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Disruptor of telomeric silencing 1-like (DOT1L) is the only non-SET domain histone lysine methyltransferase (KMT) and writer of H3K79 methylation on nucleosomes marked by H2B ubiquitination. DOT1L has elicited significant attention because of its interaction or fusion with members of the AF protein family in blood cell biology and leukemogenic transformation. Here, our goal was to extend previous structural information by performing a robust molecular dynamic study of DOT1L and its leukemogenic partners combined with mutational analysis. We show that statically and dynamically, D161, G163, E186, and F223 make frequent time-dependent interactions with SAM, while additional residues T139, K187, and N241 interact with SAM only under dynamics. Dynamics models reveal DOT1L, SAM, and H4 moving as one and show that more than twice the number of DOT1L residues interacts with these partners, relative to the static structure. Mutational analyses indicate that six of these residues are intolerant to substitution. We describe the dynamic behavior of DOT1L interacting with AF10 and AF9. Studies on the dynamics of a heterotrimeric complex of DOT1L1-AF10 illuminated describe coordinated motions that impact the relative position of the DOT1L HMT domain to the nucleosome. The molecular motions of the DOT1L-AF9 complex are less extensive and highly dynamic, resembling a swivel-like mechanics. Through molecular dynamics and mutational analysis, we extend the knowledge previous provided by static measurements. These results are important to consider when describing the biochemical properties of DOT1L, under normal and in disease conditions, as well as for the development of novel therapeutic agents.
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Carcinogénesis , N-Metiltransferasa de Histona-Lisina , Leucemia/metabolismo , Carcinogénesis/química , Carcinogénesis/metabolismo , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Simulación de Dinámica Molecular , Nucleosomas/química , Nucleosomas/metabolismo , Proteínas de Fusión Oncogénica/química , Proteínas de Fusión Oncogénica/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismoRESUMEN
HNF4α (hepatocyte nuclear factor 4α) is one of the master regulators of pancreatic ß-cell development and function, and mutations in the HNF4α gene are well-known monogenic causes of diabetes. As a member of the nuclear receptor family, HNF4α exerts its gene regulatory function through various molecular interactions; however, there is a paucity of knowledge of the different functional complexes in which HNF4α participates. Here, to find HNF4α-binding proteins in pancreatic ß-cells, we used yeast two-hybrid screening, a mammalian two-hybrid assay, and glutathione S-transferase pulldown approaches, which identified EBP1 (ErbB3-binding protein 1) as a factor that binds HNF4α in a LXXLL motif-mediated manner. In the ß-cells, EBP1 suppressed the expression of HNF4α target genes that are implicated in insulin secretion, which is impaired in HNF4α mutation-driven diabetes. The crystal structure of the HNF4α ligand-binding domain in complex with a peptide harboring the EBP1 LXXLL motif at 3.15Å resolution hinted at the molecular basis of the repression. The details of the structure suggested that EBP1's LXXLL motif competes with HNF4α coactivators for the same binding pocket and thereby prevents recruitment of additional transcriptional coactivators. These findings provide further evidence that EBP1 plays multiple cellular roles and is involved in nuclear receptor-mediated gene regulation. Selective disruption of the HNF4α-EBP1 interaction or tissue-specific EBP1 inactivation can enhance HNF4α activities and thereby improve insulin secretion in ß-cells, potentially representing a new strategy for managing diabetes and related metabolic disorders.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Factor Nuclear 4 del Hepatocito/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Regulación de la Expresión Génica/genética , Células HeLa , Factor Nuclear 4 del Hepatocito/fisiología , Humanos , Insulina/metabolismo , Secreción de Insulina/genética , Células Secretoras de Insulina/fisiología , Regiones Promotoras Genéticas/genética , Unión Proteica/fisiología , Proteínas de Unión al ARN/fisiología , Factores de TranscripciónRESUMEN
Human glucose-6-phosphatase plays a vital role in blood glucose homeostasis and holds promise as a therapeutic target for diabetes. Expression of its catalytic subunit gene 1 (G6PC1) is tightly regulated by metabolic-response transcription factors such as FoxO1 and CREB. Although at least three potential FoxO1 binding sites (insulin response elements, IREs) and one CREB binding site (cAMP response element, CRE) within the proximal region of the G6PC1 promoter have been identified, the interplay between FoxO1 and CREB and between FoxO1 bound at multiple IREs has not been well characterized. Here we present the crystal structures of the FoxO1 DNA binding domain in complex with the G6PC1 promoter. These complexes reveal the presence of a new non-consensus FoxO1 binding site that overlaps the CRE, suggesting a mutual exclusion mechanism for FoxO1 and CREB binding at the G6PC1 promoter. Additional findings include (i) non-canonical FoxO1 recognition sites, (ii) incomplete FoxO1 occupancies at the available IRE sites, and (iii) FoxO1 dimeric interactions that may play a role in stabilizing DNA looping. These findings provide insight into the regulation of G6PC1 gene transcription by FoxO1, and demonstrate a high versatility of target gene recognition by FoxO1 that correlates with its diverse roles in biology.
