RESUMEN
Drug discovery relies on efficient identification of small-molecule leads and their interactions with macromolecular targets. However, understanding how chemotypes impact mechanistically important conformational states often remains secondary among high-throughput discovery methods. Here, we present a conformational discovery pipeline integrating time-resolved, high-throughput small-angle X-ray scattering (TR-HT-SAXS) and classic fragment screening applied to allosteric states of the mitochondrial import oxidoreductase apoptosis-inducing factor (AIF). By monitoring oxidized and X-ray-reduced AIF states, TR-HT-SAXS leverages structure and kinetics to generate a multidimensional screening dataset that identifies fragment chemotypes allosterically stimulating AIF dimerization. Fragment-induced dimerization rates, quantified with time-resolved SAXS similarity analysis (kVR), capture structure-activity relationships (SAR) across the top-ranked 4-aminoquinoline chemotype. Crystallized AIF-aminoquinoline complexes validate TR-SAXS-guided SAR, supporting this conformational chemotype for optimization. AIF-aminoquinoline structures and mutational analysis reveal active site F482 as an underappreciated allosteric stabilizer of AIF dimerization. This conformational discovery pipeline illustrates TR-HT-SAXS as an effective technology for targeting chemical leads to important macromolecular states.
Asunto(s)
Factor Inductor de la Apoptosis , Dispersión del Ángulo Pequeño , Regulación Alostérica , Relación Estructura-Actividad , Factor Inductor de la Apoptosis/química , Factor Inductor de la Apoptosis/metabolismo , Difracción de Rayos X/métodos , Humanos , Descubrimiento de Drogas/métodos , Modelos Moleculares , Cinética , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Intracellular DNA sensors regulate innate immunity and can provide a bridge to adaptive immunogenicity. However, the activation of the sensors in antigen-presenting cells (APCs) by natural agonists such as double-stranded DNAs or cyclic nucleotides is impeded by poor intracellular delivery, serum stability, enzymatic degradation and rapid systemic clearance. Here we show that the hydrophobicity, electrostatic charge and secondary conformation of helical polypeptides can be optimized to stimulate innate immune pathways via endoplasmic reticulum stress in APCs. One of the three polypeptides that we engineered activated two major intracellular DNA-sensing pathways (cGAS-STING (for cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes) and Toll-like receptor 9) preferentially in APCs by promoting the release of mitochondrial DNA, which led to the efficient priming of effector T cells. In syngeneic mouse models of locally advanced and metastatic breast cancers, the polypeptides led to potent DNA-sensor-mediated antitumour responses when intravenously given as monotherapy or with immune checkpoint inhibitors. The activation of multiple innate immune pathways via engineered cationic polypeptides may offer therapeutic advantages in the generation of antitumour immune responses.
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Células Presentadoras de Antígenos , Inmunidad Innata , Péptidos , Animales , Inmunidad Innata/efectos de los fármacos , Péptidos/química , Péptidos/farmacología , Ratones , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/efectos de los fármacos , Humanos , Femenino , Cationes/química , Ratones Endogámicos C57BL , Línea Celular Tumoral , Receptor Toll-Like 9/metabolismo , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/tratamiento farmacológico , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/químicaRESUMEN
Growth factor receptor-bound protein 2 (GRB2) is a cytoplasmic adapter for tyrosine kinase signaling and a nuclear adapter for homology-directed-DNA repair. Here we find nuclear GRB2 protects DNA at stalled replication forks from MRE11-mediated degradation in the BRCA2 replication fork protection axis. Mechanistically, GRB2 binds and inhibits RAD51 ATPase activity to stabilize RAD51 on stalled replication forks. In GRB2-depleted cells, PARP inhibitor (PARPi) treatment releases DNA fragments from stalled forks into the cytoplasm that activate the cGAS-STING pathway to trigger pro-inflammatory cytokine production. Moreover in a syngeneic mouse metastatic ovarian cancer model, GRB2 depletion in the context of PARPi treatment reduced tumor burden and enabled high survival consistent with immune suppression of cancer growth. Collective findings unveil GRB2 function and mechanism for fork protection in the BRCA2-RAD51-MRE11 axis and suggest GRB2 as a potential therapeutic target and an enabling predictive biomarker for patient selection for PARPi and immunotherapy combination.
