Genome-Wide Prediction and Validation of Peptides That Bind Human Prosurvival Bcl-2 Proteins

Programmed cell death is regulated by interactions between pro-apoptotic and prosurvival members of the Bcl-2 family. Pro-apoptotic family members contain a weakly conserved BH3 motif that can adopt an alpha-helical structure and bind to a groove on prosurvival partners Bcl-x[subscript L], Bcl-w, Bcl-2, Mcl-1 and Bfl-1. Peptides corresponding to roughly 13 reported BH3 motifs have been verified to bind in this manner. Due to their short lengths and low sequence conservation, BH3 motifs are not detected using standard sequence-based bioinformatics approaches. Thus, it is possible that many additional proteins harbor BH3-like sequences that can mediate interactions with the Bcl-2 family. In this work, we used structure-based and data-based Bcl-2 interaction models to find new BH3-like peptides in the human proteome. We used peptide SPOT arrays to test candidate peptides for interaction with one or more of the prosurvival proteins Bcl-x[subscript L], Bcl-w, Bcl-2, Mcl-1 and Bfl-1. For the 36 most promising array candidates, we quantified binding to all five human receptors using direct and competition binding assays in solution. All 36 peptides showed evidence of interaction with at least one prosurvival protein, and 22 peptides bound at least one prosurvival protein with a dissociation constant between 1 and 500 nM; many peptides had specificity profiles not previously observed. We also screened the full-length parent proteins of a subset of array-tested peptides for binding to Bcl-x[subscript L] and Mcl-1. Finally, we used the peptide binding data, in conjunction with previously reported interactions, to assess the affinity and specificity prediction performance of different models.National Institutes of Health (U.S.) (Award GM084181)National Science Foundation (U.S.) (Grant 0821391)American Cancer Society (Postdoctoral Fellowship PF-12-155-01-DMC

Locating Herpesvirus Bcl-2 Homologs in the Specificity Landscape of Anti-Apoptotic Bcl-2 Proteins

Viral homologs of the anti-apoptotic Bcl-2 proteins are highly diverged from their mammalian counterparts, yet they perform overlapping functions by binding and inhibiting BH3 (Bcl-2 homology 3)-motif-containing proteins. We investigated the BH3 binding properties of the herpesvirus Bcl-2 homologs KSBcl-2, BHRF1, and M11, as they relate to those of the human Bcl-2 homologs Mcl-1, Bfl-1, Bcl-w, Bcl-xL, and Bcl-2. Analysis of the sequence and structure of the BH3 binding grooves showed that, despite low sequence identity, M11 has structural similarities to Bcl-xL, Bcl-2, and Bcl-w. BHRF1 and KSBcl-2 are more structurally similar to Mcl-1 than to the other human proteins. Binding to human BH3-like peptides showed that KSBcl-2 has similar specificity to Mcl-1, and BHRF1 has a restricted binding profile; M11 binding preferences are distinct from those of Bcl-xL, Bcl-2, and Bcl-w. Because KSBcl-2 and BHRF1 are from human herpesviruses associated with malignancies, we screened computationally designed BH3 peptide libraries using bacterial surface display to identify selective binders of KSBcl-2 or BHRF1. The resulting peptides bound to KSBcl-2 and BHRF1 in preference to Bfl-1, Bcl-w, Bcl-xL, and Bcl-2 but showed only modest specificity over Mcl-1. Rational mutagenesis increased specificity against Mcl-1, resulting in a peptide with a dissociation constant of 2.9 nM for binding to KSBcl-2 and > 1000-fold specificity over other Bcl-2 proteins, as well as a peptide with > 70-fold specificity for BHRF1. In addition to providing new insights into viral Bcl-2 binding specificity, this study will inform future work analyzing the interaction properties of homologous binding domains and designing specific protein interaction partners.National Institute of General Medical Sciences (U.S.) (R01GM110048)National Science Foundation (U.S.) (0821391

Rapid Optimization of Mcl-1 Inhibitors using Stapled Peptide Libraries Including Non-Natural Side Chains

Alpha helices form a critical part of the binding interface for many protein-protein interactions, and chemically stabilized synthetic helical peptides can be effective inhibitors of such helix-mediated complexes. In particular, hydrocarbon stapling of peptides to generate constrained helices can improve binding affinity and other peptide properties, but determining the best stapled peptide variant often requires laborious trial and error. Here, we describe the rapid discovery and optimization of a stapled-helix peptide that binds to Mcl-1, an antiapoptotic protein that is overexpressed in many chemoresistant cancers. To accelerate discovery, we developed a peptide library synthesis and screening scheme capable of identifying subtle affinity differences among Mcl-1-binding stapled peptides. We used our method to sample combinations of non-natural amino-acid substitutions that we introduced into Mcl-1 inhibitors in the context of a fixed helix-stabilizing hydrocarbon staple that increased peptide helical content and reduced proteolysis. Peptides discovered in our screen contained surprising substitutions at sites that are conserved in natural binding partners. Library-identified peptide M3d is the most potent molecule yet tested for selectively triggering mitochondrial permeabilization in Mcl-1 dependent cell lines. Our library approach for optimizing helical peptide inhibitors can be readily applied to the study of other biomedically important targets.National Institute of General Medical Sciences (U.S.) (Award R01GM110048

