Role of High-Mobility Group Box-1 in Liver Pathogenesis

High-mobility group box 1 (HMGB1) is a highly abundant DNA-binding protein that can relocate to the cytosol or undergo extracellular release during cellular stress or death. HMGB1 has a functional versatility depending on its cellular location. While intracellular HMGB1 is important for DNA structure maintenance, gene expression, and autophagy induction, extracellular HMGB1 acts as a damage-associated molecular pattern (DAMP) molecule to alert the host of damage by triggering immune responses. The biological function of HMGB1 is mediated by multiple receptors, including the receptor for advanced glycation end products (RAGE) and Toll-like receptors (TLRs), which are expressed in different hepatic cells. Activation of HMGB1 and downstream signaling pathways are contributing factors in the pathogenesis of non-alcoholic fatty liver disease (NAFLD), alcoholic liver disease (ALD), and drug-induced liver injury (DILI), each of which involves sterile inflammation, liver fibrosis, ductular reaction, and hepatic tumorigenesis. In this review, we will discuss the critical role of HMGB1 in these pathogenic contexts and propose HMGB1 as a bona fide and targetable DAMP in the setting of common liver diseases

Bacillus cereus non-haemolytic enterotoxin activates the NLRP3 inflammasome

Inflammasomes are important for host defence against pathogens and homeostasis with commensal microbes. Here, we show non-haemolytic enterotoxin (NHE) from the neglected human foodborne pathogen Bacillus cereus is an activator of the NLRP3 inflammasome and pyroptosis. NHE is a non-redundant toxin to haemolysin BL (HBL) despite having a similar mechanism of action. Via a putative transmembrane region, subunit C of NHE initiates binding to the plasma membrane, leading to the recruitment of subunit B and subunit A, thus forming a tripartite lytic pore that is permissive to efflux of potassium. NHE mediates killing of cells from multiple lineages and hosts, highlighting a versatile functional repertoire in different host species. These data indicate that NHE and HBL operate synergistically to induce inflammation and show that multiple virulence factors from the same pathogen with conserved function and mechanism of action can be exploited for sensing by a single inflammasome

The cytoskeleton in cell-autonomous immunity: structural determinants of host defence

Host cells use antimicrobial proteins, pathogen-restrictive compartmentalization and cell death in their defence against intracellular pathogens. Recent work has revealed that four components of the cytoskeleton — actin, microtubules, intermediate filaments and septins, which are well known for their roles in cell division, shape and movement — have important functions in innate immunity and cellular self-defence. Investigations using cellular and animal models have shown that these cytoskeletal proteins are crucial for sensing bacteria and for mobilizing effector mechanisms to eliminate them. In this Review, we highlight the emerging roles of the cytoskeleton as a structural determinant of cell-autonomous host defence

Russell-like bodies in plant seeds share common features with prolamin bodies and occur upon recombinant protein production

Although many recombinant proteins have been produced in seeds at high yields without adverse effects on the plant, endoplasmic reticulum (ER) stress and aberrant localization of endogenous or recombinant proteins have also been reported. The production of murine interleukin-10 (mIL-10) in Arabidopsis thaliana seeds resulted in the de novo formation of ER-derived structures containing a large fraction of the recombinant protein in an insoluble form. These bodies containing mIL-10 were morphologically similar to Russell bodies found in mammalian cells. We confirmed that the compartment containing mIL-10 was enclosed by ER membranes, and 3D electron microscopy revealed that these structures have a spheroidal shape. Another feature shared with Russell bodies is the continued viability of the cells that generate these organelles. To investigate similarities in the formation of Russell-like bodies and the plant-specific protein bodies formed by prolamins in cereal seeds, we crossed plants containing ectopic ER-derived prolamin protein bodies with a line accumulating mIL-10 in Russell-like bodies. This resulted in seeds containing only one population of protein bodies in which mIL-10 inclusions formed a central core surrounded by the prolamin-containing matrix, suggesting that both types of protein aggregates are together removed from the secretory pathway by a common mechanism. We propose that, like mammalian cells, plant cells are able to form Russell-like bodies as a self-protection mechanism, when they are overloaded with a partially transport-incompetent protein, and we discuss the resulting challenges for recombinant protein production

