Split Inteins: From Mechanistic Studies to Novel Protein Engineering Technologies

Inteins are auto-processing protein domains that carry out a post-translational process known as protein splicing. This process is characterized by excision of the intein (intervening protein) domain from within a larger polypeptide sequence with concomitant ligation of the flanking extein ( external protein) regions through a native peptide bond. Remarkably, a small subset of all inteins are naturally transcribed and translated as two fragments that efficiently associate and carry out the same biochemical process in trans, and these split inteins are potentially powerful tools for protein engineering. Recently, a split intein from the cyanobacterium Nostoc punctiforme (Npu) was discovered that can carry out protein splicing with a half-life of one minute, as opposed to hours as seen for previously characterized split and contiguous inteins. Inspired by the apparent uniqueness of this “ultrafast” splicing activity and its practical implications, we characterized several orthologous split inteins from the same family as Npu. Surprisingly, many of these inteins splice as quickly as Npu, and biochemical characterization of this family divulged sequence-activity correlations that provided insights into the molecular determinants for fast protein trans-splicing. Importantly, several of these inteins are extraordinarily efficient in their first auto-processing step, peptide bond cleavage coupled to thioester formation. Harnessing this property, along with efficient fragment association, a streamlined iteration of Expressed Protein Ligation (EPL), the most prevalent protein semi-synthesis technique, was developed. Further insights into protein splicing were obtained by the development of a novel kinetic assay that allowed for quantitative observation of a crucial intermediate in the protein splicing pathway, the branched intermediate (BI). Using this assay, BI resolution was unambiguously identified as the rate limiting step for Npu splicing. Furthermore, the roles of extein residues in individual steps along the splicing pathway were teased apart. Using protein semi-synthesis, kinetic measurements, and structural techniques, C-extein composition was found to be intimately linked to active-site dynamics and BI resolution kinetics. In addition to chemical reactivity, the fragment assembly of Npu was also characterized. Mutation of charged residues at the binding interface demonstrated that split intein binding affinity was dominated by intermolecular electrostatic interactions. By swapping charged residues between the intein fragments, a new split intein was engineered with orthogonal binding and reactivity to the wild-type Npu split intein. The wild-type and charges wapped inteins could be used in protein semi-synthesis endeavors requiring parallel selective splicing reactions in one pot. Finally, using a combination of biophysical techniques, the mechanism of split intein assembly was elucidated. Our analyses indicated that the assembly follows a unique trajectory comprised of coupled binding and folding of disordered regions of each fragment followed by a collapse of the structure into a stable functional domain. Collectively, these structural and functional studies not only provide insights into the inner workings of inteins but will also continue to aid in the development of important protein engineering technologies

Biological Importance of Intrinsically Destructured Proteins (IDPs)

[Abstract] IDPs are proteins without stable structure, which perform multiple functions, many of them, essential for the cell. Due to their structural properties, they require structural techniques different from those used in classical structure determination. They can interact specifically with many different proteins DNA and RNA. Their specific interaction with different is with low affinity, therefore, easy to reverse, giving them relevant roles in signaling and regulation. They are also important from an evolutive perspective and in drug development. Some IDPs change their folding state upon interaction through some regions, increasing the number of conformations. In addition, they may suffer different types of postranslational modifications that lead to multiple structures for a unique protein and, therefore, different functions.[Resumen] Las IDPs son proteínas sin estructura estable que cumplen una gran variedad de funciones, muchas de ellas, imprescindibles para la célula. Debido a sus características estructurales, requieren de técnicas diferentes de las clásicas para su estudio y pueden interaccionar de forma específica con numerosas proteínas, DNA y RNA. Su interacción específica con los diferentes ligandos es de baja afinidad, y por tanto, fácilmente reversible, de forma que son proteínas con gran relevancia en señalización y regulación. También son importantes desde el punto de vista evolutivo y en el desarrollo de medicamentos. En algunos casos, cambian su estado de plegamiento al interaccionar a través de ciertas regiones, aumentando así el número de conformaciones en que se encuentran. A esto hay que sumarle las diferentes modificaciones postraduccionales que pueden sufrir, dando lugar a múltiples estructuras para una misma proteína y a diferentes funciones.Traballo fin de grao (UDC.CIE). Bioloxía. Curso 2019/202

