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1.
Nucleic Acids Res ; 48(4): 2013-2025, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31930301

RESUMO

The rolling-circle replication is the most common mechanism for the replication of small plasmids carrying antibiotic resistance genes in Gram-positive bacteria. It is initiated by the binding and nicking of double-stranded origin of replication by a replication initiator protein (Rep). Duplex unwinding is then performed by the PcrA helicase, whose processivity is critically promoted by its interaction with Rep. How Rep and PcrA proteins interact to nick and unwind the duplex is not fully understood. Here, we have used magnetic tweezers to monitor PcrA helicase unwinding and its relationship with the nicking activity of Staphylococcus aureus plasmid pT181 initiator RepC. Our results indicate that PcrA is a highly processive helicase prone to stochastic pausing, resulting in average translocation rates of 30 bp s-1, while a typical velocity of 50 bp s-1 is found in the absence of pausing. Single-strand DNA binding protein did not affect PcrA translocation velocity but slightly increased its processivity. Analysis of the degree of DNA supercoiling required for RepC nicking, and the time between RepC nicking and DNA unwinding, suggests that RepC and PcrA form a protein complex on the DNA binding site before nicking. A comprehensive model that rationalizes these findings is presented.


Assuntos
Proteínas de Bactérias/genética , DNA Helicases/genética , Replicação do DNA/genética , Farmacorresistência Bacteriana/genética , Quebras de DNA de Cadeia Simples/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Geobacillus stearothermophilus/efeitos dos fármacos , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/patogenicidade , Plasmídeos/efeitos dos fármacos , Plasmídeos/genética , Ligação Proteica/genética , Domínios e Motivos de Interação entre Proteínas/genética , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Tetraciclina/farmacologia , Transativadores/genética
2.
Int J Cancer ; 146(6): 1578-1591, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-31577845

RESUMO

Breast cancer remains a leading cause of cancer-related death for women. The stepwise development of breast cancer through preinvasive to invasive disease is associated with progressive disruption of cellular and tissue organization. Apical-basal polarity is thought to be a barrier to breast cancer development, but the extent and potential mechanisms that contribute to disrupted polarity are incompletely understood. To investigate the cell polarity status of invasive breast cancers, we performed multiplex imaging of polarity markers on tissue cores from 432 patients from a spectrum of grades, stages and molecular subtypes. Apical-basal cell polarity was lost in 100% of cells in all cases studied, indicating that loss of epithelial polarity may be a universal feature of invasive breast cancer. We then analyzed genomic events from the TCGA dataset for an 18-gene set of core polarity genes. Coamplification of polarity genes with established breast oncogenes was found, which is consistent with functional cooperation within signaling amplicons. Gene-expression levels of several polarity genes were significantly associated with survival, and protein localization of Par6 correlated with higher grade, nodal metastasis and molecular subtype. Finally, multiple hotspot mutations in protein-protein interaction domains critical for cell polarity were identified. Our data indicate that genomic events likely contribute to pervasive disruption of epithelial polarity observed in invasive breast cancer.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/patologia , Polaridade Celular/genética , Células Epiteliais/patologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Variações do Número de Cópias de DNA , Conjuntos de Dados como Assunto , Intervalo Livre de Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Imagem Molecular , Mutação , Invasividade Neoplásica/genética , Domínios e Motivos de Interação entre Proteínas/genética , Transdução de Sinais/genética , Análise Serial de Tecidos
3.
PLoS Genet ; 15(11): e1008463, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31675375