In vivo analysis of endocytic and biosynthetic transport to the plant vacuole

The plasma membrane forms the interaction site between a cell and its environment. The proteins in the plasma membrane, namely translocators and receptors, allow for the exchange of nutrients and information. This, however, urges for a stringent regulation of these proteins. One mechanism to control their amount is to transport them into the lytic vacuole via the endocytic pathway. The degradative function of vacuoles depends on proteolytic enzymes, which reach that very organelle through a different route. They are synthesized in the endoplasmic reticulum and transported via the endomembrane system. Vacuolar transport of those soluble proteins depends on sorting receptors, separate them from secretory cargo. To gain a deeper understanding on the trafficking of membrane-bound and soluble cargo to the vacuole, we aimed at characterizing the machinery mediating those processes. For this, we employed nanobody-epitope interactions to create intra-cellular setups, which enabled us to perform transport- and interaction-analyses of proteins via confocal microscopy. We revealed that “Vacuolar Sorting Receptors” (VSRs) interact with their ligands in the endoplasmic reticulum and the Golgi apparatus, but not in the trans-Golgi network and the multivesicular body, by performing “Fluorescent Lifetime Imaging to measure Förster Resonance Energy Transfer” (FRET-FLIM; Künzl et al., 2016). In order to create the reporters for the compartment-specific FRET-FLIM measurements, we linked the ligand binding domain of the VSRs to marker-proteins via a nanobody-epitope interaction. We demonstrated that VSRs do indeed recycle and identified the cis-Golgi as the destination of their retrograde transport (Früholz et al., 2018). These discoveries were based on the combination of two nanobody-epitope pairs. We used those for post-translational labelling and trapping of vacuolar sorting receptors. Concerning the machinery mediating the transport of to-be-degraded plasma membrane proteins to the vacuole, we analyzed the “Endosomal Sorting Complex Required For Transport II” (ESCRT-II). Here, we employed FRETFLIM to show that “Vacuolar Protein Sorting 22” (VPS22), 25 and 36 interact to form this specific complex. We pushed the limits of nanobody-based approaches by employing membrane-anchored nanobodies in order to import the method of co-immune precipitation into living cells. This enabled us to perform in vivo studies, which showed that ESCRT-II contains two VPS25 moieties (Fäßler et al., prepared manuscript)

Towards the development of Bacillus subtilis as a cell factory for membrane proteins and protein complexes

Background: The Gram-positive bacterium Bacillus subtilis is an important producer of high quality industrial enzymes and a few eukaryotic proteins. Most of these proteins are secreted into the growth medium, but successful examples of cytoplasmic protein production are also known. Therefore, one may anticipate that the high protein production potential of B. subtilis can be exploited for protein complexes and membrane proteins to facilitate their functional and structural analysis. The high quality of proteins produced with B. subtilis results from the action of cellular quality control systems that efficiently remove misfolded or incompletely synthesized proteins. Paradoxically, cellular quality control systems also represent bottlenecks for the production of various heterologous proteins at significant concentrations. Conclusion: While inactivation of quality control systems has the potential to improve protein production yields, this could be achieved at the expense of product quality. Mechanisms underlying degradation of secretory proteins are nowadays well understood and often controllable. It will therefore be a major challenge for future research to identify and modulate quality control systems of B. subtilis that limit the production of high quality protein complexes and membrane proteins, and to enhance those systems that facilitate assembly of these proteins.

