RESUMEN
N-Boc-N-(2-(tritylthio)ethoxy)glycine has been developed as a building block for peptide ubiquitination, which is fully compatible with solid-phase Fmoc chemistry and common peptide modifications including phosphorylation, methylation, acetylation, biotinylation, and fluorescence labeling. The optimal conditions for peptide cleavage and auxiliary removal were obtained. The utility of this building block in peptide ubiquitination was demonstrated by the synthesis of seven ubiquitinated histone and Tau peptides bearing various modifications. Cys residues were well tolerated and did not require orthogonal protection. The structural integrity and folding of the synthesized ubiquitinated peptides were confirmed by enzymatic deubiquitination of a fluorescently labeled ubiquitin conjugate. The synthetic strategy using this building block provides a practical approach for the preparation of ubiquitinated peptides with diverse modifications.
Asunto(s)
Glicina , Péptidos , Péptidos/química , Ubiquitinación , Ubiquitina , Procesamiento Proteico-PostraduccionalRESUMEN
Engineered destruction of target proteins by recruitment to the cell's degradation machinery has emerged as a promising strategy in drug discovery. The majority of molecules that facilitate targeted degradation do so via a select number of ubiquitin ligases, restricting this therapeutic approach to tissue types that express the requisite ligase. Here, we describe a new strategy of targeted protein degradation through direct substrate recruitment to the 26S proteasome. The proteolytic complex is essential and abundantly expressed in all cells; however, proteasomal ligands remain scarce. We identify potent peptidic macrocycles that bind directly to the 26S proteasome subunit PSMD2, with a 2.5-Å-resolution cryo-electron microscopy complex structure revealing a binding site near the 26S pore. Conjugation of this macrocycle to a potent BRD4 ligand enabled generation of chimeric molecules that effectively degrade BRD4 in cells, thus demonstrating that degradation via direct proteasomal recruitment is a viable strategy for targeted protein degradation.
Asunto(s)
Proteínas Nucleares , Factores de Transcripción , Proteínas Nucleares/metabolismo , Microscopía por Crioelectrón , Factores de Transcripción/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Autophagy-related proteins (Atgs) drive the lysosome-mediated degradation pathway, autophagy, to enable the clearance of dysfunctional cellular components and maintain homeostasis. In humans, this process is driven by the mammalian Atg8 (mAtg8) family of proteins comprising the LC3 and GABARAP subfamilies. The mAtg8 proteins play essential roles in the formation and maturation of autophagosomes and the capture of specific cargo through binding to the conserved LC3-interacting region (LIR) sequence within target proteins. Modulation of interactions of mAtg8 with its target proteins via small-molecule ligands would enable further interrogation of their function. Here we describe unbiased fragment and DNA-encoded library (DEL) screening approaches for discovering LC3 small-molecule ligands. Both strategies resulted in compounds that bind to LC3, with the fragment hits favoring a conserved hydrophobic pocket in mATG8 proteins, as detailed by LC3A-fragment complex crystal structures. Our findings demonstrate that the malleable LIR-binding surface can be readily targeted by fragments; however, rational design of additional interactions to drive increased affinity proved challenging. DEL libraries, which combine small, fragment-like building blocks into larger scaffolds, yielded higher-affinity binders and revealed an unexpected potential for reversible, covalent ligands. Moreover, DEL hits identified possible vectors for synthesizing fluorescent probes or bivalent molecules for engineering autophagic degradation of specific targets.
Asunto(s)
Autofagia , Proteínas Asociadas a Microtúbulos , Humanos , Animales , Proteínas Asociadas a Microtúbulos/metabolismo , Ligandos , Familia de las Proteínas 8 Relacionadas con la Autofagia/química , Autofagosomas/metabolismo , Mamíferos/metabolismoRESUMEN
[This corrects the article DOI: 10.1016/j.bpr.2021.100033.].
RESUMEN
Polyubiquitination is a complex form of posttranslational modification responsible for the control of numerous cellular processes. Many ubiquitin-binding proteins recognize distinct polyubiquitin chain types, and these associations help drive ubiquitin-signaling pathways. There is considerable interest in understanding the specificity of ubiquitin-binding proteins; however, because of the multivalent nature of polyubiquitin, affinity measurements of these interactions that rely on affixing ubiquitin-binding proteins to a surface can display artifactual, method-dependent avidity, or "bridging." This artifact, which is distinct from biologically relevant, avid interactions with polyubiquitin, is commonplace in such polyubiquitin-binding measurements and can lead to dramatic overestimations of binding affinities for particular chain types, and thus, incorrect conclusions about specificity. Here, we use surface-based measurements of ubiquitin binding in three model systems to illustrate bridging and lay out practical ways of identifying and mitigating it. Specifically, we describe a simple fitting model that enables researchers to diagnose the severity of bridging artifacts, determine whether they can be minimized, and more accurately evaluate polyubiquitin-binding specificity.
