Tesis Doctorals - Departament - Bioquímica i Biomedicina Molecular

URI permanent per a aquesta col·leccióhttps://hdl.handle.net/2445/104171

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Identification of VAT1 as a regulator of lipid metabolism at mitochondria endoplasmic reticulum contact sites and implications in liver pathology
(Universitat de Barcelona, 2025-03-27) Danezi, Aikaterini; Zorzano Olarte, Antonio; Universitat de Barcelona. Departament de Bioquímica i Biomedicina Molecular
[eng] Lipid homeostasis is of vital importance for cellular health and its disruption is a hallmark of disease. Recently, our lab reported that ablation of Mitofusin 2 (MFN2) leads to liver disease, a phenotype driven by deficient phosphatidylserine (PS) transport at the mitochondria- endoplasmic reticulum (ER) contact sites (MERCs). Despite the capacity of MFN2 to extract PS from membranes and generate PS-rich domains, evidence suggested that it does not possess lipid transport properties. In this study, we propose that Vesicle amine transport protein 1 (VAT1) localises at MERCs, interacts with MFN2 and its PS transport capacity is modulated by MFN2 in vitro. Our results in cells additionally suggest a functional relationship between VAT1 and MFN2. Finally, our study reveals that ablation of hepatic VAT1 in mice leads to sex-dependent metabolic adaptations and has a detrimental effect on the lipid profile of mitochondria-associated membranes, ER and pure mitochondria.
The Kv1.3 channel: Functional consequences of cellular and spatial reorganization
(Universitat de Barcelona, 2024-12-19) Navarro Pérez, María; Felipe Campo, Antonio; Capera Aragonés, Jesusa; Universitat de Barcelona. Departament de Bioquímica i Biomedicina Molecular
[eng] The voltage-gated potassium channel Kv1.3 is a transmembrane protein that facilitates the selective flow of K+ ions across cell membranes, playing a crucial role in regulating cellular excitability and maintaining the electrochemical gradient. This channel is ubiquitously expressed within the human body and participates in several physiological processes, including proliferation, apoptosis, and leukocyte activation. The functional role of Kv1.3 is influenced by its dual localisation at the cell surface and the inner mitochondrial membrane. Hence, while Kv1.3 at the plasma membrane promotes cell proliferation, its mitochondrial counterpart is associated with the regulation of apoptosis. In this context, the present thesis aims to further explore this channel dichotomy, from the early biogenesis to the functional implications. The interaction between caveolin and Kv1.3 via a caveolin binding domain (CBD) is crucial for channel localisation at the plasma membrane. Abrogation of the CBD redirects Kv1.3 to mitochondria, where its accumulation affects organelle physiology and compromises cell survival. Thus, our findings reveal an unexpected role of the mitochondrial caveolin-Kv1.3 axis in regulating cell survival and apoptosis, with significant implications for chemotherapy resistance. Moreover, we decipher a new role for mitochondrial Kv1.3 in regulating cell proliferation by modulating the balance between mitochondrial fusion and fission. This function, extending beyond apoptosis, adds further complexity to the role of the mitochondrial channel. Finally, we describe for the first time the mitochondrial route of Kv1.3. Interestingly, the channel uses a pre-sequence independent pathway where transmembrane domains function as redundant motifs mediating the import process. The interaction with cytosolic chaperones is essential for the subsequent translocation through unconventional TIM/TOM complexes. Kv1.3 plays a crucial role in T cell activation, and channel delocalisation or increased activity is linked to the onset of diverse autoimmune diseases. For this reason, we studied the plasma membrane functions of Kv1.3 in the context of the immunological synapse (IS). In a pioneering effort to increase the resolution of Kv1.3 distribution at the IS, we reveal, for the first time, its organisation into a peripheral ring. As the contact matures, Kv1.3 migrates to the centre of the synapse, where it becomes more static to facilitate endocytosis for degradation, recycling, or exocytosis into extracellular vesicles. The internalisation process is regulated by CaMKII- dependent palmitoylation of Kv1.3, which facilitates channel ubiquitination and subsequent endocytosis. CaMKII also mediates the impact of Ca2+ fluctuations on Kv1.3 regulation, influencing the channel localisation in lipid rafts and its role T cell activation, among others. Thus, our work deepens the knowledge of Kv1.3 biology at the IS, contributing to the refinement of therapeutic strategies for autoimmune diseases characterised by channel dysregulations. Finally, we investigate alternative roles of Kv1.3 cysteine residues during channel biogenesis and sorting. Absence of palmitoylation in intracellular cysteines reduces Kv1.3 interaction with caveolin, redirecting the channel to mitochondria where it promotes apoptosis. Moreover, we identify two transmembrane cysteines that participate in the oxidative folding of Kv1.3 mediated by protein disulphide isomerase. Overall, the present thesis offers a broader view of Kv1.3 functions regarding the spatiotemporal context. We introduce novel roles and regulatory mechanisms that could contribute to the development of new therapies for cancer, obesity, and autoimmune diseases.
TP53INP2 at the crossroad of transcription, autophagy and liver metabolism
(Universitat de Barcelona, 2024-06-03) Sun Wang, Jia Liang; Zorzano Olarte, Antonio; Ivanova, Saška; Universitat de Barcelona. Departament de Bioquímica i Biomedicina Molecular
[eng] Metabolic-associated steatotic liver disease (MASLD) is the leading global liver chronic disease and it is estimated to affect 30% of the world population. MASLD is a progressive multi-stage disease characterized by an abnormal accumulation of fat in liver, also known as steatosis. Major risk factors for developing MASLD include obesity and metabolic syndrome. Patients with MASLD are at risk of developing co-morbidities such as cardiovascular disease, diabetes and chronic kidney disease. Unfortunately, there is no approved pharmacological treatment for MASLD. PPARα is a nuclear receptor that is activated upon fasting to coordinate the hepatic response against this cue. It promotes the expression of genes that are essential for fatty acid oxidation (FAO) and ketone body (KB) synthesis. It emerged as a promising therapeutic target for MASLD due to its strong protective role against the development of MASLD in mouse models. In addition, treatment of MASLD mouse models with PPARα agonists improves steatosis. In this thesis, we have identified TP53INP2 as a novel regulator of PPARα. TP53INP2 is a multifaceted protein that regulates many aspects of metabolic homeostasis through its action in different tissues. It also modulates many cellular processes, including autophagy and apoptosis. Essentially, liver-specific TP53INP2 ablation impaired the activation of PPARα upon fasting, which caused a blunted upregulation of FAO and KB synthesis genes. This resulted in increased hepatic fat accumulation. These effects were more pronounced in aged mice. By using PPARα agonists, we uncovered that TP53INP2 is required to fully activate PPARα. Interestingly, TP53INP2 ablation also impaired the transcriptional activation of autophagy during fasting. This may be a direct effect of impaired PPARα activation, as PPARα was shown to be a master regulator of hepatic autophagy in response to fasting. Autophagy has also been established to be a protective against MASLD. We used a methionine- and choline-deficient (MCD) diet to promote MASLD in control and TP53INP2-deficient mice and treated them with the PPARα agonist GW7647 to assess the potential role of TP53INP2 in the development and treatment of MASLD. Interestingly, female TP53INP2-deficient mice subjected to a one-week regime of MCD diet showed impaired activation of PPARα by the agonist GW7647. In addition, male TP53INP2-deficient mice fed with MCD diet during 3 weeks and treated chronically with GW7647 during the diet showed lower response to GW7647. To elucidate how TP53INP2 regulates PPARα activation we used the BioID technology to map the binding partners of TP53INP2. Most of the identified interactors are involved in the regulation of chromatin dynamics and epigenetics, with coregulator activity and nuclear receptor binding functions. In keeping with this, we have also shown that TP53INP2 is a chromatin-binding protein that interacts with RING1A, a ubiquitin ligase (E3) that acts as the catalytic component of the Polycomb repressive complex 1 (PRC1). The PRC1 is an epigenetic modulator that ubiquitinates histone H2A to suppress gene expression. In addition, TP53INP2 interacts with UBR5, another E3 that is involved in the turnover of nuclear receptors. These results indicate that TP53INP2 may regulate PPARα-dependent gene expression through its interaction with players of the epigenetic machinery. ChIP-Seq experiments will be key to confirm this hypothesis. In conclusion, TP53INP2 is required for the full activation of PPARα in response to fasting and to agonists. When TP53INP2 is ablated in the liver, upregulation of FAO, KB synthesis and autophagy genes in response to fasting is blunted. This renders TP53INP2- deficient hepatocytes incapable of ridding of the fat overload coming from adipose tissue lipolysis, leading to increased hepatic fat accumulation.