Respostas celulares à infeção viral: proteostase e imunidade inata

Mestrado em Biomedicina MolecularViruses are small opportunistic infectious agents. Virus entry, replication and assembly are dynamic and coordinated processes that require precise interactions with host components, often with cellular organelles. Hence, we proposed to study two different viruses affecting two distinct cellular surveillance mechanisms: Human Cytomegalovirus (HCMV) and Influenza A Virus (IAV) influence on the innate immune response and proteostasis, respectively. HCMV might be associated with additional long-term health consequences in human due to its ability to establish a lifelong persistent latent infection. HCMV encodes vMIA, an anti-apoptotic protein known to co-localize at peroxisomes and mitochondria, induce their fragmentation and inhibit the downstream cellular antiviral response that is established at both organelles. In the present work, we aimed to characterize the role of vMIA in the peroxisomal-MAVS dependent antiviral response. We proposed to map the vMIA domains responsible for the organelles’ morphology changes and innate immune response inhibition. Our results revealed that the 115-130 amino acid sequence might be important for the organelles’ fragmentation. We also found that m38.5, an analogue of vMIA in murine CMV (MCMV) seems to localize at peroxisomes, induce the organelle’s fragmentation and clearly inhibit the peroxisome-dependent antiviral immune response. These results suggest that this virus may be useful to complement our results with experiments performed in animals or in the context of a viral infection. IAV is the causative agent for most of the annual epidemic in humans. During IAV infection, it occurs the accumulation of unfolded proteins and the formation of specialized sites of viral replication, resulting in the formation of insoluble aggregates or inclusions. In this study, we proposed to determine whether and how IAV infection leads to aggresomal-prone proteins accumulation. Our preliminary results suggest aggresomes formation during viral infection, previous to the vRNP release in to the cytoplasm.Os vírus são agentes infeciosos oportunistas. Os diferentes passos de um ciclo de vida viral, incluindo a entrada do vírus na célula, a replicação do seu genoma e a formação de novas partículas virais requerem interações com os diferentes componentes celulares do hospedeiro, nomeadamente com organelos. Neste projeto, propomos estudar dois tipos diferentes de vírus que afetam dois mecanismos distintos de sobrevivência celular: a influência do Citomegalovírus de humano (HCMV) na resposta imunitária inata e o efeito do Vírus da Influenza A (IAV) na proteostase. O HCMV pode estar associado com consequências graves para a saúde da população, uma vez que tem a capacidade para estabelecer uma infeção latente e persistente no hospedeiro. Este vírus codifica para a vMIA, uma proteína anti-apoptótica que se localiza nos peroxissomas e nas mitocôndrias, induzindo a sua fragmentação e inibindo a resposta antiviral celular que é estabelecida em ambos. Com isto, sugerimos mapear os domínios da vMIA responsáveis pelas alterações na morfologia dos organelos e na inibição da resposta imune. Os nossos resultados revelaram que a sequência de aminoácidos 115-130 poderá ser importante para a fragmentação dos organelos. Também descobrimos que a proteína m38.5 do Citomegalovírus de ratinho (MCMV), análoga à vMIA, parece localizar nos peroxissomas, induzir a sua fragmentação e claramente inibir a resposta antiviral dependente deste organelo. Estes resultados sugerem que este vírus poderá ser útil para complementar os nossos resultados com experiências animais ou no contexto de infeção viral. O IAV é o agente causativo da maioria das epidemias anuais em humanos. Durante a infeção com IAV, ocorre acumulação de proteínas com conformação errada e a formação de locais especializados de replicação viral, resultando na formação de agregados insolúveis ou inclusões. Neste estudo, propusemos determinar se a infeção com IAV conduz à acumulação de proteína com pré-disponibilidade para formar agressomas. Os nossos resultados, embora preliminares, sugerem que existe formação destas estruturas durante a infeção viral, previamente à libertação do genoma viral no citoplasma

The Mechanism of ER to Cytosol Retrotranslocation of Proteins Targeted to ERAD (ER Associated Degradation)

Retro-translocation from the endoplasmic reticulum (ER) to the cytosol of secretory and membrane proteins takes place on misfolded molecules targeted for proteasomal degradation, in a process called ER associated degradation (ERAD). Because of the difficulties in clearly discriminating the fraction of molecules already retro-translocated from the ones in the ER, we took advantage of the E. coli biotin-ligase (BirA) expressed in the cytosol of mammalian cells, to specifically biotin-label proteins that undergo retro-translocation. The method was validated using four different model proteins, known to undergo retro-translocation upon different conditions: the MHC-1α chain, the non-secretory null-Hong Kong mutant of α1 antitrypsin, the immunoglobulin γH chain and calreticulin. The specific mono-biotinylation of only cytosolically dislocated molecules resulted in a novel quantitative method to determine the extent of retro-translocation. The method was used to study dislocation of CD4 and BST-2/Tetherin, two membrane proteins targeted to degradation by the HIV-1 protein Vpu. It was found that CD4 retro-translocates with oxidised intra-chain disulphide bridges that only upon proteasomal inhibition accumulates in the cytosol in reduced and de-glycosylated form. Similarly, BST-2/Tetherin is first exposed to the cytosol as a dimeric-oxidised complex, which then becomes de-glycosylated and reduced to monomers. Experiments with the non-secreted NS1 Ig-κ light chain showed that also this ERAD model protein is retro-translocated with oxidised cysteines. The role of VCP/p97-ATPase in retro-translocation was investigated. In contrast to what previously reported, it was found that it is not required to dislocate ERAD substrates from the ER lumen to the cytosol, while it is required for efficient de-glycosylation and proteasomal degradation of ERAD substrates. The results obtained indicate that complete cysteine reduction and unfolding is not strictly required for retro-translocation, suggesting alternative mechanisms of the ERAD pathway. In addition, the role of VCP/p97-ATPase was found associated to stages downstream of membrane crossing