RESUMEN
Ubiquitin specific protease 7 (USP7) regulates the protein stability of key cellular regulators in pathways ranging from apoptosis to neuronal development, making it a promising therapeutic target. Here we used an engineered, activated variant of the USP7 catalytic domain to perform structure-activity studies of electrophilic peptidomimetic inhibitors. Employing this USP7 variant, we found that inhibitors with a cyanopyrrolidine warhead unexpectedly promoted a ß-elimination reaction of the initial covalent adducts, thereby converting the active-site cysteine residue to dehydroalanine. We determined that this phenomenon is specific for the USP7 catalytic cysteine and that structural features of the inhibitor and protein microenvironment impact elimination rates. Using comprehensive docking studies, we propose that the characteristic conformational dynamics of USP7 allow access to conformations that promote the ligand-induced elimination. Unlike in conventional reversible-covalent inhibition, the compounds described here irreversibly destroy a catalytic residue while simultaneously converting the inhibitor to a nonelectrophilic byproduct. Accordingly, this unexpected finding expands the scope of covalent inhibitor modalities and offers intriguing insights into enzyme-inhibitor dynamics.
Asunto(s)
Dominio Catalítico/efectos de los fármacos , Pirrolidinas/química , Pirrolidinas/farmacología , Peptidasa Específica de Ubiquitina 7/antagonistas & inhibidores , Cisteína/química , Cisteína/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Simulación del Acoplamiento Molecular , Peptidomiméticos/química , Peptidomiméticos/farmacología , Peptidasa Específica de Ubiquitina 7/química , Peptidasa Específica de Ubiquitina 7/metabolismoRESUMEN
CARD9 and CARD11 drive immune cell activation by nucleating Bcl10 polymerization, but are held in an autoinhibited state prior to stimulation. Here, we elucidate the structural basis for this autoinhibition by determining the structure of a region of CARD9 that includes an extensive interface between its caspase recruitment domain (CARD) and coiled-coil domain. We demonstrate, for both CARD9 and CARD11, that disruption of this interface leads to hyperactivation in cells and to the formation of Bcl10-templating filaments in vitro, illuminating the mechanism of action of numerous oncogenic mutations of CARD11. These structural insights enable us to characterize two similar, yet distinct, mechanisms by which autoinhibition is relieved in the course of canonical CARD9 or CARD11 activation. We also dissect the molecular determinants of helical template assembly by solving the structure of the CARD9 filament. Taken together, these findings delineate the structural mechanisms of inhibition and activation within this protein family.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/ultraestructura , Guanilato Ciclasa/ultraestructura , Dominios Proteicos , Proteína 10 de la LLC-Linfoma de Células B/metabolismo , Proteínas Adaptadoras de Señalización CARD/genética , Proteínas Adaptadoras de Señalización CARD/inmunología , Proteínas Adaptadoras de Señalización CARD/metabolismo , Microscopía por Crioelectrón , Guanilato Ciclasa/genética , Guanilato Ciclasa/inmunología , Guanilato Ciclasa/metabolismo , Células HEK293 , Humanos , Mutación , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica en Hélice alfa , Multimerización de Proteína/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Transducción de Señal/inmunologíaRESUMEN
The caspase recruitment domain-containing protein 9 (CARD9)-B-cell lymphoma/leukemia 10 (Bcl10) signaling axis is activated in myeloid cells during the innate immune response to a variety of diverse pathogens. This signaling pathway requires a critical caspase recruitment domain (CARD)-CARD interaction between CARD9 and Bcl10 that promotes downstream activation of factors, including NF-κB and the mitogen-activated protein kinase (MAPK) p38. Despite these insights, CARD9 remains structurally uncharacterized, and little mechanistic understanding of its regulation exists. We unexpectedly found here that the CARD in CARD9 binds to Zn2+ with picomolar affinity-a concentration comparable with the levels of readily accessible Zn2+ in the cytosol. NMR solution structures of the CARD9-CARD in the apo and Zn2+-bound states revealed that Zn2+ has little effect on the ground-state structure of the CARD; yet the stability of the domain increased considerably upon Zn2+ binding, with a concomitant reduction in conformational flexibility. Moreover, Zn2+ binding inhibited polymerization of the CARD9-CARD into helical assemblies. Here, we also present a 20-Å resolution negative-stain EM (NS-EM) structure of these filamentous assemblies and show that they adopt a similar helical symmetry as reported previously for filaments of the Bcl10 CARD. Using both bulk assays and direct NS-EM visualization, we further show that the CARD9-CARD assemblies can directly template and thereby nucleate Bcl10 polymerization, a capacity considered critical to propagation of the CARD9-Bcl10 signaling cascade. Our findings indicate that CARD9 is a potential target of Zn2+-mediated signaling that affects Bcl10 polymerization in innate immune responses.
