ABSTRACT
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.
Subject(s)
Apoptosis , Mitochondria/pathology , Mitochondrial Membranes/metabolism , Protein Multimerization , bcl-2-Associated X Protein/chemistry , bcl-2-Associated X Protein/metabolism , Animals , Biological Transport , Cell Membrane Permeability , Cytosol/metabolism , Humans , Mice , Mitochondria/metabolism , Models, Molecular , Protein Conformation , Proto-Oncogene Proteins c-fosABSTRACT
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.
Subject(s)
Proteasome Endopeptidase Complex/metabolism , Ubiquitin-Activating Enzymes/metabolism , Ubiquitin/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Binding Sites , Cell Line, Tumor , Drug Design , Drug Resistance, Neoplasm , Humans , Molecular Conformation , Molecular Docking Simulation , Peptides/chemistry , Protein Binding , Structure-Activity Relationship , Ubiquitin/chemistry , Ubiquitin/genetics , UbiquitinationABSTRACT
Antibodies are essential biological research tools and important therapeutic agents, but some exhibit non-specific binding to off-target proteins and other biomolecules. Such polyreactive antibodies compromise screening pipelines, lead to incorrect and irreproducible experimental results, and are generally intractable for clinical development. Here, we design a set of experiments using a diverse naïve synthetic camelid antibody fragment (nanobody) library to enable machine learning models to accurately assess polyreactivity from protein sequence (AUC > 0.8). Moreover, our models provide quantitative scoring metrics that predict the effect of amino acid substitutions on polyreactivity. We experimentally test our models' performance on three independent nanobody scaffolds, where over 90% of predicted substitutions successfully reduced polyreactivity. Importantly, the models allow us to diminish the polyreactivity of an angiotensin II type I receptor antagonist nanobody, without compromising its functional properties. We provide a companion web-server that offers a straightforward means of predicting polyreactivity and polyreactivity-reducing mutations for any given nanobody sequence.
Subject(s)
Immunoglobulin FragmentsABSTRACT
While there are hundreds of predicted E3 ligases, characterizing their applications for targeted protein degradation has proved challenging. Here, we report a chemical biology approach to evaluate the ability of modified recombinant E3 ligase components to support neo-substrate degradation. Bypassing the need for specific E3 ligase binders, we use maleimide-thiol chemistry for covalent functionalization followed by E3 electroporation (COFFEE) in live cells. We demonstrate that electroporated recombinant von Hippel-Lindau (VHL) protein, covalently functionalized at its ligandable cysteine with JQ1 or dasatinib, induces degradation of BRD4 or tyrosine kinases, respectively. Furthermore, by applying COFFEE to SPSB2, a Cullin-RING ligase 5 receptor, as well as to SKP1, the adaptor protein for Cullin-RING ligase 1 F box (SCF) complexes, we validate this method as a powerful approach to define the activity of previously uncharacterized ubiquitin ligase components, and provide further evidence that not only E3 ligase receptors but also adaptors can be directly hijacked for neo-substrate degradation.
Subject(s)
Ubiquitin-Protein Ligases/metabolism , Cell Line , Female , Humans , Male , Recombinant Proteins/metabolismABSTRACT
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.
Subject(s)
Antineoplastic Agents/chemistry , Cell Cycle Proteins/chemistry , Oligopeptides/chemistry , Proto-Oncogene Proteins/chemistry , Tumor Suppressor Protein p53/chemistry , Amino Acid Substitution , Antineoplastic Agents/pharmacology , Binding Sites , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Humans , Molecular Docking Simulation , Oligopeptides/pharmacology , Protein Binding , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Proto-Oncogene Proteins c-mdm2/chemistry , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/metabolismABSTRACT
The BCL-2 family is composed of anti- and pro-apoptotic members that respectively protect or disrupt mitochondrial integrity. Anti-apoptotic overexpression can promote oncogenesis by trapping the BCL-2 homology 3 (BH3) "killer domains" of pro-apoptotic proteins in a surface groove, blocking apoptosis. Groove inhibitors, such as the relatively large BCL-2 drug venetoclax (868 Da), have emerged as cancer therapies. BFL-1 remains an undrugged oncogenic protein and can cause venetoclax resistance. Having identified a unique C55 residue in the BFL-1 groove, we performed a disulfide tethering screen to determine if C55 reactivity could enable smaller molecules to block BFL-1's BH3-binding functionality. We found that a disulfide-bearing N-acetyltryptophan analog (304 Da adduct) effectively targeted BFL-1 C55 and reversed BFL-1-mediated suppression of mitochondrial apoptosis. Structural analyses implicated the conserved leucine-binding pocket of BFL-1 as the interaction site, resulting in conformational remodeling. Thus, therapeutic targeting of BFL-1 may be achievable through the design of small, cysteine-reactive drugs.
