RESUMO
Chromosomes pair and synapse with their homologous partners to segregate correctly at the first meiotic division. Association of telomeres with the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex composed of SUN1 and KASH5 enables telomere-led chromosome movements and telomere bouquet formation, facilitating precise pairwise alignment of homologs. Here, we identify a direct interaction between SUN1 and Speedy A (SPDYA) and determine the crystal structure of human SUN1-SPDYA-CDK2 ternary complex. Analysis of meiosis prophase I process in SPDYA-binding-deficient SUN1 mutant mice reveals that the SUN1-SPDYA interaction is required for the telomere-LINC complex connection and the assembly of a ring-shaped telomere supramolecular architecture at the nuclear envelope, which is critical for efficient homologous pairing and synapsis. Overall, our results provide structural insights into meiotic telomere structure that is essential for meiotic prophase I progression.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Prófase Meiótica I , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Telômero/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas de Ciclo Celular/ultraestrutura , Linhagem Celular Tumoral , Cristalografia por Raios X , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/isolamento & purificação , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 2 Dependente de Ciclina/ultraestrutura , Feminino , Células HEK293 , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/ultraestrutura , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/isolamento & purificação , Proteínas Associadas aos Microtúbulos/ultraestrutura , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestruturaRESUMO
The ASCC3 subunit of the activating signal co-integrator complex is a dual-cassette Ski2-like nucleic acid helicase that provides single-stranded DNA for alkylation damage repair by the α-ketoglutarate-dependent dioxygenase AlkBH3. Other ASCC components integrate ASCC3/AlkBH3 into a complex DNA repair pathway. We mapped and structurally analyzed interacting ASCC2 and ASCC3 regions. The ASCC3 fragment comprises a central helical domain and terminal, extended arms that clasp the compact ASCC2 unit. ASCC2-ASCC3 interfaces are evolutionarily highly conserved and comprise a large number of residues affected by somatic cancer mutations. We quantified contributions of protein regions to the ASCC2-ASCC3 interaction, observing that changes found in cancers lead to reduced ASCC2-ASCC3 affinity. Functional dissection of ASCC3 revealed similar organization and regulation as in the spliceosomal RNA helicase Brr2. Our results delineate functional regions in an important DNA repair complex and suggest possible molecular disease principles.
Assuntos
DNA Helicases/genética , Reparo do DNA , Neoplasias/genética , Proteínas Nucleares/genética , Sequência de Aminoácidos , Sequência Conservada/genética , DNA Helicases/isolamento & purificação , DNA Helicases/metabolismo , Células HEK293 , Humanos , Mutação , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Ligação Proteica/genética , Conformação Proteica em alfa-Hélice/genética , Domínios Proteicos/genética , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Spliceossomos/metabolismoRESUMO
AIMP2-DX2, an exon 2-deleted splice variant of AIMP2 (aminoacyl-tRNA synthetase-interacting multifunctional protein 2), is highly expressed in lung cancer and involved in tumor progression in vivo. Oncogenic function of AIMP2-DX2 and its correlation with poor prognosis of cancer patients have been well established; however, the application of this potentially important biomarker to cancer research and diagnosis has been hampered by a lack of antibodies specific for the splice variant, possibly due to the poor immunogenicity and/or stability of AIMP2-DX2. In this study a monoclonal antibody, H5, that specifically recognizes AIMP2-DX2 and its isoforms was generated via rabbit immunization and phage display techniques, using a short peptide corresponding to the exon 1/3 junction sequence as an antigen. Furthermore, based on mutagenesis, limited cleavage, and mass spectrometry studies, it is also suggested that the endogenous isoform of AIMP2-DX2 recognized by H5 is produced by proteolytic cleavage of 33 amino acids from N-terminus and is capable of inducing cell proliferation similarly to the uncleaved protein. H5 monoclonal antibody is applicable to enzyme-linked immunosorbent assay, immunoblot, immunofluorescence, and immunohistochemistry, and expected to be a valuable tool for detecting AIMP2-DX2 with high sensitivity and specificity for research and diagnostic purposes.
