Revisiting the Training of Logic Models of Protein Signaling Networks with a Formal Approach based on Answer Set Programming

A fundamental question in systems biology is the construction and training to data of mathematical models. Logic formalisms have become very popular to model signaling networks because their simplicity allows us to model large systems encompassing hundreds of proteins. An approach to train (Boolean) logic models to high-throughput phospho-proteomics data was recently introduced and solved using optimization heuristics based on stochastic methods. Here we demonstrate how this problem can be solved using Answer Set Programming (ASP), a declarative problem solving paradigm, in which a problem is encoded as a logical program such that its answer sets represent solutions to the problem. ASP has significant improvements over heuristic methods in terms of efficiency and scalability, it guarantees global optimality of solutions as well as provides a complete set of solutions. We illustrate the application of ASP with in silico cases based on realistic networks and data

Identificação de alterações no perfil proteómico de uma estirpe industrial mutante, Saccharomyces cerevisiae PYCC 5334, capaz de consumo simultâneo de glucose e maltose

A levedura Saccharomyces cerevisiae (S. cerevisiae) é capaz de fermentar substratos industriais, tais como hidrolisados de amido, os quais são compostos por açúcares como a maltose e a glucose. No entanto, devido ao efeito repressivo pela glucose no metabolismo de fontes alternativas de carbono, todos os outros açúcares fermentáveis permanecem não utilizados pela levedura, até que a glucose no meio seja totalmente consumida. Para melhorar o desempenho fermentativo de S. cerevisiae em processos industriais, foi obtido anteriormente, por tratamento mutagénico aleatório por UV, um mutante designado PYCC 5334, capaz de co fermentar a glucose e a maltose. A caracterização do mutante mostrou que o fenótipo desreprimido desta estirpe resultou de uma falha nas vias de indução e repressão, cuja extensão apenas pode ser avaliada por abordagens de rastreio global, como é o caso da proteómica. Assim, neste trabalho, pretendeu-se identificar proteínas diferencialmente expressas entre a estirpe selvagem PYCC 5297 e as estirpes mutantes diploide PYCC 5334 e haploide PYCC 5334-6 de S. cerevisiae através de uma análise proteómica baseada em eletroforese bidimensional (2DE) acoplada a técnicas de espetrometria de massa (MS). As estirpes estudadas foram crescidas em glucose e recolhidas em final da fase exponencial. Os extratos proteicos totais foram analisados por 2DE, num gradiente de pH 4-7. Os géis 2D das frações proteicas das estirpes PYCC 5297, PYCC 5334 e PYCC 5334-6 apresentaram um total de 697, 616 e 586 spots de proteína, respetivamente. Curiosamente, os perfis proteicos de PYCC 5297 e PYCC 5334 mostraram-se bastante distintos, identificando-se apenas 53 spots comuns. Por outro lado, os proteomas de PYCC 5297 e PYCC 5334-6 revelaram-se muito semelhantes e após a sua avaliação estatística foi possível quantificar 45 spots diferenciais. Foram identificadas 16 proteínas por MS. Destas, encontraram-se proteínas diferencialmente expressas com funções importantes na resposta ao stress e em processos do metabolismo energético, tanto na fermentação como na respiração

Robustness of Nutrient Signaling Is Maintained by Interconnectivity Between Signal Transduction Pathways

Systems biology approaches provide means to study the interplay between biological processes leading to the mechanistic understanding of the properties of complex biological systems. Here, we developed a vector format rule-based Boolean logic model of the yeast S. cerevisiae cAMP-PKA, Snf1, and the Snf3-Rgt2 pathway to better understand the role of crosstalk on network robustness and function. We identified that phosphatases are the common unknown components of the network and that crosstalk from the cAMP-PKA pathway to other pathways plays a critical role in nutrient sensing events. The model was simulated with known crosstalk combinations and subsequent analysis led to the identification of characteristics and impact of pathway interconnections. Our results revealed that the interconnections between the Snf1 and Snf3-Rgt2 pathway led to increased robustness in these signaling pathways. Overall, our approach contributes to the understanding of the function and importance of crosstalk in nutrient signaling

Reconstruction and logical modeling of glucose repression signaling pathways in Saccharomyces cerevisiae