Targeting metabolic reprogramming in metastatic colorectal and prostate cancer cells to prevent therapeutic failure
(Universitat de Barcelona, 2024-03-19) Hernández Carro, Claudia; Casscante i Serratosa, Marta; Tarrado Castellarnau, Míriam Neus; Universitat de Barcelona. Departament de Bioquímica i Biomedicina Molecular
[eng] Prostate and colorectal cancer are among the cancer types with the highest recurrence and mortality rates. The emergence of drug resistance poses a considerable challenge for effective treatment, especially in the advanced stages of the disease. Metabolic reprogramming is a crucial contributor to the adaptative process of drug resistance and a key hallmark of cancer that enables tumor cells to meet the metabolic and energetic requirements for tumor survival and progression. Then, targeting metabolic reprogramming represents a promising anti-cancer strategy, and understanding the metabolic alterations acquired after drug resistance can provide valuable insights for developing effective combination therapies. In this work, we study metabolic alterations underlying Oxaliplatin, an alkylating agent that impairs DNA replication and transcription by promoting DNA damage through the generation of platinum crosslinks, and Palbociclib, a selective inhibitor of cyclin- dependent kinases 4 and 6 (CDK4/6) that arrests cells in the G1/G0 phase of the cell cycle. Both compounds are commonly employed in cancer therapies and have exhibited efficacy in several types of cancer. This work assesses the metabolic characterization of drug resistance acquisition after these treatments in advanced stages of colorectal and prostate cancer cells, focusing on the aggressive SW620 and PC-3 cell lines, respectively. Based on the metabolic characterization, transcriptomic, and respiratory data, or applying cell-line-specific metabolic reconstructions after data integration using Genome-Scale Metabolic Models (GSMM), our results identify metabolic vulnerabilities in drug-adapted phenotypes and reveal key metabolic pathways on which cancer cells rely to adapt and survive, serving as attractive targets for therapeutic interventions. These pathways include but are not limited to glycolysis, fatty acid metabolism, amino acid metabolism, and oxidative phosphorylation. Our strategy proposes rational therapeutic combinations with Oxaliplatin or Palbociclib to overcome drug resistance in advanced-stage prostate and colorectal cancers based on drug repurposing. These findings include the interaction between metabolic reprogramming and cell cycle regulation, promoting cell cycle arrest in the G1/G0 phase to address Oxaliplatin resistance, and targeting oxidative phosphorylation, mitochondrial metabolism, and reactive oxygen species (ROS) modulation to sensitize cells to Palbociclib. Notably, Palbociclib combination with a ROS modulator represents a successful approach that expands the potential of application across different colorectal models, showing efficacy in preclinical models, significantly compromising in vivo tumor progression in NOD/SCID mice with SW620 tumors and in vitro cell proliferation in both the metastatic SW620 and the primary colorectal cancer HCT116 cell lines. Based on the metabolic reprogramming underlying Oxaliplatin and Palbociclib treatments, this work provides alternative combination therapies by targeting metabolic vulnerabilities that could be further explored to improve treatment outcomes and overcome drug resistance in advanced stages of prostate and colorectal cancers.
Analysis of consensus motions in proteins through molecular dynamics simulations
(Universitat de Barcelona, 2024-02-06) Jordà Bordoy, Luis; Gelpi Buchaca, Josep Lluís; Universitat de Barcelona. Departament de Bioquímica i Biomedicina Molecular
[eng] The understanding of proteins as dynamical entities rather than static structures marked a very significant advance in the interpretation of their functional role in life. The capacity of proteins to interact with their environment, sense molecular perturbations and exert responses can be explained in an effective manner by specific dynamical events. The study of proteins from this perspective has been possible in the last decades thanks to the emergence of computational approaches. Among these techniques, Molecular Dynamics (MD) simulations have emerged as a potent tool, playing a pivotal role in investigating conformational transitions at atomic resolution across diverse biomacromolecular systems. As computational power and infrastructures keep evolving, we are increasingly able to generate longer MD simulations that are capable of capturing dynamical events at biologically relevant timescales. MD simulations typically generate an overwhelming amount of data in the form of a collection of snapshots, called a trajectory. Thus, we need to find suitable metrics to extract, quantify and present the relevant information depending on the target of the study. The scenario is even more challenging when we aim to analyze multiple trajectories and compare their similarity. Among the proposed strategies to explore the comparability between trajectories, essential dynamics analysis (EDA) approaches are a common choice, where Principal Components Analysis (PCA) or other dimensionality reduction techniques are applied to express the differential behavior between trajectories in terms of the underlying collective features of the ensemble. The work presented in this thesis delves further into this analytical field with the aim of improving the applicability of EDA in functional studies of proteins. The developed approach, termed Consensus Essential Dynamics Analysis (CEDA), introduces a protocol to integrate the information from independent PCAs and derive a consensus set of vectors, the Consensus Principal Components (CPCs). CPCs encapsulate the most representative (consensus) collective motions of an ensemble of trajectories of the system under study, allowing for sharper descriptions and comparisons of its relevant dynamical events. The framework of CEDA also facilitates the comparative study of alternative trajectory ensembles of the same system, in terms of the reference set of CPCs. The outcomes of such comparisons may be interpreted using different data analysis techniques and graphical representations. In this thesis, a strategy was proposed to evaluate the underlying similarities and differences between trajectory ensembles by comparison of their conformational profiles and application of similarity metrics between statistical distributions. The capacities of the CEDA protocol were demonstrated with the analysis of a collection of MD simulations of human erythrocyte pyruvate kinase (PKR) that covers multiple conditions of the enzymatic complex with its natural ligands, as well as a large array of human genomic missense variants of the protein. Pyruvate kinase is among the most studied proteins from the perspective of biochemistry, given both its role in glycolysis and its paradigmatic and complex set of allosteric properties. This study has provided new support for several of the proposed conformational changes that are associated with the transition between the inactive and active states of the enzyme. Following from the study of the wild-type protein, a second experimental part of the project revolved around the characterization of the functional effects of missense variants of the enzyme. Analysis with CEDA enabled detection of altered dynamical behavior in variants either with a previously validated pathogenic status or for which no functional details were previously known. The conducted research in this regard is presented in depth throughout this manuscript. The obtained results are discussed in the light of the potential application of this protocol in functional studies of proteins in general, and with a particular perspective on pathogenicity prediction studies.
