RESUMEN
KDM4 histone demethylases became an exciting target for inhibitor development as the evidence linking them directly to tumorigenesis mounts. In this study, we set out to better understand the binding cavity using an X-ray crystallographic approach to provide a detailed landscape of possible interactions within the under-investigated region of KDM4. Our design strategy was based on utilizing known KDM binding motifs, such as nicotinic acid and tetrazolylhydrazides, as core motifs that we decided to enrich with flexible tails to map the distal histone binding site. The resulting X-ray structures of the novel compounds bound to KDM4D, a representative of the KDM4 family, revealed the interaction pattern with distal residues in the histone-binding site. The most prominent protein rearrangement detected upon ligand binding is the loop movement that blocks the accessibility to the histone binding site. Apart from providing new sites that potential inhibitors can target, the novel compounds may prove helpful in exploring the capacity of ligands to bind in sites distal to the cofactor-binding site of other KDMs or 2-oxoglutarate (2OG)-dependent oxygenases. The case study proves that combining a strong small binding motif with flexible tails to probe the binding pocket will facilitate lead discovery in classical drug-discovery campaigns, given the ease of accessing X-ray quality crystals.
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Histonas , Histona Demetilasas con Dominio de Jumonji , Piridinas , Tetrazoles , Histona Demetilasas con Dominio de Jumonji/metabolismo , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Histona Demetilasas con Dominio de Jumonji/química , Tetrazoles/química , Tetrazoles/farmacología , Tetrazoles/metabolismo , Tetrazoles/síntesis química , Piridinas/química , Piridinas/farmacología , Piridinas/metabolismo , Piridinas/síntesis química , Humanos , Sitios de Unión , Cristalografía por Rayos X , Relación Estructura-Actividad , Histonas/metabolismo , Histonas/química , Estructura Molecular , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/síntesis química , Modelos Moleculares , Relación Dosis-Respuesta a DrogaRESUMEN
The phospholipase D (PLD) family is comprised of enzymes bearing phospholipase activity towards lipids or endo- and exonuclease activity towards nucleic acids. PLD3 is synthesized as a type II transmembrane protein and proteolytically cleaved in lysosomes, yielding a soluble active form. The deficiency of PLD3 leads to the slowed degradation of nucleic acids in lysosomes and chronic activation of nucleic acid-specific intracellular toll-like receptors. While the mechanism of PLD phospholipase activity has been extensively characterized, not much is known about how PLDs bind and hydrolyze nucleic acids. Here, we determined the high-resolution crystal structure of the luminal N-glycosylated domain of human PLD3 in its apo- and single-stranded DNA-bound forms. PLD3 has a typical phospholipase fold and forms homodimers with two independent catalytic centers via a newly identified dimerization interface. The structure of PLD3 in complex with an ssDNA-derived thymidine product in the catalytic center provides insights into the substrate binding mode of nucleic acids in the PLD family. Our structural data suggest a mechanism for substrate binding and nuclease activity in the PLD family and provide the structural basis to design immunomodulatory drugs targeting PLD3.
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Exodesoxirribonucleasas , Fosfolipasa D , Humanos , Lisosomas/metabolismo , Fosfolipasa D/química , Fosfolipasas , Exodesoxirribonucleasas/químicaRESUMEN
BACKGROUND: Transcription factors regulate gene expression by binding to transcription factor binding sites (TFBSs). Most models for predicting TFBSs are based on position weight matrices (PWMs), which require a specific motif to be present in the DNA sequence and do not consider interdependencies of nucleotides. Novel approaches such as Transcription Factor Flexible Models or recurrent neural networks consequently provide higher accuracies. However, it is unclear whether such approaches can uncover novel non-canonical, hitherto unexpected TFBSs relevant to human transcriptional regulation. RESULTS: In this study, we trained a convolutional recurrent neural network with HT-SELEX data for GRHL1 binding and applied it to a set of GRHL1 binding sites obtained from ChIP-Seq experiments from human cells. We identified 46 non-canonical GRHL1 binding sites, which were not found by a conventional PWM approach. Unexpectedly, some of the newly predicted binding sequences lacked the CNNG core motif, so far considered obligatory for GRHL1 binding. Using isothermal titration calorimetry, we experimentally confirmed binding between the GRHL1-DNA binding domain and predicted GRHL1 binding sites, including a non-canonical GRHL1 binding site. Mutagenesis of individual nucleotides revealed a correlation between predicted binding strength and experimentally validated binding affinity across representative sequences. This correlation was neither observed with a PWM-based nor another deep learning approach. CONCLUSIONS: Our results show that convolutional recurrent neural networks may uncover unanticipated binding sites and facilitate quantitative transcription factor binding predictions.