Asunto(s)
Replicación del ADN , Neoplasias , Animales , Humanos , Ratones , ADN , Inestabilidad Genómica , Proteína Adaptadora GRB2/genética , Proteína Adaptadora GRB2/metabolismo , Inmunidad Innata , Proteína Homóloga de MRE11/metabolismo , Neoplasias/genética , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismoRESUMEN
Acute myeloid leukemia (AML) is driven by numerous molecular events that contribute to disease progression. Herein, we identify hnRNP K overexpression as a recurrent abnormality in AML that negatively correlates with patient survival. Overexpression of hnRNP K in murine fetal liver cells results in altered self-renewal and differentiation potential. Further, murine transplantation models reveal that hnRNP K overexpression results in myeloproliferation in vivo. Mechanistic studies expose a direct functional relationship between hnRNP K and RUNX1-a master transcriptional regulator of hematopoiesis often dysregulated in leukemia. Molecular analyses show that overexpression of hnRNP K results in an enrichment of an alternatively spliced isoform of RUNX1 lacking exon 4. Our work establishes hnRNP K's oncogenic potential in influencing myelogenesis through its regulation of RUNX1 splicing and subsequent transcriptional activity.
RESUMEN
We present a Chemistry and Structure Screen Integrated Efficiently (CASSIE) approach (named for Greek prophet Cassandra) to design inhibitors for cancer biology and pathogenesis. CASSIE provides an effective path to target master keys to control the repair-replication interface for cancer cells and SARS CoV-2 pathogenesis as exemplified here by specific targeting of Poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribose glycohydrolase ARH3 macrodomains plus SARS CoV-2 nonstructural protein 3 (Nsp3) Macrodomain 1 (Mac1) and Nsp15 nuclease. As opposed to the classical massive effort employing libraries with large numbers of compounds against single proteins, we make inhibitor design for multiple targets efficient. Our compact, chemically diverse, 5000 compound Goldilocks (GL) library has an intermediate number of compounds sized between fragments and drugs with predicted favorable ADME (absorption, distribution, metabolism, and excretion) and toxicological profiles. Amalgamating our core GL library with an approved drug (AD) library, we employ a combined GLAD library virtual screen, enabling an effective and efficient design cycle of ranked computer docking, top hit biophysical and cell validations, and defined bound structures using human proteins or their avatars. As new drug design is increasingly pathway directed as well as molecular and mechanism based, our CASSIE approach facilitates testing multiple related targets by efficiently turning a set of interacting drug discovery problems into a tractable medicinal chemistry engineering problem of optimizing affinity and ADME properties based upon early co-crystal structures. Optimization efforts are made efficient by a computationally-focused iterative chemistry and structure screen. Thus, we herein describe and apply CASSIE to define prototypic, specific inhibitors for PARG vs distinct inhibitors for the related macrodomains of ARH3 and SARS CoV-2 Nsp3 plus the SARS CoV-2 Nsp15 RNA nuclease.
Asunto(s)
COVID-19 , Ácidos Nucleicos , Síndrome Respiratorio Agudo Grave , Reparación del ADN , Humanos , Simulación del Acoplamiento Molecular , SARS-CoV-2RESUMEN
Arrival of the novel SARS-CoV-2 has launched a worldwide effort to identify both pre-approved and novel therapeutics targeting the viral proteome, highlighting the urgent need for efficient drug discovery strategies. Even with effective vaccines, infection is possible, and at-risk populations would benefit from effective drug compounds that reduce the lethality and lasting damage of COVID-19 infection. The CoV-2 MacroD-like macrodomain (Mac1) is implicated in viral pathogenicity by disrupting host innate immunity through its mono (ADP-ribosyl) hydrolase activity, making it a prime target for antiviral therapy. We therefore solved the structure of CoV-2 Mac1 from non-structural protein 3 (Nsp3) and applied structural and sequence-based genetic tracing, including newly determined A. pompejana MacroD2 and GDAP2 amino acid sequences, to compare and contrast CoV-2 Mac1 with the functionally related human DNA-damage signaling factor poly (ADP-ribose) glycohydrolase (PARG). Previously, identified targetable features of the PARG active site allowed us to develop a pharmacologically useful PARG inhibitor (PARGi). Here, we developed a focused chemical library and determined 6 novel PARGi X-ray crystal structures for comparative analysis. We applied this knowledge to discovery of CoV-2 Mac1 inhibitors by combining computation and structural analysis to identify PARGi fragments with potential to bind the distal-ribose and adenosyl pockets of the CoV-2 Mac1 active site. Scaffold development of these PARGi fragments has yielded two novel compounds, PARG-345 and PARG-329, that crystallize within the Mac1 active site, providing critical structure-activity data and a pathway for inhibitor optimization. The reported structural findings demonstrate ways to harness our PARGi synthesis and characterization pipeline to develop CoV-2 Mac1 inhibitors targeting the ADP-ribose active site. Together, these structural and computational analyses reveal a path for accelerating development of antiviral therapeutics from pre-existing drug optimization pipelines.
