència de la seua contribució als nivells de glucosa en sang. El coneixement de la cinètica de la digestió del midó contribuirà a dissenyar aliments a mesura per controlar els nivells de glucosa postprandial. L'objectiu d'aquesta tesi doctoral va ser adquirir una millor comprensió de l'impacte de la microestructura en la digestió del midó i com els enzims digestius podrien ser modulats per l'ús de compostos fenòlics. Amb aquest propòsit, es va avaluar el paper de l'estructura del pa en la masticació in vivo i la digestió in vitro. Posteriorment, es van produir gels de midó de diferents fonts i es van digerir en un sistema de digestió oro-gastrointestinal in vitro per analitzar l'impacte de la microestructura del gel. Després dels estudis de microestructura en gels de midó i pa, es van explorar diferents àcids fenòlics o extractes polifenòlics d'algues com inhibidors dels enzims digestius del midó, i es va avaluar la participació de la microestructura del gel de midó en la digestió enzimàtica. La masticació i la textura de la bitla de pans torrats de blat es va veure afectada per les seues diferencies estructurals, tot i que no es van observar diferències en la percepció sensorial. El procés de panificació va oferir la possibilitat de modificar l'estructura del pa. De fet, la variació de la forma de la massa va donar lloc a pans amb diferents propietats d'estructura i textura de la molla. La digestió dels pans amb diferent estructura de molla va confirmar que es disgregaven de manera diferent, produint variacions en la posterior digestibilitat del midó. Una vegada que es va establir la importància de la microestructura de la molla en la digestió del midó, es va canviar l'enfocament per a enllaçar la microestructura dels gels de midó amb la seua digestió in vitro. Els gels obtinguts amb midó de diferent fonts botàniques van mostrar diferent digestibilitat, el que es va relacionar amb la seua microestructura, però també amb el seu contingut d'amilosa. Considerant l'acció dels enzims digestius (α-amilasa i α-glucosidasa) sobre la hidròlisi del midó, es van estudiar diferents compostos fenòlics per a comprendre les interaccions entre els fenòlics i els enzims o substrats. La forma més eficaç d'inhibir els enzims era incubar-los amb àcids fenòlics. Es va necessitar una major concentració de l'inhibidor quan els compostos fenòlics interactuaven prèviament amb el substrat, a causa de la seua retenció dins del gel de midó. L'estructura química dels àcids fenòlics controlava la inhibició de l'enzim. Així mateix, els extractes fenòlics complexos, com els extrets de l'alga A. nodosum, podrien utilitzar-se per a inhibir els enzims digestius, mostrant major efecte inhibidor quan van ser prèviament incubats amb l'enzim, a causa de l'existència de complexos carbohidrat-polifenols amb les seues diferents capacitats inhibitòries. A més, els àcids fenòlics van afectar les propietats de pegat i, per tant, la estructura i textura dels gels de midó. No obstant, estos canvis en la estructura i textura dels gels no van ser suficients per a controlar la hidròlisi enzimàtica del midó, que estava relacionada amb l'estructura química dels àcids fenòlics i les seues propietats. En general, la microestructura de la molla de pa o gel de midó pot limitar l'accessibilitat dels enzims digestius, la qual cosa reduiria la hidròlisi del midó. A més, la inclusió d'àcids fenòlics en aliments a base de midó podria ser l'alternativa per a reduir el grau de digestió del midó en inhibir aquests enzims. / [EN] Due to the increasing importance of diet on health management, it remains of utmost interest to unravel how food is processed in the human digestive system. Food structure can significantly influence food processing, affecting its performance during eating and digestion. Specifically, the digestion of carbohydrates-based foods requires further insight due to their contribution to blood glucose levels. The knowledge of starch digestion kinetics will contribute to design tailored foods for managing postprandial glucose levels. The objective of this doctoral thesis was to acquire a better understanding of the impact of microstructure on starch digestion and how digestive enzymes might be modulated by the use of phenolic compounds. With that purpose, the role of bread structure on in vivo mastication, and in vitro digestion was evaluated. Subsequently, starch gels from different sources were produced and digested in an in vitro oro-gastro-intestinal digestion system to analyze the impact of gel microstructure. After the microstructure studies on bread and starch gels, different phenolic acids or seaweed polyphenolic extracts were explored as inhibitors of starch digestive enzymes, and the involvement of starch gel microstructure on the enzymatic digestion was assessed. Mastication of toasted wheat breads was affected by their different structures, despite no differences in the sensory perception was observed. Bolus texture was also altered by bread structure and texture. The breadmaking process offered the possibility to modify the bread structure. In fact, varying dough shaping led to breads with