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Regulación de la Expresión Génica , Factores de Transcripción , Humanos , Factores de Transcripción/metabolismo , Sitios de Unión , Unión Proteica , Redes Neurales de la Computación , Nucleótidos/metabolismo , Proteínas Represoras/genéticaRESUMEN
Tryptophan hydroxylases catalyze the first and rate-limiting step in the biosynthesis of serotonin, a well-known neurotransmitter that plays an important role in multiple physiological functions. A reduction of serotonin levels, especially in the brain, can cause dysregulation leading to depression or insomnia. In contrast, overproduction of peripheral serotonin is associated with symptoms like carcinoid syndrome and pulmonary arterial hypertension. Recently, we developed a class of TPH inhibitors based on xanthine-benzimidazoles, characterized by a tripartite-binding mode spanning the binding sites of the cosubstrate pterin and the substrate tryptophan and by chelation of the catalytic iron ion. Herein, we describe the structure-based development of a second generation of xanthine-imidiazopyridines and -imidazothiazoles designed to inhibit TPH1 in the periphery while preventing the interaction with TPH2 in the brain. Lead compound 32 (TPT-004) shows superior pharmacokinetic and pharmacodynamic properties as well as efficacy in preclinical models of peripheral serotonin attenuation and colorectal tumor growth.
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Triptófano Hidroxilasa , Triptófano , Triptófano/metabolismo , Xantina , Serotonina/metabolismoRESUMEN
Studying mechanisms of bacterial biofilm generation is of vital importance to understanding bacterial cell-cell communication, multicellular cohabitation principles, and the higher resilience of microorganisms in a biofilm against antibiotics. Biofilms of the nonpathogenic, gram-positive soil bacterium Bacillus subtilis serve as a model system with biotechnological potential toward plant protection. Its major extracellular matrix protein components are TasA and TapA. The nature of TasA filaments has been of debate, and several forms, amyloidic and non-Thioflavin T-stainable have been observed. Here, we present the three-dimensional structure of TapA and uncover the mechanism of TapA-supported growth of nonamyloidic TasA filaments. By analytical ultracentrifugation and NMR, we demonstrate TapA-dependent acceleration of filament formation from solutions of folded TasA. Solid-state NMR revealed intercalation of the N-terminal TasA peptide segment into subsequent protomers to form a filament composed of ß-sandwich subunits. The secondary structure around the intercalated N-terminal strand ß0 is conserved between filamentous TasA and the Fim and Pap proteins, which form bacterial type I pili, demonstrating such construction principles in a gram-positive organism. Analogous to the chaperones of the chaperone-usher pathway, the role of TapA is in donating its N terminus to serve for TasA folding into an Ig domain-similar filament structure by donor-strand complementation. According to NMR and since the V-set Ig fold of TapA is already complete, its participation within a filament beyond initiation is unlikely. Intriguingly, the most conserved residues in TasA-like proteins (camelysines) of Bacillaceae are located within the protomer interface.