Designing helical peptide inhibitors of protein–protein interactions

Short helical peptides combine characteristics of small molecules and large proteins and provide an exciting area of opportunity in protein design. A growing number of studies report novel helical peptide inhibitors of protein-protein interactions. New techniques have been developed for peptide design and for chemically stabilizing peptides in a helical conformation, which frequently improves protease resistance and cell permeability. We summarize advances in peptide crosslinking chemistry and give examples of peptide design studies targeting coiled-coil transcription factors, Bcl-2 family proteins, MDM2/MDMX, and HIV gp41, among other targets.National Institute of General Medical Sciences (U.S.) (Award GM067681)National Institute of General Medical Sciences (U.S.) (Award GM110048)National Institute of General Medical Sciences (U.S.) (Award GM084181

Dissecting and Targeting the PUMA and OLIG2 Control Points of Tumors of Neuroectodermal Origin with Stapled Peptides

Tumors of neuroectodermal origin are among the most aggressive and treatment-refractory forms of human cancer. While such tumors arise from a variety of defects, two key targets are the transcription factors p53 and OLIG2. We have developed stabilized peptides to study and target deregulated p53 and OLIG2 pathways in neuroectodermal cancers. PUMA (p53-upregulated modulator of apoptosis) is a BH3-only member of the BCL-2 protein family that regulates apoptosis in response to p53-dependent and p53-independent stress signals. The specific interactions that mediate the pro-apoptotic activity of PUMA remain controversial. We generated stabilized alpha-helices of BCL-2 domains (SAHB) peptides modeled after the BH3 effector domain of PUMA. Structural analyses determined that PUMA SAHB contacts BAX at both the N-terminal $\alpha1/\alpha6$ trigger site and the canonical BH3 binding pocket, binding events that functionally activate BAX. Notably, both PUMA SAHB and PUMA protein pull-downs identified anti- and pro-apoptotic binding partners in a cellular context. As PUMA has been implicated in driving apoptosis in multiple neural cell types, we further demonstrated that treatment of neuroblastoma cell lines with a cell-permeable PUMA SAHB analog triggered dose-dependent apoptosis. Together, we find that the PUMA BH3 domain activates apoptosis through multimodal interactions with BCL-2 family proteins, and its mimetics may serve as prototype therapeutics in tumors of neural origin. Whereas suppression of p53 signaling and apoptosis are features of diverse tumor types, the basic helix-loop-helix (bHLH) transcription factor OLIG2 is selectively overexpressed in gliomas. Early in development, OLIG2 is responsible for maintaining progenitor cells in a replication-competent state. Tumor stem cells are believed to co-opt this OLIG2 functionality to continually repopulate glial tumors. To achieve its transcriptional function, OLIG2 must dimerize via its bHLH domain. Stabilized alpha-helices of OLIG2 (SAH-OLIG2) peptides of the OLIG2 bHLH domain were generated in an effort to disrupt this pathologic dimerization. While helical stabilization of several SAH-OLIG2 peptides was achieved, specific engagement and disruption of the native bHLH dimer did not occur, informing alternative design strategies for future targeting efforts. These studies underscored the importance of interrogating the OLIG2 dimeric structure and catalyzed the discovery of candidate OLIG2 interaction partners for therapeutic targeting

Enriching Peptide Libraries for Binding Affinity and Specificity Through Computationally Directed Library Design

Peptide reagents with high affinity or specificity for their target protein interaction partner are of utility for many important applications. Optimization of peptide binding by screening large libraries is a proven and powerful approach. Libraries designed to be enriched in peptide sequences that are predicted to have desired affinity or specificity characteristics are more likely to yield success than random mutagenesis. We present a library optimization method in which the choice of amino acids to encode at each peptide position can be guided by available experimental data or structure-based predictions. We discuss how to use analysis of predicted library performance to inform rounds of library design. Finally, we include protocols for more complex library design procedures that consider the chemical diversity of the amino acids at each peptide position and optimize a library score based on a user-specified input model.National Institute of General Medical Sciences (U.S.) (Award R01 GM110048

