Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 6.963
Filtrar
1.
Medicine (Baltimore) ; 98(52): e18449, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31876727

RESUMO

Twist and E-cadherin are crucial for the development of different types of cancer; however, their clinical significance in adenocarcinoma of the gastroesophageal junction (AGE) remains unknown. Here, we investigated the correlation between the expression of Twist and E-cadherin and their impact on the clinical outcomes and prognosis of patients with AGE and proximal gastric carcinoma (PGC).Using immunohistochemistry, we determined the expression of Twist and E-cadherin in the tissue samples of patients with AGE and PGC. The correlation of the expression of Twist and E-cadherin with the clinicopathological factors was assessed by using the chi-square test, Fisher exact test, and non-parametric Mann-Whitney U test. The Kaplan-Meier method along with the log-rank test and Cox proportional-hazards model were used to evaluate the correlation of Twist and E-cadherin expression with the overall survival (OS) of patients.Overall, 94 patients with AGE (n = 45, 47.87%) or PGC (n = 49, 52.13%) who underwent primary tumor resection were included in this study. The median follow-up period was 40.5 months. We observed a significant difference in the smoking status (P < .001) and differentiation grade (P = .004) between patients with AGE and PGC. There was a significant association of a high Twist expression with T stage (only in PGC, P = .008), lymph node metastasis (AGE, P = .075; PGC, P = .051), and advanced pathological stages (AGE, P = .019; PGC, P = .006). A low E-cadherin expression showed similar results; however, it was not significantly associated with the advanced pathological stages of AGE (P = .372). A low E-cadherin expression was significantly associated with a low differentiation grade of AGE (P = .002). In addition, a significant inverse relationship was observed between Twist and E-cadherin expression. The Kaplan-Meier survival analysis and Cox regression analysis revealed that a high Twist expression and low E-cadherin expression were independent prognostic factors for short OS of patients with AGE or PGC.A high Twist expression or low E-cadherin expression was associated with unfavorable clinicopathological factors and independently predicted short OS of patients with AGE or PGC.


Assuntos
Adenocarcinoma/metabolismo , Caderinas/metabolismo , Carcinoma/metabolismo , Junção Esofagogástrica/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Gástricas/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Adenocarcinoma/química , Adenocarcinoma/diagnóstico , Adulto , Idoso , Biomarcadores Tumorais/química , Biomarcadores Tumorais/metabolismo , Caderinas/análise , Carcinoma/química , Carcinoma/diagnóstico , Junção Esofagogástrica/química , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/química , Prognóstico , Neoplasias Gástricas/química , Neoplasias Gástricas/diagnóstico , Proteína 1 Relacionada a Twist/química
2.
Phys Chem Chem Phys ; 21(45): 25276-25289, 2019 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-31701109

RESUMO

As a member of the bromodomain and extra terminal domain (BET) protein family, bromodomain-containing protein 4 (BRD4) is an epigenetic reader and can recognize acetylated lysine residues in histones. BRD4 has been regarded as an essential drug target for cancers, inflammatory diseases and acute heart failure, and therefore the discovery of potent BRD4 inhibitors with novel scaffolds is highly desirable. In this study, the crystalline water molecules in BRD4 involved in ligand binding were analyzed first, and the simulation results suggest that several conserved crystalline water molecules are quite essential to keep the stability of the crystalline water network and therefore they need to be reserved in structure-based drug design. Then, a docking-based virtual screening workflow with the consideration of the conserved crystalline water network in the binding pocket was utilized to identify the potential inhibitors of BRD4. The in vitro fluorescence resonance energy transfer (HTRF) binding assay illustrates that 4 hits have good inhibitory activity against BRD4 in the micromolar regime, including three compounds with IC50 values below 5 µM and one below 1 µM (0.37 µM). The structural analysis demonstrates that three active compounds possess novel scaffolds. Moreover, the interaction patterns between the hits and BRD4 were characterized by molecular dynamics simulations and binding free energy calculations, and then several suggestions for the further optimization of these hits were proposed.