different crumb structure and texture properties. After stressing the importance of selecting the in vitro oral processing method used to simulate mastication, the further digestion of the bread with different crumb structure confirmed that they were differently disaggregated yielding variations on posterior starch digestibility. Once stating the importance of crumb microstructure on starch digestion, the focus was shifted to connect starch gels microstructure with its in vitro digestion. Gels obtained with a different type of starch, from cereals, pulses, or tubers, showed different digestibility, which was related to their microstructure but also their amylose content. Considering the action of digestive enzymes (α-amylase and α-glucosidase) on starch hydrolysis, different phenolic compounds were studied to understand the interactions between phenolics and either enzymes or substrates. The most effective way to inhibit enzymes was to incubate them with phenolic acids. A higher concentration of the inhibitor was needed when phenolic compounds interacted previously with the substrate, due to their retention within the starch gel. The chemical structure of phenolic acids controlled the enzyme inhibition. Similarly, complex phenolic extracts, like those extracted from A. nodosum seaweed could be used to inhibit digestive enzymes, showing greater inhibition effect when they were previously incubated with the enzyme, owing to the existence of carbohydrate-polyphenol complexes their different inhibitory capabilities. In addition, phenolic acids affected pasting properties and therefore gel microstructure and gel texture of starches. However, those changes on gels microstructure and texture were not enough to control starch enzymatic hydrolysis, which was related to the specific chemical structure of the phenolic acids and their properties. Overall, crumb or gel microstructure can limit digestive enzymes accessibility, which would reduce the starch hydrolysis. Moreover, the inclusion of phenolic acids on starch-based foods might be the alternative to reduce the extent of starch digestion by inhibiting digestive enzymes. / Authors acknowledge the financial support of the Spanish Ministry of Science, Innovation and Universities (Project RTI2018-095919-B-C21) funded by MCIN/AEI/10.13039/501100011033, “ERDF A way of making Europe” by the “European Union”, Generalitat Valenciana (Project Prometeo 2017/189) and Xunta de Galicia (ED431B 2019/01). This work is based upon the work from COST Action 18101 SOURDOMICS – Sourdough biotechnology network towards novel, healthier and sustainable food and bioprocesses, where A. Aleixandre was supported by COST (European Cooperation in Science and Technology). COST is a funding agency for research and innovation networks. / Aleixandre Agustín, A. (2022). Going from Digestion to Microstructure of Starch-Based Food Products: Understanding the Role of Polyphenols [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/181137 / TESIS / Compendio

Ceramid, ein zentrales Signalmolekül des Sphingolipidstoffwechsels, ist neben der de novo Synthese über die enzymatische Spaltung von Sphingomyelin und Glucosylceramid zugänglich. Genetische Mutationen, die eine Fehlfaltung der verantwortlichen Enzyme saure Sphingomyelinase (aSMase) und Glucocerebrosidase (GCase) begünstigen, könnten somit zu einer Dysregulation des gesamten Sphingolipidstoffwechsels und den damit verbundenen Signaltransduktionsprozessen führen. Niedermolekulare Inhibitoren können in Zellstudien einen Einblick in diese Prozesse geben und den Defekt eines Enzyms simulieren oder eine etwaige Überaktivität derselben Enzyme verhindern. Für derartige Studien ist die Möglichkeit einer zeitaufgelösten Inhibition von Vorteil. Für diese Methode müssten photolabile Schutzgruppen in eine bereits bekannte Inhibitorstruktur integriert werden. Im Fall der aSMase würden sich hierfür myo-Inosit-bisphosphat-Derivate anbieten, die starke, kompetitive Inhibitoren des Enzyms darstellen. Auf dieser Grundlage werden in der vorliegenden Arbeit die Synthese sowie die in vitro und in cellulo Wirkung des ersten zellpermeablen, photoaktivierbaren Inhibitors für die aSMase präsentiert. Kompetitive Inhibitoren können ebenso als sogenannte pharmakologische Chaperone fungieren, welche Proteine durch Herabsetzung der freien Energie des jeweiligen Faltungszustandes stabilisieren. Dies ist besonders bei von Mutationen betroffenen lysosomalen Enzymen von Interesse, um diese vor einem proteasomalen Abbau zu bewahren und einen geregelten Transport in die Lysosomen zu gewährleisten. So wurden in der vorliegenden Arbeit verschiedene myo-Inositderivate als potenzielle pharmakologische Chaperone für die aSMase und GCase synthetisiert. Um eine Verdrängung der Verbindungen vom aktiven Zentrum des Enzyms durch das natürliche Substrat zu beschleunigen, wurde eine Orthoesterfunktion in die Seitenkette der Inhibitorstruktur integriert, die im sauren Milieu der Lysosomen gespalten werden kann. / Ceramide, a central signaling molecule in sphingolipid metabolism, is in addition to the novo synthesis accessible via the enzymatic