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BCL-w is a BCL-2 family protein that promotes cell survival in tissue- and disease-specific contexts. The canonical anti-apoptotic functionality of BCL-w is mediated by a surface groove that traps the BCL-2 homology 3 (BH3) α-helices of pro-apoptotic members, blocking cell death. A distinct N-terminal portion of BCL-w, termed the BCL-2 homology 4 (BH4) domain, selectively protects axons from paclitaxel-induced degeneration by modulating IP3 receptors, a noncanonical BCL-2 family target. Given the potential of BCL-w BH4 mimetics to prevent or mitigate chemotherapy-induced peripheral neuropathy, we sought to characterize the interaction between BCL-w BH4 and the IP3 receptor, combining "staple" and alanine scanning approaches with molecular dynamics simulations. We generated and identified stapled BCL-w BH4 peptides with optimized IP3 receptor binding and neuroprotective activities. Point mutagenesis further revealed the sequence determinants for BCL-w BH4 specificity, providing a blueprint for therapeutic targeting of IP3 receptors to achieve neuroprotection.
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The average time to degree for completing a life sciences PhD in the United States is longer for single-degree than dual-degree trainees, supporting a perception that the PhD training of MD-PhDs is less rigorous or fulsome. To determine whether degree format influences the duration and impact of graduate training, we analyzed data for the 2011-2016 graduates of 3 Harvard Medical School PhD programs. Linear mixed effects models were used to determine the association between degree type (MD-PhD vs. PhD) and research outcomes, including time to degree, time to thesis defense, and publications submitted during the PhD. Although pursuing an MD-PhD was associated with a 1.5-year shorter time to PhD degree, basing this calculation on the official PhD period does not account for completion of early PhD requirements, including research rotations and qualifying coursework, during the first 2 years of medical school. There was no association between degree format and total number of first-author or overall publications, though pursuing a dual degree was associated with increased impact metrics of published papers. The results highlight that despite the seemingly shorter PhD durations of MD-PhD graduates based on graduate program enrollment period, research training is on par with their single-degree peers, rendering MD-PhD graduates well equipped to become successful scientific investigators.
Assuntos
Pesquisa Biomédica , Humanos , Estados Unidos , Pesquisa Biomédica/educação , Educação de Pós-Graduação em Medicina/métodos , Fatores de TempoRESUMO
BCL-2-associated X protein (BAX) is a promising therapeutic target for activating or restraining apoptosis in diseases of pathologic cell survival or cell death, respectively. In response to cellular stress, BAX transforms from a quiescent cytosolic monomer into a toxic oligomer that permeabilizes the mitochondria, releasing key apoptogenic factors. The mitochondrial lipid trans-2-hexadecenal (t-2-hex) sensitizes BAX activation by covalent derivatization of cysteine 126 (C126). In this study, we performed a disulfide tethering screen to discover C126-reactive molecules that modulate BAX activity. We identified covalent BAX inhibitor 1 (CBI1) as a compound that selectively derivatizes BAX at C126 and inhibits BAX activation by triggering ligands or point mutagenesis. Biochemical and structural analyses revealed that CBI1 can inhibit BAX by a dual mechanism of action: conformational constraint and competitive blockade of lipidation. These data inform a pharmacologic strategy for suppressing apoptosis in diseases of unwanted cell death by covalent targeting of BAX C126.
Assuntos
Apoptose , Proteína X Associada a bcl-2 , Proteína X Associada a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Humanos , Apoptose/efeitos dos fármacos , Cisteína/química , Cisteína/metabolismo , Animais , Aldeídos/química , Aldeídos/farmacologia , Modelos Moleculares , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismoRESUMO
The continued emergence of highly pathogenic viruses, which either thwart immune- and small molecule-based therapies or lack interventions entirely, mandates alternative approaches, particularly for prompt and facile pre- and post-exposure prophylaxis. Many highly pathogenic viruses, including coronaviruses, employ the six-helix bundle heptad repeat membrane fusion mechanism to achieve infection. Although heptad-repeat-2 decoys can inhibit viral entry by blocking six-helix bundle assembly, the biophysical and pharmacologic liabilities of peptides have hindered their clinical development. Here, we develop a chemically stapled lipopeptide inhibitor of SARS-CoV-2 as proof-of-concept for the platform. We show that our lead compound blocks infection by a spectrum of SARS-CoV-2 variants, exhibits mucosal persistence upon nasal administration, demonstrates enhanced stability compared to prior analogs, and mitigates infection in hamsters. We further demonstrate that our stapled lipopeptide platform yields nanomolar inhibitors of respiratory syncytial, Ebola, and Nipah viruses by targeting heptad-repeat-1 domains, which exhibit strikingly low mutation rates, enabling on-demand therapeutic intervention to combat viral outbreaks.