RESUMO

Nucleophosmin (NPM1) is frequently mutated or subjected to chromosomal translocation in acute myeloid leukemia (AML). NPM protein is primarily located in the nucleus, but the recurrent NPMc+ mutation, which creates a nuclear export signal, is characterized by cytoplasmic localization and leukemogenic properties. Similarly, the NPM-MLF1 translocation product favors the partial cytoplasmic retention of NPM. Regardless of their common cellular distribution, NPM-MLF1 malignancies engender different effects on hematopoiesis compared to NPMc+ counterparts, highlighting possible aberrant nuclear function(s) of NPM in NPMc+ and NPM-MLF1 AML. We performed a proteomic analysis and found that NPM and NPM-MLF1 interact with various nuclear proteins including subunits of the chromatin remodeling complexes ISWI, NuRD and P/BAF. Accordingly, NPM and NPM-MLF1 are recruited to transcriptionally active or repressed genes along with NuRD subunits. Although the overall gene expression program in NPM knockdown cells is similar to that resulting from NPMc+, NPM-MLF1 expression differentially altered gene transcription regulated by NPM. The abnormal gene regulation imposed by NPM-MLF1 can be characterized by the enhanced recruitment of NuRD to gene regulatory regions. Thus, different mechanisms would orchestrate the dysregulation of NPM function in NPMc+- versus NPM1-MLF1-associated leukemia.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Adenosina Trifosfatases/genética , Anticorpos/genética , Linhagem Celular Tumoral , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Humanos , Leucemia Mieloide Aguda/patologia , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Mutação/genética , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Domínios e Motivos de Interação entre Proteínas/genética , Proteômica/métodos , Translocação Genética/genética
4.
Elife ; 82019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31573509

RESUMO

Small heat shock proteins (sHSPs) are nature's 'first responders' to cellular stress, interacting with affected proteins to prevent their aggregation. Little is known about sHSP structure beyond its structured α-crystallin domain (ACD), which is flanked by disordered regions. In the human sHSP HSPB1, the disordered N-terminal region (NTR) represents nearly 50% of the sequence. Here, we present a hybrid approach involving NMR, hydrogen-deuterium exchange mass spectrometry, and modeling to provide the first residue-level characterization of the NTR. The results support a model in which multiple grooves on the ACD interact with specific NTR regions, creating an ensemble of 'quasi-ordered' NTR states that can give rise to the known heterogeneity and plasticity of HSPB1. Phosphorylation-dependent interactions inform a mechanism by which HSPB1 is activated under stress conditions. Additionally, we examine the effects of disease-associated NTR mutations on HSPB1 structure and dynamics, leveraging our emerging structural insights.


Assuntos
Proteínas de Choque Térmico Pequenas/genética , Proteínas de Choque Térmico/genética , Chaperonas Moleculares/genética , Agregados Proteicos/genética , Domínios e Motivos de Interação entre Proteínas/genética , Sequência de Aminoácidos/genética , Humanos , Espectrometria de Massas , Modelos Moleculares , Conformação Proteica , Multimerização Proteica/genética , Espalhamento a Baixo Ângulo
5.
Nucleic Acids Res ; 47(19): 10426-10438, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31544921

RESUMO

The Mixed Lineage Leukemia protein 1 (MLL1) plays an essential role in the maintenance of the histone H3 lysine 4 (H3K4) methylation status for gene expression during differentiation and development. The methyltransferase activity of MLL1 is regulated by three conserved core subunits, WDR5, RBBP5 and ASH2L. Here, we determined the structure of human RBBP5 and demonstrated its role in the assembly and regulation of the MLL1 complex. We identified an internal interaction between the WD40 propeller and the C-terminal distal region in RBBP5, which assisted the maintenance of the compact conformation of the MLL1 complex. We also discovered a vertebrate-specific motif in the C-terminal distal region of RBBP5 that contributed to nucleosome recognition and methylation of nucleosomes by the MLL1 complex. Our results provide new insights into functional conservation and evolutionary plasticity of the scaffold protein RBBP5 in the regulation of KMT2-family methyltransferase complexes.


Assuntos
Proteínas de Ligação a DNA/ultraestrutura , Histona-Lisina N-Metiltransferase/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Proteína de Leucina Linfoide-Mieloide/ultraestrutura , Fatores de Transcrição/genética , Diferenciação Celular/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Humanos , Conformação Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas Nucleares/genética , Ligação Proteica/genética , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Fatores de Transcrição/química
6.
Immunity ; 51(4): 766-779.e17, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31495665