Functionally Relevant Macromolecular Interactions of Disordered Proteins

Disordered proteins are relatively recent newcomers in protein science. They were first described in detail by Wright and Dyson, in their J. Mol. Biol. paper in 1999. First, it was generally thought for more than a decade that disordered proteins or disordered parts of proteins have different amino acid compositions than folded proteins, and various prediction methods were developed based on this principle. These methods were suitable for distinguishing between the disordered (unstructured) and structured proteins known at that time. In addition, they could predict the site where a folded protein binds to the disordered part of a protein, shaping the latter into a well-defined 3D structure. Recently, however, evidence has emerged for a new type of disordered protein family whose members can undergo coupled folding and binding without the involvement of any folded proteins. Instead, they interact with each other, stabilizing their structure via “mutual synergistic folding” and, surprisingly, they exhibit the same residue composition as the folded protein. Increasingly more examples have been found where disordered proteins interact with non-protein macromolecules, adding to the already large variety of protein–protein interactions. There is also a very new phenomenon when proteins are involved in phase separation, which can represent a weak but functionally important macromolecular interaction. These phenomena are presented and discussed in the chapters of this book

The Exonuclease Xrn1 : a key regulator of gene expression under physiological and perturbed conditions

The highly conserved exonuclease Xrn1 plays a dual role in gene expression by degrading cellular mRNAs and promoting their transcription initiation and elongation. In this thesis we uncover an unanticipated role of Xrn1 in translational control under physiological conditions and in cell adaptation and survival under osmotic stress conditions. Here we show that Xrn1 promotes translation of brome mosaic virus (BMV) RNAs in Saccharomyces cerevisiae. By integrating ribosome profiling analysis to functional and biochemical studies we report a broader role of Xrn1 in translation initiation of a subset of yeast mRNAs encoding membrane proteins. Xrn1-dependent yeast transcripts, as the viral ones, harbor highly structural traits around the translation initiation site (TIS) that confers a poor context for translation initiation. Interestingly, functional studies indicated that the unstructured C-terminal domain of Xrn1 interacts with components of the translation initiation machinery to facilitate protein synthesis and that Xrn1 mediates the correct localization of Xrn1-dependent mRNAs at the endoplasmic reticulum, the cellular translation compartment where membrane proteins are synthesized. Importantly, Xrn1 promotes transcription, translation and decay of the same group of mRNAs. Together, our results reveal a novel crosstalk between the three major steps of gene expression coordinated by Xrn1 to finely tune expression of membrane proteins. We surmise that this linkage has evolved to avoid toxic aggregations, as membrane proteins contain hydrophobic domains prone to aggregate. Not only is gene expression an important cellular process under physiological conditions, but it also plays a key role in the adaptation and survival of cells to changing environmental conditions. Importantly, previous studies linked Xrn1 to the regulation of yeast mRNA homeostasis in response to glucose deprivation. In this thesis we show that Xrn1 modulates cellular transcriptional and translational responses upon hyper-osmotic shock by combining genome-wide RNA sequencing with functional and biochemical analyses. Microscopy imaging revealed that Xrn1 localizes to stress-induced aggregates shortly after osmotic shock in a manner dependent on its unstructured C-terminal domain. This localization is mediated by the major signal integrator Snf1 adenosine monophosphate-activated protein kinase (AMPK). Under these conditions Xrn1 maintains a diminished exonuclease activity and assists in the transcriptional and translational activation of a subset of osmo-induced genes that are enriched for proteins interacting with Hog1, the main mitogen-activated protein kinase involved in osmoregulation. Based on the evidence provided we claim that the exonuclease Xrn1 links the Snf1 and Hog1 pathways to control gene expression upon osmotic stress. Collectively, our results point to Xrn1 as a key regulator of gene expression under physiological and perturbed conditions.La exonucleasa altamente conservada Xrn1 desempeña un doble papel en la expresión génica al degradar los ARNm celulares y promover la iniciación y el alargamiento de la transcripción de los mismos. En esta tesis descubrimos un papel no anticipado de Xrn1 en el control de la traducción de ARNm bajo condiciones fisiológicas y en la adaptación y supervivencia celular bajo estrés osmótico. Aquí mostramos que Xrn1 promueve la traducción de ARN del virus del mosaico del bromo (BMV) en Saccharomyces cerevisiae. Al integrar el análisis de perfiles de ribosomas a estudios funcionales y bioquímicos, confirmamos un papel más amplio de Xrn1 en el inicio de la traducción de un subconjunto de ARNm de levadura que codifica proteínas de membrana. Los tránscritos celulares dependientes de Xrn1, como los virales, albergan rasgos altamente estructurales alrededor del sitio de inicio de la traducción (TIS) que confieren un contexto pobre para el inicio de la traducción. Además, estudios funcionales indicaron que el dominio C-terminal no estructurado de Xrn1 interactúa con los componentes de la maquinaria de iniciación de la traducción para facilitar la síntesis de proteínas y que Xrn1 media la localización correcta de los ARNm dependientes de Xrn1 en el retículo endoplasmático, el compartimento de traducción celular donde las proteínas de membrana son sintetizadas. Es importante destacar que Xrn1 promueve la transcripción, traducción y degradación del mismo grupo de ARNm. Juntos, nuestros resultados revelan una nueva diafonía entre los tres pasos principales de la expresión génica coordinada por Xrn1 para regular la expresión de proteínas de membrana. Suponemos que este enlace ha evolucionado para evitar agregaciones tóxicas, ya que las proteínas de membrana contienen dominios hidrófobos propensos a agregarse. La expresión génica no solo es un proceso celular importante en condiciones fisiológicas, sino que también juega un papel clave en la adaptación y supervivencia de las células a condiciones ambientales cambiantes. Es importante destacar que estudios previos vincularon Xrn1 a la regulación de la homeostasis de ARNm de levadura en respuesta a la privación de glucosa. En esta tesis hemos combinado la secuenciación de ARN de todo el genoma con análisis funcionales y bioquímicos y hemos descubierto que Xrn1 modula la transcripción y traducción de ARNm tras un choque hiperosmótico. Las imágenes de microscopía revelaron que, poco después de añadir sal a las células, Xrn1 se localiza en agregados citosólicos inducidos por el estrés de una manera dependiente de su dominio C-terminal no estructurado. Esta localización está mediada por la proteína quinasa activada por AMP Snf1. En estas condiciones, Xrn1 mantiene una actividad exonucleasa disminuida y ayuda a la activación transcripcional y traduccional de un subconjunto de genes inducidos en condiciones de estrés que están enriquecidos en proteínas que interactúan con Hog1, la principal proteína quinasa activada por mitógeno involucrada en la osmoregulación. Con base en la evidencia proporcionada, afirmamos que la exonucleasa Xrn1 une las vías Snf1 y Hog1 para controlar la expresión génica bajo estrés osmótico. En conjunto, nuestros resultados apuntan a Xrn1 como un regulador clave de la expresión génica en condiciones fisiológicas y cambiantes