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Infecções por Coronavirus , Lipopeptídeos , Humanos , Lipopeptídeos/farmacologia , Lipopeptídeos/uso terapêutico , Lipopeptídeos/química , Pandemias/prevenção & controleRESUMO
MCL-1 is a high-priority target due to its dominant role in the pathogenesis and chemoresistance of cancer, yet clinical trials of MCL-1 inhibitors are revealing toxic side effects. MCL-1 biology is complex, extending beyond apoptotic regulation and confounded by its multiple isoforms, its domains of unresolved structure and function, and challenges in distinguishing noncanonical activities from the apoptotic response. We find that, in the presence or absence of an intact mitochondrial apoptotic pathway, genetic deletion or pharmacologic targeting of MCL-1 induces DNA damage and retards cell proliferation. Indeed, the cancer cell susceptibility profile of MCL-1 inhibitors better matches that of anti-proliferative than pro-apoptotic drugs, expanding their potential therapeutic applications, including synergistic combinations, but heightening therapeutic window concerns. Proteomic profiling provides a resource for mechanistic dissection and reveals the minichromosome maintenance DNA helicase as an interacting nuclear protein complex that links MCL-1 to the regulation of DNA integrity and cell-cycle progression.
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Antineoplásicos , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Apoptose , Proteômica , Antineoplásicos/farmacologia , Dano ao DNA , Linhagem Celular TumoralRESUMO
The historical lack of preclinical models reflecting the genetic heterogeneity of multiple myeloma (MM) hampers the advance of therapeutic discoveries. To circumvent this limitation, we screened mice engineered to carry eight MM lesions (NF-κB, KRAS, MYC, TP53, BCL2, cyclin D1, MMSET/NSD2 and c-MAF) combinatorially activated in B lymphocytes following T cell-driven immunization. Fifteen genetically diverse models developed bone marrow (BM) tumors fulfilling MM pathogenesis. Integrative analyses of â¼500 mice and â¼1,000 patients revealed a common MAPK-MYC genetic pathway that accelerated time to progression from precursor states across genetically heterogeneous MM. MYC-dependent time to progression conditioned immune evasion mechanisms that remodeled the BM microenvironment differently. Rapid MYC-driven progressors exhibited a high number of activated/exhausted CD8+ T cells with reduced immunosuppressive regulatory T (Treg) cells, while late MYC acquisition in slow progressors was associated with lower CD8+ T cell infiltration and more abundant Treg cells. Single-cell transcriptomics and functional assays defined a high ratio of CD8+ T cells versus Treg cells as a predictor of response to immune checkpoint blockade (ICB). In clinical series, high CD8+ T/Treg cell ratios underlie early progression in untreated smoldering MM, and correlated with early relapse in newly diagnosed patients with MM under Len/Dex therapy. In ICB-refractory MM models, increasing CD8+ T cell cytotoxicity or depleting Treg cells reversed immunotherapy resistance and yielded prolonged MM control. Our experimental models enable the correlation of MM genetic and immunological traits with preclinical therapy responses, which may inform the next-generation immunotherapy trials.
Assuntos
Mieloma Múltiplo , Camundongos , Animais , Mieloma Múltiplo/terapia , Mieloma Múltiplo/tratamento farmacológico , Linfócitos T CD8-Positivos , Evasão da Resposta Imune , Linfócitos T Reguladores , Imunoterapia/efeitos adversos , Microambiente Tumoral/genéticaRESUMO
MCL-1 is an anti-apoptotic BCL-2 family protein essential for survival of diverse cell types and is a major driver of cancer and chemoresistance. The mechanistic basis for the oncogenic supremacy of MCL-1 among its anti-apoptotic homologs is unclear and implicates physiologic roles of MCL-1 beyond apoptotic suppression. Here we find that MCL-1-dependent hematologic cancer cells specifically rely on fatty acid oxidation (FAO) as a fuel source because of metabolic wiring enforced by MCL-1 itself. We demonstrate that FAO regulation by MCL-1 is independent of its anti-apoptotic activity, based on metabolomic, proteomic, and genomic profiling of MCL-1-dependent leukemia cells lacking an intact apoptotic pathway. Genetic deletion of Mcl-1 results in transcriptional downregulation of FAO pathway proteins such that glucose withdrawal triggers cell death despite apoptotic blockade. Our data reveal that MCL-1 is a master regulator of FAO, rendering MCL-1-driven cancer cells uniquely susceptible to treatment with FAO inhibitors.