Assuntos
Simulação de Acoplamento Molecular , Proteínas Nucleares/química , Fatores de Transcrição/química , Água/química , Proteínas de Ciclo Celular , Cristalização , Transferência Ressonante de Energia de Fluorescência , Humanos , Simulação de Dinâmica Molecular , Proteínas Nucleares/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores
3.
Biochem Soc Trans ; 47(6): 1597-1608, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31769470

RESUMO

Phosphorylation by protein kinases is a fundamental mechanism of signal transduction. Many kinase families contain one or several members that, although evolutionarily conserved, lack the residues required for catalytic activity. Studies combining structural, biochemical, and functional approaches revealed that these pseudokinases have crucial roles in vivo and may even represent attractive targets for pharmacological intervention. Pseudokinases mediate signal transduction by a diversity of mechanisms, including allosteric regulation of their active counterparts, assembly of signaling hubs, or modulation of protein localization. One such pseudokinase, named Tra1 in yeast and transformation/transcription domain-associated protein (TRRAP) in mammals, is the only member lacking all catalytic residues within the phosphatidylinositol 3-kinase related kinase (PIKK) family of kinases. PIKKs are related to the PI3K family of lipid kinases, but function as Serine/Threonine protein kinases and have pivotal roles in diverse processes such as DNA damage sensing and repair, metabolic control of cell growth, nonsense-mediated decay, or transcription initiation. Tra1/TRRAP is the largest subunit of two distinct transcriptional co-activator complexes, SAGA and NuA4/TIP60, which it recruits to promoters upon transcription factor binding. Here, we review our current knowledge on the Tra1/TRRAP pseudokinase, focusing on its role as a scaffold for SAGA and NuA4/TIP60 complex assembly and recruitment to chromatin. We further discuss its evolutionary history within the PIKK family and highlight recent findings that reveal the importance of molecular chaperones in pseudokinase folding, function, and conservation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Evolução Biológica , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Animais , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ligação Proteica , Dobramento de Proteína , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Transcrição Genética
4.
Chem Pharm Bull (Tokyo) ; 67(10): 1139-1143, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31582633

RESUMO

We have discovered that ß-amino acid homooligomers with cis- or trans-amide conformation can fold themselves into highly ordered helices. Moreover, unlike α-amino acid peptides, which are significantly stabilized by intramolecular hydrogen bonding, these helical structures are autogenous conformations that are stable without the aid of hydrogen bonding and irrespective of solvent (protic/aprotic/halogenated) or temperature. A structural overlap comparison of helical cis/trans bicyclic ß-proline homooligomers with typical α-helix structure of α-amino acid peptides reveals clear differences of pitch and diameter per turn. Bridgehead substituents of the present homooligomers point outwards from the helical surface. We were interested to know whether such non-naturally occurring divergent helical molecules could mimic α-helix structures. In this study, we show that bicyclic ß-proline oligomer derivatives inhibit p53-MDM2 and p53-MDMX protein-protein interactions, exhibiting MDM2-antagonistic and MDMX-antagonistic activities.


Assuntos
Proteínas Nucleares/química , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas/química , Proteína Supressora de Tumor p53/química , Proteínas de Ciclo Celular , Humanos , Estrutura Molecular , Proteínas Nucleares/antagonistas & inibidores , Prolina/análogos & derivados , Prolina/farmacologia , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteína Supressora de Tumor p53/antagonistas & inibidores
5.
Nat Cell Biol ; 21(10): 1261-1272, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31570835

RESUMO

The repression of transposons by the Piwi-interacting RNA (piRNA) pathway is essential to protect animal germ cells. In Drosophila, Panoramix enforces transcriptional silencing by binding to the target-engaged Piwi-piRNA complex, although the precise mechanisms by which this occurs remain elusive. Here, we show that Panoramix functions together with a germline-specific paralogue of a nuclear export factor, dNxf2, and its cofactor dNxt1 (p15), to suppress transposon expression. The transposon RNA-binding protein dNxf2 is required for animal fertility and Panoramix-mediated silencing. Transient tethering of dNxf2 to nascent transcripts leads to their nuclear retention. The NTF2 domain of dNxf2 competes dNxf1 (TAP) off nucleoporins, a process required for proper RNA export. Thus, dNxf2 functions in a Panoramix-dNxf2-dependent TAP/p15 silencing (Pandas) complex that counteracts the canonical RNA exporting machinery and restricts transposons to the nuclear peripheries. Our findings may have broader implications for understanding how RNA metabolism modulates heterochromatin formation.