Subject(s)
Apoptosis/drug effects , Disulfides/pharmacology , Peptides/pharmacology , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Disulfides/chemistry , Dose-Response Relationship, Drug , Humans , Minor Histocompatibility Antigens/metabolism , Models, Molecular , Molecular Structure , Peptides/chemical synthesis , Peptides/chemistry , Proto-Oncogene Proteins c-bcl-2/metabolism , Structure-Activity Relationship , Tryptophan/analogs & derivatives , Tryptophan/chemistry , Tryptophan/pharmacologyABSTRACT
BCL-2 family proteins are high-priority cancer targets whose structures provide essential blueprints for drug design. Whereas numerous structures of anti-apoptotic BCL-2 protein complexes with α-helical BH3 peptides have been reported, the corresponding panel of apo structures remains incomplete. Here, we report the crystal structure of apo BFL-1 at 1.69-Å resolution, revealing similarities and key differences among unliganded anti-apoptotic proteins. Unlike all other BCL-2 proteins, apo BFL-1 contains a surface-accessible cysteine within its BH3-binding groove, allowing for selective covalent targeting by a NOXA BH3-based stapled peptide inhibitor. The crystal structure of this complex demonstrated the sulfhydryl bond and fortuitous interactions between the acrylamide-bearing moiety and a newly formed hydrophobic cavity. Comparison of the apo and BH3-liganded structures further revealed an induced conformational change. The two BFL-1 structures expand our understanding of the surface landscapes available for therapeutic targeting so that the apoptotic blockades of BFL-1-dependent cancers can be overcome.
Subject(s)
Antineoplastic Agents/chemistry , Apoproteins/chemistry , Minor Histocompatibility Antigens/chemistry , Peptide Fragments/chemistry , Proto-Oncogene Proteins c-bcl-2/chemistry , Proto-Oncogene Proteins/chemistry , Amino Acid Sequence , Antineoplastic Agents/chemical synthesis , Apoproteins/antagonists & inhibitors , Apoproteins/genetics , Apoproteins/metabolism , Binding Sites , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Hydrophobic and Hydrophilic Interactions , Ligands , Minor Histocompatibility Antigens/genetics , Minor Histocompatibility Antigens/metabolism , Models, Molecular , Peptide Fragments/chemical synthesis , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Proto-Oncogene Proteins/chemical synthesis , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
Cancer cells overexpress a diversity of anti-apoptotic BCL-2 family proteins, such as BCL-2, MCL-1, and BFL-1/A1, to enforce cellular immortality. Thus, intensive drug development efforts have focused on targeting this class of oncogenic proteins to overcome treatment resistance. Whereas a selective BCL-2 inhibitor has been FDA approved and several small molecule inhibitors of MCL-1 have recently entered phase I clinical testing, BFL-1/A1 remains undrugged. Here, we developed a series of stapled peptide design principles to engineer a functionally selective and cell-permeable BFL-1/A1 inhibitor that is specifically cytotoxic to BFL-1/A1-dependent human cancer cells. Because cancers harbor a diversity of resistance mechanisms and typically require multi-agent treatment, we further investigated BFL-1/A1 co-dependencies by mining a genome-scale CRISPR-Cas9 screen. We identified ataxia-telangiectasia-mutated (ATM) kinase as a BFL-1/A1 co-dependency in acute myeloid leukemia (AML), which informed the validation of BFL-1/A1 and ATM inhibitor co-treatment as a synergistic approach to subverting apoptotic resistance in cancer.
Subject(s)
Antineoplastic Agents/pharmacology , Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors , Leukemia, Myeloid, Acute/metabolism , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Ataxia Telangiectasia Mutated Proteins/metabolism , Binding Sites , Cell Line, Tumor , Drug Resistance, Neoplasm , Female , HEK293 Cells , Humans , Male , Minor Histocompatibility Antigens/chemistry , Minor Histocompatibility Antigens/metabolism , Protein Binding , Proto-Oncogene Proteins c-bcl-2/chemistry , Proto-Oncogene Proteins c-bcl-2/metabolismABSTRACT
Anti-apoptotic BCL-2 family proteins block cell death by trapping the critical α-helical BH3 domains of pro-apoptotic members in a surface groove. Cancer cells hijack this survival mechanism by overexpressing a spectrum of anti-apoptotic members, mounting formidable apoptotic blockades that resist chemotherapeutic treatment. Drugging the BH3-binding pockets of anti-apoptotic proteins has become a highest-priority goal, fueled by the clinical success of ABT-199, a selective BCL-2 inhibitor, in reactivating apoptosis in BCL-2-dependent cancers. BFL-1 is a BCL-2 homolog implicated in melanoma, lymphoma, and other cancers, and remains undrugged. A natural juxtaposition of two unique cysteines at the binding interface of the NOXA BH3 helix and BFL-1 pocket informed the development of stapled BH3 peptides bearing acrylamide warheads to irreversibly inhibit BFL-1 by covalent targeting. Given the frequent proximity of native cysteines to regulatory binding surfaces, covalent stapled peptide inhibitors provide a new therapeutic strategy for targeting pathologic protein interactions.