Assuntos
Anticorpos Monoclonais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Proteínas Nucleares/genética , Isoformas de Proteínas/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Células Cultivadas , Cricetulus , Humanos , Neoplasias Pulmonares/metabolismo , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , CoelhosRESUMO
BRG1/SMARCA4 and its paralog BRM/SMARCA2 are the ATPase subunits of human SWI/SNF chromatin remodeling complexes. These multisubunit assemblies can act as either tumor suppressors or drivers of cancer, and inhibiting both BRG1 and BRM, is emerging as an effective therapeutic strategy in diverse cancers. BRG1 and BRM contain a BRK domain. The function of this domain is unknown, but it is often found in proteins involved in transcription and developmental signaling in higher eukaryotes, in particular in proteins that remodel chromatin. We report the NMR structure of the BRG1 BRK domain. It shows similarity to the glycine-tyrosine-phenylalanine (GYF) domain, an established protein-protein interaction module. Computational peptide-binding-site analysis of the BRK domain identifies a binding site that coincides with a highly conserved groove on the surface of the protein. This sets the scene for experiments to elucidate the role of this domain, and evaluate the potential of targeting it for cancer therapy.
Assuntos
Montagem e Desmontagem da Cromatina , DNA Helicases/química , Proteínas Nucleares/química , Fatores de Transcrição/química , Cromatina/química , Cromatina/metabolismo , DNA Helicases/genética , DNA Helicases/isolamento & purificação , Humanos , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Ligação Proteica , Conformação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Domínios de Homologia de srcRESUMO
Liver sinusoidal endothelial cells play a key role maintaining the hepatic homeostasis, the disruption of which is associated with such end-stage liver diseases as hepatocellular carcinoma and cirrhosis. In the present study we investigated the role of brahma-related gene 1 (BRG1), a chromatin remodeling protein, in regulating endothelial transcription and the implication in liver fibrosis. We report that endothelial-specific deletion of BRG1 in mice attenuated liver fibrosis induced by injection with thioacetamide (TAA). Coincidently, alleviation of liver fibrosis as a result of endothelial BRG1 deletion was accompanied by an up-regulation of eNOS activity and NO bioavailability. In cultured endothelial cells, exposure to lipopolysaccharide (LPS) suppressed eNOS activity whereas BRG1 depletion with small interfering RNA restored eNOS-dependent NO production. Further analysis revealed that BRG1 was recruited to the caveolin-1 (CAV1) promoter by Sp1 and activated transcription of CAV1, which in turn inhibited eNOS activity. Mechanistically, BRG1 interacted with the H3K4 trimethyltransferase MLL1 to modulate H3K4 trimethylation surrounding the CAV1 promoter thereby contributing to LPS-induced CAV1 activation. In conclusion, our data unveil a novel role for BRG1 in the regulation of endothelial function and liver fibrosis.