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Bacillus subtilis , Proteínas Bacterianas , Proteínas Bacterianas/metabolismo , Bacillus subtilis/metabolismo , Espectroscopía de Resonancia Magnética , Estructura Secundaria de Proteína , Chaperonas Moleculares/metabolismo , BiopelículasRESUMEN
p97 is an essential AAA+ ATPase that extracts and unfolds substrate proteins from membranes and protein complexes. Through its mode of action, p97 contributes to various cellular processes, such as membrane fusion, ER-associated protein degradation, DNA repair, and many others. Diverse p97 functions and protein interactions are regulated by a large number of adaptor proteins. Alveolar soft part sarcoma locus (ASPL) is a unique adaptor protein that regulates p97 by disassembling functional p97 hexamers to smaller entities. An alternative mechanism to regulate the activity and interactions of p97 is by posttranslational modifications (PTMs). Although more than 140 PTMs have been identified in p97, only a handful of those have been described in detail. Here we present structural and biochemical data to explain how the p97-remodeling adaptor protein ASPL enables the metastasis promoting methyltransferase METTL21D to bind and trimethylate p97 at a single lysine side chain, which is deeply buried inside functional p97 hexamers. The crystal structure of a heterotrimeric p97:ASPL:METTL21D complex in the presence of cofactors ATP and S-adenosyl homocysteine reveals how structural remodeling by ASPL exposes the crucial lysine residue of p97 to facilitate its trimethylation by METTL21D. The structure also uncovers a role of the second region of homology (SRH) present in the first ATPase domain of p97 in binding of a modifying enzyme to the AAA+ ATPase. Investigation of this interaction in the human, fish, and plant reveals fine details on the mechanism and significance of p97 trimethylation by METTL21D across different organisms.
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ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas , Metiltransferasas , Animales , Humanos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina Trifosfatasas/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Lisina/metabolismo , Metilación , Unión Proteica , Procesamiento Proteico-Postraduccional , Factores de Transcripción/metabolismo , Proteína que Contiene Valosina/metabolismo , Metiltransferasas/metabolismoRESUMEN
Tryptophan hydroxylases catalyze the first and rate-limiting step in the synthesis of serotonin. Serotonin is a key neurotransmitter in the central nervous system and, in the periphery, functions as a local hormone with multiple physiological functions. Studies in genetically altered mouse models have shown that dysregulation of peripheral serotonin levels leads to metabolic, inflammatory, and fibrotic diseases. Overproduction of serotonin by tumor cells causes severe symptoms typical for the carcinoid syndrome, and tryptophan hydroxylase inhibitors are already in clinical use for patients suffering from this disease. Here, we describe a novel class of potent tryptophan hydroxylase inhibitors, characterized by spanning all active binding sites important for catalysis, specifically those of the cosubstrate pterin, the substrate tryptophan as well as directly chelating the catalytic iron ion. The inhibitors were designed to efficiently reduce serotonin in the periphery while not passing the blood-brain barrier, thus preserving serotonin levels in the brain.
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Bencimidazoles , Serotonina , Triptófano Hidroxilasa , Xantina , Animales , Bencimidazoles/química , Bencimidazoles/farmacología , Ratones , Triptófano Hidroxilasa/antagonistas & inhibidores , Xantina/química , Xantina/farmacologíaRESUMEN
The neutrophil NADPH oxidase produces both intracellular and extracellular reactive oxygen species (ROS). Although oxidase activity is essential for microbial killing, and ROS can act as signaling molecules in the inflammatory process, excessive extracellular ROS directly contributes to inflammatory tissue damage, as well as to cancer progression and immune dysregulation in the tumor microenvironment. How specific signaling pathways contribute to ROS localization is unclear. Here we used a systems pharmacology approach to identify the specific Class I PI3-K isoform p110ß, and PLD1, but not PLD2, as critical regulators of extracellular, but not intracellular ROS production in primary neutrophils. Combined crystallographic and molecular dynamics analysis of the PX domain of the oxidase component p47phox, which binds the lipid products of PI 3-K and PLD, was used to clarify the membrane-binding mechanism and guide the design of mutant mice whose p47phox is unable to bind 3-phosphorylated inositol phospholipids. Neutrophils from these K43A mutant animals were specifically deficient in extracellular, but not intracellular, ROS production, and showed increased dependency on signaling through the remaining PLD1 arm. These findings identify the PX domain of p47phox as a critical integrator of PLD1 and p110ß signaling for extracellular ROS production, and as a potential therapeutic target for modulating tissue damage and extracellular signaling during inflammation.