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Proteína Forkhead Box O1/metabolismo , Glucosa-6-Fosfatasa/genética , Regiones Promotoras Genéticas , Sitios de Unión , Dominio Catalítico/genética , Cristalografía por Rayos X , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Humanos , Factores de Transcripción/metabolismoRESUMEN
Although the liver and ventral pancreas are thought to arise from a common multipotent progenitor pool, it is unclear whether these progenitors of the hepatopancreas system are specified by a common genetic mechanism. Efforts to determine the role of Hnf1b and Wnt signaling in this crucial process have been confounded by a combination of factors, including a narrow time frame for hepatopancreas specification, functional redundancy among Wnt ligands, and pleiotropic defects caused by either severe loss of Wnt signaling or Hnf1b function. Using a novel hypomorphic hnf1ba zebrafish mutant that exhibits pancreas hypoplasia, as observed in HNF1B monogenic diabetes, we show that hnf1ba plays essential roles in regulating ß-cell number and pancreas specification, distinct from its function in regulating pancreas size and liver specification, respectively. By combining Hnf1ba partial loss of function with conditional loss of Wnt signaling, we uncover a crucial developmental window when these pathways synergize to specify the entire ventrally derived hepatopancreas progenitor population. Furthermore, our in vivo genetic studies demonstrate that hnf1ba generates a permissive domain for Wnt signaling activity in the foregut endoderm. Collectively, our findings provide a new model for HNF1B function, yield insight into pancreas and ß-cell development, and suggest a new mechanism for hepatopancreatic specification.
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Factor Nuclear 1-beta del Hepatocito/metabolismo , Hepatopáncreas/citología , Hepatopáncreas/metabolismo , Células Madre/citología , Células Madre/metabolismo , Proteínas Wnt/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Factor Nuclear 1-beta del Hepatocito/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Proteínas Wnt/genética , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
Non-small-cell lung cancer (NSCLC) is associated with diverse genetic alterations including mutation of epidermal growth factor receptor (EGFR). Isoliquiritigenin (ILQ), a chalcone derivative, possesses anticancer activities. In the present study, we investigated the effects of ILQ on the growth of tyrosine kinase inhibitor (TKI)-sensitive and -resistant NSCLC cells and elucidated its underlying mechanisms. Treatment with ILQ inhibited growth and induced apoptosis in both TKI-sensitive and -resistant NSCLC cells. ILQ-induced apoptosis was associated with the cleavage of caspase-3 and poly-(ADP-ribose)-polymerase, increased expression of Bim, and reduced expression of Bcl-2. In vitro kinase assay results revealed that ILQ inhibited the catalytic activity of both wild type and double mutant (L858R/T790M) EGFR. Treatment with ILQ inhibited the anchorage-independent growth of NIH3T3 cells stably transfected with either wild type or double-mutant EGFR with or without EGF stimulation. ILQ also reduced the phosphorylation of Akt and ERK1/2 in both TKI-sensitive and -resistant NSCLC cells, and attenuated the kinase activity of Akt1 and ERK2 in vitro. ILQ directly interacted with both wild type and double-mutant EGFR in an ATP-competitive manner. A docking model study showed that ILQ formed two hydrogen bonds (Glu-762 and Met-793) with wild type EGFR and three hydrogen bonds (Lys-745, Met-793, and Asp-855) with mutant EGFR. ILQ attenuated the xenograft tumor growth of H1975 cells, which was associated with decreased expression of Ki-67 and diminished phosphorylation of Akt and ERK1/2. Taken together, ILQ suppresses NSCLC cell growth by directly targeting wild type or mutant EGFR.