Asunto(s)
Antivirales/química , Proteasas Similares a la Papaína de Coronavirus/metabolismo , Inhibidores Enzimáticos/química , Glicósido Hidrolasas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Xantinas/química , Secuencia de Aminoácidos , Antivirales/farmacología , Dominio Catalítico , Cristalografía por Rayos X , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Humanos , Modelos Moleculares , Dominios Proteicos , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , Xantinas/farmacología , Tratamiento Farmacológico de COVID-19RESUMEN
BACKGROUND: Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an RNA-binding protein that is aberrantly expressed in cancers. We and others have previously shown that reduced hnRNP K expression downmodulates tumor-suppressive programs. However, overexpression of hnRNP K is the more commonly observed clinical phenomenon, yet its functional consequences and clinical significance remain unknown. METHODS: Clinical implications of hnRNP K overexpression were examined through immunohistochemistry on samples from patients with diffuse large B-cell lymphoma who did not harbor MYC alterations (n = 75). A novel transgenic mouse model that overexpresses hnRNP K specifically in B cells was generated to directly examine the role of hnRNP K overexpression in mice (three transgenic lines). Molecular consequences of hnRNP K overexpression were determined through proteomics, formaldehyde-RNA-immunoprecipitation sequencing, and biochemical assays. Therapeutic response to BET-bromodomain inhibition in the context of hnRNP K overexpression was evaluated in vitro and in vivo (n = 3 per group). All statistical tests were two-sided. RESULTS: hnRNP K is overexpressed in diffuse large B-cell lymphoma patients without MYC genomic alterations. This overexpression is associated with dismal overall survival and progression-free survival (P < .001). Overexpression of hnRNP K in transgenic mice resulted in the development of lymphomas and reduced survival (P < .001 for all transgenic lines; Line 171[n = 30]: hazard ratio [HR] = 64.23, 95% confidence interval [CI] = 26.1 to 158.0; Line 173 [n = 31]: HR = 25.27, 95% CI = 10.3 to 62.1; Line 177 [n = 25]: HR = 119.5, 95% CI = 42.7 to 334.2, compared with wild-type mice). Clinical samples, mouse models, global screening assays, and biochemical studies revealed that hnRNP K's oncogenic potential stems from its ability to posttranscriptionally and translationally regulate MYC. Consequently, Hnrnpk overexpression renders cells sensitive to BET-bromodomain-inhibition in both in vitro and transplantation models, which represents a strategy for mitigating hnRNP K-mediated c-Myc activation in patients. CONCLUSION: Our findings indicate that hnRNP K is a bona fide oncogene when overexpressed and represents a novel mechanism for c-Myc activation in the absence of MYC lesions.