Assuntos
DNA Helicases/metabolismo , Células Endoteliais/metabolismo , Fibrose/metabolismo , Fígado/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , DNA Helicases/deficiência , DNA Helicases/isolamento & purificação , Fibrose/induzido quimicamente , Humanos , Fígado/efeitos dos fármacos , Camundongos , Óxido Nítrico/análise , Proteínas Nucleares/deficiência , Proteínas Nucleares/isolamento & purificação , Tioacetamida , Fatores de Transcrição/deficiência , Fatores de Transcrição/isolamento & purificaçãoRESUMO
Many cell stressors block protein translation, inducing formation of cytoplasmic aggregates. These aggregates, named stress granules (SGs), are composed by translationally stalled ribonucleoproteins and their assembly strongly contributes to cell survival. Composition and dynamics of SGs are thus important starting points for identifying critical factors of the stress response. In the present study we link components of the H/ACA snoRNP complexes, highly concentrated in the nucleoli and the Cajal bodies, to SG composition. H/ACA snoRNPs are composed by a core of four highly conserved proteins -dyskerin, Nhp2, Nop10 and Gar1- and are involved in several fundamental processes, including ribosome biogenesis, RNA pseudouridylation, stabilization of small nucleolar RNAs and telomere maintenance. By taking advantage of cells overexpressing a dyskerin splice variant undergoing a dynamic intracellular trafficking, we were able to show that H/ACA snoRNP components can participate in SG formation, this way contributing to the stress response and perhaps transducing signals from the nucleus to the cytoplasm. Collectively, our results show for the first time that H/ACA snoRNP proteins can have additional non-nuclear functions, either independently or interacting with each other, thus further strengthening the close relationship linking nucleolus to SG composition.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Grânulos Citoplasmáticos/metabolismo , Proteínas Nucleares/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Células Tumorais CultivadasRESUMO
BAF250a and BAF250b are subunits of the SWI/SNF chromatin-remodeling complex that recruit the complex to chromatin allowing transcriptional activation of several genes. Despite being the central subunits of the SWI/SNF complex, the structural and functional annotation of BAF250a/b remains poorly understood. BAF250a (nearly 2200 residues protein) harbors an N-terminal DNA binding ARID (~110 residues) and a C-terminal folded region (~250 residues) of unknown structure and function, recently annotated as BAF250_C. Using hydrophobic core analysis, fold prediction and comparative modeling, here we have defined a domain boundary and associate a ß-catenin like ARM-repeat fold to the C-terminus of BAF250a that encompass BAF250_C. The N-terminal DNA-binding ARID is found in diverse domain combinations in proteins imparting unique functions. We used a comparative sequence analysis based approach to study the ARIDs from diverse domain contexts and identified conserved residue positions that are important to preserve its core structure. Supporting this, mutation of one such conserved residue valine, at position 1067, to glycine, resulted in destabilization, loss of structural integrity and DNA binding affinity of ARID. Additionally, we identified a set of conserved and surface-exposed residues unique to the ARID when it co-occurs with the ARM repeat containing BAF250_C in BAF250a. Several of these residues are found mutated in somatic cancers. We predict that these residues in BAF250a may play important roles in mediating protein-DNA and protein-protein interactions in the BAF complex.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Complexos Multienzimáticos/genética , Proteínas Nucleares/genética , Domínios Proteicos/genética , Fatores de Transcrição/genética , Diferenciação Celular , Biologia Computacional , Proteínas de Ligação a DNA , Conjuntos de Dados como Assunto , Glicina/genética , Simulação de Dinâmica Molecular , Complexos Multienzimáticos/química , Mutagênese Sítio-Dirigida , Mutação , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/química , Proteínas Nucleares/isolamento & purificação , Ligação Proteica/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/isolamento & purificação , Valina/genéticaRESUMO
The human polybromo-1 protein (BAF180) is a known driver mutation in clear cell renal cell carcinoma, where it is mutated in approximately 40% of cases. BAF180 is the chromatin-targeting subunit of the PBAF complex. BAF180 has six bromodomains, two BAH domains, and one HMG box. Bromodomains are known to recognize acetylated-lysines on histones and play a role in nucleosome recognition. BAH domains are required for ubiquitination of PCNA, a key regulator of DNA damage. The putative HMG box, if functional, may be involved in DNA-binding. While the binding specificities of individual bromodomains have been studied by our lab and others, the results have failed to reach a consensus. The acetyl-histone binding features of the full-length protein is unknown and is the motivation for this work. The hypothetical HMG and BAH domains have not been studied and the actual function of these regions is currently unknown. Thus, the precise interactions of this large and complex protein are not well-studied. Advances in understanding this large protein have been hindered by the inability to express and purify recombinant full-length BAF180 protein. Currently, only phenomenological studies using BAF180 expressed in mammalian cells have been conducted. Here, we report the successful expression, purification of full-length biologically active BAF180 protein using the GAP promoter in the heterologous host Pichia pastoris. The ability to express full-length and mutated BAF180 will allow for biophysical binding studies. Knowledge of the binding interactions is critical for us to understand the role of BAF180 in cancer development and its progression.