RESUMO

Increasing evidence indicates CD4+ T cells can recognize cancer-specific antigens and control tumor growth. However, it remains difficult to predict the antigens that will be presented by human leukocyte antigen class II molecules (HLA-II), hindering efforts to optimally target them therapeutically. Obstacles include inaccurate peptide-binding prediction and unsolved complexities of the HLA-II pathway. To address these challenges, we developed an improved technology for discovering HLA-II binding motifs and conducted a comprehensive analysis of tumor ligandomes to learn processing rules relevant in the tumor microenvironment. We profiled >40 HLA-II alleles and showed that binding motifs were highly sensitive to HLA-DM, a peptide-loading chaperone. We also revealed that intratumoral HLA-II presentation was dominated by professional antigen-presenting cells (APCs) rather than cancer cells. Integrating these observations, we developed algorithms that accurately predicted APC ligandomes, including peptides from phagocytosed cancer cells. These tools and biological insights will enable improved HLA-II-directed cancer therapies.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Vacinas Anticâncer/imunologia , Mapeamento de Epitopos/métodos , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidade Classe II/genética , Imunoterapia/métodos , Espectrometria de Massas/métodos , Neoplasias/terapia , Algoritmos , Alelos , Apresentação do Antígeno , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Conjuntos de Dados como Assunto , Antígenos HLA/genética , Antígenos HLA-D/metabolismo , Humanos , Neoplasias/imunologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Software
7.
Int J Mol Sci ; 20(14)2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31295832

RESUMO

Kv7.2 subunits encoded by the KCNQ2 gene provide a major contribution to the M-current (IKM), a voltage-gated K+ current crucially involved in the regulation of neuronal excitability. Heterozygous missense variants in Kv7.2 are responsible for epileptic diseases characterized by highly heterogeneous genetic transmission and clinical severity, ranging from autosomal-dominant Benign Familial Neonatal Seizures (BFNS) to sporadic cases of severe epileptic and developmental encephalopathy (DEE). Here, we describe a patient with neonatal onset DEE, carrying a previously undescribed heterozygous KCNQ2 c.418G > C, p.Glu140Gln (E140Q) variant. Patch-clamp recordings in CHO cells expressing the E140Q mutation reveal dramatic loss of function (LoF) effects. Multistate structural modelling suggested that the E140Q substitution impeded an intrasubunit electrostatic interaction occurring between the E140 side chain in S2 and the arginine at position 210 in S4 (R210); this interaction is critically involved in stabilizing the activated configuration of the voltage-sensing domain (VSD) of Kv7.2. Functional results from coupled charge reversal or disulfide trapping experiments supported such a hypothesis. Finally, retigabine restored mutation-induced functional changes, reinforcing the rationale for the clinical use of Kv7 activators as personalized therapy for DEE-affected patients carrying Kv7.2 LoF mutations.


Assuntos
Encefalopatias/genética , Deficiências do Desenvolvimento/genética , Predisposição Genética para Doença , Variação Genética , Canal de Potássio KCNQ2/genética , Domínios e Motivos de Interação entre Proteínas/genética , Espasmos Infantis/genética , Substituição de Aminoácidos , Biomarcadores , Encefalopatias/diagnóstico , Encefalopatias/terapia , Pré-Escolar , Deficiências do Desenvolvimento/diagnóstico , Deficiências do Desenvolvimento/terapia , Eletroencefalografia , Estudos de Associação Genética , Humanos , Lactente , Recém-Nascido , Canal de Potássio KCNQ2/química , Mutação com Perda de Função , Imagem por Ressonância Magnética , Masculino , Modelos Moleculares , Neuroimagem , Conformação Proteica , Espasmos Infantis/diagnóstico , Espasmos Infantis/terapia , Relação Estrutura-Atividade , Avaliação de Sintomas
8.
Nat Commun ; 10(1): 3205, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324802

RESUMO

Protein-protein interaction (PPI) networks (interactome networks) have successfully advanced our knowledge of molecular function, disease and evolution. While much progress has been made in quantifying errors and biases in experimental PPI datasets, it remains unknown what fraction of the error-free PPIs in the cell are completely dispensable, i.e., effectively neutral upon disruption. Here, we estimate dispensable content in the human interactome by calculating the fractions of PPIs disrupted by neutral and non-neutral mutations. Starting with the human reference interactome determined by experiments, we construct a human structural interactome by building homology-based three-dimensional structural models for PPIs. Next, we map common mutations from healthy individuals as well as Mendelian disease-causing mutations onto the human structural interactome, and perform structure-based calculations of how these mutations perturb the interactome. Using our predicted as well as experimentally-determined interactome perturbation patterns by common and disease mutations, we estimate that <~20% of the human interactome is completely dispensable.