Asunto(s)
Susceptibilidad a Enfermedades , Ribonucleoproteína Heterogénea-Nuclear Grupo K/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Linfoma de Células B/etiología , Linfoma de Células B/metabolismo , Adulto , Anciano , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Ribonucleoproteína Heterogénea-Nuclear Grupo K/química , Humanos , Linfoma de Células B/mortalidad , Linfoma de Células B/patología , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Estadificación de Neoplasias , Fenotipo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
We recently reported that SF2312 ((1,5-dihydroxy-2-oxopyrrolidin-3-yl)phosphonic acid), a phosphonate antibiotic with a previously unknown mode of action, is a potent inhibitor of the glycolytic enzyme, Enolase. SF2312 can only be synthesized as a racemic-diastereomeric mixture. However, co-crystal structures with Enolase 2 (ENO2) have consistently shown that only the (3S,5S)-enantiomer binds to the active site. The acidity of the alpha proton at C-3, which deprotonates under mildly alkaline conditions, results in racemization; thus while the separation of four enantiomeric intermediates was achieved via chiral High Performance Liquid Chromatography (HPLC) of the fully protected intermediate, deprotection inevitably nullified enantiopurity. To prevent epimerization of the C-3, we designed and synthesized MethylSF2312, ((1,5-dihydroxy-3-methyl-2-oxopyrrolidin-3-yl)phosphonic acid), which contains a fully-substituted C-3 alpha carbon. As a racemic-diastereomeric mixture, MethylSF2312 is equipotent to SF2312 in enzymatic and cellular systems against Enolase. Chiral HPLC separation of a protected MethylSF2312 precursor resulted in the efficient separation of the four enantiomers. After deprotection and inevitable re-equilibration of the anomeric C-5, (3S)-MethylSF2312 was up to 2000-fold more potent than (3R)-MethylSF2312 in an isolated enzymatic assay. This observation strongly correlates with biological activity in both human cancer cells and bacteria for the 3S enantiomer of SF2312. Novel X-ray structures of human ENO2 with chiral and racemic MethylSF2312 show that only (3S,5S)-enantiomer occupies the active site. Enolase inhibition is thus a direct result of binding by the (3S,5S)-enantiomer of MethylSF2312. Concurrent with these results for MethylSF2312, we contend that the (3S,5S)-SF2312 is the single active enantiomer of inhibitor SF2312.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Organofosfonatos/farmacología , Fosfopiruvato Hidratasa/antagonistas & inhibidores , Fosfopiruvato Hidratasa/química , Pirrolidinonas/farmacología , Sitios de Unión , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Organofosfonatos/química , Unión Proteica , Pirrolidinonas/química , Análisis Espectral , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
We and others have reported that the anticancer activity of L-asparaginase (ASNase) against asparagine synthetase (ASNS)-positive cell types requires ASNase glutaminase activity, whereas anticancer activity against ASNS-negative cell types does not. Here, we attempted to disentangle the relationship between asparagine metabolism, glutamine metabolism, and downstream pathways that modulate cell viability by testing the hypothesis that ASNase anticancer activity is based on asparagine depletion rather than glutamine depletion per se. We tested ASNase wild-type (ASNaseWT) and its glutaminase-deficient Q59L mutant (ASNaseQ59L) and found that ASNase glutaminase activity contributed to durable anticancer activity against xenografts of the ASNS-negative Sup-B15 leukemia cell line in NOD/SCID gamma mice, whereas asparaginase activity alone yielded a mere growth delay. Our findings suggest that ASNase glutaminase activity is necessary for durable, single-agent anticancer activity in vivo, even against ASNS-negative cancer types.
Asunto(s)
Asparaginasa/farmacología , Aspartatoamoníaco Ligasa/antagonistas & inhibidores , Glutaminasa/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Asparaginasa/administración & dosificación , Asparaginasa/farmacocinética , Asparagina/metabolismo , Aspartatoamoníaco Ligasa/metabolismo , Línea Celular Tumoral , Glutaminasa/administración & dosificación , Glutaminasa/farmacocinética , Glutamina/metabolismo , Humanos , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologíaRESUMEN
The mitochondrial caseinolytic protease P (ClpP) plays a central role in mitochondrial protein quality control by degrading misfolded proteins. Using genetic and chemical approaches, we showed that hyperactivation of the protease selectively kills cancer cells, independently of p53 status, by selective degradation of its respiratory chain protein substrates and disrupts mitochondrial structure and function, while it does not affect non-malignant cells. We identified imipridones as potent activators of ClpP. Through biochemical studies and crystallography, we show that imipridones bind ClpP non-covalently and induce proteolysis by diverse structural changes. Imipridones are presently in clinical trials. Our findings suggest a general concept of inducing cancer cell lethality through activation of mitochondrial proteolysis.
