RESUMEN
The discovery of a series of structurally-novel biaryl urea IDO inhibitors is described. Optimization of a micromolar hit through iterative cycles of synthesis and screening in an assay measuring IDO-mediated intracellular conversion of tryptophan to kynurenine led to potent inhibitors with favorable selectivity and metabolic stability profiles.
Asunto(s)
Ácidos Carboxílicos/farmacología , Inhibidores Enzimáticos/farmacología , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Urea/farmacología , Animales , Ácidos Carboxílicos/síntesis química , Ácidos Carboxílicos/química , Perros , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Células HeLa , Ensayos Analíticos de Alto Rendimiento , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Ratones , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Estructura Molecular , Relación Estructura-Actividad , Urea/análogos & derivados , Urea/químicaRESUMEN
N-Benzylic-substituted glycine sulfonamides that reversibly inhibit diacylglycerol (DAG) lipases are reported. Detailed herein are the structure activity relationships, profiling characteristics and physico-chemical properties for the first reported series of DAG lipase (DAGL) inhibitors that function without covalent attachment to the enzyme. Highly potent examples are presented that represent valuable tool compounds for studying DAGL inhibition and constitute important leads for future medicinal chemistry efforts.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Glicina/farmacología , Lipoproteína Lipasa/antagonistas & inhibidores , Sulfonamidas/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Glicina/análogos & derivados , Glicina/química , Humanos , Lipoproteína Lipasa/metabolismo , Estructura Molecular , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/químicaRESUMEN
XIAP (X-chromosome-linked inhibitor of apoptosis protein) is a central apoptosis regulator that blocks cell death by inhibiting caspase-3, caspase-7, and caspase-9 via binding interactions with the XIAP BIR2 and BIR3 domains (where BIR is baculovirus IAP repeat). Smac protein, in its dimeric form, effectively antagonizes XIAP by concurrently targeting both its BIR2 and BIR3 domains. Here we describe the development of highly sensitive homogeneous time-resolved fluorescence resonance energy transfer (HTRF) assays to measure binding affinities of potent bivalent peptidomimetic inhibitors of XIAP. Our results indicate that these assays can differentiate Smac-mimetic inhibitors with a wide range of binding affinities down to the picomolar range. Furthermore, we demonstrate the utility of these fluorescent tools for characterization of inhibitor off-rates, which as a crucial determinant of target engagement and cellular potency is another important parameter to guide optimization in a structure-based drug discovery effort. Our study also explores how increased inhibitor valency can lead to enhanced potency at multimeric proteins such as IAP.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Peptidomiméticos/farmacología , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidores , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Animales , Caspasa 3/metabolismo , Línea Celular , Humanos , Ratones Endogámicos BALB C , Peptidomiméticos/química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteína Inhibidora de la Apoptosis Ligada a X/químicaRESUMEN
JAK2 kinase inhibitors are a promising new class of agents for the treatment of myeloproliferative neoplasms and have potential for the treatment of other diseases possessing a deregulated JAK2-STAT pathway. X-ray structure and ADME guided refinement of C-4 heterocycles to address metabolic liability present in dialkylthiazole 1 led to the discovery of a clinical candidate, BMS-911543 (11), with excellent kinome selectivity, in vivo PD activity, and safety profile.
RESUMEN
Duchenne muscular dystrophy (DMD) is a genetic, lethal, muscle disorder caused by the loss of the muscle protein, dystrophin, leading to progressive loss of muscle fibers and muscle weakness. Drug discovery efforts targeting DMD have used two main approaches: (1) the restoration of dystrophin expression or the expression of a compensatory protein, and (2) the mitigation of downstream pathological mechanisms, including dysregulated calcium homeostasis, oxidative stress, inflammation, fibrosis, and muscle ischemia. The aim of this review is to introduce the disease, its pathophysiology, and the available research tools to a drug discovery audience. This review will also detail the most promising therapies that are currently being tested in clinical trials or in advanced preclinical models.