Proteome-wide Screening for Mitogen-Activated Protein Kinase Docking Motifs and Interactors

AbstractProteome-wide screening for mitogen-activated protein kinase docking motifs and interactors Guangda Shi 2021 Kinases catalyze the transfer of phosphate to substrates, a reaction critical for many cellular events. Mitogen-activated protein kinases (MAPKs), including ERK, p38, and JNK, phosphorylate hundreds of substrates, and each plays a pivotal role in distinct processes such as cell growth, survival, differentiation, and apoptosis. To correctly respond to external stimuli, MAPKs use several mechanisms to achieve a high degree of selectivity for their target substrates. A critical aspect of MAPK specificity comes from docking interactions occurring between sites distal from the site of catalysis and short linear sequence motifs located in MAPK interactors. These docking sequences conform to a general motif of ψ(1-3)-X(2-6)-ΦΦ-X-Φ (ψ: basic residue, X: any residue, Φ: hydrophobic residue). However, sites conforming to this motif can nonetheless bind specifically to any of the distinct MAPK subfamilies. MAPK preference for docking sites is therefore coded within sub-motifs falling within the consensus. Here I present yeast-based genetic screens to identify docking sequences selectively binding to either JNK or p38 MAPKs. First, I comprehensively characterized all possible amino acid substitutions with two known docking sites, revealing new information about amino acid residues critical for MAPK binding. Second, I screened for functional p38α and JNK1 binding sequences from the human proteome. I subsequently picked 36 peptide sequences selected in these screens and determined their binding affinity to both p38 and JNK. Over 90% of the peptides showed the predicted binding specificity, thus validating the screen results. I have also validated that hit sequences can serve to recruit MAPKs to phosphorylate protein substrates. Systematic analysis of sequences selected by each MAPK revealed key features conferring MAPK specificity. This work has provided unbiased insights into MAPK substrate specificity, and also suggests new biological processes regulated by MAPKs