Asunto(s)
Endopeptidasa Clp/genética , Endopeptidasa Clp/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/administración & dosificación , Leucemia Mieloide Aguda/tratamiento farmacológico , Mitocondrias/metabolismo , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Ensayos de Selección de Medicamentos Antitumorales , Endopeptidasa Clp/química , Femenino , Células HCT116 , Células HEK293 , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Imidazoles , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Ratones , Modelos Moleculares , Mutación Puntual , Conformación Proteica/efectos de los fármacos , Proteolisis , Piridinas , Pirimidinas , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Despite being crucial for energy generation in most forms of life, few if any microbial antibiotics specifically inhibit glycolysis. To develop a specific inhibitor of the glycolytic enzyme enolase 2 (ENO2) for the treatment of cancers with deletion of ENO1 (encoding enolase 1), we modeled the synthetic tool compound inhibitor phosphonoacetohydroxamate (PhAH) into the active site of human ENO2. A ring-stabilized analog of PhAH, in which the hydroxamic nitrogen is linked to Cα by an ethylene bridge, was predicted to increase binding affinity by stabilizing the inhibitor in a bound conformation. Unexpectedly, a structure-based search revealed that our hypothesized backbone-stabilized PhAH bears strong similarity to SF2312, a phosphonate antibiotic of unknown mode of action produced by the actinomycete Micromonospora, which is active under anaerobic conditions. Here, we present multiple lines of evidence, including a novel X-ray structure, that SF2312 is a highly potent, low-nanomolar inhibitor of enolase.
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Inhibidores Enzimáticos/farmacología , Organofosfonatos/farmacología , Fosfopiruvato Hidratasa/antagonistas & inhibidores , Pirrolidinonas/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estructura Molecular , Organofosfonatos/química , Fosfopiruvato Hidratasa/metabolismo , Pirrolidinonas/química , Relación Estructura-ActividadRESUMEN
Membrane-bound cGMP-dependent protein kinase (PKG) II is a key regulator of bone growth, renin secretion, and memory formation. Despite its crucial physiological roles, little is known about its cyclic nucleotide selectivity mechanism due to a lack of structural information. Here, we find that the C-terminal cyclic nucleotide binding (CNB-B) domain of PKG II binds cGMP with higher affinity and selectivity when compared with its N-terminal CNB (CNB-A) domain. To understand the structural basis of cGMP selectivity, we solved co-crystal structures of the CNB domains with cyclic nucleotides. Our structures combined with mutagenesis demonstrate that the guanine-specific contacts at Asp-412 and Arg-415 of the αC-helix of CNB-B are crucial for cGMP selectivity and activation of PKG II. Structural comparison with the cGMP selective CNB domains of human PKG I and Plasmodium falciparum PKG (PfPKG) shows different contacts with the guanine moiety, revealing a unique cGMP selectivity mechanism for PKG II.
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Proteína Quinasa Dependiente de GMP Cíclico Tipo II/química , Proteína Quinasa Dependiente de GMP Cíclico Tipo II/metabolismo , GMP Cíclico/metabolismo , Regulación Alostérica , Animales , Células COS , Chlorocebus aethiops , Cristalografía por Rayos X , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
Signal transducer and activator of transcription 6 (STAT6) transmits signals from cytokines IL-4 and IL-13 and is activated in allergic airway disease. We are developing phosphopeptide mimetics targeting the SH2 domain of STAT6 to block recruitment to phosphotyrosine residues on IL-4 or IL-13 receptors and subsequent Tyr641 phosphorylation to inhibit the expression of genes contributing to asthma. Structure-affinity relationship studies showed that phosphopeptides based on Tyr631 from IL-4Rα bind with weak affinity to STAT6, whereas replacing the pY+3 residue with simple aryl and alkyl amides resulted in affinities in the mid to low nM range. A set of phosphatase-stable, cell-permeable prodrug analogues inhibited cytokine-stimulated STAT6 phosphorylation in both Beas-2B human airway cells and primary mouse T-lymphocytes at concentrations as low as 100 nM. IL-13-stimulated expression of CCL26 (eotaxin-3) was inhibited in a dose-dependent manner, demonstrating that targeting the SH2 domain blocks both phosphorylation and transcriptional activity of STAT6.