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

Development of Methods for the Discovery of Small Molecule Biological Probes

Advances in combinatorial chemistry have facilitated the production of large chemical libraries that can be used as tools to discover biological probes and therapeutics. High-throughput screening (HTS) strategies have emerged as the standard method to assess the biological activity of small molecules. These screens involve the individual analysis of each small molecule in multi-well plates, often requiring expensive automated methods and development of robust assays that may not translate to physiologically relevant contexts. This problem of evaluating large numbers of reagents in physiologically relevant cell and animal models has been addressed for genetic reagents such as RNAi, CRISPR, and cDNA by creating barcoded retroviral libraries that can be used to infect target cells in culture or in animal models. Using these tools, effective reagents can be selected and decoded using a rapid and inexpensive procedure compared to testing of individual reagents one at a time in an arrayed fashion. In order to more efficiently analyze small molecules, a pooled approach would similarly be useful. This dissertation describes the studies towards developing a pooled screening strategy for small molecules in cellular contexts. Through an initial screen, we set to phenotypically profile small molecule biological activity in a pooled fashion, while simultaneously gain insight about an individual, active molecule’s mechanism of action. I first describe the design of the pooled screen and define the goals necessary for successful application. Next, I outline the steps taken and challenges encountered during the invention of each component of the technology. Finally, I discuss a computational, target-based approach to design small molecules appropriate for future applications of the new screening technology

Mechanistic details of apoptosis-regulatory proteins BAX and BFL1 at the membrane level

252 p.La homeostasis de tejidos es considerado como el equilibrio entre la muerte y proliferación celular, y es por ello que su desregulación a menudo está asociada a diversas situaciones patológicas, tales como enfermedades neurodegenerativas o el cáncer. Este equilibrio, se encuentra regulado principalmente por un mecanismo denominado apoptosis o suicidio celular. La familia de proteínas BCL2 se erige como pieza clave en la regulación de este mecanismo, principalmente, a nivel de la permeabilización de la membrana externa mitocondrial (MEM). En las últimas décadas, se han realizado avances de notoria importancia en el conocimiento sobre los mecanismos de acción e interacción de esta familia de proteínas, Sin embargo, importantes aspectos del proceso de activación de las proteinas proapoptóticas tipo BAX (BAX, BAK y quizás BOK) se encuentran vagamente descritos, desconociendo entre otros aspectos la disposición estructural exacta que la proteína BAX adopta en membrana o la cantidad mínima de subunidades de BAX necesarias para la formación del poro. Por otra parte, puesto que la membrana conforma el locus biológico de estas proteinas, el estudio del componente lipídico y de la propia membrana se encuentra actualmente en boga. Concretamente, el lípido especifico de la mitocondria, cardiolipina (CL), se ha erigido como uno de los moduladores claves de la apoptosis. Sin embargo, a pesar de su importancia, la función exacta de la CL en particular y de los lípidos en general, en las actividades de membrana de las BCL2, se encuentra vagamente descrita. Además, dilucidar el modo de acción de las proteinas antiapoptóticas o tipo BCL2, mediadoras del bloqueo de la actividad de las proapoptóticas, es considerado uno de los pilares fundamentales en el desarrollo de posibles dianas terapéuticas. El objetivo de esta Tesis Doctoral ha sido avanzar en la comprensión de los mecanismos de acción de la familia de proteínas BCL2, así como de los factores reguladores que modulan su actividad durante la apoptosis. Concretamente, nos centramos en dos miembros de la familia: la proapoptótica multidominio prototípica BAX, y la poco estudiada proteína antiapoptótica BFL1. Para la consecución de estos objetivos, se han realizado estudios a nivel de membrana tanto en sistemas modelo como en células, mediante diversas técnicas biofísicas cuantitativas. Considerando todo referente a la topología de BAX en membrana, concluimos que en ausencia de estímulo apoptótico BAX adopta una estructura globular apoyando su C-terminal en la membrana, conformación a la cual hemos denominado "lipid primed", ya que el lípido parece favorecer la exposición del bolsillo hidrofóbico que actuará como receptor en el proceso apoptótico. Sin embargo, una vez activada, BAX adopta una configuración BH3 into groove donde la inserción del dominio core, pero no del dominio latch, se antoja indispensable para la formación del poro apoptótico. Referente a BFL1 y sus actividades de membrana, concluimos que BFL1 posee un mecanismo de acción multimodal (MODO-0 o retrotranslocación de BAX, MODO-1 o formación de un hetero-complejo con la proteína proapoptótica cBID y MODO-2 interacción con la proteína BAX activa) y bifuncional (fenotipo antiapoptótico y proapoptótico) regulado por la cantidad de CL en la membrana. BFL1 ejerce su actividad antiapoptótica mediada por interacciones canónicas BH3-into-groove mientras que en membranas con alto contenido en cardiolipina, las cuales imitan los microdominios de este lípido descritos en condiciones apoptóticas, BFL1 no solo pierde la capacidad de formar heterodímeros con los miembros proapoptóticos, sino que también es capaz de homodimerizar vía una superficie no descrita previamente situada en el dominio BH4 de la proteína