Beyond hairballs: depicting complexity of a kinase-phosphatase network in the budding yeast

Les kinases et les phosphatases (KP) représentent la plus grande famille des enzymes dans la cellule. Elles régulent les unes les autres ainsi que 60 % du protéome, formant des réseaux complexes kinase-phosphatase (KP-Net) jouant un rôle essentiel dans la signalisation cellulaire. Ces réseaux caractérisés d’une organisation de type commandes-exécutions possèdent généralement une structure hiérarchique. Malgré les nombreuse études effectuées sur le réseau KP-Net chez la levure, la structure hiérarchique ainsi que les principes fonctionnels sont toujours peux connu pour ce réseau. Dans ce contexte, le but de cette thèse consistait à effectuer une analyse d’intégration des données provenant de différentes sources avec la structure hiérarchique d’un réseau KP-Net de haute qualité chez la levure, S. cerevisiae, afin de générer des hypothèses concernant les principes fonctionnels de chaque couche de la hiérarchie du réseau KP-Net. En se basant sur une curation de données d’interactions effectuée dans la présente et dans d’autres études, le plus grand et authentique réseau KP-Net reconnu jusqu’à ce jour chez la levure a été assemblé dans cette étude. En évaluant le niveau hiérarchique du KP-Net en utilisant la métrique de la centralisation globale et en élucidant sa structure hiérarchique en utilisant l'algorithme vertex-sort (VS), nous avons trouvé que le réseau KP-Net possède une structure hiérarchique ayant la forme d’un sablier, formée de trois niveaux disjoints (supérieur, central et inférieur). En effet, le niveau supérieur du réseau, contenant un nombre élevé de KPs, était enrichi par des KPs associées à la régulation des signaux cellulaire; le niveau central, formé d’un nombre limité de KPs fortement connectées les unes aux autres, était enrichi en KPs impliquées dans la régulation du cycle cellulaire; et le niveau inférieur, composé d’un nombre important de KPs, était enrichi en KPs impliquées dans des processus cellulaires diversifiés. En superposant une grande multitude de propriétés biologiques des KPs sur le réseau KP-Net, le niveau supérieur était enrichi en phosphatases alors que le niveau inférieur en était appauvri, suggérant que les phosphatases seraient moins régulées par phosphorylation et déphosphorylation que les kinases. De plus, le niveau central était enrichi en KPs représentant des « bottlenecks », participant à plus d’une voie de signalisation, codées par des gènes essentiels et en KPs qui étaient les plus strictement régulées dans l’espace et dans le temps. Ceci implique que les KPs qui jouent un rôle essentiel dans le réseau KP-Net devraient être étroitement contrôlées. En outre, cette étude a montré que les protéines des KPs classées au niveau supérieur du réseau sont exprimées à des niveaux d’abondance plus élevés et à un niveau de bruit moins élevé que celles classées au niveau inférieur du réseau, suggérant que l’expression des enzymes à des abondances élevées invariables au niveau supérieur du réseau KP-Net pourrait être importante pour assurer un système robuste de signalisation. L’étude de l’algorithme VS a montré que le degré des nœuds affecte leur classement dans les différents niveaux d’un réseau hiérarchique sans biaiser les résultats biologiques du réseau étudié. En outre, une analyse de robustesse du réseau KP-Net a montré que les niveaus du réseau KP-Net sont modérément stable dans des réseaux bruités générés par ajout d’arrêtes au réseau KP-Net. Cependant, les niveaux de ces réseaux bruités et de ceux du réseau KP-Net se superposent significativement. De plus, les propriétés topologiques et biologiques du réseau KP-Net étaient retenues dans les réseaux bruités à différents niveaux. Ces résultats indiquant que bien qu’une robustesse partielle de nos résultats ait été observée, ces derniers représentent l’état actuel de nos connaissances des réseaux KP-Nets. Finalement, l’amélioration des techniques dédiées à l’identification des substrats des KPs aideront davantage à comprendre comment les réseaux KP-Nets fonctionnent. À titre d’exemple, je décris, dans cette thèse, une stratégie que nous avons conçu et qui permet à déterminer les interactions KP-substrats et les sous-unités régulatrices sur lesquelles ces interactions dépendent. Cette stratégie est basée sur la complémentation des fragments de protéines basée sur la cytosine désaminase chez la levure (OyCD PCA). L’OyCD PCA représente un essai in vivo à haut débit qui promet une description plus précise des réseaux KP-Nets