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Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Chalconas/farmacología , Receptores ErbB/genética , Neoplasias Pulmonares/tratamiento farmacológico , Animales , Línea Celular Tumoral , Proliferación Celular , Receptores ErbB/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Mutación Missense , Células 3T3 NIH , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-akt/metabolismo , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Multiple sclerosis (MS) is an autoimmune disease in which dysregulated immune cells attack myelin in the central nervous system (CNS), leading to irreversible neuronal degeneration. Our previous studies have demonstrated that epidermal fatty acid binding protein (E-FABP), widely expressed in immune cells, in particular in dendritic cells (DCs) and T lymphocytes, fuels the overactive immune responses in the mouse model of experimental autoimmune encephalomyelitis (EAE). METHODS: In the present study, we conducted an intensive computational docking analysis to identify novel E-FABP inhibitors for regulation of immune cell functions and for treatment of EAE. RESULTS: We demonstrate that compound [2-(4-acetylphenoxy)-9,10-dimethoxy-6,7-dihydropyrimido[6,1-a]isoquinolin-4-one; designated as EI-03] bound to the lipid binding pocket of E-FABP and enhanced the expression of peroxisome proliferator-activating receptor (PPAR) γ. Further in vitro experiments showed that EI-03 regulated DC functions by inhibition of TNFα production while promoting IL-10 secretion. Moreover, EI-03 treatment counterregulated T cell balance by decreasing effector T cell differentiation (e.g. Th17, Th1) while increasing regulatory T cell development. Most importantly, mice treated with this newly identified compound exhibited reduced clinical symptoms of EAE in mouse models. CONCLUSIONS: Taken together, we have identified a new compound which displays a potential therapeutic benefit for treatment of MS by targeting E-FABP.
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Encefalomielitis Autoinmune Experimental/terapia , Proteínas de Unión a Ácidos Grasos/antagonistas & inhibidores , Terapia Molecular Dirigida , Proteínas de Neoplasias/antagonistas & inhibidores , Animales , Diferenciación Celular , Citocinas/biosíntesis , Células Dendríticas/metabolismo , Evaluación Preclínica de Medicamentos , Proteínas de Unión a Ácidos Grasos/química , Proteínas de Unión a Ácidos Grasos/metabolismo , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismoRESUMEN
HNF1α (Hepatocyte Nuclear Factor 1α) is one of the master regulators in pancreatic beta-cell development and function, and the mutations in Hnf1α are the most common monogenic causes of diabetes mellitus. As a member of the POU transcription factor family, HNF1α exerts its gene regulatory function through various molecular interactions; however, there is a paucity of knowledge in their functional complex formation. In this study, we identified the Groucho protein AES (Amino-terminal Enhancer of Split) as a HNF1α-specific physical binding partner and functional repressor of HNF1α-mediated transcription, which has a direct link to glucose-stimulated insulin secretion in beta-cells that is impaired in the HNF1α mutation-driven diabetes.
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Factor Nuclear 1-alfa del Hepatocito/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas Represoras/metabolismo , Animales , Línea Celular , Proteínas Co-Represoras , Glucosa/metabolismo , Células HeLa , Factor Nuclear 1-alfa del Hepatocito/química , Factor Nuclear 1-alfa del Hepatocito/genética , Humanos , Insulina/metabolismo , Ratones , Mutación , Dominios y Motivos de Interacción de Proteínas , Mapas de Interacción de Proteínas , Transcripción Genética , Activación TranscripcionalRESUMEN
The bacteriophage phi29 DNA packaging motor, one of the strongest biological motors characterized to date, is geared by a packaging RNA (pRNA) ring. When assembled from three RNA fragments, its three-way junction (3WJ) motif is highly thermostable, is resistant to 8 M urea, and remains associated at extremely low concentrations in vitro and in vivo. To elucidate the structural basis for its unusual stability, we solved the crystal structure of this pRNA 3WJ motif at 3.05 Å. The structure revealed two divalent metal ions that coordinate 4 nt of the RNA fragments. Single-molecule fluorescence resonance energy transfer (smFRET) analysis confirmed a structural change of 3WJ upon addition of Mg²âº. The reported pRNA 3WJ conformation is different from a previously published construct that lacks the metal coordination sites. The phi29 DNA packaging motor contains a dodecameric connector at the vertex of the procapsid, with a central pore for DNA translocation. This portal connector serves as the foothold for pRNA binding to procapsid. Subsequent modeling of a connector/pRNA complex suggests that the pRNA of the phi29 DNA packaging motor exists as a hexameric complex serving as a sheath over the connector. The model of hexameric pRNA on the connector agrees with AFM images of the phi29 pRNA hexamer acquired in air and matches all distance parameters obtained from cross-linking, complementary modification, and chemical modification interference.