Background: In the yeast Saccharomyces cerevisiae, the presence of high levels of glucose leads to an array of down-regulatory effects known as glucose repression. This process is complex due to the presence of feedback loops and crosstalk between different pathways, complicating the use of intuitive approaches to analyze the system. Results: We established a logical model of yeast glucose repression, formalized as a hypergraph. The model was constructed based on verified regulatory interactions and it includes 50 gene transcripts, 22 proteins, 5 metabolites and 118 hyperedges. We computed the logical steady states of all nodes in the network in order to simulate wildtype and deletion mutant responses to different sugar availabilities. Evaluation of the model predictive power was achieved by comparing changes in the logical state of gene nodes with transcriptome data. Overall, we observed 71% true predictions, and analyzed sources of errors and discrepancies for the remaining. Conclusion: Though the binary nature of logical (Boolean) models entails inherent limitations, our model constitutes a primary tool for storing regulatory knowledge, searching for incoherencies in hypotheses and evaluating the effect of deleting regulatory elements involved in glucose repression.Fundação para a Ciência e a TecnologiaPortuguese Ministry of Science and Technology (grant no. SFRH/BD/12435/2003

A systems biology understanding of protein constraints in the metabolism of budding yeasts

Fermentation technologies, such as bread making and production of alcoholic beverages, have been crucial for development of humanity throughout history. Saccharomyces cerevisiae provides a natural platform for this, due to its capability to transform sugars into ethanol. This, and other yeasts, are now used for production of pharmaceuticals, including insulin and artemisinic acid, flavors, fragrances, nutraceuticals, and fuel precursors. In this thesis, different systems biology methods were developed to study interactions between metabolism, enzymatic capabilities, and regulation of gene expression in budding yeasts. In paper I, a study of three different yeast species (S. cerevisiae, Yarrowia lipolytica and Kluyveromyces marxianus), exposed to multiple conditions, was carried out to understand their adaptation to environmental stress. Paper II revises the use of genome-scale metabolic models (GEMs) for the study and directed engineering of diverse yeast species. Additionally, 45 GEMs for different yeasts were collected, analyzed, and tested. In paper III, GECKO 2.0, a toolbox for integration of enzymatic constraints and proteomics data into GEMs, was developed and used for reconstruction of enzyme-constrained models (ecGEMs) for three yeast species and model organisms. Proteomics data and ecGEMs were used to further characterize the impact of environmental stress over metabolism of budding yeasts. On paper IV, gene engineering targets for increased accumulation of heme in S. cerevisiae cells were predicted with an ecGEM. Predictions were experimentally validated, yielding a 70-fold increase in intracellular heme. The prediction method was systematized and applied to the production of 102 chemicals in S. cerevisiae (Paper V). Results highlighted general principles for systems metabolic engineering and enabled understanding of the role of protein limitations in bio-based chemical production. Paper VI presents a hybrid model integrating an enzyme-constrained metabolic network, coupled to a gene regulatory model of nutrient-sensing mechanisms in S. cerevisiae. This model improves prediction of protein expression patterns while providing a rational connection between metabolism and the use of nutrients from the environment.This thesis demonstrates that integration of multiple systems biology approaches is valuable for understanding the connection of cell physiology at different levels, and provides tools for directed engineering of cells for the benefit of society

Mecanismos de regulación de transportadores de iones y nutrientes a través de proteínas de tráfico relacionadas con las arrestinas