Assuntos
Proteínas Argonauta/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Inativação Gênica , Heterocromatina/metabolismo , Proteínas Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteínas Argonauta/química , Proteínas Argonauta/metabolismo , Montagem e Desmontagem da Cromatina , Elementos de DNA Transponíveis , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Heterocromatina/ultraestrutura , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Oócitos/metabolismo , Oócitos/ultraestrutura , Ovário/citologia , Ovário/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
7.
Nat Genet ; 51(10): 1518-1529, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31570891

RESUMO

RNA modifications are emerging as key determinants of gene expression. However, compelling genetic demonstrations of their relevance to human disease are lacking. Here, we link ribosomal RNA 2'-O-methylation (2'-O-Me) to the etiology of dyskeratosis congenita. We identify nucleophosmin (NPM1) as an essential regulator of 2'-O-Me on rRNA by directly binding C/D box small nucleolar RNAs, thereby modulating translation. We demonstrate the importance of 2'-O-Me-regulated translation for cellular growth, differentiation and hematopoietic stem cell maintenance, and show that Npm1 inactivation in adult hematopoietic stem cells results in bone marrow failure. We identify NPM1 germline mutations in patients with dyskeratosis congenita presenting with bone marrow failure and demonstrate that they are deficient in small nucleolar RNA binding. Mice harboring a dyskeratosis congenita germline Npm1 mutation recapitulate both hematological and nonhematological features of dyskeratosis congenita. Thus, our findings indicate that impaired 2'-O-Me can be etiological to human disease.


Assuntos
Disceratose Congênita/genética , Epigenômica/métodos , Mutação em Linhagem Germinativa , Proteínas Nucleares/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Ribossômico/genética , Animais , Disceratose Congênita/patologia , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/química , RNA Nucleolar Pequeno , Transcriptoma
8.
Nat Commun ; 10(1): 4149, 2019 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-31515493

RESUMO

Studies of cellular mechano-signaling have often utilized static models that do not fully replicate the dynamics of living tissues. Here, we examine the time-dependent response of primary human mesenchymal stem cells (hMSCs) to cyclic tensile strain (CTS). At low-intensity strain (1 h, 4% CTS at 1 Hz), cell characteristics mimic responses to increased substrate stiffness. As the strain regime is intensified (frequency increased to 5 Hz), we characterize rapid establishment of a broad, structured and reversible protein-level response, even as transcription is apparently downregulated. Protein abundance is quantified coincident with changes to protein conformation and post-translational modification (PTM). Furthermore, we characterize changes to the linker of nucleoskeleton and cytoskeleton (LINC) complex that bridges the nuclear envelope, and specifically to levels and PTMs of Sad1/UNC-84 (SUN) domain-containing protein 2 (SUN2). The result of this regulation is to decouple mechano-transmission between the cytoskeleton and the nucleus, thus conferring protection to chromatin.


Assuntos
Núcleo Celular/metabolismo , Células-Tronco Mesenquimais/citologia , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Fenômenos Biomecânicos , Forma do Núcleo Celular , Cromatina/metabolismo , Citoesqueleto/metabolismo , Dano ao DNA , Histonas/metabolismo , Humanos , Canais Iônicos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Modelos Biológicos , Membrana Nuclear/metabolismo , Proteínas Nucleares/química , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estresse Mecânico , Resistência à Tração
9.
Fish Shellfish Immunol ; 94: 607-616, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31541777