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Fagos de Bacillus/genética , Empaquetamiento del ADN , ADN Viral/química , ARN Viral/química , Sitios de Unión , Proteínas de la Cápside/química , Cationes Bivalentes , Cristalografía por Rayos X , Transferencia Resonante de Energía de Fluorescencia , Magnesio/química , Modelos Moleculares , Simulación del Acoplamiento Molecular , Conformación de Ácido Nucleico , Estabilidad del ARN , Ensamble de VirusRESUMEN
Epithelial-mesenchymal transition (EMT) is a transdifferentiation programme. The mechanism underlying the epigenetic regulation of EMT remains unclear. In this study, we identified that Snail1 interacted with histone lysine-specific demethylase 1 (LSD1). We demonstrated that the SNAG domain of Snail1 and the amine oxidase domain of LSD1 were required for their mutual interaction. Interestingly, the sequence of the SNAG domain is similar to that of the histone H3 tail, and the interaction of Snail1 with LSD1 can be blocked by LSD1 enzymatic inhibitors and a histone H3 peptide. We found that the formation of a Snail1-LSD1-CoREST ternary complex was critical for the stability and function of these proteins. The co-expression of these molecules was found in cancer cell lines and breast tumour specimens. Furthermore, we showed that the SNAG domain of Snail1 was critical for recruiting LSD1 to its target gene promoters and resulted in suppression of cell migration and invasion. Our study suggests that the SNAG domain of Snail1 resembles a histone H3-like structure and functions as a molecular hook for recruiting LSD1 to repress gene expression in metastasis.
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Lisina/metabolismo , Monoaminooxidasa/metabolismo , Péptidos/metabolismo , Animales , Diferenciación Celular , Histonas/metabolismo , Lisina/química , Monoaminooxidasa/química , Péptidos/químicaRESUMEN
Maple syrup urine disease (MSUD) is a disorder of branched-chain amino acid metabolism caused by a defect in the branched-chain α-ketoacid dehydrogenase (BCKD) complex (OMIM #248600). The hallmark presentation is encephalopathic crisis in neonates, but can also present with metabolic decompensation, developmental delays, and feeding difficulties. Biochemical evidence for MSUD includes elevated branched-chain amino acids (BCAA) and the pathognomonic presence of alloisoleucine. The BCKD complex contains several subunits associated with autosomal recessive MSUD, while its regulatory proteins have less well-defined disease associations. We report on two families with the same BCKDK variant (c.1115C>G (p.Thr372Arg)). Probands were detected on newborn screening and demonstrated biochemical evidence of MSUD. The variant was identified in reportedly asymptomatic parents and additional family members who had elevated BCAA and alloisoleucine, following an autosomal dominant pattern of inheritance. To better define the functional effect of the variant on the kinase, we completed molecular modeling using sequence-based (2D), structural-based (3D), and dynamic-based (4D) analyses. The BCKDK variant modeling indicated a gain-of-function which leads to impaired BCAA catabolism consistent with the biochemical evidence in this cohort. Combining the evidence gained from molecular modeling with the absence of metabolic decompensation in our patients and several adult family members, despite encountering stressors typically problematic in classic MSUD, we suggest that heterozygous gain-of-function variants in BCKDK may represent a novel biochemical phenotype of MSUD with a benign clinical course.