Assuntos
Biologia Computacional/métodos , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Proteínas de Transporte/genética , Doença/genética , Regulação da Expressão Gênica , Ontologia Genética , Humanos , Modelos Moleculares , Mutação , Domínios e Motivos de Interação entre Proteínas/genética , Mapas de Interação de Proteínas/genética , Proteínas/genética , Proteínas/metabolismo
9.
Mol Cell ; 75(2): 238-251.e5, 2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31348879

RESUMO

BRCT domains support myriad protein-protein interactions involved in genome maintenance. Although di-BRCT recognition of phospho-proteins is well known to support the genotoxic response, whether multi-BRCT domains can acquire distinct structures and functions is unclear. Here we present the tetra-BRCT structures from the conserved yeast protein Rtt107 in free and ligand-bound forms. The four BRCT repeats fold into a tetrahedral structure that recognizes unmodified ligands using a bi-partite mechanism, suggesting repeat origami enabling function acquisition. Functional studies show that Rtt107 binding of partner proteins of diverse activities promotes genome replication and stability in both distinct and concerted manners. A unified theme is that tetra- and di-BRCT domains of Rtt107 collaborate to recruit partner proteins to chromatin. Our work thus illustrates how a master regulator uses two types of BRCT domains to recognize distinct genome factors and direct them to chromatin for constitutive genome protection.


Assuntos
Instabilidade Genômica/genética , Proteínas Nucleares/ultraestrutura , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Saccharomyces cerevisiae/genética , Cromatina/genética , Dano ao DNA/genética , Ligantes , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosforilação , Ligação Proteica , Domínios Proteicos/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
10.
RNA ; 25(9): 1177-1191, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31221726

RESUMO

Uridine insertion deletion editing in kinetoplastid protozoa requires a complex machinery, a primary component of which is the RNA editing substrate binding complex (RESC). RESC contains two modules termed GRBC (guide RNA binding complex) and REMC (RNA editing mediator complex), although how interactions between these modules and their mRNA and gRNA binding partners are controlled is not well understood. Here, we demonstrate that the ARM/HEAT repeat containing RESC protein, MRB10130, controls REMC association with mRNA- and gRNA-loaded GRBC. High-throughput sequencing analyses show that MRB10130 functions in both initiation and 3' to 5' progression of editing through gRNA-defined domains. Editing intermediates that accumulate upon MRB10130 depletion significantly intersect those in cells depleted of another RESC organizer, MRB7260, but are distinct from those in cells depleted of specific REMC proteins. We present a model in which MRB10130 coordinates numerous protein-protein and protein-RNA interactions during editing progression.


Assuntos
Edição de RNA/genética , Animais , Linhagem Celular , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas de Protozoários/genética , Interferência de RNA/fisiologia , RNA Guia/genética , RNA Mensageiro/genética , RNA de Protozoário/genética , Trypanosoma brucei brucei/genética , Uridina/genética
11.
Nat Commun ; 10(1): 2636, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201318

RESUMO

The leading cause of cystic fibrosis (CF) is the deletion of phenylalanine 508 (F508del) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR). The mutation affects the thermodynamic stability of the domain and the integrity of the interface between NBD1 and the transmembrane domain leading to its clearance by the quality control system. Here, we develop nanobodies targeting NBD1 of human CFTR and demonstrate their ability to stabilize both isolated NBD1 and full-length protein. Crystal structures of NBD1-nanobody complexes provide an atomic description of the epitopes and reveal the molecular basis for stabilization. Furthermore, our data uncover a conformation of CFTR, involving detachment of NBD1 from the transmembrane domain, which contrast with the compact assembly observed in cryo-EM structures. This unexpected interface rearrangement is likely to have major relevance for CF pathogenesis but also for the normal function of CFTR and other ABC proteins.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Modelos Moleculares , Cristalografia por Raios X , Regulador de Condutância Transmembrana em Fibrose Cística/isolamento & purificação , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas/genética , Estabilidade Proteica , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Anticorpos de Domínio Único/metabolismo
12.
EMBO J ; 38(11)2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31036554