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Descubrimiento de Drogas , Distrofina/metabolismo , Distrofia Muscular de Duchenne/tratamiento farmacológico , Animales , Antioxidantes/uso terapéutico , Benzoxazoles/uso terapéutico , Calcio/metabolismo , Modelos Animales de Enfermedad , Distrofina/genética , Expresión Génica/efectos de los fármacos , Humanos , Distrofia Muscular de Duchenne/fisiopatología , Oxadiazoles/uso terapéuticoRESUMEN
Inhibition of signal transduction downstream of the IL-23 receptor represents an intriguing approach to the treatment of autoimmunity. Using a chemogenomics approach marrying kinome-wide inhibitory profiles of a compound library with the cellular activity against an IL-23-stimulated transcriptional response in T lymphocytes, a class of inhibitors was identified that bind to and stabilize the pseudokinase domain of the Janus kinase tyrosine kinase 2 (Tyk2), resulting in blockade of receptor-mediated activation of the adjacent catalytic domain. These Tyk2 pseudokinase domain stabilizers were also shown to inhibit Tyk2-dependent signaling through the Type I interferon receptor but not Tyk2-independent signaling and transcriptional cellular assays, including stimulation through the receptors for IL-2 (JAK1- and JAK3-dependent) and thrombopoietin (JAK2-dependent), demonstrating the high functional selectivity of this approach. A crystal structure of the pseudokinase domain liganded with a representative example showed the compound bound to a site analogous to the ATP-binding site in catalytic kinases with features consistent with high ligand selectivity. The results support a model where the pseudokinase domain regulates activation of the catalytic domain by forming receptor-regulated inhibitory interactions. Tyk2 pseudokinase stabilizers, therefore, represent a novel approach to the design of potent and selective agents for the treatment of autoimmunity.
Asunto(s)
Modelos Moleculares , Transducción de Señal , Linfocitos T/enzimología , TYK2 Quinasa/química , Cristalografía por Rayos X , Estabilidad de Enzimas , Humanos , Janus Quinasa 1/genética , Janus Quinasa 1/metabolismo , Janus Quinasa 3/genética , Janus Quinasa 3/metabolismo , Estructura Terciaria de Proteína , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/metabolismo , Receptores de Trombopoyetina/genética , Receptores de Trombopoyetina/metabolismo , TYK2 Quinasa/genéticaRESUMEN
Recent genetic evidence suggests that the diacylglycerol lipase (DAGL-α) isoform is the major biosynthetic enzyme for the most abundant endocannabinoid, 2-arachidonoyl-glycerol (2-AG), in the central nervous system. Revelation of its essential role in regulating retrograde synaptic plasticity and adult neurogenesis has made it an attractive therapeutic target. Therefore, it has become apparent that selective inhibition of DAGL-α enzyme activity with a small molecule could be a strategy for the development of novel therapies for the treatment of disease indications such as depression, anxiety, pain, and cognition. In this report, the authors present the identification of small-molecule inhibitor chemotypes of DAGL-α, which were selective (≥10-fold) against two other lipases, pancreatic lipase and monoacylglycerol lipase, via high-throughput screening of a diverse compound collection. Seven chemotypes of interest from a list of 185 structural clusters, which included 132 singletons, were initially selected for evaluation and characterization. Selection was based on potency, selectivity, and chemical tractability. One of the chemotypes, the glycine sulfonamide series, was prioritized as an initial lead for further medicinal chemistry optimization.