RESUMO

Akirin, which are members of the NF-κB signaling pathway, play critical roles in regulating the expression of antimicrobial peptides. In the present study, the Akirin gene from Penaeus monodon was identified from a transcriptome database and designated as PmAkirin. The complete sequence of the PmAkirin cDNA was 1508 bp, encoding a protein of 213 amino acids, and it showed 99% amino acid identity to the Litopenaeus vannamei Akirin. Two predicted nuclear localization signals (NLSs) were found, and the amino acid sequence alignments showed that PmAkirin was highly conserved at the N-terminus and C-terminus. PmAkirin expression was found to be the highest in the hemolymph, followed by the heart, gill, stomach, hepatopancreas, intestine, and muscle. When challenged with Vibrio parahaemolyticus infection, the PmAkirin mRNA and three antimicrobial peptides (AMPs: PmALF2, PmALF3, and PmCrus4) were upregulated. However, another five AMPs (PmALF6, PmCrus1, PmPEN3a, PmPEN3b, and PmPEN5) were downregulated by V. parahaemolyticus infection. Silencing PmAkirin by dsRNA significantly decreased the expression of the eight AMPs, which lead to an increase in the blood concentration of V. parahaemolyticus and higher mortality in the shrimp. In contrast, the overexpression of PmAkirin significantly increased the expression of the eight AMPs, which led to a reduction in the blood concentration of V. parahaemolyticus and promoted the survival of the shrimp. Taken together, we concluded that PmAkirin plays an important role in regulating the expression of AMPs in black tiger shrimp to defend against V. parahaemolyticus infection.


Assuntos
Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Penaeidae/genética , Penaeidae/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Perfilação da Expressão Gênica , Proteínas Nucleares/química , Filogenia , Domínios Proteicos , Alinhamento de Sequência , Vibrio parahaemolyticus/fisiologia
10.
Nat Commun ; 10(1): 3967, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31481669

RESUMO

N6-threonyl-carbamoylation of adenosine 37 of ANN-type tRNAs (t6A) is a universal modification essential for translational accuracy and efficiency. The t6A pathway uses two sequentially acting enzymes, YRDC and OSGEP, the latter being a subunit of the multiprotein KEOPS complex. We recently identified mutations in genes encoding four out of the five KEOPS subunits in children with Galloway-Mowat syndrome (GAMOS), a clinically heterogeneous autosomal recessive disease characterized by early-onset steroid-resistant nephrotic syndrome and microcephaly. Here we show that mutations in YRDC cause an extremely severe form of GAMOS whereas mutations in GON7, encoding the fifth KEOPS subunit, lead to a milder form of the disease. The crystal structure of the GON7/LAGE3/OSGEP subcomplex shows that the intrinsically disordered GON7 protein becomes partially structured upon binding to LAGE3. The structure and cellular characterization of GON7 suggest its involvement in the cellular stability and quaternary arrangement of the KEOPS complex.


Assuntos
Adenosina/análogos & derivados , Proteínas de Ligação ao GTP/genética , Hérnia Hiatal/genética , Proteínas Intrinsicamente Desordenadas/genética , Microcefalia/genética , Nefrose/genética , Proteínas Nucleares/genética , RNA de Transferência/genética , Proteínas de Ligação a RNA/genética , Adenosina/genética , Criança , Feminino , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Masculino , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo
11.
Nucleic Acids Res ; 47(18): 9619-9636, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31392992

RESUMO

Connections between epigenetic reprogramming and transcription or splicing create novel mechanistic networks that can be targeted with tailored therapies. Multiple subunits of the chromatin remodeling BAF complex, including ARID1A, play a role in oncogenesis, either as tumor suppressors or oncogenes. Recent work demonstrated that EWS-FLI1, the oncogenic driver of Ewing sarcoma (ES), plays a role in chromatin regulation through interactions with the BAF complex. However, the specific BAF subunits that interact with EWS-FLI1 and the precise role of the BAF complex in ES oncogenesis remain unknown. In addition to regulating transcription, EWS-FLI1 also alters the splicing of many mRNA isoforms, but the role of splicing modulation in ES oncogenesis is not well understood. We have identified a direct connection between the EWS-FLI1 protein and ARID1A isoform protein variant ARID1A-L. We demonstrate here that ARID1A-L is critical for ES maintenance and supports oncogenic transformation. We further report a novel feed-forward cycle in which EWS-FLI1 leads to preferential splicing of ARID1A-L, promoting ES growth, and ARID1A-L reciprocally promotes EWS-FLI1 protein stability. Dissecting this interaction may lead to improved cancer-specific drug targeting.