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Fosfatidilinositol 3-Quinasa Clase I , NADPH Oxidasas , Neutrófilos , Especies Reactivas de Oxígeno , Animales , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Activación Enzimática , Inflamación , Ratones , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Neutrófilos/enzimología , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
Jumonji domain-containing lysine demethylase (KDM) enzymes are encoded by genes of the KDM superfamily. Activities of the KDM4 subfamily promote aggressive phenotypes associated with prostate cancer (PCa). Previously, we discovered a benzimidazole pyrazole molecule that inhibited KDM4 isoforms with properties tractable for development. Here, we demonstrate that a benzyl-substituted variant of this inhibitor exhibits improved potency in biochemical assays, is cell-permeable, and kills PCa cells at low micromolar concentrations. By X-ray crystallography and kinetics-based assays, we demonstrate that the mechanism of inhibition is complex, proceeding via competition with the enzyme for binding of active-site Fe2+ and by populating a distal site on the enzyme surface. Furthermore, we provide evidence that the inhibitor's cytostatic properties arise from direct intracellular inhibition of KDM4 enzymes. PCa cells treated with the inhibitor exhibit reduced expression of genes regulated by the androgen receptor, an outcome accompanied by epigenetic maintenance of a heterochromatic state.
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Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Bencimidazoles , Sitios de Unión/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Modelos Moleculares , Estructura Molecular , Pirazoles , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
The CCCH-type zinc finger (ZnF) containing ZC3H12 ribonucleases are crucial in post-transcriptional immune homoeostasis with ZC3H12A being the only structurally studied member of the family. In this study, we present a structural-biochemical characterization of ZC3H12C, which is linked with chronic immune disorders like psoriasis. We established that the RNA substrate is cooperatively recognized by the PIN and ZnF domains of ZC3H12C and analyzed the crystal structure of ZC3H12C bound to a single-stranded RNA substrate. The RNA engages in hydrogen-bonded contacts and stacking interactions with the PIN and ZnF domains simultaneously. The ZC3H12 ZnF shows unprecedented structural features not previously observed in any member of the CCCH-ZnF family and utilizes stacking interactions via a unique combination of spatially conserved aromatic residues to align the target transcript in a bent conformation onto the ZnF scaffold. Further comparative structural analysis of ZC3H12 CCCH-ZnF suggests that a trinucleotide sequence is recognized by ZC3H12 ZnF in target RNA. Our work not only describes the initial structure-biochemical study on ZC3H12C, but also provides the first molecular insight into RNA recognition by a ZC3H12 family member. Finally, our work points to an evolutionary code for RNA recognition adopted by CCCH-type ZnF proteins.
Asunto(s)
ARN/química , Ribonucleasas/química , Regiones no Traducidas 3' , Animales , Cristalografía por Rayos X , Células HEK293 , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Magnesio , Ratones , Modelos Moleculares , Unión Proteica , Dominios Proteicos , ARN/metabolismo , Ribonucleasas/metabolismo , Dedos de ZincRESUMEN
Pathogenic microorganisms often reside in glycan-based biofilms. Concentration and chain length distribution of these mostly anionic exopolysaccharides (EPS) determine the overall biophysical properties of a biofilm and result in a highly viscous environment. Bacterial communities regulate this biofilm state via intracellular small-molecule signaling to initiate EPS synthesis. Reorganization or degradation of this glycan matrix, however, requires the action of extracellular glycosidases. So far, these were mainly described for bacteriophages that must degrade biofilms for gaining access to host bacteria. The plant pathogen Pantoea stewartii (P. stewartii) encodes the protein WceF within its EPS synthesis cluster. WceF has homologs in various biofilm forming plant pathogens of the Erwinia family. In this work, we show that WceF is a glycosidase active on stewartan, the main P. stewartii EPS biofilm component. WceF has remarkable structural similarity with bacteriophage tailspike proteins (TSPs). Crystal structure analysis showed a native trimer of right-handed parallel ß-helices. Despite its similar fold, WceF lacks the high stability found in bacteriophage TSPs. WceF is a stewartan hydrolase and produces oligosaccharides, corresponding to single stewartan repeat units. However, compared with a stewartan-specific glycan hydrolase of bacteriophage origin, WceF showed lectin-like autoagglutination with stewartan, resulting in notably slower EPS cleavage velocities. This emphasizes that the bacterial enzyme WceF has a role in P. stewartii biofilm glycan matrix reorganization clearly different from that of a bacteriophage exopolysaccharide depolymerase.