complexes. En l’appliquant pour déterminer les substrats de la kinase cycline-dépendante de type 1 (Cdk1, appelée aussi Cdc28) chez la levure et l’implication des cyclines dans la phosphorylation de ces substrats par Cdk1, l’essai OyCD PCA a montré un comportement compensatoire collectif des cyclines pour la majorité des substrats. De plus, cet essai a montré que la tubuline- γ est phosphorylée spécifiquement par Clb3-Cdk1, établissant ainsi le moment pendant lequel cet événement contrôle l'assemblage du fuseau mitotique.Kinases and phosphatases (KP) form the largest family of enzymes in living cells. They regulate each other and 60 % of the proteome forming complex kinase-phosphatase networks (KP-Net) essential for cell signaling. Such networks having the command-execution aspect tend to have a hierarchical structure. Despite the extensive study of the KP-Net in the budding yeast, the hierarchical structure as well as the functional principles of this network are still not known. In this context, this thesis aims to perform an integrative analysis of multi-omics data with the hierarchical structure of a bona fide KP-Net in the budding yeast Saccharomyces cerevisiae, in order to generate hypotheses about the functional principles of each layer in the KP-Net hierarchy. Based on a literature curation effort accomplished in this and in other studies, the largest bona fide KP-Net of the S. cerevisiae known to date was assembled in this thesis. By assessing the hierarchical level of the KP-Net using the global reaching centrality and by elucidating the its hierarchical structure using the vertex-sort (VS) algorithm, we found that the KP-Net has a moderate hierarchical structure made of three disjoint layers (top, core and bottom) resembling a bow tie shape. The top layer having a large size was found enriched for signaling regulation; the core layer made of few strongly connected KPs was found enriched mostly for cell cycle regulation; and the bottom layer having a large size was found enriched for diverse biological processes. On overlaying a wide range of KP biological properties on top of the KP-Net hierarchical structure, the top layer was found enriched for and the bottom layer was found depleted for phosphatases, suggesting that phosphatases are less regulated by phosphorylation and dephosphoryation interactions (PDI) than kinases. Moreover, the core layer was found enriched for KPs representing bottlenecks, pathway-shared components, essential genes and for the most tightly regulated KPs in time and space, implying that KPs playing an essential role in the KP-Net should be firmly controlled. Interestingly, KP proteins in the top layer were found more abundant and less noisy than those of the bottom layer, suggesting that availability of enzymes at invariable protein expression level at the top of the network might be important to ensure a robust signaling. Analysis of the VS algorithm showed that node degrees affect their classification in the different layers of a network hierarchical structure without biasing biological results of the sorted network. Robustness analysis of the KP-Net showed that KP-Net layers are moderately stable in noisy networks generated by adding edges to the KP-Net. However, layers of these noisy overlap significantly with those of the KP-Net. Moreover, topological and biological properties of the KP-Net were retained in the noisy networks to different levels. These findings indicate that despite the observed partial robustness of our results, they mostly represent our current knowledge about KP-Nets. Finally, enhancement of techniques dedicated to identify KPs substrates will enhance our understanding about how KP-Nets function. As an example, I describe here a strategy that we devised to help in determining KP-substrate interactions and the regulatory subunits on which these interactions depend. The strategy is based on a protein-fragment complementation assay based on the optimized yeast cytosine deaminase (OyCD PCA). The OyCD PCA represents a large scale in vivo screen that promises a substantial improvement in delineating the complex KP-Nets. We applied the strategy to determine substrates of the cyclin-dependent kinase 1 (Cdk1; also called Cdc28) and cyclins implicated in phosphorylation of these substrates by Cdk1 in S. cerevisiae. The OyCD PCA showed a wide compensatory behavior of cyclins for most of the substrates and the phosphorylation of γ-tubulin specifically by Clb3-Cdk1, thus establishing the timing of the latter event in controlling assembly of the mitotic spindle