RESUMO

To characterize keratin intermediate filament assembly mechanisms at atomic resolution, we determined the crystal structure of wild-type human keratin-1/keratin-10 helix 1B heterotetramer at 3.0 Å resolution. It revealed biochemical determinants for the A11 mode of axial alignment in keratin filaments. Four regions on a hydrophobic face of the K1/K10-1B heterodimer dictated tetramer assembly: the N-terminal hydrophobic pocket (defined by L227K1, Y230K1, F231K1, and F234K1), the K10 hydrophobic stripe, K1 interaction residues, and the C-terminal anchoring knob (formed by F314K1 and L318K1). Mutation of both knob residues to alanine disrupted keratin 1B tetramer and full-length filament assembly. Individual knob residue mutant F314AK1, but not L318AK1, abolished 1B tetramer formation. The K1-1B knob/pocket mechanism is conserved across keratins and many non-keratin intermediate filaments. To demonstrate how pathogenic mutations cause skin disease by altering filament assembly, we additionally determined the 2.39 Å structure of K1/10-1B containing a S233LK1 mutation linked to epidermolytic palmoplantar keratoderma. Light scattering and circular dichroism measurements demonstrated enhanced aggregation of K1S233L/K10-1B in solution without affecting secondary structure. The K1S233L/K10-1B octamer structure revealed S233LK1 causes aberrant hydrophobic interactions between 1B tetramers.


Assuntos
Proteínas de Filamentos Intermediários/química , Proteínas de Filamentos Intermediários/metabolismo , Queratina-10 , Queratina-1 , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica/fisiologia , Substituição de Aminoácidos , Dicroísmo Circular , Cristalografia por Raios X , Citoesqueleto/química , Citoesqueleto/metabolismo , Difusão Dinâmica da Luz , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Filamentos Intermediários/genética , Queratina-1/química , Queratina-1/genética , Queratina-1/metabolismo , Queratina-10/química , Queratina-10/genética , Queratina-10/metabolismo , Modelos Moleculares , Mutação de Sentido Incorreto , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas/genética , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Dermatopatias/genética , Dermatopatias/metabolismo , Dermatopatias/patologia
13.
Basic Clin Pharmacol Toxicol ; 125(3): 259-270, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30861618

RESUMO

The Keap1-Nrf2-ARE system serves as a premier defence mechanism to curb oxidative stress, which remains as one of the major causes of ageing and pathogenesis in various diseases. Nrf2 is the principal master regulator of the cellular defence system, and its activation remains the prospective therapeutic approach against chronic diseases. One of the recent strategies is to disrupt Keap1-Nrf2 protein-protein interaction (PPI) that alters the docking of Keap1 with Nrf2 by compounds occupying a position in the Keap1 blocking the interface with Nrf2. In this study, we made an attempt to identify the compounds with anticancer, antioxidant and anti-inflammatory properties to disrupt Keap1a/b-Nrf2 PPI through in silico molecular docking in zebrafish. The phylogenetic analysis of Keap1 proteins revealed the existence of orthologous Keap1-Nrf2-ARE system in lower vertebrates that includes zebrafish. The DGR domains of zebrafish Keap1a and Keap1b were modelled with Modeller 9.19 using Keap1 of Mus musculus (PDB ID:5CGJ) as template. Based on the docking calculations, top hit compounds were identified to disrupt both Keap1a and Keap1b interaction with Nrf2 which include quercetin 3,4'-diglucoside, flavin adenine dinucleotide disodium salt hydrate, salvianolic acid A, tunicamycin and esculin. The LC50 of esculin in 3 dpf zebrafish larvae is 5 mmol/L, and the qRT-PCR results showed that esculin significantly increased the transcription of Nrf2 target genes-Gstpi, Nqo1, Hmox1a and Prdx1 in 3 dpf zebrafish larvae. These potential hits could serve as safer Nrf2 activators due to their non-covalent disruption of Keap1-Nrf2 PPI and be developed into efficacious preventive/therapeutic agents for various diseases.


Assuntos
Antioxidantes/farmacologia , Descoberta de Drogas , Fator 2 Relacionado a NF-E2/agonistas , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Proteínas de Peixe-Zebra/agonistas , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Embrião não Mamífero , Esculina/farmacologia , Dose Letal Mediana , Ligantes , Simulação de Acoplamento Molecular , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Filogenia , Ligação Proteica/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas/genética , Transcrição Genética/efeitos dos fármacos , Peixe-Zebra , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
14.
PLoS One ; 14(2): e0212492, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30768627