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Fosfopéptidos/farmacología , Factor de Transcripción STAT6/efectos de los fármacos , Dominios Homologos src/efectos de los fármacos , Animales , Asma/genética , Linfocitos T CD4-Positivos/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-13/biosíntesis , Interleucina-4/biosíntesis , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Profármacos , Ratas , Receptores de Interleucina-3/efectos de los fármacos , Receptores de Interleucina-4/efectos de los fármacos , Relación Estructura-Actividad , Activación Transcripcional/efectos de los fármacos , Tirosina/química , Tirosina/metabolismoRESUMEN
Members of the plakophilin-catenin sub-family (Pkp-1, -2, and -3) facilitate the linkage of desmosome junctional components to each other (e.g. desmosomal cadherins to desmoplakin) and the intermediate-filament cytoskeleton. Pkps also contribute to desmosomal stabilization and the trafficking of its components. The functions of Pkps outside of the desmosome are less well studied, despite evidence suggesting their roles in mRNA regulation, small-GTPase modulation (e.g. mid-body scission) during cell division, and cell survival following DNA damage. Pkp-catenins are further believed to have roles in the nucleus given their nuclear localization in some contexts and the known nuclear roles of structurally related catenins, such as beta-catenin and p120-catenin. Further, Pkp-catenin activities in the nuclear compartment have become of increased interest with the identification of interactions between Pkp2-catenin and RNA Pol III and Pkp1 with single-stranded DNA. Consistent with earlier reports suggesting possible nuclear roles in development, we previously demonstrated prominent nuclear localization of Pkp3 in Xenopus naïve ectoderm ("animal cap") cells and recently resolved a similar localization in mouse embryonic stem cells. Here, we report the association and positive functional interaction of Pkp3 with a transcription factor, Ets variant gene 1 (ETV1), which has critical roles in neural development and prominent roles in human genetic disease. Our results are the first to report the interaction of a sequence-specific transcription factor with any Pkp. Using Xenopus laevis embryos and mammalian cells, we provide evidence for the Pkp3:ETV1 complex on both biochemical and functional levels.
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Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/genética , Placofilinas/metabolismo , Factores de Transcripción/metabolismo , Animales , Cartilla de ADN/genética , Proteínas de Unión al ADN/genética , Embrión no Mamífero/metabolismo , Técnica del Anticuerpo Fluorescente , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Inmunoprecipitación , Hibridación in Situ , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción/genética , Técnicas del Sistema de Dos Híbridos , Xenopus laevisRESUMEN
Hfq is a posttranscriptional riboregulator and RNA chaperone that binds small RNAs and target mRNAs to effect their annealing and message-specific regulation in response to environmental stressors. Structures of Hfq-RNA complexes indicate that U-rich sequences prefer the proximal face and A-rich sequences the distal face; however, the Hfq-binding sites of most RNAs are unknown. Here, we present an Hfq-RNA mapping approach that uses single tryptophan-substituted Hfq proteins, all of which retain the wild-type Hfq structure, and tryptophan fluorescence quenching (TFQ) by proximal RNA binding. TFQ properly identified the respective distal and proximal binding of A15 and U6 RNA to Gram-negative Escherichia coli (Ec) Hfq and the distal face binding of (AA)3A, (AU)3A and (AC)3A to Gram-positive Staphylococcus aureus (Sa) Hfq. The inability of (GU)3G to bind the distal face of Sa Hfq reveals the (R-L)n binding motif is a more restrictive (A-L)n binding motif. Remarkably Hfq from Gram-positive Listeria monocytogenes (Lm) binds (GU)3G on its proximal face. TFQ experiments also revealed the Ec Hfq (A-R-N)n distal face-binding motif should be redefined as an (A-A-N)n binding motif. TFQ data also demonstrated that the 5'-untranslated region of hfq mRNA binds both the proximal and distal faces of Ec Hfq and the unstructured C-terminus.