Regulation of protein transport into the ER by phosphorylation of Sbh1/Sec61β

The ER protein translocation channel subunit Sbh1 is non-essential, but contains multiple phosphorylation sites suggesting a regulatory role in ER protein import. It has been already shown that mutating two N-terminal, proline-flanked, phosphorylation sites in the Sbh1 cytosolic domain phenocopies the temperature-sensitivity of a yeast strain lacking SBH1/SBH2, and results in reduced translocation into the ER of an Sbh1-dependent substrate, Gls1. In the present work I characterized the sbh1 mutant strains. I also identified targeting signals that are Sbh1-dependent, Phospho-Sbh1 dependent, or Ess1-dependent. In a high content microscopic screen, I identified about 12% of secretory proteins assayed as Sbh1-dependent and I found that Sbh1-dependent proteins have suboptimal ER targeting sequences, with lower hydrophobicity and frequently without or with an inverse charge bias. A smaller fraction of proteins was dependent on N-terminal phosphorylation of Sbh1. I also developed and optimized different screens for finding the kinase responsible for S3/T5-Sbh1 phosphorylation, with no conclusive result. I conclude that Sbh1 promotes ER import of substrates with suboptimal targeting sequences and its activity can be regulated by a conformational change induced by N-terminal phosphorylation and I suggested a model for ER protein translocation regulation.Die ER-Protein-Translokationskanal-Untereinheit Sbh1 ist nicht essentiell, enthält aber mehrere Phosphorylierungsstellen, was auf eine regulatorische Rolle beim ER-Proteinimport hindeutet. Es wurde bereits gezeigt, dass die Mutation von zwei N-terminalen, Prolin-flankierten Phosphorylierungsstellen in der zytosolischen Sbh1-Domäne die Temperaturempfindlichkeit eines Hefestamms, dem SBH1/SBH2 fehlt, phänokopiert und zu einer verringerten Translokation in das ER eines Sbh1-Abhängigen führt Substrat, Gls1. In der vorliegenden Arbeit habe ich die sbh1-Mutantenstämme charakterisiert. Ich habe auch Targeting-Signale identifiziert, die Sbh1-abhängig, Phospho-Sbh1-abhängig oder Ess1-abhängig sind. In einem mikroskopischen High-Content-Screen identifizierte ich etwa 12 % der sekretorischen Proteine, die als Sbh1-abhängig getestet wurden, und ich fand heraus, dass Sbh1-abhängige Proteine suboptimale ER-Targeting-Sequenzen mit geringerer Hydrophobizität und häufig ohne oder mit einer inversen Ladungsverzerrung aufweisen. Ein kleinerer Teil der Proteine war von der N-terminalen Phosphorylierung von Sbh1 abhängig. Ich habe auch verschiedene Screens entwickelt und optimiert, um die für die S3/T5-Sbh1-Phosphorylierung verantwortliche Kinase zu finden, ohne schlüssiges Ergebnis. Ich schlussfolgere, dass Sbh1 den ER-Import von Substraten mit suboptimalen Targeting-Sequenzen fördert und seine Aktivität durch eine durch N-terminale Phosphorylierung induzierte Konformationsänderung reguliert werden kann, und ich schlug ein Modell für die Regulation der ER-Proteintranslokation vor

Determining the Role of Nudt7 in the Regulation of Cellular CoA Levels and Metabolism

Coenzyme A (CoA) is an essential cofactor required for hundreds of metabolic processes. Because it is such a critical cofactor, CoA levels are tightly regulated. In the fasted state and in diabetic mice, the concentration of CoA increases dramatically in the liver. This phenotype is associated with constitutively low CoA degradation, a process that is emerging as a potentially important mechanism for CoA regulation. Nudt7 and Nudt19 are two mammalian peroxisomal enzymes with CoA-degrading activity, which are highly expressed in the liver and kidney, respectively. Limited information is available on the biochemistry of Nudt7 and Nudt19; the structural basis for their distinct features and the extent to which Nudt7 contributes to maintaining homeostatic CoA levels in vivo are currently unknown. We used a combination of techniques including mutagenesis, molecular modeling, and enzymatic assays on purified proteins, plus metabolomics and measurement of fatty acid oxidation in whole tissue homogenates and intact hepatocytes to: 1) characterize the biochemical, structural, and regulatory properties of Nudt7 and Nudt19 and 2) determine the effects that manipulations of Nudt7 expression have on CoA levels and lipid metabolism in mouse liver. This research establishes the importance of Nudt7-dependent CoA degradation in the regulation of select acyl-CoA species and the output of peroxisomal metabolic pathways such as bile acid synthesis and peroxisomal fatty acid oxidation