Asunto(s)
Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/farmacología , Lipoproteína Lipasa/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas , Línea Celular , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Humanos , Cinética , Lipoproteína Lipasa/metabolismo , Reproducibilidad de los Resultados , Especificidad por SustratoRESUMEN
Depolarization-induced suppression of inhibition (DSI) is a prevailing form of endocannabinoid signalling. However, several discrepancies have arisen regarding the roles played by the two major brain endocannabinoids, 2-arachidonoylglycerol (2-AG) and anandamide, in mediating DSI. Here we studied endocannabinoid signalling in the prefrontal cortex (PFC), where several components of the endocannabinoid system have been identified, but endocannabinoid signalling remains largely unexplored. In voltage clamp recordings from mouse PFC pyramidal neurons, depolarizing steps significantly suppressed IPSCs induced by application of the cholinergic agonist carbachol. DSI in PFC neurons was abolished by extra- or intracellular application of tetrahydrolipstatin (THL), an inhibitor of the 2-AG synthesis enzyme diacylglycerol lipase (DAGL). Moreover, DSI was enhanced by inhibiting 2-AG degradation, but was unaffected by inhibiting anandamide degradation. THL, however, may affect other enzymes of lipid metabolism and does not selectively target the α (DAGLα) or ß (DAGLß) isoforms of DAGL. Therefore, we studied DSI in the PFC of DAGLα(-/-) and DAGLß(-/-) mice generated via insertional mutagenesis by gene-trapping with retroviral vectors. Gene trapping strongly reduced DAGLα or DAGLß mRNA levels in a locus-specific manner. In DAGLα(-/-) mice cortical levels of 2-AG were significantly decreased and DSI was completely abolished, whereas DAGLß deficiency did not alter cortical 2-AG levels or DSI. Importantly, cortical levels of anandamide were not significantly affected in DAGLα(-/-) or DAGLß(-/-) mice. The chronic decrease of 2-AG levels in DAGLα(-/-) mice did not globally alter inhibitory transmission or the response of cannabinoid-sensitive synapses to cannabinoid receptor stimulation, although it altered some intrinsic membrane properties. Finally, we found that repetitive action potential firing of PFC pyramidal neurons suppressed synaptic inhibition in a DAGLα-dependent manner. These results show that DSI is a prominent form of endocannabinoid signalling in PFC circuits. Moreover, the close agreement between our pharmacological and genetic studies indicates that 2-AG synthesized by postsynaptic DAGLα mediates DSI in PFC neurons.
Asunto(s)
Ácidos Araquidónicos/fisiología , Moduladores de Receptores de Cannabinoides/fisiología , Endocannabinoides , Glicéridos/fisiología , Potenciales Postsinápticos Inhibidores/fisiología , Lipoproteína Lipasa/fisiología , Inhibición Neural/fisiología , Corteza Prefrontal/fisiología , Animales , Carbacol/farmacología , Agonistas Colinérgicos/farmacología , Inhibidores Enzimáticos/farmacología , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/deficiencia , Isoenzimas/fisiología , Lipoproteína Lipasa/antagonistas & inhibidores , Lipoproteína Lipasa/deficiencia , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Inhibición Neural/efectos de los fármacos , Técnicas de Placa-Clamp , Alcamidas Poliinsaturadas , Corteza Prefrontal/efectos de los fármacos , Células Piramidales/efectos de los fármacos , Células Piramidales/fisiologíaRESUMEN
Diacylglycerol lipase α is the key enzyme in the formation of the most prevalent endocannabinoid, 2-arachidonoylglycerol in the brain. In this study we identified the catalytic triad of diacylglycerol lipase α, consisting of serine 472, aspartate 524 and histidine 650. A truncated version of diacylglycerol lipase α, spanning residues 1-687 retains complete catalytic activity suggesting that the C-terminal domain is not required for catalysis. We also report the discovery and the characterization of fluorogenic and chromogenic substrates for diacylglycerol lipase α. Assays performed with these substrates demonstrate equipotent inhibition of diacylglycerol lipase α by tetrahydrolipastatin and RHC-20867 as compared to reactions performed with the native diacylglycerol substrate. Thus, confirming the utility of assays using these substrates for identification and kinetic characterization of inhibitors from pharmaceutical collections.
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Lipoproteína Lipasa/química , Catálisis , Membrana Celular/enzimología , Compuestos Cromogénicos/química , Ciclohexanonas/química , Fluorescencia , Células HEK293 , Humanos , Lactonas/química , Lipoproteína Lipasa/genética , Mutación , Orlistat , Especificidad por SustratoRESUMEN
Classical enzymology has been used for generations to understand the interactions of inhibitors with their enzyme targets. Enzymology tools enabled prediction of the biological impact of inhibitors as well as the development of novel, more potent, ones. Experiments designed to examine the competition between the tested inhibitor and the enzyme substrate(s) are the tool of choice to identify inhibitors that bind in the active site. Competition between an inhibitor and a substrate is considered a strong evidence for binding of the inhibitor in the active site, while the lack of competition suggests binding to an alternative site. Nevertheless, exceptions to this notion do exist. Active site-binding inhibitors can display non-competitive inhibition patterns. This unusual behavior has been observed with enzymes utilizing an exosite for substrate binding, isomechanism enzymes, enzymes with multiple substrates and/or products and two-step binding inhibitors. In many of these cases, the mechanisms underlying the lack of competition between the substrate and the inhibitor are well understood. Tools like alternative substrates, testing the enzyme reaction in the reverse direction and monitoring inhibition time dependence can be applied to enable distinction between 'badly behaving' active site binders and true exosite inhibitors.