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Neoplasias , Proteômica , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Ácidos Graxos , Glucose , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Neoplasias/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Despite continuing advances in the development of novel cellular-, antibody-, and chemotherapeutic-based strategies to enhance immune reactivity, the presence of regulatory T cells (Treg cells) remains a complicating factor for their clinical efficacy. To overcome dosing limitations and off-target effects from antibody-based Treg cell deletional strategies or small molecule drugging, we investigated the ability of hydrocarbon stapled alpha-helical (SAH) peptides to target FOXP3, the master transcription factor regulator of Treg cell development, maintenance, and suppressive function. Using the crystal structure of the FOXP3 homodimer as a guide, we developed SAHs in the likeness of a portion of the native FOXP3 antiparallel coiled-coil homodimerization domain (SAH-FOXP3) to block this key FOXP3 protein-protein interaction (PPI) through molecular mimicry. We describe the design, synthesis, and biochemical evaluation of single- and double-stapled SAHs covering the entire coiled-coil expanse. We show that lead SAH-FOXP3s bind FOXP3, are cell permeable and nontoxic to T cells, induce dose-dependent transcript and protein level alterations of FOXP3 target genes, impede Treg cell function, and lead to Treg cell gene expression changes in vivo consistent with FOXP3 dysfunction. These results demonstrate a proof of concept for rationally designed FOXP3-directed peptide therapeutics that could be used as approaches to amplify endogenous immune responsiveness.
Assuntos
Fatores de Transcrição Forkhead , Linfócitos T Reguladores , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Peptídeos/metabolismo , Conformação Proteica em alfa-HéliceRESUMO
Very long-chain acyl-CoA dehydrogenase (VLCAD) is an inner mitochondrial membrane enzyme that catalyzes the first and rate-limiting step of long-chain fatty acid oxidation. Point mutations in human VLCAD can produce an inborn error of metabolism called VLCAD deficiency that can lead to severe pathophysiologic consequences, including cardiomyopathy, hypoglycemia, and rhabdomyolysis. Discrete mutations in a structurally-uncharacterized C-terminal domain region of VLCAD cause enzymatic deficiency by an incompletely defined mechanism. Here, we conducted a structure-function study, incorporating X-ray crystallography, hydrogen-deuterium exchange mass spectrometry, computational modeling, and biochemical analyses, to characterize a specific membrane interaction defect of full-length, human VLCAD bearing the clinically-observed mutations, A450P or L462P. By disrupting a predicted α-helical hairpin, these mutations either partially or completely impair direct interaction with the membrane itself. Thus, our data support a structural basis for VLCAD deficiency in patients with discrete mutations in an α-helical membrane-binding motif, resulting in pathologic enzyme mislocalization.
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Erros Inatos do Metabolismo Lipídico , Doenças Mitocondriais , Acil-CoA Desidrogenase de Cadeia Longa/genética , Acil-CoA Desidrogenase de Cadeia Longa/metabolismo , Síndrome Congênita de Insuficiência da Medula Óssea/genética , Humanos , Erros Inatos do Metabolismo Lipídico/genética , Erros Inatos do Metabolismo Lipídico/metabolismo , Doenças Mitocondriais/genética , Doenças MuscularesRESUMO
Peptide and protein bioconjugation technologies have revolutionized our ability to site-specifically or chemoselectively install a variety of functional groups for applications in chemical biology and medicine, including the enhancement of bioavailability. Here, we introduce a site-specific bioconjugation strategy inspired by chemical ligation at serine that relies on a noncanonical amino acid containing a 1-amino-2-hydroxy functional group and a salicylaldehyde ester. More specifically, we harness this technology to generate analogues of glucagon-like peptide-1 that resemble Semaglutide, a long-lasting blockbuster drug currently used in the clinic to regulate glucose levels in the blood. We identify peptides that are more potent than unmodified peptide and equipotent to Semaglutide in a cell-based activation assay, improve the stability in human serum, and increase glucose disposal efficiency in vivo. This approach demonstrates the potential of "serine ligation" for various applications in chemical biology, with a particular focus on generating stabilized peptide therapeutics.