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Proteínas Bacterianas/química , Biopelículas/crecimiento & desarrollo , Glicósido Hidrolasas/química , Pantoea/enzimología , Polisacáridos Bacterianos/química , Proteínas de la Cola de los Virus/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteriófagos/química , Bacteriófagos/enzimología , Sitios de Unión , Secuencia de Carbohidratos , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Modelos Moleculares , Oligosacáridos/química , Oligosacáridos/metabolismo , Pantoea/genética , Plantas/microbiología , Polisacáridos Bacterianos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología Estructural de Proteína , Proteínas de la Cola de los Virus/genética , Proteínas de la Cola de los Virus/metabolismoRESUMEN
The cold-shock domain has a deceptively simple architecture but supports a complex biology. It is conserved from bacteria to man and has representatives in all kingdoms of life. Bacterial cold-shock proteins consist of a single cold-shock domain and some, but not all are induced by cold shock. Cold-shock domains in human proteins are often associated with natively unfolded protein segments and more rarely with other folded domains. Cold-shock proteins and domains share a five-stranded all-antiparallel ß-barrel structure and a conserved surface that binds single-stranded nucleic acids, predominantly by stacking interactions between nucleobases and aromatic protein sidechains. This conserved binding mode explains the cold-shock domains' ability to associate with both DNA and RNA strands and their limited sequence selectivity. The promiscuous DNA and RNA binding provides a rationale for the ability of cold-shock domain-containing proteins to function in transcription regulation and DNA-damage repair as well as in regulating splicing, translation, mRNA stability and RNA sequestration.
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Dishevelled (Dvl) proteins are important regulators of the Wnt signalling pathway, interacting through their PDZ domains with the Wnt receptor Frizzled. Blocking the Dvl PDZ-Frizzled interaction represents a potential approach for cancer treatment, which stimulated the identification of small-molecule inhibitors, among them the anti-inflammatory drug Sulindac and Ky-02327. Aiming to develop tighter binding compounds without side effects, we investigated structure-activity relationships of sulfonamides. X-ray crystallography showed high complementarity of anthranilic acid derivatives in the GLGF loop cavity and space for ligand growth towards the PDZ surface. Our best binding compound inhibits Wnt signalling in a dose-dependent manner as demonstrated by TOP-GFP assays (IC50â¼50⯵M) and Western blotting of ß-catenin levels. Real-time PCR showed reduction in the expression of Wnt-specific genes. Our compound interacted with Dvl-1 PDZ (KD=2.4⯵M) stronger than Ky-02327 and may be developed into a lead compound interfering with the Wnt pathway.
RESUMEN
Transcription factors (TFs) bind DNA in a sequence-specific manner and thereby regulate target gene expression. TF binding and its regulatory activity is highly context dependent, and is not only determined by specific cell types or differentiation stages but also relies on other regulatory mechanisms, such as DNA and chromatin modifications. Interactions between TFs and their DNA binding sites are critical mediators of phenotypic variation and play important roles in the onset of disease. A continuously growing number of studies therefore attempts to elucidate TF:DNA interactions to gain knowledge about regulatory mechanisms and disease-causing variants. Here we summarize how TF-binding characteristics and the impact of variants can be investigated, how bioinformatic tools can be used to analyze and predict TF:DNA binding, and what additional information can be obtained from the TF protein structure.