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Inhibidores Enzimáticos/química , Enzimas/química , Unión Competitiva , Dominio Catalítico , Inhibidores Enzimáticos/farmacología , Enzimas/metabolismoRESUMEN
Histone acetyl transferases are important regulators of cellular homeostasis. This study describes a sensitive acetyl transferase electrophoretic mobility shift assay applicable both for kinetic analysis of acetyl transferase inhibitors and for high-throughput testing. Application of the assay for human GCN5L2 enabled dissection of inhibitor competition with respect to acetyl coenzyme A. Furthermore, we demonstrated that the assay can detect time-dependent inhibition of human GCN5L2 by reactive inhibitors.
Asunto(s)
Ensayo de Cambio de Movilidad Electroforética/métodos , Histona Acetiltransferasas/antagonistas & inhibidores , Histona Acetiltransferasas/metabolismo , Acetilcoenzima A/metabolismo , Animales , Línea Celular , Humanos , CinéticaRESUMEN
The amyloid-beta (Abeta) peptide, which likely plays a key role in Alzheimer disease, is derived from the amyloid-beta precursor protein (APP) through consecutive proteolytic cleavages by beta-site APP-cleaving enzyme and gamma-secretase. Unexpectedly gamma-secretase inhibitors can increase the secretion of Abeta peptides under some circumstances. This "Abeta rise" phenomenon, the same inhibitor causing an increase in Abeta at low concentrations but inhibition at higher concentrations, has been widely observed. Here we show that the Abeta rise depends on the beta-secretase-derived C-terminal fragment of APP (betaCTF) or C99 levels with low levels causing rises. In contrast, the N-terminally truncated form of Abeta, known as "p3," formed by alpha-secretase cleavage, did not exhibit a rise. In addition to the Abeta rise, low betaCTF or C99 expression decreased gamma-secretase inhibitor potency. This "potency shift" may be explained by the relatively high enzyme to substrate ratio under conditions of low substrate because increased concentrations of inhibitor would be necessary to affect substrate turnover. Consistent with this hypothesis, gamma-secretase inhibitor radioligand occupancy studies showed that a high level of occupancy was correlated with inhibition of Abeta under conditions of low substrate expression. The Abeta rise was also observed in rat brain after dosing with the gamma-secretase inhibitor BMS-299897. The Abeta rise and potency shift are therefore relevant factors in the development of gamma-secretase inhibitors and can be evaluated using appropriate choices of animal and cell culture models. Hypothetical mechanisms for the Abeta rise, including the "incomplete processing" and endocytic models, are discussed.