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Receptor do Peptídeo Semelhante ao Glucagon 1 , Serina , Peptídeo 1 Semelhante ao Glucagon , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Glucose , Humanos , Hipoglicemiantes , Peptídeos/farmacologiaRESUMO
Glucose metabolism modulates the islet ß cell responses to diabetogenic stress, including inflammation. Here, we probed the metabolic mechanisms that underlie the protective effect of glucose in inflammation by interrogating the metabolite profiles of primary islets from human donors and identified de novo glutathione synthesis as a prominent glucose-driven pro-survival pathway. We find that pyruvate carboxylase is required for glutathione synthesis in islets and promotes their antioxidant capacity to counter inflammation and nitrosative stress. Loss- and gain-of-function studies indicate that pyruvate carboxylase is necessary and sufficient to mediate the metabolic input from glucose into glutathione synthesis and the oxidative stress response. Altered redox metabolism and cellular capacity to replenish glutathione pools are relevant in multiple pathologies beyond obesity and diabetes. Our findings reveal a direct interplay between glucose metabolism and glutathione biosynthesis via pyruvate carboxylase. This metabolic axis may also have implications in other settings where sustaining glutathione is essential.
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Glucose/metabolismo , Glutationa/biossíntese , Piruvato Carboxilase/metabolismo , Adulto , Animais , Antioxidantes/fisiologia , Feminino , Glutationa/metabolismo , Humanos , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Oxirredução , Estresse Oxidativo/fisiologia , Cultura Primária de CélulasRESUMO
BAX is a pro-apoptotic member of the BCL-2 family, which regulates the balance between cellular life and death. During homeostasis, BAX predominantly resides in the cytosol as a latent monomer but, in response to stress, transforms into an oligomeric protein that permeabilizes the mitochondria, leading to apoptosis. Because renegade BAX activation poses a grave risk to the cell, the architecture of BAX must ensure monomeric stability yet enable conformational change upon stress signaling. The specific structural features that afford both stability and dynamic flexibility remain ill-defined and represent a critical control point of BAX regulation. We identify a nexus of interactions involving four residues of the BAX core α5 helix that are individually essential to maintaining the structure and latency of monomeric BAX and are collectively required for dimeric assembly. The dual yet distinct roles of these residues reveals the intricacy of BAX conformational regulation and opportunities for therapeutic modulation.
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Aminoácidos/genética , Apoptose/genética , Mutação , Transdução de Sinais/genética , Proteína X Associada a bcl-2/genética , Aminoácidos/química , Aminoácidos/metabolismo , Animais , Sítios de Ligação/genética , Células Cultivadas , Citosol/metabolismo , Humanos , Camundongos Knockout , Mitocôndrias/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismoRESUMO
The number of approved peptide therapeutics, as well as those in development, has been increasing in recent years. Frequently, the biological activity of such peptides is elicited through the adoption of secondary structural elements upon interaction with their cellular target. However, many therapeutic peptides are unstructured in solution and accordingly exhibit a poor bioavailability due to rapid proteolysis in vivo. To combat this degradation, numerous naturally occurring peptides with therapeutic properties contain stabilizing features, such as N-to-C cyclization or disulfide bonds. Recently, hydrocarbon stapling via non-native amino acid substitution followed by ring-closing metathesis has been shown to induce a dramatic stabilization of α-helical peptides. Identifying the ideal staple location along the peptide backbone is a critical developmental step, and methods to streamline this optimization are needed. Mass spectrometry-based methods such as ion mobility (IM) and hydrogen-deuterium exchange (HDX) can detect multiple discrete peptide conformations, a significant advantage over bulk spectroscopic techniques. In this study we use IM-MS and HDX-MS to demonstrate that the native 36-residue enfuvirtide peptide is highly dynamic in solution and the conformational ensemble populated by stabilized constructs depends heavily on the staple location. Further, our measurements yielded results that correlate well with the average α-helical content measured by circular dichroism. The MS-based approaches described herein represent sensitive and potentially high-throughput methods for characterizing and identifying optimally stapled peptides.