Asunto(s)
Proteína 10 de la LLC-Linfoma de Células B/metabolismo , Proteínas Adaptadoras de Señalización CARD/metabolismo , Zinc/metabolismo , Proteína 10 de la LLC-Linfoma de Células B/química , Proteína 10 de la LLC-Linfoma de Células B/genética , Proteínas Adaptadoras de Señalización CARD/química , Proteínas Adaptadoras de Señalización CARD/genética , Cristalografía por Rayos X , Humanos , FN-kappa B/genética , FN-kappa B/metabolismo , Polimerizacion , Unión Proteica , Dominios Proteicos , Transducción de Señal , Zinc/química , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Ubiquitin-specific protease 7 (USP7) deubiquitinase activity is controlled by a number of regulatory factors, including stimulation by intramolecular accessory domains. Alone, the USP7 catalytic domain (USP7cd) shows limited activity and apo USP7cd crystal structures reveal a disrupted catalytic triad. By contrast, ubiquitin-conjugated USP7cd structures demonstrate the canonical cysteine protease active-site geometry; however, the structural features of the USP7cd that stabilize the inactive conformation and the mechanism of transition between inactive and active states remain unclear. Here we use comparative structural analyses, molecular dynamics simulations, and in silico sequence re-engineering via directed sampling by RosettaDesign to identify key molecular determinants of USP7cd activation and successfully engineer USP7cd for improved activity. Full kinetic analysis and multiple X-ray crystal structures of our designs indicate that electrostatic interactions in the distal "switching loop" region and local packing in the hydrophobic core mediate subtle but significant conformational changes that modulate USP7cd activation.
Asunto(s)
Inhibidores Enzimáticos/química , Mutación , Peptidomiméticos/química , Peptidasa Específica de Ubiquitina 7/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Dominio Catalítico , Clonación Molecular , Cristalografía por Rayos X , Activación Enzimática , Inhibidores Enzimáticos/síntesis química , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Simulación de Dinámica Molecular , Peptidomiméticos/síntesis química , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Electricidad Estática , Especificidad por Sustrato , Termodinámica , Peptidasa Específica de Ubiquitina 7/antagonistas & inhibidores , Peptidasa Específica de Ubiquitina 7/genética , Peptidasa Específica de Ubiquitina 7/metabolismoRESUMEN
Pyroptosis is an inflammatory form of cell death that not only protects multicellular organisms from invading pathogenic bacteria and microbial infections, but can also lead to sepsis and lethal septic shock if overactivated. Here, we present an overview of recent developments within the pyroptosis field, beginning with the discovery of Gasdermin D (GSDMD) as a substrate of caspase-1 and caspase-11 upon detection of cytosolic lipopolysaccharide (LPS). Cleavage releases the N-terminal domain of GSDMD, causing it to form cytotoxic pores in the plasma membrane of cells. We further discuss the implications for the rest of the gasdermin (GSDM) family, which are emerging as mediators of programmed cell death in a variety of processes that regulate cellular differentiation and proliferation.