Voltage-dependent anion channels: different isoforms for different functions

The Voltage-dependent anion channel (VDAC) is the most abundant protein of the outer mitochondrial membrane (OMM) and mediates the flow of ions and metabolites between the cytoplasm and the mitochondrial network. Here we reveal novel and unexpected roles of this protein in the regulation of Ca2+ signaling, cell death and autophagy, throwing light on the differential contribution of the three mammalian isoforms in these cellular processes. In particular, we show that: i) VDAC is physically linked to the endoplasmic reticulum Ca2+ release channel inositol-1,4,5-trisphosphate receptor (IP3R), through the molecular chaperone grp75 and the functional coupling of these channel directly enhances Ca2+ accumulation in mitochondria; ii) the different VDAC isoforms share common Ca2+ channelling properties in living cells but VDAC1 is the only isotype selectively coupled to the ER Ca2+ releasing machinery, thus laying the foundations for a preferential route specifically transmitting Ca2+-mediated cell death signals between the two organelles; iii) VDAC2 is selectively required for the induction of the autophagic process through the establishment of specific protein-protein interactions and the consequent assembly of macromolecular complexes at the OMM level involved in nutrient sensing mediated by the mammalian Target Of Rapamycin (mTOR) signaling pathway. These data highlight the pleiotropic functions of VDAC and its role as central regulator of cell patho-physiology

Understanding and Drugging the Bcl-2 Transmembrane Interactome for Tumor Treatment