[EN] Any living cell needs to maintain adequate levels of nutrients and ions to ensure the continuity of its functions. The first step in the maintenance of both ion and nutrient homeostasis is their uptake from the extrenal environment, where plasma membrane transporter proteins play an important role. The regulation of both the activity and abundance of permeases in the plasma membrane is a pivotal aspect of the cellular response to different environmental conditions. Therefore, deciphering the pathways that regulate the function or the presence of these transport proteins is important for a better understanding of cell physiology and stress responses. Transcriptional regulation is a contributor to the control of the abundance of plasma membrane transporters, and more specifically in the yeast model Saccharomyces cerevisiae a strong transcriptional regulation of the hexose transporters has been demonstrated. However, there are other important levels for the control and maintenance of glucose homeostasis, such as post-translational regulation of glucose transporters, which are still being defined. Ubiquitination, as a signal for endocytosis of membrane proteins, mediated by the E3 ubiquitin ligase Rsp5 has been demonstrated for many transporters, including the HXT family. In recent years, several studies have shown the involvement of the ART protein family (Arrestin-Releated Trafficking proteins) in this process, acting as adapters for Rsp5 and providing specificity to the ubiquitination of permeases in response to changes in environmental conditions. In turn, the members of this family of adapter proteins are targets of post-translational modifications, such as phosphorylation or ubiquitination, affecting their activity and adding an additional level in the regulation of transporter interactions. Previous studies have demonstrated the existence of a genetic interaction between the high affinity glucose transporter Hxt6 and the Rsp5 adapter protein, Rod1 (Art4). In turn, the Snf1 kinase was shown to be involved in the phosphorylation of this adapter protein, suggesting a possible role in the regulation of its activity. With this background, and taking into consideration the interaction of 14-3-3 proteins with other adapter proteins, in this study the biochemical characterization of the Art4-Snf1-14-3-3 signaling pathway involved in the regulation of the endocytosis of the glucose transporter Hxt6 and the effect of Snf1 and 14-3-3 proteins (Bmh2) on intracellular traffic of the Hxt6-Art4 complex will be investigated. Similarly, the effect of Art4, and its paralogue Rog3 (Art7), on Hxt6 levels and the phenotypes of other glucose transporter mutants, such as hxt1 and hxt3 will be analyzed to determine whetherthese transporters display a common regulatory mechanism also involving Art4 and Art7. In short, this thesis aims to provide new data on the post-translational regulation of the HXT transporters through the Rsp5 adapter family of ART proteins, with emphasis on biochemical aspects, such as phosphorylation, and molecular and cellular aspects, such as intracellular trafficking and permease stability[ES] Cualquier célula viva necesita mantener niveles adecuados de iones y nutrientes para asegurar la continuidad de sus funciones. El primer paso en la conservación de la homeostasis de iones como el potasio o nutrientes como la glucosa, es la toma de los mismos desde el medio externo, teniendo los transportadores de membrana un papel básico en este proceso. La regulación de estos transportadores tiene una función fundamental en la respuesta de las células a diferentes condiciones ambientales, siendo esenciales tanto la modulación de su actividad como la abundancia de permeasas en la membrana plasmática. Por esto, el estudio de las rutas que regulan la función o la presencia de dichos transportadores en la membrana plasmática es importante para un mejor conocimiento de la fisiología celular y la respuesta de las células al estrés. La regulación transcripcional supone un nivel importante en el control de la abundancia de transportadores en la membrana plasmática, y más concretamente en la levadura modelo Saccharomyces cerevisiae se ha demostrado que los transportadores de hexosas poseen una fuerte regulación a nivel transcripcional. Sin embargo, existen otros niveles importantes para el control y el mantenimiento de la homeóstasis de hexosas, como la regulación postraduccional, y que en el caso de los transportadores de glucosa es menos conocida. La ubiquitinación como señal para la endocitosis de proteínas de membrana a través de la E3 ubiquitín ligasa Rsp5 ha sido demostrada para gran número de transportadores, incluidos los de la familia HXT. En los últimos años, varios estudios han demostrado la implicación de la familia de proteínas ART (Arrestin Releated Trafficking proteins) en este proceso, actuando como adaptadores de Rsp5 y aportando especificidad a la ubiquitinación de transportadores en respuesta a cambios en las condiciones del medio. A su vez, esta familia de proteínas adaptadoras es diana de modificaciones postraduccionales, como la ubiquitinación o la fosforilación, que repercuten en su actividad y suman un nivel adicional en la regulación de los transportadores con los que