Cellular response to external signals in S. cerevisiae

En esta tesis, se utilizó como organismo modelo la levadura Saccharomyces cerevisiae para obtener conocimiento fundamental sobre una variedad de mecanismos moleculares utilizados por la célula para responder y adaptarse a los estímulos externos. En respuesta al aumento de la osmolaridad externa, la ruta de señalización MAPK High Osmolarity Glycerol (HOG) se activa para mediar cambios en diversas funciones celulares, incluyendo la reprogramación global de la transcripción y traducción, con el fin último de lograr la adaptación. Tras la detección de alta osmolaridad externa, la señal se transduce a través de dos ramas funcionalmente redundantes pero mecanísticamente distintas, SLN1 y SHO1, para finalmente activar el efector MAPK Hog1. Aunque múltiples interacciones entre sensores, proteínas adaptadoras y los componentes de señalización de la rama SHO1 se han descrito anteriormente, esta tesis caracteriza aún más la complejidad de los perfiles de las interacciones para dilucidar cómo se propaga la señal con eficacia y cómo se logra la fidelidad de la señal. En este sentido, hemos utilizado el nuevo método M-track que detecta tanto las interacciones entre proteínas de corta duración como las estables y hemos realizado varias observaciones interesantes. Una vez que se transduce la señal de respuesta a estrés, Hog1 activado entra en el núcleo de la célula y, entre sus más destacados funciones aguas abajo, modula los cambios de expresión génica global. El complejo de unión a la caperuza CBC (por cap-binding complex), que está formado por las proteínas de unión a RNA Cbc1 y Cbc2, se asocia con la caperuza en 5' del mensajero co-transcripcionalmente y se ha descrito su función en diversos aspectos de la vida de mensajero, incluyendo la transcripción. Utilizando técnicas de biología molecular y genómica, describimos Cbc1 como regulador de la transcripción global, tanto en condiciones de estrés y no estrés, para mediar la expresión génica de manera que se consigan niveles altos de una forma rápida. En respuesta a feromonas del tipo sexual contrario, la ruta de MAPK de feromonas de células de levadura haploides se activa para mediar una serie de cambios fisiológicos en preparación para el apareamiento, que incluyen la reprogramación de la expresión génica, la parada del ciclo celular, la formación de una proyección sexual denomina ¨shmoo¨, y en última instancia la fusión celular de los tipos de células de apareamiento contrario. El factor de traducción eIF5A, esencial y conservado evolutivamente, se ha descrito recientemente que funciona en la traducción de proteínas que contienen tres o más residuos de prolina consecutivos (polyPro), a través de su unión a ribosomas para aliviar el estancamiento del ribosoma durante la formación del enlace péptidico entre prolinas. La activación de eIF5A requiere una modificación post-traduccional única, la hipusinación, donde el residuo de hipusina se deriva de la espermidina, un factor esencial para la fertilidad de los mamíferos y que se requiere para el apareamiento de la levadura. Aquí se investigó eIF5A como regulador de la respuesta a feromonas a través de la traducción de proteínas polyPro con funciones en el apareamiento.In this thesis, we utilised the model organism the budding yeast Saccharomyces cerevisiae to gain fundamental knowledge on a variety of molecular mechanisms employed by the cell to respond and adapt to external stimuli. In response to increased external osmolarity, the yeast high osmolarity glycerol (HOG) MAPK pathway becomes activated to mediate changes to various cellular functions, including changes in glycerol accumulation, cell-cycle arrest, re-establishment of ion homeostasis and global reprogrammation of transcription and translation of the whole genome, in order to achieve adaptation. Upon detection of high external osmolarity, signal is transduced via two functionally redundant but mechanistically distinct branches, SLN1 and SHO1, to finally activate the effector MAPK Hog1. Although multiple interactions between osmosensors, adaptor proteins and signalling components of the SHO1 branch have been previously described, this thesis further characterises the complexity of their interaction profiles to elucidate how signal is effectively propagated and how signal fidelity is achieved. Here, we utilised the novel M-track method, which detects both short-lived and stable protein interactions, and made several interesting observations. Once the osmostress signal is transduced, activated Hog1 enters the cell nucleus and, amongst its most prominent downstream functions, modulates global gene expression changes. Within minutes of shock, global transcription rate rapidly decreases by 50 %, however the transcription machinery is reallocated to specific genes implicated in osmostress cellular protection and their transcription is strongly and rapidly induced. The cap-binding complex (CBC), consisting of mRNA-binding proteins Cbc1 and Cbc2, associates with the 5´ mRNA cap co-transcriptionally and has been described to function in various aspects of the mRNA life, including transcription. Utilising both genomic and molecular biology techniques, we describe Cbc1 as a global transcription regulator, both under stress and non-stress conditions, to mediate high and timely gene expression. In response to pheromones of the opposite mating type, the yeast pheromone MAPK pathway is activated to mediate a series of physiological changes in preparation for mating; including reprogrammation of gene expression, cell-cycle arrest, formation of a sexual projection termed ¨shmoo¨, and ultimately cell fusion of mating partners. The evolutionarily conserved and essential translation factor eIF5A has recently been described to promote translation of proteins containing three or more consecutive proline residues (polyPro) by binding to ribosomes and alleviating ribosome stalling during the formation of the Pro-Pro peptide bond. The activation of eIF5A requires the addition of a unique post-translational modification, a hypusine residue, which is derived from spermidine, an essential factor for mammalian fertility and required for yeast mating. Here we investigated eIF5A as a regulator of the pheromone response through the translation of polyPro proteins with roles in mating