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Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Regulación Enzimológica de la Expresión Génica , Animales , Encéfalo/metabolismo , Butiratos/farmacología , Línea Celular , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Hidrocarburos Halogenados/farmacología , Ratones , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Especificidad por SustratoAsunto(s)
Trastornos de la Coagulación Sanguínea/sangre , Coagulación Sanguínea , Factor XIIa/metabolismo , Factor XIa/metabolismo , Quininógeno de Alto Peso Molecular/metabolismo , Calicreína Plasmática/metabolismo , Precalicreína/metabolismo , Animales , Coagulación Sanguínea/genética , Deficiencia del Factor XI/sangre , Factor XIa/genética , Retroalimentación Fisiológica , Genotipo , Humanos , FenotipoRESUMEN
11beta-hydroxysteroid dehydrogenase 1 regulates the tissue availability of cortisol by interconverting cortisone and cortisol. It is capable of functioning as both a reductase and a dehydrogenase depending upon the surrounding milieu. In this work, we have studied the reaction mechanism of a soluble form of human 11beta-hydroxysteroid dehydrogenase 1 and its mode of inhibition by potent and selective inhibitors belonging to three different structural classes. We found that catalysis follows an ordered addition with NADP(H) binding preceding the binding of the steroid. While all three inhibitors tested bound to the steroid binding pocket, they differed in their interactions with the cofactor NADP(H). Compound A, a pyridyl amide bound more efficiently to the NADPH-bound form of 11beta-hydroxysteroid dehydrogenase 1. Compound B, an adamantyl triazole, was unaffected by NADP(H) binding and the sulfonamide, Compound C, showed preferential binding to the NADP+ -bound form of 11beta-hydroxysteroid dehydrogenase 1. These differences were found to augment significant selectivity towards inhibition of the reductase reaction versus the dehydrogenase reaction. This selectivity may translate to differences in the in vivo effects of 11beta-hydroxysteroid dehydrogenase 1 inhibitors.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , 11-beta-Hidroxiesteroide Deshidrogenasas/metabolismo , Piridinas/farmacología , Sulfonamidas/farmacología , Triazoles/farmacología , Humanos , Cinética , NADP/metabolismoRESUMEN
In this study, we tested the hypothesis that factor XI (FXI) activation occurs in plasma following activation of the extrinsic pathway by thrombin-mediated feedback activation. We used two different assays: (i) a direct measurement of activated FXI by ELISA and (ii) a functional assay that follows the activation of the coagulation cascade in the presence or absence of a FXI inhibiting antibody by monitoring thrombin activity. We failed to detect any FXI activation or functional contribution to the activation of the coagulation cascade in platelet poor or platelet-rich plasma, when activation was initiated by thrombin or tissue factor. Additionally, we found that, in the absence of a contact system inhibitor during blood draw, contact activation of FXI can mistakenly appear as thrombin- or tissue-factor-dependent activation. Thus, activation of FXI by thrombin in solution or on the surface of activated platelets does not appear to play a significant role in a plasma environment. These results call for reevaluation of the physiological role of the contact activation system in blood coagulation.
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Factor XI/metabolismo , Trombina/metabolismo , Plaquetas/metabolismo , Deficiencia del Factor XI/metabolismo , Retroalimentación Fisiológica , Humanos , Plasma/metabolismo , Agregación Plaquetaria , Tromboplastina/metabolismoRESUMEN
Methyltransferases form a large class of enzymes, most of which use S-adenosylmethionine as the methyl donor. In fact, S-adenosylmethionine is second only to ATP in the variety of reactions for which it serves as a cofactor. Several methods to measure methyltransferase activity have been described, most of which are applicable only to specific enzymes and/or substrates. In this work we describe a sensitive liquid chromatography/mass spectroscopy-based methyltransferase assay. The assay monitors the conversion of S-adenosylmethionine to S-adenosylhomocysteine and can be applied to any methyltransferase and substrate of interest. We used the well-characterized enzyme catechol O-methyltransferase to demonstrate that the assay can monitor activity with a variety of substrates, can identify new substrates, and can be used even with crude preparation of enzyme. Furthermore, we demonstrate the utility of the assay for kinetic characterization of enzymatic activity.