interaccionan. Estudios anteriores demostraron la existencia de una interacción genética entre el transportador de glucosa de alta afinidad Hxt6 y la proteína adaptadora de Rsp5, Rod1 (Art4). A su vez, la quinasa Snf1 fue implicada en la fosforilación de esta proteína adaptadora, sugiriendo una posible función en la regulación de su actividad. Con estos precedentes, y teniendo en consideración la interacción de las proteínas 14-3-3 con otras proteínas adaptadoras, en el presente estudio se llevará a cabo la caracterización bioquímica de la ruta de señalización Art4-Snf1-14-3-3 implicada en la regulación por endocitosis del transportador de glucosa Hxt6 y se estudiará el efecto de Snf1 y las proteínas 14-3-3 (Bmh2) sobre el tráfico intracelular del complejo Hxt6-Art4. Del mismo modo, se analizará el efecto de Art4, y de su parálogo Rog3 (Art7), sobre los niveles de Hxt6 y se caracterizarán los fenotipos de mutantes para otros transportadores de glucosa, como Hxt1 y Hxt3, por la posibilidad de que presenten un mecanismo regulador común que implique también a Art4 y Art7. En definitiva, la presente tesis doctoral pretende aportar nuevos datos acerca de la regulación postraduccional de transportadores de la familia HXT a través de las proteínas ART, adaptadoras de Rsp5, haciendo hincapié en aspectos bioquímicos como la fosforilación y la ubiquitinación, y en aspectos moleculares y celulares, como el tráfico intracelular o la estabilidad de permeasas.[CA] Qualsevol cèl-lula viva necessita mantindre nivells adequats d'ions i nutrients per tal d'assegurar la continuïtat de les seues funcions. El primer pas en la conservació de l'homeòstasi d'ions com el potassi o nutrients com la glucosa, és la pressa dels mateixos des del medi extern, tot i tenint els transportadors de membrana un paper bàsic en este procés. De manera directa, la regulació d'aquests transportadors té una funció fonamental en la resposta de les cèl-lules a diferents condicions ambientals, tot i sent processos essencials tant la modulació de la seua activitat com l'abundància de permeases a la membrana plasmàtica. Per això, entendre les rutes que regulen la funció o la presència dels transportadors a la membrana és important per a un millor coneixement de la fisiologia cel-lular i la resposta a l'estrés. La regulació transcripcional suposa un nivell important en el control de l'abundància de transportadors a la membrana plasmàtica, i més concretament en el rent model Saccharomyces cerevisiae s'ha demostrat que les permeases d'hexoses tenen una forta regulació a nivell transcripcional. A banda d'aquest tipus de regulació, existeixen altres nivells importants per al control i el manteniment de l'homeòstasi de la glucosa, com la regulació postraduccional, i , que en el cas dels transportadors de glucosa, és menys coneguda. L'ubiquitinació com a senyal per a l'endocitosi de proteïnes de membrana a través de l'E3 ubiquitina ligasa Rsp5, ha sigut demostrada per a un gran nombre de transportadors, inclosos els de la família HXT. En els últims anys, diversos estudis han posat de manifest la implicació de la família de proteïnes ART (Arrestin Releated Trafficking proteins) en este procés, tot i actuant com adaptadores de Rsp5 i aportant especificitat a l'ubiquitinació de transportadors en resposta a canvis en les condicions del medi. Al mateix temps, aquesta família de proteïnes és diana de modificacions postraduccionals, com l'ubiquitinació o la fosforilació, que tenen un efecte en la seua activitat i sumen un nivell addicional a la regulació dels transportadors amb els quals interaccionen. Estudis anteriors demostraren l'existència d'una interacció genètica entre el transportador de glucosa d'alta afinitat Hxt6 i la proteïna adaptadora de Rsp5, Rod1 (Art4). De la mateixa manera, la quinasa Snf1 va ser involucrada en la fosforilació d'aquesta proteïna adaptadora, tot i suggerint una possible funció en la regulació de la seua activitat. Amb estos precedents, i tenint en compte la interacció de les proteïnes 14-3-3 amb altres proteïnes adaptadores, en el present estudi es durà a terme la caracterització bioquímica de la ruta de senyalització Art4-Snf1-14-3-3 implicada en la regulació per endocitosi del transportador de glucosa Hxt6, i s'estudiarà l'efecte de Snf1 i les proteïnes 14-3-3 (Bmh2) sobre el tràfic intracel-lular del complex Hxt6-Art4. Per altra banda, s'analitzarà l'efecte de Art4, i del seu paràleg Rog3 (Art7), sobre els nivells de Hxt6 i es caracteritzaran els fenotips de mutants per a altres transportadors de glucosa, tals com Hxt1 i Hxt3, per la possibilitat que presenten un mecanisme regulador comú que també implique a Art4 i Art7. En definitiva, la present tesi doctoral pretén aportar noves dades al voltant de la regulació postraduccional dels transportadors de la família HXT a través de les proteïnes ART, adaptadores de Rsp5, tot i aprofundint en aspectes bioquímics com la fosforilació i l'ubiquitinació, i en aspectes moleculars i cel-lulars, com el tràfic intracel-lular o l'estabilitat de permeasesLlopis Torregrosa, V. (2016). Mecanismos de regulación de transportadores de iones y nutrientes a través de proteínas de tráfico relacionadas con las arrestinas [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/60149TESI