Asunto(s)
Infecciones/inmunología , Inflamación/inmunología , Piroptosis/inmunología , Choque Séptico/inmunología , Animales , Caspasa 1/metabolismo , Caspasas/metabolismo , Caspasas Iniciadoras , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Proteínas de Neoplasias/metabolismo , Proteínas de Unión a FosfatoRESUMEN
Gasdermin-D (GsdmD) is a critical mediator of innate immune defense because its cleavage by the inflammatory caspases 1, 4, 5, and 11 yields an N-terminal p30 fragment that induces pyroptosis, a death program important for the elimination of intracellular bacteria. Precisely how GsdmD p30 triggers pyroptosis has not been established. Here we show that human GsdmD p30 forms functional pores within membranes. When liberated from the corresponding C-terminal GsdmD p20 fragment in the presence of liposomes, GsdmD p30 localized to the lipid bilayer, whereas p20 remained in the aqueous environment. Within liposomes, p30 existed as higher-order oligomers and formed ring-like structures that were visualized by negative stain electron microscopy. These structures appeared within minutes of GsdmD cleavage and released Ca(2+) from preloaded liposomes. Consistent with GsdmD p30 favoring association with membranes, p30 was only detected in the membrane-containing fraction of immortalized macrophages after caspase-11 activation by lipopolysaccharide. We found that the mouse I105N/human I104N mutation, which has been shown to prevent macrophage pyroptosis, attenuated both cell killing by p30 in a 293T transient overexpression system and membrane permeabilization in vitro, suggesting that the mutants are actually hypomorphs, but must be above certain concentration to exhibit activity. Collectively, our data suggest that GsdmD p30 kills cells by forming pores that compromise the integrity of the cell membrane.
Asunto(s)
Proteínas de Neoplasias/fisiología , Piroptosis , Animales , Caspasas/metabolismo , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular , Liposomas , Ratones , Mutación , Proteínas de Unión a FosfatoRESUMEN
Protein ubiquitination patterns are an important component of cellular signaling. The WD-repeat protein WDR48 (USP1-associated factor UAF-1) stimulates activity of ubiquitin-specific proteases USP1, USP12, and USP46. To understand how WDR48 exerts its effect on the USP scaffold, we determined structures of the ternary WDR48:USP46:ubiquitin complex. WDR48 interacts with the USP46 fingers subdomain via a relatively small, highly polar surface on the top center of the WDR48 ß propeller. In addition, WDR48 has a novel ancillary domain and a C-terminal SUMO-like domain encircling the USP46-bound ubiquitin. Mutation of residues involved in the WDR48:USP46 interaction abrogated both binding and deubiquitinase activity of the complex. An analogous mutation in USP1 similarly blocked WDR48-dependent activation. Our data suggest a possible mechanism of deubiquitinase stimulation via stabilization and prolonged residence time of substrate. The unprecedented mode of interaction between the USP fingers domain and the WD-repeat ß propeller serves as a prototypical example for this family of deubiquitinases.
Asunto(s)
Endopeptidasas/química , Proteínas/química , Secuencia de Aminoácidos , Sitios de Unión , Endopeptidasas/genética , Endopeptidasas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Mutación , Unión Proteica , Proteínas/genética , Proteínas/metabolismoRESUMEN
Cellular inhibitor of apoptosis 1 (cIAP1) is a ubiquitin ligase with critical roles in the control of programmed cell death and NF-κB signaling. Under normal conditions, the protein exists as an autoinhibited monomer, but proapoptotic signals lead to its dimerization, activation and proteasomal degradation. This view of cIAP1 as a binary switch has been informed by static structural studies that cannot access the protein's dynamics. Here, we use NMR spectroscopy to study micro- and millisecond motions of specific domain interfaces in human cIAP1 and use time-resolved small-angle X-ray scattering to observe the global conformational changes necessary for activation. Although motions within each interface of the 'closed' monomer are insufficient to activate cIAP1, they enable associations with catalytic partners and activation factors. We propose that these internal motions facilitate rapid peptide-induced opening and dimerization of cIAP1, which undergoes a dramatic spring-loaded structural transition.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis/química , Proteínas Inhibidoras de la Apoptosis/metabolismo , Humanos , Cinética , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Dispersión del Ángulo Pequeño , Ubiquitina/metabolismo , Enzimas Activadoras de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas , Difracción de Rayos XRESUMEN
Tank-binding kinase 1 (TBK1) serves as an important component of multiple signaling pathways. While the majority of research on TBK1 has focused on its role in innate immunity, critical functions for TBK1 in autophagy and cancer are beginning to emerge. This review highlights recent structural and biochemical studies that provide insights into the molecular mechanism of TBK1 activation and summarizes what is known to date about TBK1 substrate selection. Growing evidence suggests that both processes rely on TBK1 subcellular localization, with a variety of adaptor proteins each directing TBK1 to discrete signaling complexes for different cellular responses. Further study of TBK1-mediated pathways will require careful consideration of TBK1 mechanisms of activation and specificity for proper dissection of these distinct signaling cascades.