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Complex neural circuitry requires stable connections formed by lengthy axons. To maintain these functional circuits, fast transport delivers RNAs to distal axons where they undergo local translation. However, the mechanism that enables long-distance transport of RNA granules is not yet understood. Here, we demonstrate that a complex containing RNA and the RNA-binding protein (RBP) SFPQ interacts selectively with a tetrameric kinesin containing the adaptor KLC1 and the motor KIF5A. We show that the binding of SFPQ to the KIF5A/KLC1 motor complex is required for axon survival and is impacted by KIF5A mutations that cause Charcot-Marie Tooth (CMT) disease. Moreover, therapeutic approaches that bypass the need for local translation of SFPQ-bound proteins prevent axon degeneration in CMT models. Collectively, these observations indicate that KIF5A-mediated SFPQ-RNA granule transport may be a key function disrupted in KIF5A-linked neurologic diseases and that replacing axonally translated proteins serves as a therapeutic approach to axonal degenerative disorders.
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Transporte Axonal , Axônios/metabolismo , Cinesinas/metabolismo , Fator de Processamento Associado a PTB/metabolismo , RNA/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Grânulos Citoplasmáticos/metabolismo , Gânglios Espinais/metabolismo , Células HEK293 , Humanos , Proteínas Associadas aos Microtúbulos , Mitocôndrias/metabolismo , Mutação/genética , Peptídeos/metabolismo , Fosforilação , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Células Receptoras Sensoriais/metabolismoRESUMO
The ubiquitin-proteasome system (UPS) is a highly regulated protein disposal process critical to cell survival. Inhibiting the pathway induces proteotoxic stress and can be an effective cancer treatment. The therapeutic window observed upon proteasomal blockade has motivated multiple UPS-targeting strategies, including preventing ubiquitination altogether. E1 initiates the cascade by transferring ubiquitin to E2 enzymes. A small molecule that engages the E1 ATP-binding site and derivatizes ubiquitin disrupts enzymatic activity and kills cancer cells. However, binding-site mutations cause resistance, motivating alternative approaches to block this promising target. We identified an interaction between the E2 N-terminal alpha-1 helix and a pocket within the E1 ubiquitin-fold domain as a potentially druggable site. Stapled peptides modeled after the E2 alpha-1 helix bound to the E1 groove, induced a consequential conformational change and inhibited E1 ubiquitin thiotransfer, disrupting E2 ubiquitin charging and ubiquitination of cellular proteins. Thus, we provide a blueprint for a distinct E1-targeting strategy to treat cancer.
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Complexo de Endopeptidases do Proteassoma/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos , Humanos , Conformação Molecular , Simulação de Acoplamento Molecular , Peptídeos/química , Ligação Proteica , Relação Estrutura-Atividade , Ubiquitina/química , Ubiquitina/genética , UbiquitinaçãoRESUMO
Apoptosis is regulated by BCL-2 family proteins. Anti-apoptotic members suppress cell death by deploying a surface groove to capture the critical BH3 α-helix of pro-apoptotic members. Cancer cells hijack this mechanism by overexpressing anti-apoptotic BCL-2 family proteins to enforce cellular immortality. We previously identified and harnessed a unique cysteine (C55) in the groove of anti-apoptotic BFL-1 to selectively neutralize its oncogenic activity using a covalent stapled-peptide inhibitor. Here, we find that disulfide bonding between a native cysteine pair at the groove (C55) and C-terminal α9 helix (C175) of BFL-1 operates as a redox switch to control the accessibility of the anti-apoptotic pocket. Reducing the C55-C175 disulfide triggers α9 release, which promotes mitochondrial translocation, groove exposure for BH3 interaction and inhibition of mitochondrial permeabilization by pro-apoptotic BAX. C55-C175 disulfide formation in an oxidative cellular environment abrogates the ability of BFL-1 to bind BH3 domains. Thus, we identify a mechanism of conformational control of BFL-1 by an intramolecular redox switch.