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This study investigates the functional significance of assorted variants of uncertain significance (VUS) in euchromatic histone lysine methyltransferase 1 (EHMT1), which is critical for early development and normal physiology. EHMT1 mutations cause Kleefstra syndrome and are linked to various human cancers. However, accurate functional interpretation of these variants are yet to be made, limiting diagnoses and future research. To overcome this, we integrate conventional tools for variant calling with computational biophysics and biochemistry to conduct multi-layered mechanistic analyses of the SET catalytic domain of EHMT1, which is critical for this protein function. We use molecular mechanics and molecular dynamics (MD)-based metrics to analyze the SET domain structure and functional motions resulting from 97 Kleefstra syndrome missense variants within this domain. Our approach allows us to classify the variants in a mechanistic manner into SV (Structural Variant), DV (Dynamic Variant), SDV (Structural and Dynamic Variant), and VUS (Variant of Uncertain Significance). Our findings reveal that the damaging variants are mostly mapped around the active site, substrate binding site, and pre-SET regions. Overall, we report an improvement for this method over conventional tools for variant interpretation and simultaneously provide a molecular mechanism of variant dysfunction.
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Histone H3 lysine 9 methylation (H3K9me), which is written by the Euchromatic Histone Lysine Methyltransferases EHMT1 and EHMT2 and read by the heterochromatin protein 1 (HP1) chromobox (CBX) protein family, is dysregulated in many types of cancers. Approaches to inhibit regulators of this pathway are currently being evaluated for therapeutic purposes. Thus, knowledge of the complexes supporting the function of these writers and readers during the process of cell proliferation is critical for our understanding of their role in carcinogenesis. Here, we immunopurified each of these proteins and used mass spectrometry to define their associated non-histone proteins, individually and at two different phases of the cell cycle, namely G1/S and G2/M. Our findings identify novel binding proteins for these writers and readers, as well as corroborate known interactors, to show the formation of distinct protein complex networks in a cell cycle phase-specific manner. Furthermore, there is an organizational switch between cell cycle phases for interactions among specific writer-reader pairs. Through a multi-tiered bioinformatics-based approach, we reveal that many interacting proteins exhibit histone mimicry, based on an H3K9-like linear motif. Gene ontology analyses, pathway enrichment, and network reconstruction inferred that these comprehensive EHMT and CBX-associated interacting protein networks participate in various functions, including transcription, DNA repair, splicing, and membrane disassembly. Combined, our data reveals novel complexes that provide insight into key functions of cell cycle-associated epigenomic processes that are highly relevant for better understanding these chromatin-modifying proteins during cell cycle and carcinogenesis.
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Histonas , Lisina , Humanos , Histonas/genética , Histonas/metabolismo , Lisina/metabolismo , Ciclo Celular , División Celular , Carcinogénesis , Antígenos de Histocompatibilidad , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismoRESUMEN
This study investigates the functional significance of assorted variants of uncertain significance (VUS) in euchromatic histone lysine methyltransferase 1 (EHMT1), which is critical for early development and normal physiology. EHMT1 mutations cause Kleefstra syndrome and are linked to various human cancers. However, accurate functional interpretations of these variants are yet to be made, limiting diagnoses and future research. To overcome this, we integrate conventional tools for variant calling with computational biophysics and biochemistry to conduct multi-layered mechanistic analyses of the SET catalytic domain of EHMT1, which is critical for this protein function. We use molecular mechanics and molecular dynamics (MD)-based metrics to analyze the SET domain structure and functional motions resulting from 97 Kleefstra syndrome missense variants within the domain. Our approach allows us to classify the variants in a mechanistic manner into SV (Structural Variant), DV (Dynamic Variant), SDV (Structural and Dynamic Variant), and VUS (Variant of Uncertain Significance). Our findings reveal that the damaging variants are mostly mapped around the active site, substrate binding site, and pre-SET regions. Overall, we report an improvement for this method over conventional tools for variant interpretation and simultaneously provide a molecular mechanism for variant dysfunction.