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Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Transducción de Señal , Secuencia de Aminoácidos , Sitios de Unión/genética , Activación Enzimática/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Especificidad por SustratoRESUMEN
Tank-binding kinase (TBK)1 plays a central role in innate immunity: it serves as an integrator of multiple signals induced by receptor-mediated pathogen detection and as a modulator of IFN levels. Efforts to better understand the biology of this key immunological factor have intensified recently as growing evidence implicates aberrant TBK1 activity in a variety of autoimmune diseases and cancers. Nevertheless, key molecular details of TBK1 regulation and substrate selection remain unanswered. Here, structures of phosphorylated and unphosphorylated human TBK1 kinase and ubiquitin-like domains, combined with biochemical studies, indicate a molecular mechanism of activation via transautophosphorylation. These TBK1 structures are consistent with the tripartite architecture observed recently for the related kinase IKKß, but domain contributions toward target recognition appear to differ for the two enzymes. In particular, both TBK1 autoactivation and substrate specificity are likely driven by signal-dependent colocalization events.
Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Catálisis , Activación Enzimática , Humanos , Modelos Moleculares , Fosforilación , Conformación Proteica , Proteínas Serina-Treonina Quinasas/químicaRESUMEN
Inhibitor of apoptosis (IAP) proteins are negative regulators of cell death. IAP family members contain RING domains that impart E3 ubiquitin ligase activity. Binding of endogenous or small-molecule antagonists to select baculovirus IAP repeat (BIR) domains within cellular IAP (cIAP) proteins promotes autoubiquitination and proteasomal degradation and so releases inhibition of apoptosis mediated by cIAP. Although the molecular details of antagonist-BIR domain interactions are well understood, it is not clear how this binding event influences the activity of the RING domain. Here biochemical and structural studies reveal that the unliganded, multidomain cIAP1 sequesters the RING domain within a compact, monomeric structure that prevents RING dimerization. Antagonist binding induces conformational rearrangements that enable RING dimerization and formation of the active E3 ligase.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Proteínas Inhibidoras de la Apoptosis/química , Secuencia de Aminoácidos , Animales , Línea Celular , Línea Celular Tumoral , Clonación Molecular , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas Inhibidoras de la Apoptosis/metabolismo , Ratones , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Complejo de la Endopetidasa Proteasomal/metabolismo , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Secundaria de Proteína , Dispersión del Ángulo Pequeño , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Ubiquitinadas/química , Proteínas Ubiquitinadas/metabolismo , UbiquitinaciónRESUMEN
The complexity of protein ubiquitination signals derives largely from the variety of polyubiquitin linkage types that can modify a target protein, each imparting distinct functional consequences. Free ubiquitin chains of uniform linkages and length are important tools in understanding how ubiquitin-binding proteins specifically recognize these different polyubiquitin modifications. While some free ubiquitin chain species are commercially available, mutational analyses and labeling schemes are limited to select, marketed stocks. Furthermore, the multimilligram quantities of material required for detailed biophysical and/or structural studies often makes these reagents cost prohibitive. To address these limitations, we have optimized known methods for the synthesis and purification of linear, K11-, K48-, and K63-linked ubiquitin dimers, trimers, and tetramers on a preparative scale. The high purity and relatively high yield of these proteins readily enables material-intensive experiments and provides flexibility for engineering specialized ubiquitin chain reagents, such as fluorescently labeled chains of discrete lengths.
Asunto(s)
Poliubiquitina/biosíntesis , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/biosíntesis , Clonación Molecular , Escherichia coli/genética , Colorantes Fluorescentes/química , Vectores Genéticos , Poliubiquitina/química , Poliubiquitina/aislamiento & purificación , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Ubiquitina/biosíntesis , Ubiquitina/químicaRESUMEN
Vismodegib (GDC-0449) is is an orally available selective Hedgehog pathway inhibitor in development for cancer treatment. The drug is ≥95% protein bound in plasma at clinically relevant concentrations and has an approximately 200-fold longer single dose half-life in humans than rats. We have identified a strong linear relationship between plasma drug concentrations and α-1-acid glycoprotein (AAG) in a phase I study. Biophysical and cellular techniques have been used to reveal that vismodegib strongly binds to human AAG (K(D) = 13 µM) and binds albumin with lower affinity (K(D) = 120 µM). Additionally, binding to rat AAG is reduced â¼20-fold relative to human, whereas the binding affinity to rat and human albumin was similar. Molecular docking studies reveal the reason for the signficiant species dependence on binding. These data highlight the utility of biophysical techniques in creating a comprehensive picture of protein binding across species.