Assuntos
Histonas/metabolismo , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Pichia/genética , Fatores de Transcrição/genética , Sítios de Ligação , Clonagem Molecular , Proteínas de Ligação a DNA , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Histonas/química , Histonas/genética , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/isolamento & purificação , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Pichia/metabolismo , Regiões Promotoras Genéticas , Análise Serial de Proteínas , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismoRESUMO
Histone deacetylases (HDACs) represent a family of enzymes that are targets for epigenetic modulation of genomic activity and may be beneficial in the treatment of many diseases, including cancer and central nervous system disorders. In animal models, HDAC inhibitors have neuroprotective, antiepileptogenic, and antidepressant effects. Assaying HDAC activity provides a robust method for identifying HDAC inhibitors and for assessing their effects under various physiological conditions or after pathological insults. In this unit, a simple and sensitive assay for measuring HDAC activity is described. HDAC activity in tissue lysates can be assessed fluorometrically using a Boc-Lys(Ac) HDAC activity kit. HDACs catalyze the deacetylation of the substrate Boc-Lys(Ac)-AMC. Addition of a trypsin-containing developer converts the deacetylated product to a quantifiable fluorophore that can be used both as a screening method to identify putative HDAC inhibitors and to assess the effects of these inhibitors on tissue and animal epigenetic-modulated phenotypes. © 2018 by John Wiley & Sons, Inc.
Assuntos
Encéfalo/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Animais , Encéfalo/metabolismo , Lesões Encefálicas Traumáticas/metabolismo , Camundongos , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismoRESUMO
Nitric oxide (NO) is an important signaling molecule involved in various plant physiological processes. The main effect of NO arises from its reaction with proteins. S-Nitrosation is the most studied NO-mediated protein posttranslational modification in plants. Via S-nitrosation, NO derivatives react with thiol groups (SHs) of protein cysteine residues and produce nitrosothiol groups (SNOs). From the time of discovering the biological function of NO in plants, an interesting case of study has been the detection of the endogenous S-nitrosated proteins in different plants, tissues, organelles, and various conditions. Maps of S-nitrosated proteins provide hints for deeper studies on the function of this modification in specific proteins, biochemical pathways, and physiological processes. Many functions of NO have been found to be related to plant defense; on the other hand the involvement of nuclear proteins in regulation of plant defense reactions is well studied. Here, an approach is described in which the Arabidopsis cell cultures first are treated with P. syringae, afterward their bioactive nuclear proteins are extracted, then the nuclear proteins are subjected to biotin switch assay in which S-nitrosated proteins are specifically converted to S-biotinylated proteins. The biotin switch technique (BST) which was introduced by Jaffrey et al. (Nat Cell Biol 3:193-197, 2001) solves the instability issue of SNOs. Additionally, it provides detection and purification of biotinylated proteins by anti-biotin antibody and affinity chromatography, respectively.
Assuntos
Arabidopsis/metabolismo , Biotina , Proteínas Nucleares/metabolismo , Células Vegetais/metabolismo , Processamento de Proteína Pós-Traducional , Biotina/química , Biotinilação , Técnicas de Cultura de Células , Células Cultivadas , Óxido Nítrico/metabolismo , Nitrosação , Proteínas Nucleares/química , Proteínas Nucleares/isolamento & purificação , S-Nitrosotióis/metabolismoRESUMO
Nucleophosmin (NPM1) is a multifunctional phosphoprotein which plays important roles in diverse biological processes. NPM1 can form homo- or hetero-oligomers through its N-terminal region, and bind DNA and RNA through its C-terminal region. However, the monomer-oligomer distribution of NPM1, and the extent of NPM1 binding and unbinding to RNA in living cells, are not fully understood. In this work, we analysed molecular complexes of NPM1 using size exclusion chromatography. We found that a substantial fraction of NPM1 behaves as an oligomer in HeLa cells. Furthermore, we identified three distinct oligomeric states of NPM1 using molecular characterization techniques such as subcellular localization and RNA binding. Finally, we found that heterozygous expression of a leukemia-associated NPM1 mutant significantly decreases the RNA binding level. Our data demonstrate that size exclusion chromatography provides a powerful tool for analysing NPM1 oligomers.