RESUMO

BACKGROUND: B-cell lymphoma/leukaemia 11A (BCL11A) is a C2H2-type zinc-finger transcription factor protein that is a critical modulator of haemoglobin switching and suppresses the production of foetal haemoglobin. Variation in the BCL11A gene ameliorates the severity of sickle cell disease (SCD) and ß-thalassemia (ß-thal). The BCL11A gene is located on chromosome 2p16.1 and encodes an 835-amino acid protein. METHOD: Using state-of-the-art in silico tools, this study examined the most pathogenic non-synonymous single nucleotide polymorphisms (nsSNPs) that disrupt the BCL11A protein and mediate foetal-to-adult globin switching. A total of 11,463 SNPs were retrieved from the Single Nucleotide Polymorphism database (dbSNP). These included 799 in the 5' untranslated region (UTR), 486 in the 3' UTR, and 266 non-synonymous, 189 coding synonymous, six nonsense, and six stop-gained SNPs. RESULTS AND DISCUSSION: In silico tools (SIFT, SNAP, PolyPhen-2, PANTHER, I-Mutant, PROVEAN, SNPs&GO, mCSM, and PhD-SNP) predicted the five most-deleterious nsSNPs: rs61742690, rs62142605, rs17028351, rs115666026, and rs74987258. Molecular dynamic simulation and homology modelling of the mutated proteins (S783N, D643N, G451S, K670R, and M313L) of the most deleterious nsSNPs revealed their functional and structural impact. nsSNP rs61742690 was predicted to be the most deleterious, as supported by eight of the nine in silico tools. CONCLUSIONS: Complete failure in the protein-protein interactions with functional partners (KLF1 and others) and significant changes (±100% variation) in the interface energy revealed that rs61742690 (S783N) in the zinc-finger domain is a suitable target for disrupting BCL11A-mediated foetal-to-adult globin switching.


Assuntos
Proteínas de Transporte/genética , Globinas/genética , Proteínas Nucleares/genética , Algoritmos , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Simulação por Computador , Bases de Dados de Ácidos Nucleicos , Hemoglobina Fetal/biossíntese , Hemoglobina Fetal/genética , Globinas/metabolismo , Humanos , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleotídeo Único , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas Repressoras , Homologia de Sequência de Aminoácidos , Dedos de Zinco/genética
15.
Mutat Res ; 814: 7-14, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30659944

RESUMO

For night blindness, a detailed structural exploration of the interactions among G-protein receptor rhodopsin, transducin and arrestin was performed. Rhodopsin is responsible for dim light vision while a point mutation (G90→D90) results in an adverse change in its photo-transduction. The validated 3D models of the three proteins were utilized, and upon mutation and interactions, rhodopsin attained higher stability (evaluated through thermodynamic energy calculations, electrostatic surface potential and solvent accessible area), thereby participating strongly with transducin. Conformational switches in mutated rhodopsin also depicted a firm conformation with few 310 helices accompanied by increased percentage of pure α-helices and sheets. All evaluations were corroborated through paired T-tests. Glu33 (glycosylated unit in the N-terminal zone) of rhodopsin plays a chief role in the overall interaction pattern. Arg69 and Glu33 from wild-type rhodopsin participated in ionic interactions, while the latter set of ionic interaction remained preserved even after mutation. Cys323 (C-terminal residue) and Arg69 formed H-bonds from the wild-type rhodopsin. Cys323 exceptionally supports cellular signaling pattern in the non-mutated situation and for the non-sufferers of night-blindness. Ser297 and Tyr43 from mutated rhodopsin reside in helices and interact with Thr32 of transducin, preserving the steady conformation in activated interacted state, even in the dark. Ser297 lies adjoined to Lys296 (retinal attachment site), which resides in NPXXY motif (an "activation switch" for signal transduction). Thus, the molecular facet for involvement of photo-transduction, which holds a paramount zone in ophthalmology, was dealt with. This might instigate the future prospect for drug discovery to prevent such mutations.