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Proteínas de Escherichia coli/química , Proteína de Factor 1 del Huésped/química , ARN/metabolismo , Triptófano/química , Regiones no Traducidas 5' , Secuencias de Aminoácidos , Sitios de Unión , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fluorescencia , Proteína de Factor 1 del Huésped/genética , Proteína de Factor 1 del Huésped/metabolismo , Listeria monocytogenes , Modelos Moleculares , Mutación , Unión Proteica , ARN/química , Staphylococcus aureusRESUMEN
OBJECTIVE: Due to the rolling manufacturing process, most advanced high-strength steels (AHSS) demonstrate in-plane anisotropic material behavior. This study investigates the effects of material orientation on the axial crush behavior and fracture of AHSS with axial crush tests and computer simulations. METHODS: Crush simulation models considering material anisotropy and damage evolution were developed in LS-DYNA based on the drop-tower crush test results and coupon characterization test data for DP780 steel. The modified Mohr-Coulomb (MMC) isotropic fracture model was employed in the crush simulation models for fracture prediction. RESULTS: The 12-sided components fabricated in the transverse (T) direction of the sheet exhibited slightly higher crush loads and reduced crush distances compared to those in the longitudinal (L) direction. The crush behavior in each direction was generally proportional to ultimate tensile strength. All of the materials investigated in this study showed some cracking in the crush tests for both component orientations, but only DP780 showed significant anisotropy in fracture behavior with more cracking for the T direction compared to the L direction. Overall, the amount of cracking observed in the tests had little or no significant effect on the axial crush performance. The MMC fracture loci in both the L and T directions were determined using a reverse engineering approach, and the stress-strain curves beyond the uniform elongation point were extended using an optimization method. Both material models MAT103 and MAT224 predicted the crush and fracture behavior with reasonably good accuracy. CONCLUSIONS: The predicted fracture mode and force-displacement curves agreed well with the test data for both the L and T directions in axial crush tests of the 12-sided components. The simple isotropic material model MAT224 is adequate for crush simulations to predict material orientation effects on AHSS component crush performance and fracture behavior.
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Accidentes de Tránsito , Modelos Teóricos , Acero , Estrés Mecánico , Anisotropía , Simulación por Computador , Humanos , Reproducibilidad de los ResultadosRESUMEN
HipA is a bacterial serine/threonine protein kinase that phosphorylates targets, bringing about persistence and multidrug tolerance. Autophosphorylation of residue Ser150 is a critical regulatory mechanism of HipA function. Intriguingly, Ser150 is not located on the activation loop, as are other kinases; instead, it is in the protein core, where it forms part of the ATP-binding "P loop motif." How this buried residue is phosphorylated and regulates kinase activity is unclear. Here, we report multiple structures that reveal the P loop motif's exhibition of a remarkable "in-out" conformational equilibrium, which allows access to Ser150 and its intermolecular autophosphorylation. Phosphorylated Ser150 stabilizes the "out state," which inactivates the kinase by disrupting the ATP-binding pocket. Thus, our data reveal a mechanism of protein kinase regulation that is vital for multidrug tolerance and persistence, as kinase inactivation provides the critical first step in allowing dormant cells to revert to the growth phenotype and to reinfect the host.