Assuntos
Biopolímeros/química , Cromatografia em Gel/métodos , Proteínas Nucleares/química , Anticorpos Monoclonais/imunologia , Biopolímeros/imunologia , Biopolímeros/isolamento & purificação , Western Blotting , Linhagem Celular , Humanos , Peso Molecular , Proteínas Nucleares/imunologia , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Nucleofosmina , Ligação Proteica , RNA/metabolismoRESUMO
DNA end resection initiates the largely accurate repair of DNA double-strand breaks (DSBs) by homologous recombination. Specifically, recombination requires the formation of 3' overhangs at DSB sites, which is carried out by nucleases that specifically degrade 5'-terminated DNA. In most cases, DNA end resection is a two-step process, comprising of initial short-range followed by more processive long-range resection. In this chapter, we describe selected assays that reconstitute both the short- and long-range pathways. First, we define methods to study the exonuclease and endonuclease activities of the MRE11-RAD50-NBS1 (MRN) complex in conjunction with phosphorylated cofactor CtIP. This reaction is particularly important to initiate processing of DNA breaks and to recruit components belonging to the subsequent long-range pathway. Next, we describe assays that reconstitute the concerted reactions of Bloom (BLM) or Werner (WRN) helicases that function together with the DNA2 nuclease-helicase, and which are as a complex capable to resect DNA of kilobases in length. The reconstituted reactions allow us to understand how the resection pathways function at the molecular level. The assays will be invaluable to define regulatory mechanisms and to identify inhibitory compounds, which may be valuable in cancer therapy.
Assuntos
Técnicas de Cultura de Células/métodos , Quebras de DNA de Cadeia Dupla , Ensaios Enzimáticos/métodos , Proteínas Recombinantes/isolamento & purificação , Reparo de DNA por Recombinação , Hidrolases Anidrido Ácido , Animais , Baculoviridae/genética , Soluções Tampão , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Técnicas de Cultura de Células/instrumentação , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas de Ciclo Celular/metabolismo , DNA Helicases/isolamento & purificação , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/isolamento & purificação , Enzimas Reparadoras do DNA/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Poliacrilamida/instrumentação , Eletroforese em Gel de Poliacrilamida/métodos , Endodesoxirribonucleases , Ensaios Enzimáticos/instrumentação , Humanos , Proteína Homóloga a MRE11/isolamento & purificação , Proteína Homóloga a MRE11/metabolismo , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Oligonucleotídeos/metabolismo , RecQ Helicases/isolamento & purificação , RecQ Helicases/metabolismo , Proteínas Recombinantes/metabolismo , Proteína de Replicação A/isolamento & purificação , Proteína de Replicação A/metabolismo , Células Sf9 , Spodoptera , Transfecção/métodos , Helicase da Síndrome de Werner/isolamento & purificação , Helicase da Síndrome de Werner/metabolismoRESUMO
Deviations from the normal program of gene expression during early pregnancy can lead to early embryonic loss as well as dysfunctional placentation, which can cause significant morbidity and mortality. Proline rich 15 (PRR15) is a low molecular weight nuclear protein expressed by the trophoblast during early gestation. Lentivirus-mediated knockdown of PRR15 mRNA in ovine trophectoderm led to demise of the embryo by gestational day 15, providing compelling evidence that PRR15 expression is critical during this precarious window of development. Our objective was to determine the effect of PRR15 knockdown on trophoblast gene expression, proliferation, and survival. The first-trimester human trophoblast cell line, ACH-3P, was infected with control lentivirus or a lentivirus expressing a short hairpin (sh)RNA to target PRR15 mRNA for degradation, resulting in a 68% reduction in PRR15 mRNA. Microarray analysis of these cell lines revealed differential expression of genes related to cancer, focal adhesion, and p53 signaling. These changes included significant up-regulation of GDF15, a cytokine increased in pregnancies with preeclampsia. Viability and proliferation decreased in PRR15-deficient cells, which was consistent with down-regulation of cell cycle-related genes CCND1 and CDK6 and an up-regulation of CCNG2 and CDKN1A in the PRR15-deficient cells. TNFSF10, a tumor necrosis factor superfamily member known to induce apoptosis increased significantly in the PRR15-deficient cells. Migration through a basement membrane matrix decreased and an increased population of apoptotic cells was present when treated with shRNA to target PRR15. These results suggest that PRR15 enhances trophoblast viability and survival during early implantation and placentation.