Assuntos
Carcinogênese/genética , Proteínas Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Proteína Proto-Oncogênica c-fli-1/genética , Proteína EWS de Ligação a RNA/genética , Sarcoma de Ewing/genética , Fatores de Transcrição/genética , Processamento Alternativo/genética , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Epigênese Genética/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Nucleares/química , Proteínas de Fusão Oncogênica/química , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Estabilidade Proteica , Proteína Proto-Oncogênica c-fli-1/química , Proteína EWS de Ligação a RNA/química , Sarcoma de Ewing/patologia , Fatores de Transcrição/química
12.
Chem Commun (Camb) ; 55(68): 10128-10131, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31386708

RESUMO

Fueled by the therapeutic potential of the epigenetic machinery, BET bromodomains have seen high interest as drug targets. Herein, we introduce different linkers to a BET bromodomain benzodiazepine ligand (I-BET762) to gauge its implications in the development of hybrid drugs, imaging probes and small molecule drug conjugates. Biophysical studies confirmed minimal disruption to binding of the BRD4 cavity by the synthesized entities, which includes imaging probes. Target engagement was confirmed in a cellular context, but poor membrane diffusion was found despite efficient localization in the nuclei after membrane disruption. Our study highlights challenges and opportunities for the successful design of benzodiazepine-derived drug-delivery systems.


Assuntos
Benzodiazepinas/farmacologia , Fluoresceínas/farmacologia , Corantes Fluorescentes/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Benzodiazepinas/síntese química , Benzodiazepinas/química , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Desenho de Drogas , Fluoresceínas/síntese química , Fluoresceínas/química , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Humanos , Ligantes , Estrutura Molecular , Proteínas Nucleares/química , Domínios Proteicos
13.
J Exp Clin Cancer Res ; 38(1): 350, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31409387

RESUMO

BACKGROUND: Ubiquitin E3 ligase CUL4A plays important oncogenic roles in the development of cancers. DTL, one of the CUL4-DDB1 associated factors (DCAFs), may involve in the process of cancer development. Programmed cell death 4 (PDCD4) is a tumor suppressor gene involved in cell apoptosis, transformation, invasion and tumor progression. METHODS: Affinity-purification mass spectrometry was used to identify potential DTL interaction proteins. Co-immunoprecipitation (Co-IP) was performed to verify protein interaction between DTL and PDCD4. mRNA levels in cancer cells and tissues were detected by Quantitative real-time PCR. Lentivirus was used to establish stable overexpression and knocking down cell lines for DTL and PDCD4. Transwell and wound healing assays were used to determine migration ability of cancer cells. Matrigel assay was used to determine invasion ability of cancer cells. MTT and colony formation assays were used to evaluate proliferation of cancer cells. RESULTS: In this study, programmed cell death 4 (PDCD4) was identified as a potential substrate of DTL. Co-IP and immunofluorescence assays further confirmed the interaction between DTL and PDCD4. Moreover, DTL overexpression decreased the protein level and accelerated the degradation rate of PDCD4. Through in vitro ubiquitination experiment, we proved that PDCD4 was degraded by DTL through ubiquitination. Clinically DTL was significantly up-regulated in cancer tissues than that in normal tissues. The survival curves showed that cancer patients with higher DTL expression owned lower survival rate. Functional experiments showed that DTL not only enhanced the proliferation and migration abilities of cancer cells, but also promoted the tumorigenesis in nude mice. Rescued experiment results demonstrated that silencing PDCD4 simultaneous with DTL recovered the phenotypes defect caused by DTL knocking down. CONCLUSIONS: Our results elucidated that DTL enhanced the motility and proliferation of cancer cells through degrading PDCD4 to promote the development of cancers.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ubiquitinas/metabolismo , Animais , Apoptose , Proteínas Reguladoras de Apoptose/química , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Biologia Computacional/métodos , Modelos Animais de Doenças , Xenoenxertos , Humanos , Camundongos , Neoplasias/genética , Neoplasias/patologia , Proteínas Nucleares/química , Proteínas Nucleares/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteínas de Ligação a RNA/química , Relação Estrutura-Atividade , Ubiquitinação
14.
Mol Cell ; 75(4): 700-710.e6, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31442422