Asunto(s)
Catecol O-Metiltransferasa/metabolismo , Cromatografía Liquida , Espectrometría de Masas en Tándem , Secuencia de Aminoácidos , Catecol O-Metiltransferasa/química , Catecol O-Metiltransferasa/fisiología , Activación Enzimática , Humanos , Cinética , Datos de Secuencia Molecular , S-Adenosilhomocisteína/química , S-Adenosilhomocisteína/metabolismoRESUMEN
Factor XIa is a serine protease which participates in both the extrinsic and intrinsic pathways of blood coagulation. In this work we used active site directed inhibitors to study the mechanism of factor IX activation by factor XIa. To this end, we developed a new sensitive method for the detection of factor IXa based on its affinity to antithrombin III. Using this assay, we found that the peptidic inhibitors, leupeptin and aprotinin, exhibited similar potencies in inhibiting factor IX activation and the cleavage of a tripeptidic chromogenic substrate by factor XIa. As expected, leupeptin and aprotinin were competitive with respect to the tripeptidic chromogenic substrate. However, the inhibition of factor IX activation was best described by mixed-type inhibition with the affinity of leupeptin and aprotinin to the factor XIa-factor IX complex only approximately 10-fold lower than their affinity toward factor XIa. These results, consistent with previous factor XI domain analyses, suggest that the active site of factor XIa does not contribute significantly to the affinity of factor XIa toward factor IX. The competitive component of the inhibition of factor IX activation suggests that binding of factor IX to factor XIa heavy chain affects the interactions of leupeptin and aprotinin with the active site.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Factor XIa/antagonistas & inhibidores , Inhibidores de Proteasas/metabolismo , Aprotinina/metabolismo , Sitios de Unión , Factor IX/metabolismo , Factor XIa/química , Factor XIa/metabolismo , Leupeptinas/metabolismo , Modelos BiológicosRESUMEN
Gamma-secretase is a unique protease which cleaves within the transmembrane domain of several substrate proteins. Among gamma-secretase substrates are members of the Notch family of receptors and the amyloid precursor protein. In this study we used a cell-free Notch-cleavage assay and specific gamma-secretase inhibitors to study the cleavage of Notch by gamma-secretase. Using this assay, we found that, in contrast to previous reports, the presence of valine at the P1(') position of Notch1 is not required for gamma-secretase cleavage. Our results suggest that the presence of valine at the N-terminus of the Notch intracellular domain cleavage product is important for its stability. Thus it appears that Notch cleavage is very similar to APP cleavage with respect to the lack of sequence specificity.
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Acetilcisteína/análogos & derivados , Endopeptidasas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación , Receptores de Superficie Celular , Factores de Transcripción , Acetilcisteína/metabolismo , Secretasas de la Proteína Precursora del Amiloide , Animales , Ácido Aspártico Endopeptidasas , Línea Celular , Sistema Libre de Células , Células Cultivadas , Inhibidores de Cisteína Proteinasa/metabolismo , Fibroblastos/citología , Fibroblastos/fisiología , Humanos , Ratones , Ratones Noqueados , Estructura Molecular , Estructura Terciaria de Proteína , Receptor Notch1 , Valina/metabolismoRESUMEN
In S. cerevisiae, posttranslational modification by the ubiquitin-like Smt3/SUMO-1 protein is essential for survival, but functions and cellular targets for this modification are largely unknown. We find that one function associated with the Smt3/SUMO-1 isopeptidase Smt4 is to control chromosome cohesion at centromeric regions and that a key Smt3/SUMO-1 substrate underlying this function is Top2, DNA Topoisomerase II. Top2 modification by Smt3/SUMO-1 is misregulated in smt4 strains, and top2 mutants resistant to Smt3/SUMO-1 modification suppress the smt4 cohesion defect. top2 mutants display aberrant chromatid stretching at the centromere in response to mitotic spindle tension and altered chromatid reassociation following microtubule depolymerization. These results suggest Top2 modification by Smt3/SUMO-1 regulates a component of chromatin structure or topology required for centromeric cohesion.
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Centrómero/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Endopeptidasas/metabolismo , Proteína SUMO-1/metabolismo , Proteínas de Ciclo Celular , Proteínas Cromosómicas no Histona , ADN-Topoisomerasas de Tipo II/genética , Endopeptidasas/genética , Proteínas Fúngicas , Proteínas Nucleares/metabolismo , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , CohesinasRESUMEN
Analysis of meiotic recombination by functional genomic approaches reveals prominent spatial and functional interactions among diverse organizational determinants. Recombination occurs between chromatin loop sequences; however, these sequences are spatially tethered to underlying chromosome axes via their recombinosomes. Meiotic chromosomal protein, Red1, localizes to chromosome axes; however, Red1 loading is modulated by R/G-bands isochores and thus by bulk chromatin state. Recombination is also modulated by isochore determinants: R-bands differentially favor double-strand break (DSB) formation but disfavor subsequent loading of meiotic RecA homolog, Dmc1. Red1 promotes DSB formation in both R- and G-bands and then promotes Dmc1 loading, specifically counteracting disfavoring R-band effects. These complexities are discussed in the context of chiasma formation as a series of coordinated local changes at the DNA and chromosome-axis levels.