Assuntos
Proteínas/fisiologia , Trofoblastos/fisiologia , Apoptose/genética , Western Blotting , Ciclo Celular/genética , Linhagem Celular , Proliferação de Células/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Silenciamento de Genes , Humanos , Proteínas Nucleares/isolamento & purificação , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
CapG is an actin-binding protein, which is overexpressed in a variety of tumors, i.e. breast, ovarian, pancreatic and lung carcinoma. We successfully expressed human CapG in the wild type strain X-33 of the methylotrophic yeast Pichia pastoris (P. pastoris), which does not express endogenous CapG, in order to characterize this protein in more detail. After mechanical cell lysis, debris was centrifuged and the soluble protein was precipitated with ammonium sulfate. This protein pellet was dialyzed and used for CapG purification. Ca2+-dependent exposure of hydrophobic sites allowed single step and selective elution from a Phenyl Sepharose™ matrix. 3.5 mg CapG/10 g wet biomass were isolated and showed a Ca2+-sensitive and dose-dependent capping activity of actin in a fluorometric assay. In P. pastoris, CapG is located at actin patches, actin cables and arranges along the budding neck. The proliferation rate and morphology of the yeast cells are not influenced by the interaction of CapG with actin. The modification pattern of human CapG from P. pastoris and human carcinoma cells is highly similar. We validated most of the known post-translational modifications and found three new phosphorylation and nine new acetylation sites by mass spectrometry. The N-terminus is acetylated or truncated. Truncated CapG is not phosphorylated at the residues S10, T212 and S337. First mutagenesis experiments indicate an N-terminal acetylation dependent C-terminal phosphorylation.
Assuntos
Expressão Gênica , Proteínas dos Microfilamentos , Proteínas Nucleares , Pichia/metabolismo , Processamento de Proteína Pós-Traducional , Acetilação , Linhagem Celular Tumoral , Humanos , Proteínas dos Microfilamentos/biossíntese , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/isolamento & purificação , Proteínas Nucleares/biossíntese , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Fosforilação , Pichia/genética , Proteínas RecombinantesRESUMO
Besides their primary role in protein synthesis, aminoacyl-tRNA synthetases (AARSs) are involved in several non-canonical processes such as apoptosis, inflammation and angiogenesis through their interactions with various cellular proteins. Nine of these AARSs interact with three aminoacyl-tRNA synthetase interacting multifunctional proteins (AIMPs), forming a multi-synthetase complex (MSC) in eukaryotes. Among the three AIMPs, AIMP2 is involved in controlling cell proliferation and apoptosis. However, a splicing variant of AIMP2 lacking exon 2, referred to as AIMP2-DX2, is oncogenic and compromises the pro-apoptotic activity of AIMP2 by competing with it for p53 and TRAF2. AIMP2-DX2 is also an inhibitor of p14arf activity. Thus, there is a pressing need for structural insight into the oncogenic role of AIMP2-DX2. In this study, we expressed and purified human AIMP2-DX2 using a SUMO tag to more than 95% purity and a yield of 10 mg/L. We have used size exclusion chromatography, glutaraldehyde cross-linking, dynamic light scattering and nuclear magnetic resonance spectroscopy to characterize its biophysical properties. These data indicate monomer-dimer equilibrium of AIMP2-DX2 in solution. These results form the basis for the structure-function study of oncogenic AIMP2-DX2.