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Farmacorresistencia Bacteriana Múltiple/fisiología , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Proteínas Serina-Treonina Quinasas/química , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Secuencias de Aminoácidos , Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Fosforilación/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Serina/química , Serina/metabolismoRESUMEN
Hfq is a small, highly abundant hexameric protein that is found in many bacteria and plays a critical role in mRNA expression and RNA stability. As an "RNA chaperone," Hfq binds AU-rich sequences and facilitates the trans annealing of small RNAs (sRNAs) to their target mRNAs, typically resulting in the down-regulation of gene expression. Hfq also plays a key role in bacterial RNA decay by binding tightly to polyadenylate [poly(A)] tracts. The structural mechanism by which Hfq recognizes and binds poly(A) is unknown. Here, we report the crystal structure of Escherichia coli Hfq bound to the poly(A) RNA, A(15). The structure reveals a unique RNA binding mechanism. Unlike uridine-containing sequences, which bind to the "proximal" face, the poly(A) tract binds to the "distal" face of Hfq using 6 tripartite binding motifs. Each motif consists of an adenosine specificity site (A site), which is effected by peptide backbone hydrogen bonds, a purine nucleotide selectivity site (R site), and a sequence-nondiscriminating RNA entrance/exit site (E site). The resulting implication that Hfq can bind poly(A-R-N) triplets, where R is a purine nucleotide and N is any nucleotide, was confirmed by binding studies. Indeed, Hfq bound to the oligoribonucleotides (AGG)(8), (AGC)(8), and the shorter (A-R-N)(4) sequence, AACAACAAGAAG, with nanomolar affinities. The abundance of (A-R-N)(4) and (A-R-N)(5) triplet repeats in the E. coli genome suggests additional RNA targets for Hfq. Further, the structure provides insight into Hfq-mediated sRNA-mRNA annealing and the role of Hfq in RNA decay.
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Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Proteína de Factor 1 del Huésped/química , ARN Mensajero/metabolismo , Secuencias de Aminoácidos , Cristalografía por Rayos X , Proteínas de Escherichia coli/metabolismo , Proteína de Factor 1 del Huésped/metabolismo , Poliadenilación , Unión Proteica , Conformación Proteica , Estabilidad del ARNRESUMEN
Homeodomain (HD) transcriptional activities are tightly regulated during embryogenesis and require protein interactions for their spatial and temporal activation. The chromatin-associated high mobility group protein (HMG-17) is associated with transcriptionally active chromatin, however its role in regulating gene expression is unclear. This report reveals a unique strategy in which, HMG-17 acts as a molecular switch regulating HD transcriptional activity. The switch utilizes the Wnt/beta-catenin signaling pathway and adds to the diverse functions of beta-catenin. A high-affinity HMG-17 interaction with the PITX2 HD protein inhibits PITX2 DNA-binding activity. The HMG-17/PITX2 inactive complex is concentrated to specific nuclear regions primed for active transcription. beta-Catenin forms a ternary complex with PITX2/HMG-17 to switch it from a repressor to an activator complex. Without beta-catenin, HMG-17 can physically remove PITX2 from DNA to inhibit its transcriptional activity. The PITX2/HMG-17 regulatory complex acts independently of promoter targets and is a general mechanism for the control of HD transcriptional activity. HMG-17 is developmentally regulated and its unique role during embryogenesis is revealed by the early embryonic lethality of HMG-17 homozygous mice. This mechanism provides a new role for canonical Wnt/beta-catenin signaling in regulating HD transcriptional activity during development using HMG-17 as a molecular switch.
Asunto(s)
Regulación de la Expresión Génica , Proteína HMGN2/metabolismo , Proteínas de Homeodominio/metabolismo , Factores de Transcripción/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animales , Células CHO , Línea Celular , Núcleo Celular/química , Cromatina/química , Cricetinae , Cricetulus , ADN/metabolismo , Proteína HMGN2/análisis , Proteína HMGN2/antagonistas & inhibidores , Proteínas de Homeodominio/antagonistas & inhibidores , Proteínas de Homeodominio/química , Humanos , Estructura Terciaria de Proteína , Interferencia de ARN , Proteínas Represoras/metabolismo , Transducción de Señal , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/química , Técnicas del Sistema de Dos Híbridos , Proteína del Homeodomínio PITX2RESUMEN
Recent studies on Hfq have provided a deeper understanding of the multiple functions of this pleiotropic post-transcriptional regulator. Insights into the mechanism of Hfq action have come from a variety of approaches. A key finding was the characterization of two RNA binding sites: the Proximal Site, which binds sRNA and mRNA; and the Distal Site, which binds poly(A) tails. Hfq was shown to interact with PAP I, PNP and RNase E, proteins that are involved in mRNA decay and in vitro, was shown to form fibres, the physiological significance of which is unknown. Fluorescence resonance energy transfer (FRET) studies directly demonstrated the role of Hfq as a chaperone that facilitates the interaction between sRNAs and target mRNAs. There are still, however, some unresolved questions.