[ES] La familia de proteínas Bcl-2 regula la apoptosis a través de una compleja red de interacciones. Las células tumorales suelen presentar mutaciones que afectan a su expresión o sus interacciones para mejorar la progresión tumoral. Además, alteraciones en su regulación también promueven la migración de células cancerígenas, la invasión y la metástasis. Para llevar a cabo sus funciones, las proteínas Bcl 2 interaccionan entre sí tanto en el citoplasma como en las membranas intracelulares. Los equilibrios de interacción de los dominios Bcl citosólicos se han investigado ampliamente y recientemente, se han propuesto como dianas terapéuticas. Sin embargo, el interactoma de los dominios transmembrana (TMD, del inglés transmembrane domains) sigue siendo poco conocido. Por ello, un conocimiento profundo de la biología de las proteínas Bcl-2 es necesario para explotar eficientemente sus superficies de unión en el tratamiento del cáncer. Para llevar a cabo este objetivo, nos hemos centrado en tres áreas: 1. La comprensión detallada de la contribución del TMD de Mcl-1 a su interactoma en membrana y su función. 2. El descubrimiento de nuevos inhibidores de Mcl-1 que actúen sobre su TMD y que permitan desarrollar una clase de drogas anticancerígenas aún por explorar. 3. La caracterización molecular de mutaciones relacionadas con el cáncer descritas en los TMD de Bcl-2 y Bcl-xL y sus implicaciones en la supervivencia de las células tumorales. La proteína antiapoptótica Mcl-1 inhibe a los miembros proapoptóticos Bak, Bax, Bok, Noxa, etc. Aunque se ha estudiado en detalle su actividad promoviendo la supervivencia celular, el mecanismo molecular por el cuál previene la apoptosis mediada por Bok aún no está claro. Además, el conocimiento de las actividades de Mcl-1, descritas hasta ahora, se basa exclusivamente en las estructuras resueltas de las regiones solubles en agua y en estudios centrados en los dominios citosólicos. Por primera vez, hemos demostrado la relevancia del TMD de Mcl-1 en su equilibrio de interacción. En este trabajo describimos su capacidad específica para homo- y hetero-oligomerizar con el TMD de Bok. También ponemos de manifiesto la influencia de estas interacciones en la modulación de apoptosis y resaltamos la relevancia clínica de los mutantes del TMD de Mcl-1 identificados en pacientes con cáncer. Muchos tumores hematológicos y sólidos sobre-expresan Mcl-1 como mecanismo para adquirir quimiorresistencia. Se han desarrollado miméticos de BH3 específicos para modular su actividad antiapoptótica en células cancerosas. Sin embargo, aún no disponemos de datos científicos que informen sobre su toxicidad y eficacia en humanos. En este trabajo, proponemos la novedosa interacción de los TMDs de Mcl-1 y Bok como un nuevo sitio de acción de fármacos quimioterapéuticos. Hemos identificado tres inhibidores de esta interacción con características que los hacen prometedores candidatos para el desarrollo farmacéutico, así como buenas herramientas moleculares para estudiar la interacción de los TMDs de Mcl-1 y Bok. Para modular la apoptosis, las células tumorales también presentan versiones mutadas de las proteínas antiapoptóticas Bcl-2 y Bcl-xL. En nuestro conocimiento, este es el primer estudio que analiza mutaciones somáticas de sus TMDs. Nuestro trabajo demuestra cómo estas mutaciones alteran el equilibrio en membrana de las proteínas. Además, nuestros resultados explican la influencia que algunos mutantes somáticos ejercen en la regulación de la apoptosis. En general, los resultados científicos que aparecen en esta tesis resaltan el papel de los Bcl TMDs en el interactoma de las proteínas Bcl-2. Estos hallazgos corroboran que las interacciones laterales entre los TMDs son específicas y contribuyen activamente a la funcionalidad de la proteína. Por lo tanto, comprender los Bcl TMDs puede proporcionar nuevos conocimientos sobre la biología de las proteínas Bcl.[CA] La família de proteïnes Bcl-2 regula l'apoptosi a través d'una complexa xarxa d'interaccions. Les cèl·lules tumorals solen presentar mutacions que afecten la seua expressió o les seues interaccions per a millorar la progressió tumoral. A més, alteracions en la seua regulació també promouen la migració de cèl·lules cancerígenes, la invasió i la metàstasi. Per a dur a terme les seues funcions, les proteïnes Bcl-2 interaccionen entre si tant en el citoplasma com en les membranes intracel·lulars. Els equilibris d'interacció dels dominis Bcl citosòlics s'han investigat àmpliament i recentment, s'han proposat com a dianes terapèutiques. No obstant això, l'interactoma dels dominis transmembrana (TMD, de l'anglés transmembrane domains) continua sent poc conegut. Per això, un coneixement profund de la biologia de les proteïnes Bcl-2 és necessari per a explotar eficientment les seues superfícies d'unió en el tractament del càncer. Per a dur a terme aquest objectiu, ens hem centrat en tres àrees: 1. La comprensió detallada de la contribució del TMD de Mcl-1 al seu interactoma en membrana i la seua funció. 2. El descobriment de nous inhibidors de Mcl-1 que actuen sobre el seu TMD i que permeten desenvolupar una classe de drogues anticanceroses encara per explorar. 