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Battling metastasis through inhibition of cell motility is considered a promising approach to support cancer therapies. In this context, Ena/VASP-depending signaling pathways, in particular interactions with their EVH1 domains, are promising targets for pharmaceutical intervention. However, protein-protein interactions involving proline-rich segments are notoriously difficult to address by small molecules. Hence, structure-based design efforts in combination with the chemical synthesis of additional molecular entities are required. Building on a previously developed nonpeptidic micromolar inhibitor, we determined 22 crystal structures of ENAH EVH1 in complex with inhibitors and rationally extended our library of conformationally defined proline-derived modules (ProMs) to succeed in developing a nanomolar inhibitor ([Formula: see text] Da). In contrast to the previous inhibitor, the optimized compounds reduced extravasation of invasive breast cancer cells in a zebrafish model. This study represents an example of successful, structure-guided development of low molecular weight inhibitors specifically and selectively addressing a proline-rich sequence-recognizing domain that is characterized by a shallow epitope lacking defined binding pockets. The evolved high-affinity inhibitor may now serve as a tool in validating the basic therapeutic concept, i.e., the suppression of cancer metastasis by inhibiting a crucial protein-protein interaction involved in actin filament processing and cell migration.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Moléculas de Adhesión Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Femenino , Humanos , Células Jurkat , Prolina/metabolismo , Unión Proteica/efectos de los fármacos , Pez CebraRESUMEN
The tyrosine phosphatase SHP2 controls the activity of pivotal signaling pathways, including MAPK, JAK-STAT, and PI3K-Akt. Aberrant SHP2 activity leads to uncontrolled cell proliferation, tumorigenesis, and metastasis. SHP2 signaling was recently linked to drug resistance against cancer medications such as MEK and BRAF inhibitors. In this work, we present the development of a novel class of azaindole SHP2 inhibitors. We applied scaffold hopping and bioisosteric replacement concepts to eliminate unwanted structural motifs and to improve the inhibitor characteristics of the previously reported pyrazolone SHP2 inhibitors. The most potent azaindole 45 inhibits SHP2 with an IC50 = 0.031 µM in an enzymatic assay and with an IC50 = 2.6 µM in human pancreas cells (HPAF-II). Evaluation in a series of cellular assays for metastasis and drug resistance demonstrated efficient SHP2 blockade. Finally, 45 inhibited proliferation of two cancer cell lines that are resistant to cancer drugs and diminished ERK signaling.
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Inhibidores Enzimáticos/farmacología , Indoles/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Pirazolonas/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Dominio Catalítico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Humanos , Indoles/síntesis química , Indoles/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Estructura Molecular , Unión Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 11/química , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Pirazolonas/síntesis química , Pirazolonas/metabolismo , Relación Estructura-ActividadRESUMEN
Complete genome sequencing of the kinetoplastid protozoans Trypanosoma cruzi, Trypanosoma brucei and Leishmania major (Tritryp), published in 2005, opened up new perspectives for drug development targeting Chagas disease, African sleeping sickness and Leishmaniasis, neglected diseases affecting millions of most economically disadvantaged people. Still, half of the Tritryp genes code for proteins of unknown function. Moreover, almost 50% of conserved eukaryotic protein domains are missing in the Tritryp genomes. This suggests that functional and structural characterization of proteins of unknown function could reveal novel protein folds used by the trypanosomes for common cellular processes. Furthermore, proteins without homologous counterparts in humans may provide potential targets for therapeutic intervention. Here we describe the crystal structure of the T. cruzi protein Q4D6Q6, a conserved and kinetoplastid-specific protein essential for cell viability. Q4D6Q6 is a representative of a family of 20 orthologs, all annotated as proteins of unknown function. Q4D6Q6 monomers adopt a ßßαßßαßß topology and form a propeller-like tetramer. Oligomerization was verified in solution using NMR, SAXS, analytical ultra-centrifugation and gel filtration chromatography. A rigorous search for similar structures using the DALI server revealed similarities with propeller-like structures of several different functions. Although a Q4D6Q6 function could not be inferred from such structural comparisons, the presence of an oxidized cysteine at position 69, part of a cluster with phosphorylated serines and hydrophobic residues, identifies a highly reactive site and suggests a role of this cysteine as a nucleophile in a post-translational modification reaction.