Assuntos
Adaptação à Escuridão/genética , Cegueira Noturna/genética , Mutação Puntual , Domínios e Motivos de Interação entre Proteínas/genética , Rodopsina/genética , Substituição de Aminoácidos , Ácido Aspártico/genética , Cristalografia por Raios X , Análise Mutacional de DNA , Glicina/genética , Humanos , Modelos Moleculares , Cegueira Noturna/metabolismo , Ligação Proteica/genética , Mapas de Interação de Proteínas , Estrutura Secundária de Proteína/genética , Rodopsina/química , Rodopsina/metabolismo , Transdução de Sinais/genética , Relação Estrutura-Atividade
16.
RNA Biol ; 16(2): 185-195, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30672374

RESUMO

Snu114, a component of the U5 snRNP, plays a key role in activation of the spliceosome. It controls the action of Brr2, an RNA-stimulated ATPase/RNA helicase that disrupts U4/U6 snRNA base-pairing prior to formation of the spliceosome's catalytic centre. Snu114 has a highly conserved domain structure that resembles that of the GTPase EF-2/EF-G in the ribosome. It has been suggested that the regulatory function of Snu114 in activation of the spliceosome is mediated by its C-terminal region, however, there has been only limited characterisation of the interactions of the C-terminal domains. We show a direct interaction between protein phosphatase PP1 and Snu114 domain 'IVa' and identify sequence 'YGVQYK' as a PP1 binding motif. Interestingly, this motif is also required for Cwc21 binding. We provide evidence for mutually exclusive interaction of Cwc21 and PP1 with Snu114 and show that the affinity of Cwc21 and PP1 for Snu114 is influenced by the different nucleotide-bound states of Snu114. Moreover, we identify a novel mutation in domain IVa that, while not affecting vegetative growth of yeast cells, causes a defect in splicing transcripts of the meiotic genes, SPO22, AMA1 and MER2, thereby inhibiting an early stage of meiosis.


Assuntos
Regulação da Expressão Gênica , Miose/genética , Mutagênese , Domínios e Motivos de Interação entre Proteínas/genética , Processamento de RNA , Ribonucleoproteína Nuclear Pequena U5/genética , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Ciclo Celular/genética , Epistasia Genética , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Miose/metabolismo , Mutação , Ligação Proteica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U5/química
17.
Genes Dev ; 33(3-4): 236-252, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30692204

RESUMO

The multisubunit CCR4-NOT mRNA deadenylase complex plays important roles in the posttranscriptional regulation of gene expression. The NOT4 E3 ubiquitin ligase is a stable component of the CCR4-NOT complex in yeast but does not copurify with the human or Drosophila melanogaster complex. Here we show that the C-terminal regions of human and D. melanogaster NOT4 contain a conserved sequence motif that directly binds the CAF40 subunit of the CCR4-NOT complex (CAF40-binding motif [CBM]). In addition, nonconserved sequences flanking the CBM also contact other subunits of the complex. Crystal structures of the CBM-CAF40 complex reveal a mutually exclusive binding surface for NOT4 and Roquin or Bag of marbles mRNA regulatory proteins. Furthermore, CAF40 depletion or structure-guided mutagenesis to disrupt the NOT4-CAF40 interaction impairs the ability of NOT4 to elicit decay of tethered reporter mRNAs in cells. Together with additional sequence analyses, our results reveal the molecular basis for the association of metazoan NOT4 with the CCR4-NOT complex and show that it deviates substantially from yeast. They mark the NOT4 ubiquitin ligase as an ancient but nonconstitutive cofactor of the CCR4-NOT deadenylase with potential recruitment and/or effector functions.


Assuntos
Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas/fisiologia , Receptores CCR4/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Sequência Conservada , Cristalização , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/química , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Estrutura Quaternária de Proteína , Estabilidade de RNA/genética , Receptores CCR4/química , Fatores de Transcrição/genética
18.
Mutat Res ; 814: 1-6, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30648609

RESUMO

HNF4α is a culprit gene product for a monogenic and dominantly-inherited form of diabetes, referred to as MODY1 (Maturity Onset Diabetes of the Young type 1). Reduced HNF4α activities have been linked to impaired insulin secretion and ß-cell function. Numerous mutations have been identified from the patients and they have been instructive as to the individual residue's role in protein structure-function and dysfunction. As a member of the nuclear receptor (NR) superfamily, HNF4α is made of characteristic modular domains and it functions exclusively as a homodimer despite its sequence homology to RXR, a common heterodimer partner of non-steroidal NRs. Transcription factors commonly dimerize to enhance their molecular functions mainly by facilitating the recognition of double helix target DNAs that display an intrinsic pseudo-2-fold symmetry and the recruitment of the remainder of the main transcriptional machinery. HNF4α is no exception and its dimerization is maintained by the ligand binding domain (LBD) mainly through the leucine-zipper-like interactions at the stalk of two interacting helices. Although many MODY1 mutations have been previously characterized, including DNA binding disruptors, ligand binding disruptors, coactivator binding disruptors, and protein stability disruptors, protein dimerization disruptors have not been formally reported. In this report, we present a set of data for the two MODY1 mutations found right at the dimerization interface (L332 P and L328del mutations) which clearly exhibit the disruptive effects of directly affecting dimerization, protein stability, and transcriptional activities. These data reinforced the fact that MODY mutations are loss-of-function mutations and HNF4α dimerization is essential for its optimal function and normal physiology.