Estudio de la interacción de hCNT3 y QDPR como nexo funcional entre la recuperación de nucleósidos y sus vías biosintéticas
(Universitat de Barcelona, 2023-12-15) Prieto Carruyo, Andrea Valentina; Pastor Anglada, Marçal; Pérez Torras, Sandra; Universitat de Barcelona. Departament de Bioquímica i Biomedicina Molecular
[spa] Aún queda mucho por conocer sobre la regulación de la función de los transportadores concentrativos de nucleósidos hCNTs. A pesar de que se expresan en muchos tipos de tejidos, su localización no es ubicua y varía según el tipo de transportador. Además, su distribución en la membrana celular tampoco es homogénea y pueden conseguirse principalmente en la membrana apical de los epitelios polarizados, desde donde favorecen el movimiento vectorial de nucleósidos. Esta expresión y ubicación de los hCNTs debe ser finamente regulada en función a los factores ambientales y las necesidades celulares para asegurar la disponibilidad de nutrientes y mantener la homeostasis. Los distintos hCNTs comparten importantes características estructurales y de localización. Sin embargo, que cada uno de ellos tenga diferencias en la especificidad de sustrato abre la posibilidad a que cumplan diferentes papeles en la regulación de los niveles de nucleós(t)idos y que además puedan regularse entre ellos. Por sus características y por estar estrechamente vinculados al salvamento de nucleósidos, como hipótesis de esta tesis se plantea que la expresión de los hCNTs podría responder a cambios metabólicos producto de alteraciones extra- e intracelulares en los niveles de estas moléculas, tal como se ha observado en otros transportadores de nutrientes. Para entender mejor la participación de los hCNTs en la regulación de la disponibilidad nucleósidos es importante conocer las diferentes proteínas con las que podrían establecer interacciones. En nuestro grupo se han identificado proteínas del interactoma de hCNT3 vinculadas al metabolismo de nucleótidos o a alguna de las vías metabólicas que lo alimentan. Con base en estas observaciones se planteó como objetivo principal estudiar el papel de hCNT3 en la regulación del metabolismo de purinas y de pirimidinas, evaluándolo a través de su interacción con otras proteínas vinculadas a estas vías. Para lograrlo, los siguientes objetivos específicos fueron planteados: I. Estudiar la regulación de los transportadores concentrativos de pirimidinas hCNT1 y hCNT3 en respuesta a la modulación del metabolismo de los nucleótidos. II. Estudiar los factores que afectan la interacción y regulación entre hCNT3 y QDPR. III. Estudiar las posibles implicaciones de la patología asociada a la deficiencia de QDPR en el metabolismo de nucleótidos.
Regulatory subunits controlling the Kv1.3 channelosome
(Universitat de Barcelona, 2023-12-15) Cassinelli, Silvia; Felipe Campo, Antonio; Universitat de Barcelona. Departament de Bioquímica i Biomedicina Molecular
[eng] The Kv1.3 voltage-dependent potassium channel plays a crucial role in the immune response participating in various cellular functions like proliferation, activation, and apoptosis. The aberrant expression of this channel is associated with autoimmune diseases, highlighting the need for precise regulation in leukocyte physiology. Kvβ proteins, the first identified modulators of Kv channels, have been extensively studied in their regulation of α-subunit kinetics and traffic. However, limited information is available regarding their own biology. Despite their cytosolic distribution, Kvβ subunits show spatial localization near plasma membrane-Kv channels for an effective immune response. Our study focused on the structural elements influencing Kvβ distribution. We discovered that Kvβ peptides could target the cell surface independently of Kv channels. Additionally, Kvβ2.1, but not Kvβ1.1, targeted lipid raft microdomains via S-acylation of two C-terminal cysteines (C301/C311), concomitantly with the peptide localization at the immunological synapse. Moreover, growth factor-dependent proliferation increased the Kvβ2.1 surface targeting, whereas PKC activation disrupted lipid raft localization, but PSD95 counteracted this action. These findings elucidate the mechanisms by which Kvβ2 clusters within immunological synapses during leukocyte activation. Kvβ peptides, interacting with Kv channels, exhibited a suggested α4/β4 conformation. While Kvβ2 and Kvβ1 can form homo- and heterotetramers with similar affinities, only Kvβ2.1 forms tetramers independently of α subunits. Thus, Kvβ oligomers stoichiometry fine-tunes hetero-oligomeric Kv channel complexes. Similar to Kvβ1.1, Kvβ1.1/Kvβ2.1 heteromers did not target lipid rafts. Therefore, because Kvβ2 is an active partner of the Kv1.3-TCR complex at the immunological synapse, an association with Kvβ1 would alter its location, impacting on immune responses. Differential regulation of Kvβs influences the traffic and architecture of Kvβ heterotetramers, modulating Kvβ-dependent physiological responses. Regulatory KCNE subunits are expressed in the immune system and KCNE4 tightly regulates Kv1.3. KCNE4 modifies Kv1.3 currents altering kinetics and retaining the channel at the endoplasmic reticulum (ER). This function affects in turn membrane localization of the channel. Our research showed that KCNE4 can dimerize via the juxtamembrane tetraleucine carboxyl-terminal domain of KCNE4. This cluster serves as a competitive structural platform for Kv1.3, Ca2+/calmodulin (CaM) and KCNE4 dyads. While KCNE4 is typically retained in the ER, the association with CaM leads to COP-II-dependent forward trafficking. Consequently, CaM plays a vital role in controlling the dimerization and membrane targeting of KCNE4, affecting the regulation of Kv1.3 and, subsequently, leukocyte physiology. Kv1.3, localized in membrane lipid rafts, accumulates at immunological synapses during cell activation, influencing membrane potential and downstream calcium-signalling pathways. KCNE4 acts as a dominant negative regulatory subunit on Kv1.3, causing intracellular retention. Palmitoylation, a reversible post- translational modification, enhances protein hydrophobicity, facilitating membrane association, protein interactions, and subcellular trafficking. Our data demonstrated the S-acylation of KCNE4, resulting in spatial rearrangements that reduce ER distribution, which in turn affects Kv1.3 regulation. KCNE4 partially traffics to the cell surface with Kv1.3 in activated dendritic cells but alters immunological synapse targeting. This highlights the significance of KCNE4 palmitoylation in regulating protein subcellular localization and oligomeric state, subsequently affecting channel membrane expression. Given the role of Kv1.3 as an immunomodulatory target, these findings offer insights for future clinical and pharmacological studies.