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Introduction: Kleefstra Syndrome type 2 (KLEFS-2) is a genetic, neurodevelopmental disorder characterized by intellectual disability, infantile hypotonia, severe expressive language delay, and characteristic facial appearance, with a spectrum of other distinct clinical manifestations. Pathogenic mutations in the epigenetic modifier type 2 lysine methyltransferase KMT2C have been identified to be causative in KLEFS-2 individuals. Methods: This work reports a translational genomic study that applies a multidimensional computational approach for deep variant phenotyping, combining conventional genomic analyses, advanced protein bioinformatics, computational biophysics, biochemistry, and biostatistics-based modeling. We use standard variant annotation, paralog annotation analyses, molecular mechanics, and molecular dynamics simulations to evaluate damaging scores and provide potential mechanisms underlying KMT2C variant dysfunction. Results: We integrated data derived from the structure and dynamics of KMT2C to classify variants into SV (Structural Variant), DV (Dynamic Variant), SDV (Structural and Dynamic Variant), and VUS (Variant of Uncertain Significance). When compared with controls, these variants show values reflecting alterations in molecular fitness in both structure and dynamics. Discussion: We demonstrate that our 3D models for KMT2C variants suggest distinct mechanisms that lead to their imbalance and are not predictable from sequence alone. Thus, the missense variants studied here cause destabilizing effects on KMT2C function by different biophysical and biochemical mechanisms which we adeptly describe. This new knowledge extends our understanding of how variations in the KMT2C gene cause the dysfunction of its methyltransferase enzyme product, thereby bearing significant biomedical relevance for carriers of KLEFS2-associated genomic mutations.
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The histone demethylase KDM6A has recently elicited significant attention because its mutations are associated with a rare congenital disorder (Kabuki syndrome) and various types of human cancers. However, distinguishing KDM6A mutations that are deleterious to the enzyme and their underlying mechanisms of dysfunction remain to be fully understood. Here, we report the results from a multi-tiered approach evaluating the impact of 197 KDM6A somatic mutations using information derived from combining conventional genomics data with computational biophysics. This comprehensive approach incorporates multiple scores derived from alterations in protein sequence, structure, and molecular dynamics. Using this method, we classify the KDM6A mutations into 136 damaging variants (69.0%), 32 tolerated variants (16.2%), and 29 variants of uncertain significance (VUS, 14.7%), which is a significant improvement from the previous classification based on the conventional tools (over 40% VUS). We further classify the damaging variants into 15 structural variants (SV), 88 dynamic variants (DV), and 33 structural and dynamic variants (SDV). Comparison with variant scoring methods used in current clinical diagnosis guidelines demonstrates that our approach provides a more comprehensive evaluation of damaging potential and reveals mechanisms of dysfunction. Thus, these results should be taken into consideration for clinical assessment of the damaging potential of each mutation, as they provide hypotheses for experimental validation and critical information for the development of mutant-specific drugs to fight diseases caused by KDM6A dysfunctions.
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We have obtained precatalytic (enzyme-substrate complex) and postcatalytic (enzyme-product complex) crystal structures of an active full-length hammerhead RNA that cleaves in the crystal. Using the natural satellite tobacco ringspot virus hammerhead RNA sequence, the self-cleavage reaction was modulated by substituting the general base of the ribozyme, G12, with A12, a purine variant with a much lower pKa that does not significantly perturb the ribozyme's atomic structure. The active, but slowly cleaving, ribozyme thus permitted isolation of enzyme-substrate and enzyme-product complexes without modifying the nucleophile or leaving group of the cleavage reaction, nor any other aspect of the substrate. The predissociation enzyme-product complex structure reveals RNA and metal ion interactions potentially relevant to transition-state stabilization that are absent in precatalytic structures.
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Conformación de Ácido Nucleico , ARN Catalítico/química , ARN Catalítico/metabolismo , Secuencia de Bases , Catálisis , Cristalografía por Rayos X , Estabilidad de Enzimas , Datos de Secuencia Molecular , Estructura Molecular , Nepovirus/enzimología , Nepovirus/genética , ARN Catalítico/genética , Especificidad por SustratoRESUMEN
The crystallographic phase problem is the primary bottleneck encountered when attempting to solve macromolecular structures for which no close crystallographic structural homologues are known. Typically, isomorphous "heavy-atom" replacement and/or anomalous dispersion methods must be used in such cases to obtain experimentally-determined phases. Even three-dimensional NMR structures of the same macromolecule are often not sufficient to solve the crystallographic phase problem. RNA crystal structures present additional challenges due to greater difficulty in obtaining suitable heavy-atom derivatives. We present a unique approach to solve the phase problem for novel RNA crystal structures that has enjoyed a reasonable degree of success. This approach involves modeling only those portions of the RNA sequence whose structure can be predicted readily, i.e., the individual A-form helical regions and well-known stem-loop sub-structures. We have found that no prior knowledge of how the helices and other structural elements are arranged with respect to one another in three-dimensional space, or in some cases, even the sequence, is required to obtain a useable solution to the phase problem, using simultaneous molecular replacement of a set of generic helical RNA fragments.