Assuntos
Diabetes Mellitus Tipo 2/genética , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Mutação , Domínios e Motivos de Interação entre Proteínas/genética , Multimerização Proteica , Dimerização , Células HeLa , Fator 4 Nuclear de Hepatócito/química , Humanos , Mutação com Perda de Função/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Multimerização Proteica/genética , Estabilidade Proteica , Estrutura Quaternária de Proteína/genética , Ativação Transcricional/genética
19.
Nat Commun ; 10(1): 253, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30651555

RESUMO

The upregulation of PPARγ/RXRα transcriptional activity has emerged as a key event in luminal bladder tumors. It renders tumor cell growth PPARγ-dependent and modulates the tumor microenvironment to favor escape from immuno-surveillance. The activation of the pathway has been linked to PPARG gains/amplifications resulting in PPARγ overexpression and to recurrent activating point mutations of RXRα. Here, we report recurrent mutations of PPARγ that also activate the PPARγ/RXRα pathway, conferring PPARγ-dependency and supporting a crucial role of PPARγ in luminal bladder cancer. These mutations are found throughout the protein-including N-terminal, DNA-binding and ligand-binding domains-and most of them enhance protein activity. Structure-function studies of PPARγ variants with mutations in the ligand-binding domain allow identifying structural elements that underpin their gain-of-function. Our study reveals genomic alterations of PPARG that lead to pro-tumorigenic PPARγ/RXRα pathway activation in luminal bladder tumors and may open the way towards alternative options for treatment.


Assuntos
PPAR gama/genética , Receptor X Retinoide alfa/genética , Transdução de Sinais/genética , Neoplasias da Bexiga Urinária/genética , Linhagem Celular Tumoral , Estudos de Coortes , Cristalografia por Raios X , Feminino , Mutação com Ganho de Função , Células HEK293 , Humanos , Masculino , Simulação de Dinâmica Molecular , PPAR gama/química , PPAR gama/metabolismo , Domínios e Motivos de Interação entre Proteínas/genética , Receptor X Retinoide alfa/metabolismo , Análise de Sequência de DNA , Relação Estrutura-Atividade , Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/patologia
20.
Dev Cell ; 48(3): 329-344.e5, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30595538

RESUMO

Frequent SPOP mutation defines the molecular feature underlying one of seven sub-types of human prostate cancer (PrCa). However, it remains largely elusive how SPOP functions as a tumor suppressor in PrCa. Here, we report that SPOP suppresses stem cell traits of both embryonic stem cells and PrCa cells through promoting Nanog poly-ubiquitination and subsequent degradation. Mechanistically, Nanog, but not other pluripotency-determining factors including Oct4, Sox2, and Klf4, specifically interacts with SPOP via a conservative degron motif. Importantly, cancer-derived mutations in SPOP or at the Nanog-degron (S68Y) disrupt SPOP-mediated destruction of Nanog, leading to elevated cancer stem cell traits and PrCa progression. Notably, we identify the Pin1 oncoprotein as an upstream Nanog regulator that impairs its recognition by SPOP and thereby stabilizes Nanog. Thus, Pin1 inhibitors promote SPOP-mediated destruction of Nanog, which provides the molecular insight and rationale to use Pin1 inhibitor(s) for targeted therapies of PrCa patients with wild-type SPOP.


Assuntos
Proliferação de Células/fisiologia , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Repressoras/metabolismo , Células-Tronco/citologia , Proteínas Culina/metabolismo , Progressão da Doença , Humanos , Masculino , Mutação/genética , Proteínas Nucleares/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas Repressoras/genética , Ubiquitinação
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