3. La caracterització molecular de mutacions relacionades amb el càncer descrites en els TMD de Bcl-2 i Bcl-xL i les seues implicacions en la supervivència de les cèl·lules tumorals. La proteïna anti apoptòtica Mcl-1 inhibeix als membres pro apoptòtics Bak, Bax, Bok, Noxa, etc. Encara que s'ha estudiat detalladament la seua activitat promovent la supervivència cel·lular, el mecanisme molecular pel qual prevé l'apoptosi mediada per Bok encara no és clar. A més, el coneixement de les activitats de Mcl-1, descrites fins ara, es basa exclusivament en les estructures resoltes solubles en aigua i en estudis centrats en els dominis externs a la membrana. Per primera vegada, hem demostrat la rellevància del TMD de Mcl-1 el seu equilibri d'interacció. En aquest treball descrivim la seua capacitat específica per a unir-se amb si mateix i per a hetero-oligomeritzar amb el TMD de Bok. També expliquem la influència d'aquestes interaccions en l'apoptosi i ressaltem la rellevància clínica dels mutants del TMD de Mcl-1 identificats en pacients amb càncer. Molts tumors hematològics i sòlids sobre-expressen Mcl-1 com un mecanisme per a adquirir quimioresistència. S'han desenvolupat mimètics de BH3 específics per a modular la seua activitat anti apoptòtica en cèl·lules canceroses. No obstant això, encara no disposem de dades científiques que informen sobre la seua toxicitat i eficàcia en humans. Per això, proposem la nova interacció dels TMDs de Mcl-1 i Bok com un lloc d'actuació de fàrmacs quimioterapèutiques. Hem identificat tres inhibidors d'aquesta interacció amb característiques que els fan prometedors candidats per al desenvolupament farmacèutic, així com bones eines moleculars per a estudiar la interacció dels TMDs de Mcl-1 i Bok. Per a modular l'apoptosi, les cèl·lules tumorals també presenten versions mutades de les proteïnes anti apoptòtiques Bcl-2 i Bcl-xL. En el nostre coneixement, aquest és el primer estudi que analitza mutacions somàtiques de les seues TMDs. El nostre treball demostra com aquestes mutacions alteren l'equilibri en membrana de les proteïnes. A més, els nostres resultats expliquen la influència que alguns mutants somàtics exerceixen en la regulació de l'apoptosi. En general, els resultats científics que apareixen en aquesta tesi ressalten el paper dels Bcl TMDs en l'interactoma de les proteïnes Bcl-2. Aquestes troballes corroboren que les interaccions laterals entre els TMDs són específiques de la seqüència i contribueixen activament a la funcionalitat de la proteïna. Per tant, comprendre els Bcl TMDs pot proporcionar nous coneixements sobre la biologia de les proteïnes Bcl[EN] The family of the Bcl-2 proteins modulates the apoptotic pathway by a complex network of interactions. Tumor cells frequently present mutations that affect Bcl-2 proteins expression or interactions to enhance cancer progression. Dysregulation of these proteins also promotes cancer cell migration, invasion, and metastasis. To execute their functions, Bcl-2 proteins interact in both the cytosol and intracellular membranes. Binding equilibria of Bcl extramembrane domains has been largely investigated and recently proposed as chemotherapeutic targets. However, the interactome of transmembrane domains (TMDs) remains poorly understood. In this scenario, a deep knowledge of the biology of Bcl-2 proteins is needed to exploit efficiently their binding surfaces for cancer treatment. To address this aim, our research focuses on three areas: 1. The detailed comprehension of the TMD contribution to both the Mcl-1 membrane interactome and protein functionality. 2. The discovery of new Mcl-1 inhibitors that target the transmembrane surface to develop a class of anticancer drugs currently unexplored. 3. The molecular characterization of cancer-related mutations within the Bcl-2 and Bcl-xL TMDs and their implications for the survival of cancer cells. Antiapoptotic Mcl-1 protein inhibits the proapoptotic members Bak, Bax, Bok, and Noxa, among others. Although its prosurvival activity has been well studied, the molecular mechanism to prevent Bok-mediated apoptosis remains unclear. Furthermore, understanding of Mcl-1 activities described to date is only based on water-soluble structures and studies focused on extramembrane domains. For the first time, we uncover the relevance of the Mcl-1 TMD in the interaction equilibria of the protein. In the present work, we describe its specific capacity to self-associate and hetero-oligomerize with the Bok TMD. We also explain the influence of these interactions in the apoptotic pathway and highlight the clinical relevance of Mcl-1 TMD mutants identified in tumor patients. Many hematological and solid malignancies overexpress Mcl-1 as an acquired chemoresistance mechanism. To modulate its antiapoptotic activity in cancer cells, specific BH3 mimetics have been developed; however, there is no scientific data yet regarding human toxicity and efficacy. In this work, we propose the novel Mcl-1 and Bok TMDs interaction interface as a drugging site in the development of chemotherapeutics. We identify three potential inhibitors of such molecular interface with promising features to become both drug candidates for pharmaceutical development and research toosl for the molecular study of the Mcl-1 and Bok TMDs interaction. To take advantage of apoptosis modulation, tumor cells also present mutated versions of the antiapoptotic members Bcl-2 and Bcl-xL. To our knowledge, this is the first study that analyzes patient-derived mutations within Bcl-2 and Bcl-xL TMDs and demonstrates how said mutations alter the membrane equilibria of these proteins. The results presented here also explain the functional influence of some somatic mutants in apoptosis regulation. Overall, the scientific results exhibited in this Thesis highlight the role of Bcl TMDs in the interactome of Bcl-2 proteins. These findings corroborate that lateral interactions between TMDs are sequence-specific and actively contribute to protein functionality. Therefore, understanding of Bcl transmembrane segments may provide new insights into the biology of Bcl 2 proteins for their pharmaceutical modulation in antitumoral therapy.The student has been granted with a PhD fellowship and a short-term fellowship from the Generalitat Valenciana (Subvenciones para la contratación de personal investigador de carácter predoctoral, 2016-2019, and Grant for predoctoral stays out of the Comunitat Valenciana, 2019). This work has been supported by the Spanish Ministry of Economy and Competitiveness (projects SAF2014-52614-R and SAF2017-84689-RLucendo Gutiérrez, E. (2020). Understanding and Drugging the Bcl-2 Transmembrane Interactome for Tumor Treatment [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/155914TESI