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Anilidas/metabolismo , Proteínas Hedgehog/antagonistas & inhibidores , Piridinas/metabolismo , Anilidas/administración & dosificación , Anilidas/farmacocinética , Animales , Biofisica , Línea Celular , Semivida , Proteínas Hedgehog/metabolismo , Humanos , Unión Proteica , Piridinas/administración & dosificación , Piridinas/farmacocinética , Ratas , Transducción de Señal/efectos de los fármacos , Especificidad de la Especie , TermodinámicaRESUMEN
Microtubules have pivotal roles in fundamental cellular processes and are targets of antitubulin chemotherapeutics. Microtubule-targeted agents such as Taxol and vincristine are prescribed widely for various malignancies, including ovarian and breast adenocarcinomas, non-small-cell lung cancer, leukaemias and lymphomas. These agents arrest cells in mitosis and subsequently induce cell death through poorly defined mechanisms. The strategies that resistant tumour cells use to evade death induced by antitubulin agents are also unclear. Here we show that the pro-survival protein MCL1 (ref. 3) is a crucial regulator of apoptosis triggered by antitubulin chemotherapeutics. During mitotic arrest, MCL1 protein levels decline markedly, through a post-translational mechanism, potentiating cell death. Phosphorylation of MCL1 directs its interaction with the tumour-suppressor protein FBW7, which is the substrate-binding component of a ubiquitin ligase complex. The polyubiquitylation of MCL1 then targets it for proteasomal degradation. The degradation of MCL1 was blocked in patient-derived tumour cells that lacked FBW7 or had loss-of-function mutations in FBW7, conferring resistance to antitubulin agents and promoting chemotherapeutic-induced polyploidy. Additionally, primary tumour samples were enriched for FBW7 inactivation and elevated MCL1 levels, underscoring the prominent roles of these proteins in oncogenesis. Our findings suggest that profiling the FBW7 and MCL1 status of tumours, in terms of protein levels, messenger RNA levels and genetic status, could be useful to predict the response of patients to antitubulin chemotherapeutics.
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Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Moduladores de Tubulina/farmacología , Tubulina (Proteína)/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Proteínas de Ciclo Celular/genética , Línea Celular , Línea Celular Tumoral , Transformación Celular Neoplásica/efectos de los fármacos , Resistencia a Antineoplásicos , Proteínas F-Box/genética , Proteína 7 que Contiene Repeticiones F-Box-WD , Fibroblastos , Humanos , Ratones , Mitosis/efectos de los fármacos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Paclitaxel/farmacología , Farmacogenética , Fosforilación/efectos de los fármacos , Poliploidía , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/deficiencia , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ubiquitina-Proteína Ligasas/deficiencia , Ubiquitina-Proteína Ligasas/genética , Vincristina/farmacologíaRESUMEN
Unlike bacteria, many eukaryotes initiate DNA replication from genomic sites that lack apparent sequence conservation. These loci are identified and bound by the origin recognition complex (ORC), and subsequently activated by a cascade of events that includes recruitment of an additional factor, Cdc6. Archaeal organisms generally possess one or more Orc1/Cdc6 homologs, belonging to the Initiator clade of ATPases associated with various cellular activities (AAA(+)) superfamily; however, these proteins recognize specific sequences within replication origins. Atomic resolution studies have shown that archaeal Orc1 proteins contact double-stranded DNA through an N-terminal AAA(+) domain and a C-terminal winged-helix domain (WHD), but use remarkably few base-specific contacts. To investigate the biochemical effects of these associations, we mutated the DNA-interacting elements of the Orc1-1 and Orc1-3 paralogs from the archaeon Sulfolobus solfataricus, and tested their effect on origin binding and deformation. We find that the AAA(+) domain has an unpredicted role in controlling the sequence selectivity of DNA binding, despite an absence of base-specific contacts to this region. Our results show that both the WHD and ATPase region influence origin recognition by Orc1/Cdc6, and suggest that not only DNA sequence, but also local DNA structure help define archaeal initiator binding sites.