Assuntos
Proteínas Nucleares , Multimerização Proteica , Humanos , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/biossíntese , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Relação Estrutura-AtividadeRESUMO
During the cell cycle, duplicated sister chromatids become physically connected during S phase through a process called sister-chromatid cohesion. Cohesion is terminated during the metaphase-to-anaphase transition to trigger sister-chromatid segregation. The establishment and dissolution of cohesion are highly regulated by the cohesin complex and its multitude of regulators. In particular, the cohesin regulator Wapl promotes the release of cohesin from chromosomes during both interphase and mitosis. Here, we describe in vitro protein binding assays between Wapl and a cohesin subcomplex, and cellular assays in human cells that probe the functions of Wapl in cohesin release.
Assuntos
Proteínas de Transporte/isolamento & purificação , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas Cromossômicas não Histona/isolamento & purificação , Segregação de Cromossomos/genética , Biologia Molecular/métodos , Proteínas Nucleares/isolamento & purificação , Proteínas Proto-Oncogênicas/isolamento & purificação , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Cromátides/genética , Proteínas Cromossômicas não Histona/genética , Humanos , Mitose/genética , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/genética , Saccharomyces cerevisiae/genética , CoesinasRESUMO
BACKGROUND & OBJECTIVES: Mutation of nucleophosmin (NPM1) gene in the absence of FLT3-ITD (FMS related tyrosine kinase 3 - internal tandem duplications) mutation carries a good prognosis in cytogenetically normal acute myeloid leukaemia (AML). NPM1, a multifunctional nucleolar phosphoprotein that shuttles between nucleus and cytoplasm, gets trapped in the cytoplasm when mutated. Immunohistochemical (IHC) demonstration of its aberrant cytoplasmic location (NPMc+) has been suggested as a simple substitute for the standard screening molecular method. This study was aimed to assess the diagnostic utility of IHC on formalin fixed bone marrow biopsies in comparison with the reference molecular method (allele specific oligonucleotide - polymerase chain reaction; ASO-PCR) to predict NPM1 mutation status in AML patients. METHODS: NPM protein IHC was performed using mouse anti-NPM monoclonal antibody on 35 paraffin-embedded bone marrow biopsies of patients with primary AML of any French-American-British (FAB) subtype. Results of IHC were compared with those of ASO-PCR. RESULTS: Of the 35 AML patients, 21 (60%) were positive for NPM1 exon 12 gene mutation by ASO-PCR, 19 (90.47%) of these 21 were NPMc+. Thirteen of the 35 patients were negative by both the methods. One NPMc+ patient was not detected by ASO-PCR. IHC had a sensitivity and specificity of 90 and 93 per cent, respectively, compared to the molecular screening gold standard. INTERPRETATION & CONCLUSIONS: Mutation of NPM1 determined by the widely available and inexpensive IHC agrees closely with results of the standard molecular methods. Thus, technically and financially not well endowed laboratories can provide the prognostically and potentially therapeutically important information on NPM1 mutation using IHC.
Assuntos
Imuno-Histoquímica , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/genética , Adolescente , Adulto , Anticorpos Monoclonais/genética , Criança , Análise Mutacional de DNA , Feminino , Humanos , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/isolamento & purificação , NucleofosminaRESUMO
Multicolor flow cytometry (MFC) and real-time quantitative PCR (RQ-PCR) are important independent techniques to determine minimal residual disease (MRD) in acute myeloid leukemia (AML). MFC is the standard method, but may be unreliable. Therefore, MFC-based determination of MRD with an RQ-PCR-based approach targeting the nucleophosmin 1 (NPM1) type A mutation was set out to compare. Since most current NPM1 RQ-PCR MRD protocols suffer from clear definitions of quantifiability, we sought to define quantifiability in a reproducible and standardized manner. The limit of quantifiability of our RQ-PCR protocol for the NPM1 type A mutation varied between 0.002% and 0.04% residual leukemic cells depending on the features of the standard curve for each PCR experiment. The limit of detection was close to 0.001% leukemic cells. The limit of detection by MFC ranged from 0.01% to 1% depending on the phenotype of the leukemic cells as compared with non-leukemic bone marrow cells. Forty-five MRD samples from 15 patients using both NPM1 mutation specific RQ-PCR and MFC were analyzed. In 32 of the 45 samples (71%), an MRD-signal could be detected with RQ-PCR. A quantifiable NPM1 mutation signal was found in 15 samples (33%) (range 0.003%-2.6% leukemic cells). By contrast, only two follow-up samples (4%) showed residual leukemic cells (0.04% and 0.3%, respectively) by MFC. Thus, RQ-PCR of the NPM1 type A mutation was more sensitive and reliable than MFC for determination of MRD, which might have clinical implications. © 2016 Wiley Periodicals, Inc.