Protein Interactions of Epithelial Neutral Amino Acid Transporters

Neutral amino acids are essential for protein synthesis, energy homeostasis, and many vital biochemical pathways. As several neutral amino acids are essential and cannot be synthesised by humans in vivo, their uptake and reuptake by the body’s absorbing epithelial layers is of major importance for systemic amino acid homeostasis and human health. Two of the major pathways for the absorption of neutral amino acids in the small intestine and kidney are the Broad Neutral Amino acid Transporters 1 and 3 (B0AT1 and B0AT3). B0AT1 is of medical interest as an indirect regulator of blood glycemia via cellular amino acid starvation response pathways. Absence of B0AT1 results in improved glycemic control and other metabolic effects, revealing it as a potential pharmacological target to treat type II diabetes. Essential to understanding the role of B0AT1 as a global metabolic regulator is its requirement for plasma membrane expression with heteromeric protein partners: collectrin in the kidney and Angiotensin Converting Enzyme 2 (ACE2) in the small intestine. Both proteins are membrane-anchored by a single trans-membrane domain and are homologous. B0AT3 also requires collectrin or ACE2 for membrane expression. Beyond this requirement for plasma membrane expression, however, little is known about the underlying mechanisms of the interaction. This thesis demonstrates additional protein partners of B0AT1, namely Aminopeptidase N (APN), syntaxin 1A, and syntaxin 3. These proteins facilitate changes in the kinetic parameters of neutral amino acid transport, in addition to regulating membrane expression. Moreover, I demonstrate that collectrin is required for catalytic activation of both B0AT1 and B0AT3, while both APN and ACE2 increase B0AT1 and B0AT3 substrate affinity, respectively. ACE2 and APN also form large complexes with B0AT1 at the small intestine brush border membrane. A binding site in both transporters for collectrin is identified as lying in the hydrophobic pocket between TM 5 and 7 of the transporters. Using a mixture of experimental and bioinformatics tools, I was able to map the interacting domains of collectrin, which involve multiple regions of the protein, including the TM domain and large regions of the N-terminus. The ability of collectrin and B0AT1 orthologs to cross-react with each other and the homologous region of ACE2, suggests a highly conserved structure-function relationship between them. In conclusion, B0AT1 and B0AT3 form large, stable protein complexes with collectrin, ACE2 and other proteins, the binding site and function of which are highly conserved. The necessity of these interactions mean these complexes should be thought of as heteromultimeric transporter metabolons – multimeric protein units facilitating efficient neutral amino acid transport in epithelial cells. There is increasing evidence that the formation of stable heteromeric membrane transport complexes is a common theme underlying the function of many transporters initially studied in isolation. Understanding the physiology and structure-function relationships of transporters in their native multimeric states is fundamental to understanding relevant biological roles. Especially as membrane transporters are heavily over-represented as causative agents of human disease and as therapeutic drug targets. Targeting stable, intrinsic protein-protein interactions presents a still unexplored field of drug treatment in biomedical science