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Cristalografía por Rayos X/métodos , ARN/química , Algoritmos , Cristalización , Programas InformáticosRESUMEN
BACKGROUND: Kabuki syndrome is a genetic disorder that affects several body systems and presents with variations in symptoms and severity. The syndrome is named for a common phenotype of faces resembling stage makeup used in a Japanese traditional theatrical art named kabuki. The most frequent cause of this syndrome is mutations in the H3K4 family of histone methyltransferases while a smaller percentage results from genetic alterations affecting the histone demethylase, KDM6A. Because of the rare presentation of the latter form of the disease, little is known about how missense changes in the KDM6A protein sequence impact protein function. RESULTS: In this study, we use molecular mechanic and molecular dynamic simulations to enhance the annotation and mechanistic interpretation of the potential impact of eleven KDM6A missense variants found in Kabuki syndrome patients. These variants (N910S, D980V, S1025G, C1153R, C1153Y, P1195L, L1200F, Q1212R, Q1248R, R1255W, and R1351Q) are predicted to be pathogenic, likely pathogenic or of uncertain significance by sequence-based analysis. Here, we demonstrate, for the first time, that although Kabuki syndrome missense variants are found outside the functionally critical regions, they could affect overall function by significantly disrupting global and local conformation (C1153R, C1153Y, P1195L, L1200F, Q1212R, Q1248R, R1255W and R1351Q), chemical environment (C1153R, C1153Y, P1195L, L1200F, Q1212R, Q1248R, R1255W and R1351Q), and/or molecular dynamics of the catalytic domain (all variants). In addition, our approaches predict that many mutations, in particular C1153R, could allosterically disrupt the key enzymatic interactions of KDM6A. CONCLUSIONS: Our study demonstrates that the KDM6A Kabuki syndrome variants may impair histone demethylase function through various mechanisms that include altered protein integrity, local environment, molecular interactions and protein dynamics. Molecular dynamics simulations of the wild type and the variants are critical to gain a better understanding of molecular dysfunction. This type of comprehensive structure- and MD-based analyses should help develop improved impact scoring systems to interpret the damaging effects of variants in this protein and other related proteins as well as provide detailed mechanistic insight that is not currently predictable from sequence alone.
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Enfermedades Hematológicas , Histona Demetilasas/genética , Enfermedades Vestibulares , Anomalías Múltiples , Cara/anomalías , Enfermedades Hematológicas/genética , Humanos , Simulación de Dinámica Molecular , Mutación , Enfermedades Vestibulares/genéticaRESUMEN
Hepatocyte nuclear factor 4alpha (HNF4alpha) is a novel nuclear receptor that participates in a hierarchical network of transcription factors regulating the development and physiology of such vital organs as the liver, pancreas, and kidney. Among the various transcriptional coregulators with which HNF4alpha interacts, peroxisome proliferation-activated receptor gamma (PPARgamma) coactivator 1alpha (PGC-1alpha) represents a novel coactivator whose activation is unusually robust and whose binding mode appears to be distinct from that of canonical coactivators such as NCoA/SRC/p160 family members. To elucidate the potentially unique molecular mechanism of PGC-1alpha recruitment, we have determined the crystal structure of HNF4alpha in complex with a fragment of PGC-1alpha containing all three of its LXXLL motifs. Despite the presence of all three LXXLL motifs available for interactions, only one is bound at the canonical binding site, with no additional contacts observed between the two proteins. However, a close inspection of the electron density map indicates that the bound LXXLL motif is not a selected one but an averaged structure of more than one LXXLL motif. Further biochemical and functional studies show that the individual LXXLL motifs can bind but drive only minimal transactivation. Only when more than one LXXLL motif is involved can significant transcriptional activity be measured, and full activation requires all three LXXLL motifs. These findings led us to propose a model wherein each LXXLL motif has an additive effect, and the multiple binding modes by HNF4alpha toward the LXXLL motifs of PGC-1alpha could account for the apparent robust activation by providing a flexible mechanism for combinatorial recruitment of additional coactivators and mediators.