cleavage of sphingomyelin and glucosylceramide. Genetic mutations that promote misfolding of the responsible enzymes acid sphingomyelinase (aSMase) and glucocerebrosidase (GCase) could thus lead to a dysregulation of the entire sphingolipid metabolism and the associated signal transduction processes. Small molecule inhibitors can provide insight into these processes in cell studies and simulate the defect of an enzyme or prevent eventual overactivity of the same enzyme. For such studies, the possibility of a time-resolved inhibition would be advantageous. For this method, photolabile protecting groups would have to be integrated into the structure of a known inhibitor. In the case of aSMase, myo-inositol-diphosphate derivatives, which represent strong, competitive inhibitors of the enzyme, would be suitable for this purpose. On this basis, the synthesis as well as the in vitro and in cellulo effects of the first cell-permeable photocaged inhibitor for acid sphingomyelinase are presented in this work. Competitive inhibitors can also act as so-called pharmacological chaperones, which stabilize proteins by reducing the free energy of the respective folding state. This is of particular interest in the case of lysosomal enzymes affected by mutations, in order to protect them from proteasomal degradation and to ensure regulated transport into the lysosomes. In the present work, various myo-inositol derivatives were synthesized as potential pharmacological chaperones for aSMase and GCase. To accelerate displacement of the compounds from the enzyme's active site by the natural substrate, an orthoester function was integrated into the side chain of the inhibitor structure, which can be cleaved in the acidic environment of the lysosome.

No description available.

N-glycosylation is the most common eukaryotic post-translational modification, impacting on protein stability, folding, and protein-protein interactions. More broadly, N-glycans play biological roles in reaction kinetics modulation, intracellular protein trafficking, and cell-cell communications. The machinery responsible for the initial stages of N-glycan assembly and processing is found on the membrane of the endoplasmic reticulum. Following N-glycan transfer to a nascent glycoprotein, the enzyme Processing α-Glucosidase I (GluI) catalyzes the selective removal of the terminal glucose residue. GluI is a highly substrate-specific enzyme, requiring a minimum glucotriose for catalysis; this glycan is uniquely found in biology in this pathway. The structural basis of the high substrate selectivity and the details of the mechanism of hydrolysis of this reaction have not been characterized. Understanding the structural foundation of this unique relationship forms the major aim of this work. To approach this goal, the S. cerevisiae homolog soluble protein, Cwht1p, was investigated. Cwht1p was expressed and purified in the methyltrophic yeast P. pastoris, improving protein yield to be sufficient for crystallization screens. From Cwht1p crystals, the structure was solved using mercury SAD phasing at a resolution of 2 Å, and two catalytic residues were proposed based upon structural similarity with characterized enzymes. Subsequently, computational methods using a glucotriose ligand were applied to predict the mode of substrate binding. From these results, a proposed model of substrate binding has been formulated, which may be conserved in eukaryotic GluI homologs.

N-glycosylation is the most common eukaryotic post-translational modification, impacting on protein stability, folding, and protein-protein interactions. More broadly, N-glycans play biological roles in reaction kinetics modulation, intracellular protein trafficking, and cell-cell communications. The machinery responsible for the initial stages of N-glycan assembly and processing is found on the membrane of the endoplasmic reticulum. Following N-glycan transfer to a nascent glycoprotein, the enzyme Processing α-Glucosidase I (GluI) catalyzes the selective removal of the terminal glucose residue. GluI is a highly substrate-specific enzyme, requiring a minimum glucotriose for catalysis; this glycan is uniquely found in biology in this pathway. The structural basis of the high substrate selectivity and the details of the mechanism of hydrolysis of this reaction have not been characterized. Understanding the structural foundation of this unique relationship forms the major aim of this work. To approach this goal, the S. cerevisiae homolog soluble protein, Cwht1p, was investigated. Cwht1p was expressed and purified in the methyltrophic yeast P. pastoris, improving protein yield to be sufficient for crystallization screens. From Cwht1p crystals, the structure was solved using mercury SAD phasing at a resolution of 2 Å, and two catalytic residues were proposed based upon structural similarity with characterized enzymes. Subsequently, computational methods using a glucotriose ligand were applied to predict the mode of substrate binding. From these results, a proposed model of substrate binding has been formulated, which may be conserved in eukaryotic GluI homologs.