Assuntos
Leucemia Mieloide Aguda/diagnóstico , Neoplasia Residual/diagnóstico , Proteínas Nucleares/genética , Adulto , Idoso , Feminino , Citometria de Fluxo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Neoplasia Residual/genética , Neoplasia Residual/patologia , Proteínas Nucleares/isolamento & purificação , Nucleofosmina , Reação em Cadeia da Polimerase em Tempo RealRESUMO
LAP2-emerin-MAN1 (LEM)-domain proteins are modular proteins characterized by the presence of a conserved motif of about 50 residues. Most LEM-domain proteins localize at the inner nuclear membrane, but some are also found in the endoplasmic reticulum or nuclear interior. Their architecture has been analyzed by predicting the limits of their globular domains, determining the 3D structure of these domains and in a few cases calculating the 3D structure of specific domains bound to biological targets. The LEM domain adopts an α-helical fold also found in SAP and HeH domains of prokaryotes and unicellular eukaryotes. The LEM domain binds to BAF (barrier-to-autointegration factor; BANF1), which interacts with DNA and tethers chromatin to the nuclear envelope. LAP2 isoforms also share an N-terminal LEM-like domain, which binds DNA. The structure and function of other globular domains that distinguish LEM-domain proteins from each other have been characterized, including the C-terminal dimerization domain of LAP2α and C-terminal WH and UHM domains of MAN1. LEM-domain proteins also have large intrinsically disordered regions that are involved in intra- and intermolecular interactions and are highly regulated by posttranslational modifications in vivo.
Assuntos
Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Membrana/isolamento & purificação , Proteínas Nucleares/isolamento & purificação , Cromatografia Líquida , Proteínas de Ligação a DNA/química , Escherichia coli , Humanos , Proteínas de Membrana/química , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de ProteínaRESUMO
RecQ helicases, also addressed as a gatekeeper of genome, are an inevitable family of genome scrutiny proteins conserved from prokaryotes to eukaryotes and play a vital role in DNA metabolism. The deficiencies of three RecQ proteins out of five are involved in genetic abnormalities like Bloom syndrome (BS), Werner syndrome (WS), and Rothmund-Thomson syndrome (RTS). It is noteworthy that Plasmodium falciparum contains only two members of the RecQ family as opposed to five members present in the host Homo sapiens. In the present study, we report the biochemical characterization of the homologue of Werner (Wrn) helicase from P. falciparum 3D7 strain. Although there are significant sequence conservations between Wrn helicases of both H. sapiens and P. falciparum as well as among all the other Plasmodium species, they contain some peculiar differences also. In silico studies reveal that PfWrn is evolutionarily close to the bacterial RecQ protein. The N-terminal fragment (PfWrnN) contains all the helicase motifs along with all the functional domains and the predicted structure resembles with the human RecQ1 protein, whereas the C-terminal fragment (PfWrnC) contains no significant domain. Biochemical characterization further revealed that purified recombinant PfWrnN shows ATPase and DNA helicase activity in 3' to 5' direction, but PfWrnC lacks the ATPase and helicase activities. Immunofluorescence study shows that PfWrn is expressed in all the stages of intraerythrocytic development of the P. falciparum 3D7 strain and localizes distinctly in the nucleus. This study can be used for further characterization of RecQ helicases that will aid in understanding the physiological significance of these helicases in the malaria parasite.