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Apoptose , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Cisteína/química , Cisteína/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Células HEK293 , Humanos , Camundongos , Antígenos de Histocompatibilidade Menor/química , Mitocôndrias/metabolismo , Modelos Moleculares , Oxirredução , Conformação Proteica , Conformação Proteica em alfa-Hélice , Proteínas Proto-Oncogênicas c-bcl-2/químicaRESUMO
Chronic inflammation is linked to diverse disease processes, but the intrinsic mechanisms that determine cellular sensitivity to inflammation are incompletely understood. Here, we show the contribution of glucose metabolism to inflammation-induced changes in the survival of pancreatic islet ß-cells. Using metabolomic, biochemical and functional analyses, we investigate the protective versus non-protective effects of glucose in the presence of pro-inflammatory cytokines. When protective, glucose metabolism augments anaplerotic input into the TCA cycle via pyruvate carboxylase (PC) activity, leading to increased aspartate levels. This metabolic mechanism supports the argininosuccinate shunt, which fuels ureagenesis from arginine and conversely diminishes arginine utilization for production of nitric oxide (NO), a chief mediator of inflammatory cytotoxicity. Activation of the PC-urea cycle axis is sufficient to suppress NO synthesis and shield cells from death in the context of inflammation and other stress paradigms. Overall, these studies uncover a previously unappreciated link between glucose metabolism and arginine-utilizing pathways via PC-directed ureagenesis as a protective mechanism.
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Arginina/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Inflamação/prevenção & controle , Células Secretoras de Insulina/efeitos dos fármacos , Distúrbios Congênitos do Ciclo da Ureia/patologia , Ureia/metabolismo , Adolescente , Adulto , Idoso , Ácido Aspártico/metabolismo , Sobrevivência Celular , Ciclo do Ácido Cítrico/efeitos dos fármacos , Feminino , Humanos , Inflamação/patologia , Células Secretoras de Insulina/patologia , Masculino , Metabolômica , Pessoa de Meia-Idade , Óxido Nítrico/metabolismo , Piruvato Carboxilase/metabolismo , Distúrbios Congênitos do Ciclo da Ureia/metabolismo , Adulto JovemRESUMO
BAX is a pro-apoptotic protein that transforms from a cytosolic monomer into a toxic oligomer that permeabilizes the mitochondrial outer membrane. How BAX monomers assemble into a higher-order conformation, and the structural determinants essential to membrane permeabilization, remain a mechanistic mystery. A key hurdle has been the inability to generate a homogeneous BAX oligomer (BAXO) for analysis. Here, we report the production and characterization of a full-length BAXO that recapitulates physiologic BAX activation. Multidisciplinary studies revealed striking conformational consequences of oligomerization and insight into the macromolecular structure of oligomeric BAX. Importantly, BAXO enabled the assignment of specific roles to particular residues and α helices that mediate individual steps of the BAX activation pathway, including unexpected functionalities of BAX α6 and α9 in driving membrane disruption. Our results provide the first glimpse of a full-length and functional BAXO, revealing structural requirements for the elusive execution phase of mitochondrial apoptosis.
Assuntos
Apoptose , Mitocôndrias/patologia , Membranas Mitocondriais/metabolismo , Multimerização Proteica , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismo , Animais , Transporte Biológico , Permeabilidade da Membrana Celular , Citosol/metabolismo , Humanos , Camundongos , Mitocôndrias/metabolismo , Modelos Moleculares , Conformação Proteica , Proteínas Proto-Oncogênicas c-fosRESUMO
p53 is a critical tumor-suppressor protein that guards the human genome against mutations by inducing cell-cycle arrest or apoptosis. Cancer cells subvert p53 by deletion, mutation, or overexpression of the negative regulators HDM2 and HDMX. For tumors that retain wild-type p53, its reactivation by pharmacologic targeting of HDM2 and/or HDMX represents a promising strategy, with a series of selective small-molecule HDM2 inhibitors and a dual HDM2/HDMX stapled-peptide inhibitor being evaluated in clinical trials. Because selective HDM2 targeting can cause hematologic toxicity, selective HDMX inhibitors could provide an alternative p53-reactivation strategy, but clinical candidates remain elusive. Here, we applied a mutation-scanning approach to uncover p53-based stapled peptides that are selective for HDMX. Crystal structures of stapled-peptide/HDMX complexes revealed a molecular mechanism for the observed specificity, which was validated by HDMX mutagenesis. Thus, we provide a blueprint for the development of HDMX-selective inhibitors to dissect and target the p53/HDMX interaction.