RESUMO

Microrchidia (MORC) ATPases are critical for gene silencing and chromatin compaction in multiple eukaryotic systems, but the mechanisms by which MORC proteins act are poorly understood. Here, we apply a series of biochemical, single-molecule, and cell-based imaging approaches to better understand the function of the Caenorhabditis elegans MORC-1 protein. We find that MORC-1 binds to DNA in a length-dependent but sequence non-specific manner and compacts DNA by forming DNA loops. MORC-1 molecules diffuse along DNA but become static as they grow into foci that are topologically entrapped on DNA. Consistent with the observed MORC-1 multimeric assemblies, MORC-1 forms nuclear puncta in cells and can also form phase-separated droplets in vitro. We also demonstrate that MORC-1 compacts nucleosome templates. These results suggest that MORCs affect genome structure and gene silencing by forming multimeric assemblages to topologically entrap and progressively loop and compact chromatin.


Assuntos
Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans/química , DNA de Helmintos/química , Proteínas Nucleares/química , Conformação de Ácido Nucleico , Nucleossomos/química , Multimerização Proteica , Animais , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/ultraestrutura , DNA de Helmintos/metabolismo , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura
15.
Nat Commun ; 10(1): 3757, 2019 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-31434876

RESUMO

Nuclear structure and function are governed by lamins, which are intermediate filaments that mostly consist of α-helices. Different lamin assembly models have been proposed based on low resolution and fragmented structures. However, their assembly mechanisms are still poorly understood at the molecular level. Here, we present the crystal structure of a long human lamin fragment at 3.2 Å resolution that allows the visualization of the features of the full-length protein. The structure shows an anti-parallel arrangement of the two coiled-coil dimers, which is important for the assembly process. We further discover an interaction between the lamin dimers by using chemical cross-linking and mass spectrometry analysis. Based on these two interactions, we propose a molecular mechanism for lamin assembly that is in agreement with a recent model representing the native state and could explain pathological mutations. Our findings also provide the molecular basis for assembly mechanisms of other intermediate filaments.


Assuntos
Laminas/química , Proteínas Nucleares/química , Domínios Proteicos , Sequência de Aminoácidos , Sítios de Ligação , Reagentes para Ligações Cruzadas/química , Cristalografia por Raios X , Humanos , Filamentos Intermediários/metabolismo , Laminas/genética , Laminas/ultraestrutura , Modelos Moleculares , Matriz Nuclear/metabolismo , Proteínas Nucleares/ultraestrutura , Fragmentos de Peptídeos/química , Conformação Proteica em alfa-Hélice , Proteínas Recombinantes , Análise de Sequência de Proteína
16.
Mol Genet Genomics ; 294(6): 1527-1534, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31363903

RESUMO

Primary ovarian insufficiency (POI) affects ~ 1-3, 7% of women under forty and is a public health problem. Most causes are unknown, but an increasing number of genetic causes have been identified recently. The identification of such causes is essential for genetic and therapeutic counseling in patients and their families. We performed whole exome sequencing in two Caucasian sisters displaying non syndromic POI and their unaffected mother. We identified two novel pathogenic variants in STAG3 encoding a meiosis-specific subunit of the cohesin ring, which ensures correct sister chromatid cohesion: a c.3052delC truncating mutation in exon 28 yielding p.Arg1018Aspfs*14, and a c.659T > G substitution in exon seven yielding p.Leu220Arg. Leu220, highly conserved throughout species, belongs to the STAG domain conserved with other mitotic subunits of the cohesion complex STAG1 and 2. In silico analysis reveals that this substitution markedly impacts the structure of this domain. The truncation removes the last 206 C-terminal residues, not conserved in STAG1 and 2, supporting an important specific role in STAG3, especially meiosis. This is the first occurrence of STAG3 mutations in a Caucasian family. Very little is known about the function of STAG proteins domains. The "knock out-like" phenotype described here supports the crucial role of a single residue in the STAG domain and of the C-terminal region in STAG3 function. In conclusion, this observation shows the necessity to perform the genetic study of POI worldwide including STAG3. This could lead to appropriate genetic counseling and long term follow-up since these patients may develop ovarian tumors.