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Proteínas Protozoarias/ultraestructura , Trypanosoma cruzi/ultraestructura , Animales , Humanos , Leishmania major/ultraestructura , Modelos Moleculares , Proteínas Protozoarias/genética , Dispersión del Ángulo Pequeño , Trypanosoma brucei brucei/ultraestructura , Trypanosoma cruzi/genética , Difracción de Rayos XRESUMEN
Cyclic-di-GMP (c-di-GMP) synthesized by diguanylate cyclases has been an important and ubiquitous secondary messenger in almost all bacterial systems. In Vibrio cholerae, c-di-GMP plays an intricate role in the production of the exopolysaccharide matrix, and thereby, in biofilm formation. The formation of the surface biofilm enables the bacteria to survive in aquatic bodies, when not infecting a human host. Diguanylate cyclases are the class of enzymes which synthesize c-di-GMP from two molecules of GTP and are endowed with a GGDEF or, a GGEEF signature domain. The VC0395_0300 protein from V. cholerae, has been established as a diguanylate cyclase with a necessary role in biofilm formation. Here we present the structure of an N-terminally truncated form of VC0395_0300, which retains the active GGEEF domain for diguanylate cyclase activity but lacks 160 residues from the poorly organized N-terminal domain. X-ray diffraction data was collected from a crystal of VC0395_0300(161-321) to a resolution of 1.9 Å. The structure displays remarkable topological similarity with diguanylate cyclases from other bacterial systems, but lacks the binding site for c-di-GMP present in its homologues. Finally, we demonstrate the ability of the truncated diguanylate cyclase VC0395_0300(161-321) to produce c-di-GMP, and its role in biofilm formation for the bacteria.
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Proteínas Bacterianas/química , Proteínas de Escherichia coli/química , Liasas de Fósforo-Oxígeno/química , Vibrio cholerae/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Dominio Catalítico , Cristalografía por Rayos X , GMP Cíclico/análogos & derivados , GMP Cíclico/biosíntesis , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Modelos Moleculares , Liasas de Fósforo-Oxígeno/genética , Liasas de Fósforo-Oxígeno/metabolismo , Dominios Proteicos , Sistemas de Mensajero Secundario , Solubilidad , Electricidad Estática , Vibrio cholerae/genética , Vibrio cholerae/fisiologíaRESUMEN
Tight junctions are complex supramolecular entities composed of integral membrane proteins, membrane-associated and soluble cytoplasmic proteins engaging in an intricate and dynamic system of protein-protein interactions. Three-dimensional structures of several tight-junction proteins or their isolated domains have been determined by X-ray crystallography, nuclear magnetic resonance spectroscopy, and cryo-electron microscopy. These structures provide direct insight into molecular interactions that contribute to the formation, integrity, or function of tight junctions. In addition, the known experimental structures have allowed the modeling of ligand-binding events involving tight-junction proteins. Here, we review the published structures of tight-junction proteins. We show that these proteins are composed of a limited set of structural motifs and highlight common types of interactions between tight-junction proteins and their ligands involving these motifs.
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Proteínas de Uniones Estrechas/química , Secuencia de Aminoácidos , Animales , Microscopía por Crioelectrón , Cristalografía por Rayos X , Humanos , Ligandos , Modelos Moleculares , Dominios PDZ , Conformación Proteica , Multimerización de Proteína , Alineación de Secuencia , Proteínas de Uniones Estrechas/metabolismo , Proteínas de Uniones Estrechas/ultraestructuraRESUMEN
The hexameric ring structure of the type II AAA+ ATPases is considered as stable and permanent. Recently, the UBX domain-containing cofactors Arabidopsis thaliana PUX1 and human alveolar soft part sarcoma locus (ASPL) were reported to bind and disassemble the cognate AAA+ ATPases AtCDC48 and human p97. Here, we present two crystal structures related to these complexes: a truncated AtCDC48 (AtCDC48-ND1) and a hybrid complex containing human p97-ND1 and the UBX domain of plant PUX1 (p97-ND1:PUX1-UBX). These structures reveal close similarity between the human and plant AAA+ ATPases, but also highlight differences between disassembling and non-disassembling AAA+ ATPase cofactors. Based on an AtCDC48 disassembly assay with PUX1 and known crystal structures of the p97-bound human cofactor ASPL, we propose a general ATPase disassembly model. Thus, our structural and biophysical investigations provide detailed insight into the mechanism of AAA+ ATPase disassembly by UBX domain cofactors and suggest a general mode of regulating the cellular activity of these molecular machines.