Network-based methods for biological data integration in precision medicine
(Universitat de Barcelona, 2023-11-14) Núñez Carpintero, Iker; Valencia Herrero, Alfonso; Cirillo, Davide; Universitat de Barcelona. Departament de Bioquímica i Biomedicina Molecular
[eng] The vast and continuously increasing volume of available biomedical data produced during the last decades opens new opportunities for large-scale modeling of disease biology, facilitating a more comprehensive and integrative understanding of its processes. Nevertheless, this type of modelling requires highly efficient computational systems capable of dealing with such levels of data volumes. Computational approximations commonly used in machine learning and data analysis, namely dimensionality reduction and network-based approaches, have been developed with the goal of effectively integrating biomedical data. Among these methods, network-based machine learning stands out due to its major advantage in terms of biomedical interpretability. These methodologies provide a highly intuitive framework for the integration and modelling of biological processes. This PhD thesis aims to explore the potential of integration of complementary available biomedical knowledge with patient-specific data to provide novel computational approaches to solve biomedical scenarios characterized by data scarcity. The primary focus is on studying how high-order graph analysis (i.e., community detection in multiplex and multilayer networks) may help elucidate the interplay of different types of data in contexts where statistical power is heavily impacted by small sample sizes, such as rare diseases and precision oncology. The central focus of this thesis is to illustrate how network biology, among the several data integration approaches with the potential to achieve this task, can play a pivotal role in addressing this challenge provided its advantages in molecular interpretability. Through its insights and methodologies, it introduces how network biology, and in particular, models based on multilayer networks, facilitates bringing the vision of precision medicine to these complex scenarios, providing a natural approach for the discovery of new biomedical relationships that overcomes the difficulties for the study of cohorts presenting limited sample sizes (data-scarce scenarios). Delving into the potential of current artificial intelligence (AI) and network biology applications to address data granularity issues in the precision medicine field, this PhD thesis presents pivotal research works, based on multilayer networks, for the analysis of two rare disease scenarios with specific data granularities, effectively overcoming the classical constraints hindering rare disease and precision oncology research. The first research article presents a personalized medicine study of the molecular determinants of severity in congenital myasthenic syndromes (CMS), a group of rare disorders of the neuromuscular junction (NMJ). The analysis of severity in rare diseases, despite its importance, is typically neglected due to data availability. In this study, modelling of biomedical knowledge via multilayer networks allowed understanding the functional implications of individual mutations in the cohort under study, as well as their relationships with the causal mutations of the disease and the different levels of severity observed. Moreover, the study presents experimental evidence of the role of a previously unsuspected gene in NMJ activity, validating the hypothetical role predicted using the newly introduced methodologies. The second research article focuses on the applicability of multilayer networks for gene priorization. Enhancing concepts for the analysis of different data granularities firstly introduced in the previous article, the presented research provides a methodology based on the persistency of network community structures in a range of modularity resolution, effectively providing a new framework for gene priorization for patient stratification. In summary, this PhD thesis presents major advances on the use of multilayer network-based approaches for the application of precision medicine to data-scarce scenarios, exploring the potential of integrating extensive available biomedical knowledge with patient-specific data.
Expresión y Funcionalidad de Receptores de Cannabinoides y de Adenosina y de sus Heterómeros en modelos de Alzheimer y de Esclerosis Múltiple
(Universitat de Barcelona, 2023-11-17) Pérez Olives, Catalina; Franco Fernández, Rafael; Navarro Brugal, Gemma; Universitat de Barcelona. Departament de Bioquímica i Biomedicina Molecular
[eng] Adenosine and endocannabinoids are regulatory molecules that are present in all organ and physiological systems. They act via specific receptors belonging to the G protein-coupled receptor (GPCR) superfamily. There are four adenosine receptors, A1, A2A, A2B and A3, that, in the central nervous system, are expressed in both neurons and glia. There are two cannabinoid receptors, CB1 that is much more expressed in neurons than in glia, and CB2 that is mainly expressed in glia. GPCR may form receptor-receptor complexes, both homo and heteromers, with specific properties. The aim of this PhD was to get insight into the potential of cannabinoid, of adenosine receptors and of their heteromers to become targets of Alzheimer’s disease and of multiple sclerosis. Both expression and functionality of some receptors and of some receptor heteromers were addressed. In addition, a novel technique aimed at assessing the percentage of receptors that are forming heteromers was used to demonstrate, in a heterologous system, a differential effect of adenosine and caffeine on the expression of A1R, A2AR and A1-A2A receptor heteromers on the cell surface. The technique also allowed to show increased heteromer expression in primary cultures of activated astroglial cells (versus resting cells). Astroglia showed expression of the A1-CB1 receptor heteromer whose functionality was characterized by a negative crosstalk and a crossantagonism; crossantagonism due to the A1-CB1 receptor heteromer was also found in primary cortical neurons. Finally, the heteromer formed by CB1R and the N-methyl-D-aspartate ionotropic glutamate receptor (NMDAR) was identified and characterized in both heterologous systems and primary neurons. The results showed that CB1R activation blocks NMDAR function and crossantagonism is detected. Increased expression of the CB1R-NMDAR heteromer in primary cortical neurons treated with β-amyloid peptide suggests that targeting the heteromer may provide benefits in combating the detrimental effect of amyloid aggregation.
Involvement of Alternative Splicing Regulation in Adipose Tissue Thermogenic Induction
(Universitat de Barcelona, 2023-09-29) Castellá Giner, Moisés; Villarroya i Gombau, Francesc; Cereijo Téllez, Rubén; Universitat de Barcelona. Departament de Bioquímica i Biomedicina Molecular; Adipose tissues
[eng] Alternative splicing (AS) is a gene expression regulatory mechanism that enables generation of multiple mRNA isoforms from a single gene which commonly are translated into proteins. This mechanism is tightly regulated by cis- and trans-acting factors. Among the latter, there are splicing factors, which form part of spliceosome, and splicing regulators, which can enhance or inhibit spliceosome activity. Therefore, AS is a mechanism that can have a dramatic impact in the regulation of protein activity, which can aﬀect other relevant cellular mechanisms and even tissue functions. AS has been studied extensively in some diseases but the knowledge in adipometabolic pathology is still scarce but growing evidence highlights AS as a regulator of gene expression in adipose tissue. Besides energy-storing white adipose tissue, AS is especially unexplored in brown adipose tissue (BAT), which can dissipate energy in form of heat and thus increase energy expenditure. Considering the potential therapeutic approach of using rediscovered BAT in human adults to assist in the treatment of obesity due to its special energy-consuming properties, we decided to study the possible involvement of AS in brown/beige adipose tissue thermogenic function. We ﬁrst determined the transcriptomic signature associated with the acquisition of a beige phenotype in human adipose tissue by analysing adipose browning occurring in patients aﬀected by pheochromocytoma using deep RNAseq analysis and last generation bioinformatic tools. We identiﬁed a massive down regulation of splicing machinery- associated genes occurring in association with human adipose browning and in diﬀerentiating human beige cells in culture. In addition, these splicing modiﬁcations may act by means of direct modulation of the enzymatic machinery involved in key metabolic and thermogenic functions and/or more indirectly aﬀecting the isoform expression of master regulators and functional molecular actors of browning. This aﬀectation of splicing machinery-associated genes in adipose browning led us to investigate the possible role of two candidates: SF3B1, a key component of the spliceosome, and RBFOX2, a splicing factor/regulator of AS, in brown adipocytes. We discovered that SF3B1 was up-regulated in vivo in murine BAT after thermogenic activation in response to acute cold, and in in vitro brown adipocytes in response to cAMP treatment, the main intracellular mediator of the noradrenergic stimuli. We further explored the eﬀects of blocking SF3B1 using siRNA-mediated interference and a pharmacological inhibitor, pladienolide-B. Blockage of this splicing factor actions reduced both the expression of prominent thermogenesis-associated transcripts and respiration in brown adipocytes in vitro. Next, we assessed the eﬀects of silencing RBFOX2 in brown adipocytes in vitro through shRNA-mediated interference. Repression of RBFOX2 caused alterations in transcript levels of thermogenesis-related genes and in alternative splicing-driven mRNA isoforms involved in brown adipocyte function. Moreover, signs of impaired respiration and lipolysis, together with the aforementioned changes, suggested that RBFOX2 activity is required for appropriate brown adipocyte thermogenic function. Finally, we obtained preliminary results of adipose tissue-speciﬁc knock out of RBFOX2 in mice model which indicated mild eﬀects on thermogenesis, with RBFOX2-KO mice showing enhanced cold exposure adaptation by increasing their body temperature faster that controls, but further research is required to full-characterize the role of RBFOX2 in vivo during adaptative thermogenesis. Taken together, our results indicate that AS is a critical process in brown adipose tissue functions that can have a deep impact in systemic energy homeostasis, suggesting further studies on the potential applications energy-consuming brown fat AS regulation- based therapies to counter obesity, type 2 diabetes and its associated comorbidties.
Development and application of methodologies and infrastructures for cancer genome analysis within Personalized Medicine
(Universitat de Barcelona, 2023-03-31) Royo Garrido, Romina; Torrents Arenales, David; Gelpí Buchaca, Josep Lluís; Universitat de Barcelona. Departament de Bioquímica i Biomedicina Molecular
[eng] Next-generation sequencing (NGS) has revolutionized biomedical sciences, especially in the area of cancer. It has nourished genomic research with extensive collections of sequenced genomes that are investigated to untangle the molecular bases of disease, as well as to identify potential targets for the design of new treatments. To exploit all this information, several initiatives have emerged worldwide, among which the Pan-Cancer project of the ICGC (International Cancer Genome Consortium) stands out. This project has jointly analyzed thousands of tumor genomes of different cancer types in order to elucidate the molecular bases of the origin and progression of cancer. To accomplish this task, new emerging technologies, including virtualization systems such as virtual machines or software containers, were used and had to be adapted to various computing centers. The portability of this system to the supercomputing infrastructure of the BSC (Barcelona Supercomputing Center) has been carried out during the first phase of the thesis. In parallel, other projects promote the application of genomics discoveries into the clinics. This is the case of MedPerCan, a national initiative to design a pilot project for the implementation of personalized medicine in oncology in Catalonia. In this context, we have centered our efforts on the methodological side, focusing on the detection and characterization of somatic variants in tumors. This step is a challenging action, due to the heterogeneity of the different methods, and an essential part, as it lays at the basis of all downstream analyses. On top of the methodological section of the thesis, we got into the biological interpretation of the results to study the evolution of chronic lymphocytic leukemia (CLL) in a close collaboration with the group of Dr. Elías Campo from the Hospital Clínic/IDIBAPS. In the first study, we have focused on the Richter transformation (RT), a transformation of CLL into a high-grade lymphoma that leads to a very poor prognosis and with unmet clinical needs. We found that RT has greater genomic, epigenomic and transcriptomic complexity than CLL. Its genome may reflect the imprint of therapies that the patients received prior to RT, indicating the presence of cells exposed to these mutagenic treatments which later expand giving rise to the clinical manifestation of the disease. Multiple NGS- based techniques, including whole-genome sequencing and single-cell DNA and RNA sequencing, among others, confirmed the pre-existence of cells with the RT characteristics years before their manifestation, up to the time of CLL diagnosis. The transcriptomic profile of RT is remarkably different from that of CLL. Of particular importance is the overexpression of the OXPHOS pathway, which could be used as a therapeutic vulnerability. Finally, in a second study, the analysis of a case of CLL in a young adult, based on whole genome and single-cell sequencing at different times of the disease, revealed that the founder clone of CLL did not present any somatic driver mutations and was characterized by germline variants in ATM, suggesting its role in the origin of the disease, and highlighting the possible contribution of germline variants or other non-genetic mechanisms in the initiation of CLL.
Noves evidències de l’efecte neuroprotector del Sistema Cannabinoide a nivell del Sistema Nerviós Central
(Universitat de Barcelona, 2023-03-17) Lillo Jové, Jaume; Franco Fernández, Rafael; Navarro Brugal, Gemma; Universitat de Barcelona. Departament de Bioquímica i Biomedicina Molecular
[cat] El Sistema Cannabinoide, que inclou els dos receptors de cannabinoides CB1 i CB2, els seus lligands endògens i els enzims de síntesi i degradació d’aquests, és un sistema molt complex que controla un gran nombre de processos biològics. Aquest sistema té un paper important tant a nivell del Sistema Nerviós Central com Perifèric, tenint una implicació directa en la plasticitat neuronal i la neuroprotecció entre d’altres. Els resultats presentats en aquesta Tesi Doctoral aporten noves evidències del rol neuroprotector del Sistema Cannabinoide vers diverses patologies del Sistema Nerviós Central. El fet que el receptor CB2 es trobi sobreexpressat en episodis de neuroinflamació posa de manifest la seva importància a l’hora d’oferir una resposta neuroprotectora. Aquest efecte l’hem observat en els resultats que es presenten en aquesta Tesi Doctoral en diferents models animals i en diferents patologies. En el model animal de ratolí d’ictus isquèmic, els individus afectats presentaven uns nivells elevats d’expressió de l’heteròmer CB2R-5HT1AR. És més, la progènie de ratolines alimentades amb dieta alta en greixos també presentaven una sobreexpressió de CB2R, aquest cop interaccionant amb el receptor GHS-R1a. En la patologia del Parkinson, s’ha demostrat una elevada expressió de l’heteròmer CB2R-GPR55 en macacos Parkinsonians. Tot i que el CB2R és el receptor cannabinoide associat més directament amb la neuroprotecció, també hem observat que el CB1R pot tenir un paper important. És cert que la sobreexpressió de CB1R no és tan evident com la de CB2R en la patologia de l’Alzheimer, on els assaigs de qPCR indiquen que en ratolins 5xFAD l’expressió de CB2R pot incrementar-se fins a gairebé 10 vegades, mentre que el CB1R es duplica. En aquest sentit hem detectat que l’heteròmer CB1R-GHR1a es troba sobreexpressat en un model d’obesitat, mentre que el CB1R-GPR55 ho fa en la patologia de Parkinson. El CBD, el segon component més abundant de la planta del cànnabis també juga un paper important en el tractament de les patologies amb un component inflamatori. Els resultats presentats en aquesta Tesi Doctoral demostren que el CBD pot modular el fenotip de la micròglia activada, a favor d’un fenotip neuroprotector en detriment d’un fenotip proinflamatori. Aquest efecte sobre la micròglia l’observem en seccions de ratolins isquèmics i en seccions de cervell de ratolins model de la patologia d’Alzheimer, on el tractament amb CBD incrementa la immunoreactivitat de marcadors neuroprotectors com l’Arginasa I i disminueix els de marcadors inflamatoris com iNOS. Potser un dels efectes més atractius del CBD és la seva capacitat per inhibir el transport axonal de proteïnes associades a la patologia de l’Alzheimer i el Parkinson entre neurones, limitant l’afectació a neurones veïnes i alentint el progrés de la malaltia. Els dipòsits d’Aβ i pTau són tòxics per les neurones i mitjançant la tècnica d’immunocitoquímica, hem observat que afecten també a la formació de neurites. Sorprenentment, el tractament amb CBD és capaç de recuperar gairebé els nivells basals de neurites que presenta una neurona sana, revertint la pèrdua patològica d’aquestes neurites.
Protein kinase-dependent Kv1.3 biology
(Universitat de Barcelona, 2023-01-20) Estadella Pérez, Irene; Felipe Campo, Antonio; Universitat de Barcelona. Departament de Bioquímica i Biomedicina Molecular
[eng] The voltage gated potassium channel Kv1.3 is a transmembrane protein that selectively drives potassium ions participating in the electrochemical gradient of cell membranes. This channel presents a wide distribution within the body, thereby playing an important role in several physiological processes, such as regulating the cell volume, proliferation and apoptosis, and leukocyte activation. Of particular interest in this dissertation is the role of Kv1.3 in the immune system, where its activity is crucial to initiate the immune response. Moreover, an increased and/or delocalized expression of the channel is observed at the onset of autoimmune diseases pointing Kv1.3 as a potential therapeutic target. In this contest, the study of the mechanisms involved in the modulation of the amount of Kv1.3 at the plasma membrane deserves considerable attention. Kv1.3 activity mostly relies in its abundance and proper plasma membrane location, which is tightly regulated by a balance between the forward trafficking and the endocytic machinery. Thus, the control of channel internalization and degradation influences the inflammatory response. The endocytosis of Kv1.3 has been extensively studied in our laboratory and we claim that ubiquitination mediates the internalization and further lysosomal degradation of Kv1.3 via both PKC and EGFR activation. However, the specific residues, among all Kv1.3 intracellular lysines, which play a major role in channel turnover are still unknown. Moreover, although the ubiquitin ligase Nedd4-2 has been proposed to downregulate Kv1.3 activity, there is no clue about how the interaction takes place. In this context, adenosine (ADO), an endogenous key mediator in the immune response, triggers Kv1.3 endocytosis via PKC activation. However, via its A1 and A2A receptors, ADO not only activates PKC but also PKA. The ADO-dependent Kv1.3 modulation via such differential mechanisms had not been explored yet in immune cells. For that reason, in the present work we aimed to shed light to the mechanism involved in Kv1.3 turnover and thus, provide new knowledge of the molecular physiology of the immune system. We have deciphered the molecular determinants involved in Ser/Thr kinase (PKC)- and Tyrosine kinase (EGF)-mediated Kv1.3 turnover. Activation of either pathway internalized the channel by the specific ubiquitination of the lysines K70, 84, 476, 498 and 519 which form two clusters at the amino and carboxy terminal domain of the channel. Moreover, our results suggested that these two clusters are also involved in the association between Kv1.3 and Nedd4-2. We confirmed that the Kv1.3-Nedd4-2 interaction is not direct but, a physical proximity (< 40 nm) between the proteins suggested the participation of adaptor proteins. We have also characterized the PKA-mediated Kv1.3 downregulation which, unlike PKC, did not triggered the endocytosis of the channel targeting Kv1.3 to proteasomal degradation. Moreover, the activation of PKC and PKA pathways using specific ADO receptors agonists efficiently modulate Kv1.3-mediated leukocyte physiology. Thus, ADO exerts an efficient anti-inflammatory response by the activation of two complementary and synergic signalling pathways. In conclusion, this thesis further expands the knowledge of the molecular mechanisms involved in Kv1.3 turnover. We provide an insight from the molecular determinants to the functional consequences of Kv1.3 downregulation controlling the immune response and the leukocyte physiology. Our results are of considerably physiological interest due to the combination of the multitherapeutic potential of Kv1.3 and ADO.
Obesitat severa i ateroesclerosi subclínica: biologia molecular, marcadors i efecte de la cirurgia bariàtrica
(Universitat de Barcelona, 2022-10-14) Carmona Maurici, Júlia; Pardina Arrese, Eva; Universitat de Barcelona. Departament de Bioquímica i Biomedicina Molecular
[cat] La predicció del risc de patir un esdeveniment cardiovascular en les persones amb obesitat severa és imprecisa. A més, els mecanismes que interrelacionen l’obesitat amb l’ateroesclerosi no es coneixen amb suficient profunditat. Es creu que els factors que determinen l’aparició de l’ateroesclerosi subclínica (asimptomàtica) en l’obesitat severa s’estenen més enllà dels factors de risc tradicionals. Per això proposem que algunes molècules plasmàtiques podrien relacionar l’obesitat amb la presència de lesió vascular subclínica i, per tant, permetrien detectar amb antelació aquelles persones amb major risc de desenvolupar esdeveniments cardiovasculars. Tenint en compte aquest context, es formulen els següents objectius: 1. Estudiar si hi ha paràmetres antropomètrics, bioquímics, genètics o fisiològics diferencials entre individus amb ateroma i sense, que puguin explicar perquè individus amb perfil d’obesitat severa similar, tenen diferent risc cardiovascular. Per això analitzarem paràmetres relacionats amb la inflamació, l’estrès oxidatiu i la funció endotelial vascular en una cohort d’individus amb obesitat severa agrupats segons la presència o absència de placa. 2. Analitzar l’efecte de la cirurgia bariàtrica sobre l’evolució de l’ateroesclerosi subclínica i el risc cardiovascular. Per això mesurarem els paràmetres relacionats amb el risc cardiovascular abans mencionats un mes abans de la BS i també al cap de 6 i 12 mesos en la mateixa cohort. 3. Establir si entre els paràmetres que resultin rellevants en el desenvolupament del procés ateromatós hi ha candidats per a l’ús com a biomarcadors amb bona capacitat predictiva per a l’ateroesclerosi subclínica i/o indicadors de l’evolució del risc cardiovascular.
Regulation of hepatic metabolism by the autophagic protein TP53INP2
(Universitat de Barcelona, 2022-10-28) Frager, Petra; Zorzano Olarte, Antonio; Universitat de Barcelona. Departament de Bioquímica i Biomedicina Molecular
[eng] Decreased physical activity and increased consumption of energy-rich food are hallmark lifestyle modifications that developed and amplified over the course of only a few decades. These changes in our daily behavior are the culprits for the rise of various metabolic diseases, collectively regarded to as the metabolic syndrome, that has reached the magnitude of a global epidemic. The hepatic manifestation of the metabolic syndrome is the non-alcoholic fatty liver disease (NAFLD), and it is estimated to affect every third adult person by 2030. Although considered relatively benign, NAFLD can progress to more severe liver diseases, up to the point of liver failure, through insufficiently understood processes. Different studies have shown a connection between autophagy and the development of steatosis. However, the different genetic models used in those studies report that steatosis can be ameliorated or enhanced by impaired autophagy and is dependent on the model as well as the experimental conditions. In the present project we aimed to analyze the impact of liver-specific depletion of a positive regulator of autophagy termed TP53INP2 on liver metabolism and delineate its potential role in the development of steatosis. We found that the hepatic expression of TP53INP2 in mice is modulated by nutrient deprivation as well as the presence of abundant nutrients. Mice specifically lacking TP53INP2 in the liver, show impaired hepatic free fatty acid oxidation and ketogenesis, evoked from a compromised transcriptional activity of the transcription factors PPARα and LXRα. We have observed that the hepatocyte-specific ablation of TP53INP2 enhances fasting- and dietary-induced steatosis and impacts cholesterol and bile acid metabolism upon consumption of diets rich in lipids and cholesterol. Furthermore, we have detected a role of TP53INP2 in the release of cholesterol from lysosomes, potentially involving the lysosome-associated membrane protein 1 (LAMP1). Together, our data indicate that TP53INP2 is a key regulator of hepatic lipid metabolism through the modulation of PPARα and LXRα activity. We propose that such a modulation relies on the promotion of ligand availability, perhaps dependent of autophagy.
Parmbsc2: Development of an accurate force field for nucleic acids simulations
(Universitat de Barcelona, 2022-09-29) Gallego Pérez, Diego; Orozco López, Modesto; Dans Puiggròs, Pablo D.; Universitat de Barcelona. Departament de Bioquímica i Biomedicina Molecular
[eng] Molecular dynamics (MD) simulations are the equivalent to a computational microscope allowing us to interrogate molecules and their behaviour in complex systems. They are recognised as a valuable and accurate tool in reproducing experimental evidence and predicting biomolecules flexibility at a resolution level which experimental techniques can’t usually reach. Running a MD simulation implies the propagation of Newton’s equations of motion of a system composed by beads connected by bonds. The core of MD is the energy functional: the force field, a classical Hamiltonian, defined by a set of empirical parameters defining how the energy of the system change as a consequence of geometrical alterations. Highly accurate force fields are nowadays available for proteins, nucleic acids, membranes, organic and inorganic ligands, materials, and many other systems. Focusing on nucleic acids, state-of-the-art force fields for DNA (parmbsc1 and OL15) are now able to predict B-DNA structure and flexibility with the same accuracy as NMR and X-ray experiments, and correctly reproduce unusual secondary motifs, alternative DNA forms, DNA binding with numerous partners, folding and melting processes, etc. On the contrary, RNA structure and flexibility is more difficult to capture, and the maturity of RNA force fields is far from being as “good” as the one reached for DNA, and consensus exists that a new force- field able to capture the rich conformational freedom of RNA should be developed. A substantial part of this thesis focuses on this precise problematic. This thesis is transversal to many fields, going from applied structural bioinformatics to pure physics-based modelling using classical and quantum approximations. We created the R package veriNA3d to systematize the process of handling PDB contents in diverse formats, including facilities to analyse large structures like ribosomes. VeriNA3d showed to be particularly useful for extracting detailed information on DNA-protein interactions, analysing any desired conformational space sampled experimentally, or generating, as for the purpose of this thesis, a specific dataset of all stacked nucleobase arrangements (over 3x105 structures). To reach a deep understanding of stacking interaction at both structural and energetic level, high-level QM approximations were used, and a Machine Learning algorithm was developed that showed to accurately predict expected stacking energies. We also proposed a new set of non-bonded parameters in Lifson’s force field framework, meant to be part of the future parmbsc2. Parmbsc2 is currently under beta- testing for both DNA and RNA systems. The thesis is written in monographic form. It covers a range of topics in nucleic acid structure and modelling introduced in chapter 1 with a general overview. The introduction leads to the objectives of this work in chapter 2. Chapter 3 focuses on methodological details covering quantum mechanics, molecular mechanics, and a detailed explanation of the force field parameterization scheme for non-bonded terms and dihedral angles. Chapter 4 is reserved for the results, and chapter 5 for the general discussion. The chapter 6 closes this thesis with the conclusions.
Obtención de aptámeros específicos para enzimas de la vía del metileritritol fosfato de microorganismos
(Universitat de Barcelona, 2022-07-04) Roca Martínez, Carlota; Imperial Ródenas, Santiago; Fernàndez Busquets, Xavier; Universitat de Barcelona. Departament de Bioquímica i Biomedicina Molecular
[spa] FUNDAMENTO: Microorganismos patógenos como las bacterias Mycobacterium tuberculosis y Pseudomonas aeruginosa, y los protozoos del filo Apicomplexa, incluyendo los agentes causantes de la malaria y la toxoplasmosis, sintetizan los precursores isoprenoides isopentenil difosfato (IPP) y dimetilalil difosfato (DMAPP), por la vía del metileritritol fosfato (MEP). Esta vía es esencial para la mayoría de las bacterias y Apicomplexa, pero no está presente en los seres humanos, que sintetizan IPP y DMAPP por la vía alternativa del mevalonato. El papel esencial de la vía MEP y su distribución en diferentes organismos hacen que sus enzimas sean objetivos atractivos para el desarrollo de nuevos agentes antiinfecciosos. Aquí, nos centramos en el desarrollo de aptámeros contra enzimas clave de la vía MEP. Los aptámeros son oligonucleótidos monocatenarios que se comportan como "anticuerpos químicos" y pueden unirse de manera específica y eficiente a una molécula diana determinada. MÉTODOS: Se han optimizado varios métodos, tales como: (i) la producción de enzimas de la vía MEP, (ii) el desarrollo de aptámeros a través de la evolución sistemática de ligandos por enriquecimiento exponencial, un proceso de selección in vitro basado en ciclos iterativos de unión, partición y amplificación de oligonucleótidos a partir de un conjunto de secuencias variantes, (iii) la clonación de aptámeros, (iv) el establecimiento de un ensayo de cambio de motilidad electroforética (EMSA) para la identificación de interacciones entre aptámeros seleccionados y sus enzimas diana, y (v) métodos para la evaluación in vitro de la actividad enzimática. RESULTADOS: Se informa de la identificación de un aptámero de ADN (D10) que se une específicamente a la enzima 1-desoxi-D-xilulosa-5- fosfato reductoisomerasa (DXR) de Plasmodium falciparum, que cataliza la segunda etapa de la vía del metileritritol fosfato (MEP) de biosíntesis de isoprenoides. Se ha demostrado que el aptámero D10 interacciona con la DXR de Plasmodium y también con la de Escherichia coli. Además, produce un efecto inhibidor sobre la actividad enzimática de las preparaciones de la enzima de E. coli. Los resultados obtenidos por microscopía confocal y citometría de flujo demuestran que el aptámero D10 marca específicamente los eritrocitos infectados por P. falciparum tanto en las fases iniciales como tardías y que su capacidad para discriminar los eritrocitos infectados de los no infectados es equiparable a la de otros marcadores específicos. En la segunda parte de la tesis se han obtenido aptámeros contra el tercer enzima de la vía del MEP, la 4-difosfocitidil-2-C-metil-D-eritritol sintasa de E. coli combinando el método de selección convencional y una estrategia de selección basada en el enfoque de esfera única. CONCLUSIONES: Los experimentos demuestran que el aptámero D10 podría ser utilizado como un nuevo aptasensor para la localización del apicoplasto, un orgánulo importante como diana de compuestos antimaláricos; y además como un candidato potencial para el desarrollo de nuevos agentes terapéuticos y para el diseño de nuevos sistemas de diagnóstico.
Molecular determinants of the Kv1.3/KCNE4 interaction
(Universitat de Barcelona, 2022-04-08) Sastre Martinez, Daniel; Felipe Campo, Antonio; Universitat de Barcelona. Departament de Bioquímica i Biomedicina Molecular
[eng] Kv1.3 plays a crucial role in leukocytes, where it regulates activation and proliferation. In these cells, Kv1.3 is coexpressed with modulatory subunit KCNE4. This ancillary peptide acts as a dominant negative regulator of the channel with a dual function. On the one hand, KCNE4 triggers the intracellular retention of Kv1.3 reducing K+ currents. On the other, KCNE4 enhances their C- type inactivation kinetics. Thus, the aim of this thesis was to characterize the molecular mechanisms of Kv1.3/KCNE4 association and modulation in the context of the immune system physiology. To do so, the thesis is structured in three chapters. Chapter I (KCNE4 in the immune system). We confirmed the physiological role of KCNE4 in immune cells like T lymphocytes and dendritic cells (Contribution 1). By manipulating the expression of KCNE4 in these cells, we were the first to describe a relationship between KCNE4 and the leukocyte function. Our results highlight a possible therapeutic role for KCNE4. Chapter II (A protein interaction hub (L69-72) in the C-terminus of KCNE4). We analysed the relationship between Kv1.3, KCNE4 and calmodulin (CaM). We identified a tetraleucine motif in the C-terminus of KCNE4 (L69-72) that determines association to Kv1.3 (Contribution 2). In a nearby position, an ER retention motif (ERRM) triggers intracellular retention of the channel. These results were replicated in a peptide containing just 22 residues of KCNE4, including both clusters. This peptide partially replicated the inhibitory effects of KCNE4 and thus, a potential pharmacological interest. We are also the first to propose a structural model for the Kv1.3/KCNE4 association. On the other hand, L69-72 in KCNE4 also determines CaM binding. The role of Ca2+/CaM is relevant in the context of the immune system physiology, where Kv1.3 potentiates the calcium signalling. Furthermore, we are the first to report dimerization of KCNE4 (Contribution 3), which modulates the traffic of KCNE4. Dimerization was also mediated by the leucine cluster, acting as a protein interaction hub. We propose CaM binding as a mechanism to fine-tune interactions with Kv1.3 and dimerization. Chapter III (Structural relationships of the Kv1.3/KCNE4 complex). We focused on the structural Kv1.3/KCNE4 association. A series of chimaeras between KCNE2 and KCNE4 allowed us to decipher roles for each domain of KCNE4 (Contribution 4). Thus, we confirmed that the C- terminus of KCNE4 mediates association and intracellular retention of Kv1.3. Moreover, for the first time we characterized the molecular determinants of KCNE4-dependent kinetic modulation of Kv1.3. These results were confirmed with Gibbs free energy calculations and the Kv1.3/KCNE4 structural model. Using alanine-scanning mutagenesis, we mapped some residues implicated in this process. Moreover, we found that, similar to other Kv/KCNE associations, Kv1.3/KCNE4 associated in an open stoichiometry with up to four KCNE4 subunits per complex (Contribution 5). The number of KCNE4 peptides in the channelosome was proportional to the effects on activation kinetics and current amplitude. However, the accentuation of Kv1.3 inactivation was independent of stoichiometry. The similarities in stoichiometry and docking to Kv channels suggest a conserved mechanism among KCNE peptides. Finally, because KCNE4 caused structural Kv1.3 changes, we studied the effect of KCNE4 on Kv1.3 pharmacology (Contribution 6). We studied margatoxin and Psora-4, two well-characterized Kv1.3 inhibitors with different binding sites. Our results point out the relevance of considering auxiliary subunits in the development of therapeutic approaches. In conclusion, this thesis deepens the knowledge of the molecular mechanisms of Kv1.3 modulation by KCNE4. We provide a structural insight into Kv channel modulation while considering the physiological context. Our results are of considerably physiological interest due to the multitherapeutic potential of Kv1.3.
Characterization of the role of TKTL1 in Acute Monocytic Leukaemia
(Universitat de Barcelona, 2022-04-07) Baptista, Inês do Carmo Viegas; Cascante i Serratosa, Marta; Marin Martínez, Silvia; Guenther, Ulrich; Universitat de Barcelona. Departament de Bioquímica i Biomedicina Molecular
[eng] Leukaemia is one of the types of cancer where treatment resistance is prevalent. Better understanding of leukemic cells metabolism opens possibilities for new therapeutic strategies and better prognosis stratification. Leukaemia arises from many different genetic alterations, that affect distinct cellular processes, all driving leukemogenesis. Many of these result in a phenotype that grants metabolic advantages amidst the hypoxic conditions of the haematopoietic niche of the bone marrow, the point of origin of all types of this cancer. In order to understand the metabolic reprogramming that grants these advantages, we performed metabolic characterization in vitro of cell lines of acute monocytic leukaemia (THP1) and chronic myeloid leukaemia (HAP1), with focus on the role of Transketolase-like 1 (TKTL1) and ten-to-eleven Methylcytosine dioxygenase 2 (TET2), respectively, on both normoxic and hypoxic experimental settings. We revealed that TKTL1 is a key enzyme in the metabolic reprogramming of the hypoxia adaptation, driving proliferation, higher glycolytic rates, higher glutamine consumption and subsequent glutamate production. Our results also showed the altered metabolism of many amino acids and biogenic amines, that grant more substrates for nucleotides synthesis, higher stress tolerance and manipulation of the immune response of the microenvironment. It also highlighted the vulnerabilities that arise from focusing on targeting TKTL1 for therapy and possible secondary targetable metabolic pathways for future therapies. Additionally, we demonstrated the effects of the loss of TET2 in the leukemic cells through a tracer-based metabolomics approach, showing how its mutation primes the cells to shift their metabolism at a higher cost for their ROS homeostasis, a known hallmark of cancer which increases the risk of more mutations occurring due to genomic instability. This allowed us to create a metabolic map of the changes induced by the loss of TET2, as a blueprint for new therapeutic venues in leukaemias that have this mutational hit. Together, the thesis presented here contributes to the knowledge of the mechanisms underlying metabolic reprogramming of leukaemia according to specific mutational genotypes and how they open new possible therapies for patients that develop resistance.
Mitochondrial dynamics as a hub in the control of muscle inflammation
(Universitat de Barcelona, 2021-12-03) Irazoqui Guimon, Andrea; Zorzano Olarte, Antonio; Gumà i Garcia, Anna Maria; Universitat de Barcelona. Departament de Bioquímica i Biomedicina Molecular
[eng] Some forms of mitochondrial dysfunction induce sterile inflammation through mitochondrial DNA (mtDNA) recognition by intracellular DNA sensors. However, our understanding of the processes operating this activation is partial. Here we have analyzed the participation of mitochondrial dynamics in the control of inflammatory responses. We document that mitochondrial fragmentation causes NFκB-dependent inflammation, whereas mitochondrial elongation activates both NFκB and type I interferon (IFN) inflammatory responses in muscle cells. This differential response is a consequence of activation of the DNA sensors TLR9 or cGAS. Surprisingly, we also document that Mfn1 deficiency-induced inflammation is associated with an enhanced encounter of mitochondria with early endosomes, which requires the participation of the early endosomal protein Rab5C. Mfn1 ablation in mouse skeletal muscles promoted NFκB activation, muscle atrophy, reduced physical performance and enhanced IL6 response to exercise, which were improved or rescued upon chronic anti- inflammatory treatment. Taken together, our data demonstrate that mitochondrial dynamics is key in mitigating different inflammatory responses through mtDNA mislocation. We also demonstrate that muscle inflammation caused by mitochondrial fragmentation precedes the development of muscle atrophy and impaired physical performance. Therefore, we propose that inflammatory muscle disorders characterized by triggering of DNA sensors can be underpinned by therapeutic strategies promoting balanced mitochondrial dynamics.

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