Assuntos
Mutação , Proteínas Nucleares/genética , Insuficiência Ovariana Primária/genética , Adolescente , Grupo com Ancestrais do Continente Europeu/genética , Feminino , Humanos , Modelos Moleculares , Proteínas Nucleares/química , Insuficiência Ovariana Primária/etnologia
17.
Cancer Sci ; 110(10): 3275-3287, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31368616

RESUMO

p97/VCP is an endoplasmic reticulum (ER)-associated protein that belongs to the AAA (ATPases associated with diverse cellular activities) ATPase family. It has a variety of cellular functions including ER-associated protein degradation, autophagy, and aggresome formation. Recent studies have shown emerging roles of p97/VCP and its potential as a therapeutic target in several cancer subtypes including multiple myeloma (MM). We conducted a cell-based compound screen to exploit novel small compounds that have cytotoxic activity in myeloma cells. Among approximately 2000 compounds, OSSL_325096 showed relatively strong antiproliferative activity in MM cell lines (IC50 , 100-500 nmol/L). OSSL_325096 induced apoptosis in myeloma cell lines, including a bortezomib-resistant cell line and primary myeloma cells purified from patients. Accumulation of poly-ubiquitinated proteins, PERK, CHOP, and IREα, was observed in MM cell lines treated with OSSL_325096, suggesting that it induces ER stress in MM cells. OSSL_325096 has a similar chemical structure to DBeQ, a known p97/VCP inhibitor. Knockdown of the gene encoding p97/VCP induced apoptosis in myeloma cells, accompanied by accumulation of poly-ubiquitinated protein. IC50 of OSSL_325096 to myeloma cell lines were found to be lower (0.1-0.8 µmol/L) than those of DBeQ (2-5 µmol/L). In silico protein-drug-binding simulation suggested possible binding of OSSL_325096 to the ATP binding site in the D2 domain of p97/VCP. In cell-free ATPase assays, OSSL_325096 showed dose-dependent inhibition of p97/VCP ATPase activity. Finally, OSSL_325096 inhibited the growth of subcutaneous myeloma cell tumors in vivo. The present data suggest that OSSL_325096 exerts anti-myeloma activity, at least in part through p97/VCP inhibition.


Assuntos
Adenosina Trifosfatases/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/administração & dosagem , Mieloma Múltiplo/tratamento farmacológico , Proteínas Nucleares/metabolismo , Bibliotecas de Moléculas Pequenas/administração & dosagem , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/química , Animais , Sítios de Ligação , Bortezomib/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático , Endorribonucleases/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Camundongos , Modelos Moleculares , Mieloma Múltiplo/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/química , Proteínas Serina-Treonina Quinases/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Fator de Transcrição CHOP/metabolismo , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto , eIF-2 Quinase/metabolismo
18.
Oxid Med Cell Longev ; 2019: 7318796, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31428229

RESUMO

Ankrd2 (ankyrin repeats containing domain 2) or Arpp (ankyrin repeat, PEST sequence, and proline-rich region) is a member of the muscle ankyrin repeat protein family. Ankrd2 is mostly expressed in skeletal muscle, where it plays an intriguing role in the transcriptional response to stress induced by mechanical stimulation as well as by cellular reactive oxygen species. Our studies in myoblasts from Emery-Dreifuss muscular dystrophy 2, a LMNA-linked disease affecting skeletal and cardiac muscles, demonstrated that Ankrd2 is a lamin A-binding protein and that mutated lamins found in Emery-Dreifuss muscular dystrophy change the dynamics of Ankrd2 nuclear import, thus affecting oxidative stress response. In this review, besides describing the latest advances related to Ankrd2 studies, including novel discoveries on Ankrd2 isoform-specific functions, we report the main findings on the relationship of Ankrd2 with A-type lamins and discuss known and potential mechanisms involving defective Ankrd2-lamin A interplay in the pathogenesis of muscular laminopathies.


Assuntos
Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular de Emery-Dreifuss/patologia , Proteínas Nucleares/metabolismo , Estresse Oxidativo , Proteínas Repressoras/metabolismo , Humanos , Lamina Tipo A/metabolismo , Mecanotransdução Celular , Proteínas Musculares/química , Distrofia Muscular de Emery-Dreifuss/metabolismo , Miocárdio/metabolismo , Proteínas Nucleares/química , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/química
19.
Mol Cell Biochem ; 461(1-2): 171-182, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31428904

RESUMO

The BAF complex (SWI/SNF) is an ATP-dependent chromatin remodeler that adapts the structural organization of the chromatin. Despite a growing understanding of the composition of BAF in different cell types, the interaction network within the BAF complex is poorly understood. Here, we characterized an isoform of the BRG1/SMARCA4 ATPase expressed in human neural progenitor cells. By electron microscopy and image processing, the neural BRG1/SMARCA4 shows an elongated globular structure, which provides a considerably larger surface than anticipated. We show that neural BRG1/SMARCA4 binds to BAF57/SMARCE1 and BAF60A/SMARCD1, two further components of BAF. Moreover, we demonstrate an interaction between the neural BRG1/SMARCA4 isoform and the central neurodevelopmental transcriptional repressor REST/NRSF. Our results provide insights into the assembly of a central transcriptional repressor complex, link the structure of the neural BRG1/SMARCA4 to its role as a protein-protein interaction platform and suggest BRG1/SMARCA4 as a key determinant that directs the BAF complex to specific DNA sites by interacting with transcription factors and regulators.


Assuntos
DNA Helicases/metabolismo , Células-Tronco Neurais/metabolismo , Proteínas Nucleares/metabolismo , Subunidades Proteicas/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Proteínas Cromossômicas não Histona/metabolismo , DNA Helicases/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Biológicos , Proteínas Nucleares/química , Ligação Proteica , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Fatores de Transcrição/química
20.
Mol Biol (Mosk) ; 53(4): 663-673, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31397440

RESUMO

Malignant cutaneous melanoma (CM) is an extremely aggressive cancer characterized by a high level of metastatic activity and unfavorable prognosis due to a high incidence of relapses, as well as resistance to standard chemotherapy. Cutaneous melanoma accounts for 80% of deaths from malignant skin tumors. Nucleolin/C23 and nucleophosmin/B23, which constitute altogether ~70% of the nucleolus volume, are promising targets for molecular therapy of melanoma. These proteins perform many important functions in the cell, so disruption of the NCL and/or NPM gene structure and abnormal expression of the C23 and B23 proteins they encode, can lead to unlimited cell proliferation and progression of a tumor. Therefore, investigation of the structure and expression of these genes is a topical problem, which is important for understanding the mechanisms of CM carcinogenesis and for the development of new therapeutic approaches. This paper describes new NCL and NPM polymorphisms, as well as the levels of C23 and B23 expression in normal tissues, CM and mucosal melanoma.


Assuntos
Melanoma/genética , Melanoma/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Nucléolo Celular/química , Nucléolo Celular/metabolismo , Proliferação de Células , Humanos , Melanoma/tratamento farmacológico , Terapia de Alvo Molecular , Proteínas Nucleares/biossíntese , Proteínas Nucleares/química , Fosfoproteínas/biossíntese , Fosfoproteínas/química , Polimorfismo Genético , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/química , Neoplasias Cutâneas/tratamento farmacológico
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA