RESUMEN
BACKGROUND: The salivary glands of female ticks degenerate rapidly by apoptosis and autophagy after feeding. Bcl-2 family proteins play an important role in the apoptosis pathways, but the functions of these proteins in ticks are unclear. We studied Bcl-2 and Bax homologs from Rhipicephalus haemaphysaloides and determined their functions in the degeneration of the salivary glands. METHODS: Two molecules containing conserved BH (Bcl-2 family homology) domains were identified and named RhBcl-2 and RhBax. After protein purification and mouse immunization, specific polyclonal antibodies (PcAb) were created in response to the recombinant proteins. Reverse transcription quantitative PCR (RT-qPCR) and western blot were used to detect the presence of RhBcl-2 and RhBax in ticks. TUNEL assays were used to determine the level of apoptosis in the salivary glands of female ticks at different feeding times after gene silencing. Co-transfection and GST pull-down assays were used to identify interactions between RhBcl-2 and RhBax. RESULTS: The RT-qPCR assay revealed that RhBax gene transcription increased significantly during feeding at all tick developmental stages (engorged larvae, nymphs, and adult females). Transcriptional levels of RhBcl-2 and RhBax increased more significantly in the female salivary glands than in other tissues post engorgement. RhBcl-2 silencing significantly inhibited tick feeding. In contrast, RhBax interference had no effect on tick feeding. TUNEL staining showed that apoptosis levels were significantly reduced after interference with RhBcl-2 expression. Co-transfection and GST pull-down assays showed that RhBcl-2 and RhBax could interact but not combine in the absence of the BH3 domain. CONCLUSIONS: This study identified the roles of RhBcl-2 and RhBax in tick salivary gland degeneration and finds that the BH3 domain is a key factor in their interactions.
Asunto(s)
Proteínas Proto-Oncogénicas/aislamiento & purificación , Rhipicephalus/metabolismo , Proteína X Asociada a bcl-2/aislamiento & purificación , Animales , Apoptosis , Femenino , Etiquetado Corte-Fin in Situ , Ratones , Proteínas Proto-Oncogénicas/fisiología , Glándulas Salivales/metabolismo , Glándulas Salivales/patología , Proteína X Asociada a bcl-2/fisiologíaRESUMEN
The 5-methylcytosine (5mC) oxidation pathway mediated by TET proteins involves step-wise oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC can be removed from DNA by base excision repair and the completion of this pathway results in "demethylation" of 5mC by converting the modified base back into cytosine. In vitro studies with TET proteins aimed at analyzing their DNA substrate specificities and their activity within defined chromatin templates are relatively limited. Here we describe purification methods for mammalian TET proteins based on expression in insect cells or in 293T cells. We also briefly summarize a method that can be used to monitor 5-methylcytosine oxidase activity of the purified TET proteins in vitro.
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5-Metilcitosina/análogos & derivados , 5-Metilcitosina/química , Metilación de ADN , ADN/análisis , Oxigenasas de Función Mixta/aislamiento & purificación , Oxigenasas de Función Mixta/metabolismo , Proteínas Proto-Oncogénicas/aislamiento & purificación , Proteínas Proto-Oncogénicas/metabolismo , ADN/química , ADN/genética , Humanos , Oxigenasas de Función Mixta/genética , Oxidación-Reducción , Proteínas Proto-Oncogénicas/genética , Especificidad por SustratoRESUMEN
Introduction: Despite some of the oncogenic driver mutations that have been associated with increased expression of programmed death-ligand 1 (PD-L1), the correlation between PD-L1 expression and ROS1 fusion in NSCLC cells, especially for those with Crizotinib resistance has not been fully addressed. Materials and Methods: The expression of PD-L1 in 30 primary NSCLC tumors with/without ROS1-fusion protein was evaluated by immunohistochemical (IHC) analysis. To assess the correlation between ROS1 fusion and PD-L1 expression, we down-regulated ROS1 with RNA interference or specific inhibitor (Crizotinib) in ROS1-fusion positive NSCLC cell line HCC78; or up-regulate ROS1-fusion gene in an immortalized human bronchial epithelial cell line (HBE). Mouse xenograft models were also used to determine the effect of ROS1 expression on PD-L1 expression in vivo. Crizotinib-resistant cell line was generated for measuring the association between Crizotinib resistance and PD-L1 expression. Results: ROS1-rearrangement in primary NSCLC tumor was significantly associated with up-regulated PD-L1 expression. PD-L1 expression was significantly up-regulated in bronchial epithelial cells after forced expression of ROS1 fusion and was eliminated when HCC78 xenograft mouse models were treated with Crizotinib. We found PD-L1 expression was modulated by MEK-ERK pathway signaling in both parental and Crizotinib-resistant NSCLC cells with ROS1 fusion. Conclusions: The correlation between ROS1-fusion and PD-L1 overexpression suggested that PD-L1/PD-1 blockade could be the second-line treatment option for the Crizotinib-resistant NSCLC with ROS1 rearrangement.
Asunto(s)
Antígeno B7-H1 , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Sistema de Señalización de MAP Quinasas , Proteínas de Fusión Oncogénica , Proteínas Tirosina Quinasas , Proteínas Proto-Oncogénicas , Animales , Antígeno B7-H1/biosíntesis , Antígeno B7-H1/genética , Antígeno B7-H1/inmunología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Xenoinjertos , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/aislamiento & purificación , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/aislamiento & purificaciónRESUMEN
Rationale: A number of guanine nucleotide exchange factors (GEFs) including epithelial cell transforming factor ECT2 are believed to drive carcinogenesis through activating distinct oncogenic GTPases. Yet, whether GEF-independent activity of ECT2 also plays a role in tumorigenesis remains unclear. Methods: Immunohistochemical (IHC) staining, colony formation and xenograft assays were used to examine the role of ECT2 in breast carcinogenesis. Co-immunoprecipitation, immunofluorescent stainings, in vivo deubiquitination and in vitro deubiquitination experiments were performed to examine the physical and functional interaction between ECT2 and ubiquitin-specific protease USP7. High-throughput RNA sequencing, quantitative reverse transcription-PCR and Western blotting were employed to investigate the biological significance of the interplay between ECT2 and USP7. Results: We report that ECT2 plays a tumor-promoting role in breast cancer, and GEF activity-deficient ECT2 is able to alleviate ECT2 depletion associated growth defects in breast cancer cells. Mechanistically, we demonstrated that ECT2 physically interacts with ubiquitin-specific protease USP7 and functionally facilitates USP7 intermolecular self-association, -deubiquitination and -stabilization in a GEF activity-independent manner. USP7 in turn, deubiquitinates and stabilizes ECT2, resulting in a feedforward regulatory circuit that ultimately sustains the expression of oncogenic protein MDM2. Conclusion: Our study uncovers a GEF-independent role of ECT2 in promoting survival of breast cancer cells, provides a molecular insight for the reciprocal regulation of ECT2 and USP7, and supports the pursuit of ECT2/USP7 as potential targets for breast cancer intervention.
Asunto(s)
Neoplasias de la Mama/genética , Carcinogénesis/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas/metabolismo , Peptidasa Específica de Ubiquitina 7/metabolismo , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular/genética , Pruebas de Enzimas , Retroalimentación Fisiológica , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Ratones , Unión Proteica/genética , Estabilidad Proteica , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/aislamiento & purificación , RNA-Seq , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Peptidasa Específica de Ubiquitina 7/genética , Peptidasa Específica de Ubiquitina 7/aislamiento & purificación , Ubiquitinación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The protein ALL1 fused from chromosome 1q (AF1q) is overexpressed in a variety of cancers and acts to activate several signaling pathways that lead to oncogenesis. For example, AF1q has been shown to interact with T-cell Factor 7 (TCF7; also known as TCF1) from the Wnt/ß-catenin pathway resulting in the transcriptional activation of the CD44 and the enhancement of breast cancer metastasis. Despite the importance of AF1q in facilitating oncogenesis and metastasis, the structural and biophysical properties of AF1q remain largely unexplored due to the absence of a viable method for producing recombinant protein. Here, we report the overexpression of AF1q in E. coli as a fusion to a N-terminal His6-tag, which forms inclusion bodies (IBs) during expression. The AF1q protein was purified from IBs under denaturing conditions by immobilized metal affinity chromatography followed by a successful one-step dialysis refolding. Refolded AF1q was further purified to homogeneity by gel filtration chromatography resulting in an overall yield of 35â¯mg/L culture. Our nuclear magnetic resonance (NMR) and analytical ultracentrifugation (AUC) measurements reveal AF1q interacts with TCF7, specifically with TCF7's high-mobility group (HMG) domain (residues 154-237), which is, to our knowledge, the first biophysical characterization of the AF1q and TCF7 interaction.
Asunto(s)
Proteínas de Neoplasias/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Recombinantes/genética , Factor 1 de Transcripción de Linfocitos T/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Cromatografía de Afinidad , Clonación Molecular , Escherichia coli , Regulación Bacteriana de la Expresión Génica , Humanos , Espectroscopía de Resonancia Magnética , Proteínas de Neoplasias/aislamiento & purificación , Proteínas de Neoplasias/metabolismo , Unión Proteica , Conformación Proteica , Proteínas Proto-Oncogénicas/aislamiento & purificación , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , UltracentrifugaciónRESUMEN
The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these epigenetic marks is accomplished by sequential oxidation by ten-eleven translocation dioxygenases (TET1-3), followed by the thymine-DNA glycosylase-dependent base excision repair. Inactivation of the TET2 gene due to genetic mutations or by other epigenetic mechanisms is associated with a poor prognosis in patients with diverse cancers, especially hematopoietic malignancies. Here, we describe an efficient single step purification of enzymatically active untagged human TET2 dioxygenase using cation exchange chromatography. We further provide a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach that can separate and quantify the four normal DNA bases (A, T, G, and C), as well as the four modified cytosine bases (5-methyl, 5-hydroxymethyl, 5-formyl, and 5-carboxyl). This assay can be used to evaluate the activity of wild type and mutant TET2 dioxygenases.
Asunto(s)
Cromatografía Liquida , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Pruebas de Enzimas/métodos , Proteínas Proto-Oncogénicas/aislamiento & purificación , Proteínas Proto-Oncogénicas/metabolismo , Espectrometría de Masas en Tándem , 5-Metilcitosina/análisis , 5-Metilcitosina/metabolismo , Citosina/análogos & derivados , Citosina/análisis , ADN/química , Desmetilación del ADN , Dioxigenasas , HumanosRESUMEN
5-Methylcytosine within CpG islands in DNA plays a crucial role in epigenetic transcriptional regulation during metazoan development. Recently, it has been established that the Ten-Eleven Translocation (TET) family, Fe(II)- and 2-oxoglutarate (2OG/αKG)-dependent oxygenases initiate 5-methylcytosine demethylation by iterative oxidation reactions. Mutations in the TET2 gene are frequently detected in patients with myeloid malignancies. Here, we describe the cloning of untagged human TET2 demethylase using Gateway technology and its efficient expression in E. coli. The untagged TET2 enzyme was purified using cation exchange and heparin sepharose chromatography. In addition, a reliable quantitative liquid chromatography-tandem mass spectrometry-based assay was utilized to analyze the activity of TET2 oxygenase. This assay was further used to analyze the activity of a number of clinical TET2 variants with mutations in the 2OG binding sites. Our results demonstrate that the activity of one TET2 mutant, TET2-R1896S, can be restored using an excess of 2OG in the reaction mixture. These studies suggest that dietary 2OG supplements, which are commonly used for several other conditions, may be used to treat some patients with myeloid malignancies harboring TET2-R1896S mutation. Results described in this paper serve as a foundation for better characterization of wild type as well as mutant TET2 demethylases.
Asunto(s)
Proteínas de Unión al ADN , Expresión Génica , Oxidorreductasas , Proteínas Proto-Oncogénicas , Cromatografía Liquida , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Dioxigenasas , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Espectrometría de Masas , Oxidorreductasas/biosíntesis , Oxidorreductasas/química , Oxidorreductasas/genética , Oxidorreductasas/aislamiento & purificación , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
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.
Asunto(s)
Proteínas Portadoras/aislamiento & purificación , Proteínas de Ciclo Celular/aislamiento & purificación , Proteínas Cromosómicas no Histona/aislamiento & purificación , Segregación Cromosómica/genética , Biología Molecular/métodos , Proteínas Nucleares/aislamiento & purificación , Proteínas Proto-Oncogénicas/aislamiento & purificación , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Cromátides/genética , Proteínas Cromosómicas no Histona/genética , Humanos , Mitosis/genética , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas/genética , Saccharomyces cerevisiae/genética , CohesinasRESUMEN
The Wnt signaling pathway plays a predominant role in aberrant proliferation in myriad of cancers. In non-cancerous cells, Wnts are blocked by the secreted frizzled-related proteins (sFRPs) that are generally downregulated in cancer cells. We have purified and characterized bacterially expressed glutathione S-transferase-tagged SFRP4 from a novel clone generated from human cell origin. Cervical cancer (HeLa) and lung cancer (A549) cells, in which Wnt and associated genes were found to be expressed, were treated with the purified recombinant sFRP4, which revealed a significant dose-dependent cell growth inhibition up to 40 %. The current investigation on functionality of this bacterially produced recombinant sFRP4 in arresting cancer cell proliferation is the first of its kind, where G2/M phase arrest and early apoptosis were evident. Increase in phosphorylated ß-catenin in sFRP4 treatment indicated inhibition of Wnt pathway, which was further confirmed by downregulation of pro-proliferative genes, namely cyclin D1, c-myc, and survivin. Functional activity of recombinant sFRP4 was further exploited in co-therapy module with chemotherapeutic drugs to decipher molecular events. Collectively, our study on purified recombinant sFRP4 from bacterial host holds great promise in targeting Wnt signaling for exploring new strategies to combat cancer.
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Antineoplásicos , División Celular/efectos de los fármacos , Fase G2/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Proteínas Proto-Oncogénicas , Vía de Señalización Wnt/efectos de los fármacos , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Escherichia coli , Células HEK293 , Células HeLa , Humanos , Proteínas de Neoplasias/biosíntesis , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/aislamiento & purificación , Proteínas Proto-Oncogénicas/farmacología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacologíaRESUMEN
Human neutrophil gelatinase associated lipocalin (NGAL) is a secretory glycoprotein initially isolated from neutrophils. It is thought to be involved in the incidence and development of immunological diseases and cancers. Urinary and serum levels of NGAL have been investigated as a new biomarker of acute kidney injury (AKI), for an earlier and more accurate detection method than with creatinine level. However, expressing high-quality recombinant NGAL is difficult both in Escherichia coli and mammalian cells for the low yield. Here, we cloned and fused NGAL to the C-terminus of signal peptides of human NGAL, human interleukin-2 (IL2), gaussia luciferase (Gluc), human serum albumin preproprotein (HSA) or an hidden Markov model-generated signal sequence (HMM38) respectively for transient expression in Expi293F suspension cells to screen for their ability to improve the secretory expression of recombinant NGAL. The best results were obtained with signal peptide derived from HSA. The secretory recombinant protein could react specifically with NGAL antibody. For scaled production, we used HSA signal peptide to establish stable Chinese hamster ovary cell lines. Then we developed a convenient colony-selection system to select high-expression, stable cell lines. Moreover, we purified the NGAL with Ni-Sepharose column. The recombinant human NGAL displayed full biological activity. We provide a method to enhance the secretory expression of recombinant human NGAL by using the HSA signal peptide and produce the glycoprotein in mammalian cells.
Asunto(s)
Proteínas de Fase Aguda/genética , Lipocalinas/genética , Ingeniería de Proteínas , Proteínas Proto-Oncogénicas/genética , Proteínas de Fase Aguda/química , Proteínas de Fase Aguda/aislamiento & purificación , Proteínas de Fase Aguda/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Lipocalina 2 , Lipocalinas/química , Lipocalinas/aislamiento & purificación , Lipocalinas/metabolismo , Lysobacter/genética , Lysobacter/metabolismo , Pliegue de Proteína , Señales de Clasificación de Proteína , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/aislamiento & purificación , Proteínas Proto-Oncogénicas/metabolismo , Albúmina Sérica/química , Albúmina Sérica/genéticaRESUMEN
Polo-like kinaseâ 1 (Plk1), a validated cancer target, harbors a protein-protein interaction domain referred to as the polo-box domain (PBD), in addition to its enzymatic domain. Although functional inhibition either of the enzymatic domain or of the PBD has been shown to inhibit Plk1, so far there have been no reports of bifunctional agents with the potential to target both protein domains. Here we report the development of Plk1 inhibitors that incorporate both an ATP-competitive ligand of the enzymatic domain, derived from BIâ 2536, and a functional inhibitor of the PBD, based either on the small molecule poloxin-2 or on a PBD-binding peptide. Although these bifunctional agents do not seem to bind both protein domains simultaneously, the most potent compound displays low-nanomolar activity against the Plk1 PBD, with excellent selectivity over the PBDs of Plk2 and Plk3. Our data provide insights into challenges and opportunities relating to the optimization of Plk1 PBD ligands as potent Plk1 inhibitors.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Péptidos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas de Ciclo Celular/aislamiento & purificación , Proteínas de Ciclo Celular/metabolismo , Relación Dosis-Respuesta a Droga , Polarización de Fluorescencia , Humanos , Estructura Molecular , Péptidos/síntesis química , Péptidos/química , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/aislamiento & purificación , Proteínas Proto-Oncogénicas/metabolismo , Relación Estructura-Actividad , Quinasa Tipo Polo 1RESUMEN
Centrosomes are major microtubule-organizing centers in eukaryotic cells and play a critical role in embryonic development and asymmetric cell division. Centrosomes comprise a pair of centrioles surrounded by an amorphous proteinaceous meshwork called the pericentriolar material (PCM). Robust deposition of PCM around the centrioles is essential for a centrosome to achieve full microtubule nucleating potential. Despite the wealth of information on PCM composition and function, the mechanism and regulation of PCM assembly have been difficult to ascertain, due in part to the lack of an in vitro system. Here, we describe methods to establish an in vitro system to study PCM assembly in Caenorhabditis elegans. Specifically, we describe (1) how to express and purify the C. elegans PCM proteins SPD-5, SPD-2, and PLK-1 from baculovirus-infected insect cells, (2) how to assemble these proteins into PCM-like structures in vitro, and (3) how to quantify this assembly process in a semiautomated fashion.
Asunto(s)
Proteínas de Caenorhabditis elegans/química , Proteínas de Ciclo Celular/química , Centriolos/química , Animales , Proteínas de Caenorhabditis elegans/aislamiento & purificación , Proteínas de Ciclo Celular/aislamiento & purificación , Cromatografía de Afinidad , Proteínas de Drosophila/química , Proteínas de Drosophila/aislamiento & purificación , Proteínas Fluorescentes Verdes/química , Microscopía Fluorescente , Multimerización de Proteína , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/aislamiento & purificación , Células Sf9 , Quinasa Tipo Polo 1RESUMEN
OBJECTIVE: To construct a prokaryotic expression vector of human neutrophil gelatinase associated lipocalin (NGAL) and identify the bioactivity of the fusion protein. METHODS: The cDNA of human NGAL obtained from GenBank was linked to a cloning vector to construct the prokaryotic expression vector pCold-NGAL. Then the vector was transformed into E.coli BL21(DE3) plysS. Under the optimal induction condition, the recombinant NGAL (rNGAL) was expressed and purified by Ni Sepharose 6 Fast Flow affinity chromatography. The purity and activity of the rNGAL were respectively identified by SDS-PAGE and Western blotting combined with NGAL reagent (Latex enhanced immunoturbidimetry). RESULTS: Restriction enzyme digestion and nucleotide sequencing proved that the expression vector pCold-NGAL was successfully constructed. Under the optimal induction condition that we determined, the rNGAL was expressed in soluble form in E.coli BL21(DE3) plysS. The relative molecular mass of the rNGAL was 25 000, and its purity was more than 98.0%. Furthermore, Western blotting and immunoturbidimetry indicated that the rNGAL reacted with NGAL mAb specifically. CONCLUSION: Human rNGAL of high purity and bioactivity was successfully constructed in E.coli BL21(DE3) plysS using the expression vector pCold-NGAL.
Asunto(s)
Proteínas de Fase Aguda/genética , Proteínas de Fase Aguda/inmunología , Escherichia coli/genética , Ingeniería Genética/métodos , Lipocalinas/genética , Lipocalinas/inmunología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/inmunología , Proteínas de Fase Aguda/aislamiento & purificación , Clonación Molecular , Expresión Génica , Vectores Genéticos/genética , Humanos , Lipocalina 2 , Lipocalinas/aislamiento & purificación , Proteínas Proto-Oncogénicas/aislamiento & purificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
Methylation of lysine 4 on histone H3 (H3K4) at promoters is tightly linked to transcriptional regulation in human cells. At least six different COMPASS-like multisubunit (SET1/MLL) complexes that contain methyltransferase activity for H3K4 have been described, but a comprehensive and quantitative analysis of these SET1/MLL complexes is lacking. We applied label-free quantitative mass spectrometry to determine the subunit composition and stoichiometry of the human SET1/MLL complexes. We identified both known and novel, unique and shared interactors and determined their distribution and stoichiometry over the different SET1/MLL complexes. In addition to being a core COMPASS subunit, the Dpy30 protein is a genuine subunit of the NURF chromatin remodeling complex. Furthermore, we identified the Bod1 protein as a discriminator between the SET1B and SET1A complexes, and we show that the H3K36me-interactor Psip1 preferentially binds to the MLL2 complex. Finally, absolute protein quantification in crude lysates mirrors many of the observed SET1/MLL complex stoichiometries. Our findings provide a molecular framework for understanding the diversity and abundance of the different SET1/MLL complexes, which together establish the H3K4 methylation landscape in human cells.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas de Neoplasias/metabolismo , Subunidades de Proteína/metabolismo , Proteínas Adaptadoras Transductoras de Señales/aislamiento & purificación , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/aislamiento & purificación , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Cromatografía de Afinidad , Proteínas de Unión al ADN/aislamiento & purificación , Células HeLa , N-Metiltransferasa de Histona-Lisina/aislamiento & purificación , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteína de la Leucemia Mieloide-Linfoide/aislamiento & purificación , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas de Neoplasias/aislamiento & purificación , Proteínas Nucleares/aislamiento & purificación , Proteínas Nucleares/metabolismo , Mapeo de Interacción de Proteínas , Subunidades de Proteína/aislamiento & purificación , Proteínas Proto-Oncogénicas/aislamiento & purificación , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/aislamiento & purificación , Factores de Transcripción/metabolismoRESUMEN
O-linked N-acetylglucosamine (O-GlcNAc) transferase (Ogt) activity is essential for embryonic stem cell (ESC) viability and mouse development. Ogt is present both in the cytoplasm and the nucleus of different cell types and catalyzes serine and threonine glycosylation. We have characterized the biochemical features of nuclear Ogt and identified the ten-eleven translocation (TET) proteins Tet1 and Tet2 as stable partners of Ogt in the nucleus of ESCs. We show at a genome-wide level that Ogt preferentially associates with Tet1 to genes promoters in close proximity of CpG-rich transcription start sites. These regions are characterized by low levels of DNA modification, suggesting a link between Tet1 and Ogt activities in regulating CpG island methylation. Finally, we show that Tet1 is required for binding of Ogt to chromatin affecting Tet1 activity. Taken together, our data characterize how O-GlcNAcylation is recruited to chromatin and interacts with the activity of 5-methylcytosine hydroxylases.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/enzimología , N-Acetilglucosaminiltransferasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Sitios de Unión , Núcleo Celular/metabolismo , Células Cultivadas , Cromatina , Islas de CpG , Proteínas de Unión al ADN/aislamiento & purificación , Dioxigenasas , Células Madre Embrionarias/metabolismo , Regulación de la Expresión Génica , Inmunoprecipitación , Redes y Vías Metabólicas/genética , Ratones , N-Acetilglucosaminiltransferasas/aislamiento & purificación , Regiones Promotoras Genéticas , Unión Proteica , Transporte de Proteínas , Proteínas Proto-Oncogénicas/aislamiento & purificación , Transducción de Señal/genética , Sitio de Iniciación de la TranscripciónRESUMEN
One of the most exciting recent advances in the epigenetic field is the discovery that 5-methylcytosine (5mC) in DNA can be iteratively oxidized by a family of proteins known as Tet proteins to generate 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). These 5mC derivatives can be further processed by thymine-DNA glycosylase (TDG) followed by base excision repair or by replication-dependent dilution leading to DNA demethylation. Given the similarity between 5mC and its oxidation derivatives, many of the conventional techniques used for 5mC analysis cannot distinguish between 5mC and 5hmC/5fC/5caC. Here, we describe 2D-TLC and mass spectrometry methods that we have successfully used in differentiating 5mC from its oxidative derivatives as well as in characterizing the enzymatic activity of Tet proteins both in vitro and in vivo.
Asunto(s)
5-Metilcitosina/química , Metilación de ADN , Proteínas de Unión al ADN/química , Pruebas de Enzimas , Proteínas Proto-Oncogénicas/química , 5-Metilcitosina/aislamiento & purificación , Animales , Secuencia de Bases , Cromatografía en Capa Delgada , ADN/química , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/aislamiento & purificación , Dioxigenasas , Células HEK293 , Humanos , Ratones , Oxidación-Reducción , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/aislamiento & purificación , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
Tumor-derived mutant KRAS (v-Ki-ras-2 Kirsten rat sarcoma viral oncogene) oncoprotein is a critical driver of cancer phenotypes and a potential biomarker for many epithelial cancers. Targeted mass spectrometry analysis by multiple reaction monitoring (MRM) enables selective detection and quantitation of wild-type and mutant KRAS proteins in complex biological samples. A recently described immunoprecipitation approach (Proc. Nat. Acad. Sci.2011, 108, 2444-2449) can be used to enrich KRAS for MRM analysis, but requires large protein inputs (2-4 mg). Here, we describe sodium dodecyl sulfate-polyacrylamide gel electrophoresis-based enrichment of KRAS in a low molecular weight (20-25 kDa) protein fraction prior to MRM analysis (GeLC-MRM). This approach reduces background proteome complexity, thus, allowing mutant KRAS to be reliably quantified in low protein inputs (5-50 µg). GeLC-MRM detected KRAS mutant variants (G12D, G13D, G12V, G12S) in a panel of cancer cell lines. GeLC-MRM analysis of wild-type and mutant was linear with respect to protein input and showed low variability across process replicates (CV = 14%). Concomitant analysis of a peptide from the highly similar HRAS and NRAS proteins enabled correction of KRAS-targeted measurements for contributions from these other proteins. KRAS peptides were also quantified in fluid from benign pancreatic cysts and pancreatic cancers at concentrations from 0.08 to 1.1 fmol/µg protein. GeLC-MRM provides a robust, sensitive approach to quantitation of mutant proteins in complex biological samples.
Asunto(s)
Proteínas Mutantes/metabolismo , Quiste Pancreático/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas ras/metabolismo , Línea Celular Tumoral , Electroforesis en Gel de Poliacrilamida , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/aislamiento & purificación , Fragmentos de Péptidos/química , Proteolisis , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/aislamiento & purificación , Proteínas Proto-Oncogénicas p21(ras) , Estándares de Referencia , Espectrometría de Masas en Tándem/normas , Proteínas ras/química , Proteínas ras/genética , Proteínas ras/aislamiento & purificaciónRESUMEN
Activation of hepatic stellate cells (HSCs) and subsequent uncontrolled accumulation of altered extracellular matrix (ECM) underpin liver fibrosis, a wound healing response to chronic injury, which can lead to organ failure and death. We sought to catalogue the components of fibrotic liver ECM to obtain insights into disease etiology and aid identification of new biomarkers. Cell-derived ECM was isolated from the HSC line LX-2, an in vitro model of liver fibrosis, and compared to ECM from human foreskin fibroblasts (HFFs) as a control. Mass spectrometry analyses of cell-derived ECMs identified, with ≥99% confidence, 61 structural ECM or secreted proteins (48 and 31 proteins for LX-2 and HFF, respectively). Gene ontology enrichment analysis confirmed the enrichment of ECM proteins, and hierarchical clustering coupled with protein-protein interaction network analysis revealed a subset of proteins enriched to fibrotic ECM, highlighting the existence of cell type-specific ECM niches. Thirty-six proteins were enriched to LX-2 ECM as compared to HFF ECM, of which Wnt-5a and CYR61 were validated by immunohistochemistry in human and murine fibrotic liver tissue. Future studies will determine if these and other components may play a role in the etiology of hepatic fibrosis, serve as novel disease biomarkers, or open up new avenues for drug discovery.
Asunto(s)
Proteína 61 Rica en Cisteína/metabolismo , Matriz Extracelular/metabolismo , Células Estrelladas Hepáticas/metabolismo , Cirrosis Hepática/metabolismo , Proteoma/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Wnt/metabolismo , Animales , Línea Celular , Análisis por Conglomerados , Proteína 61 Rica en Cisteína/aislamiento & purificación , Fibroblastos/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , Proteoma/aislamiento & purificación , Proteómica , Proteínas Proto-Oncogénicas/aislamiento & purificación , Proteínas Wnt/aislamiento & purificación , Proteína Wnt-5aRESUMEN
Herein we report a new strategy for highly sensitive and selective colorimatric assay for genotyping of single-nucleotide polymorphisms (SNPs). It is based on the use of a specific gap ligation reaction, horseradish peroxidase (HRP) for signal amplification, and magnetic beads for the easy separation of the ligated product. Briefly, oligonucleotide capture probe functionalized magnetic beads are first hybridized to a target DNA. Biotinylated oligonucleotide detection probes are then allowed to hybridize to the already captured target DNA. A subsequent ligation at the mutation point joins the two probes together. The introduction of streptavidin-conjugated HRP and a simple magnetic separation allow colorimetric genotyping of SNPs. The assay is able to discriminate one copy of mutant in 1000 copies of wild-type KRAS oncogene at 30 picomolar. The detection limit of the assay is further improved to 1 femtomolar by incorporating a ligation chain reaction amplification step, offering an excellent opportunity for the development of a simple and highly sensitive diagnostic tool.
Asunto(s)
Técnicas Biosensibles/métodos , Colorimetría/métodos , Técnicas de Genotipaje/métodos , Proteínas Proto-Oncogénicas , Proteínas ras , Biotina/química , Humanos , Límite de Detección , Imanes/química , Mutación , Sondas de Oligonucleótidos/química , Polimorfismo de Nucleótido Simple , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/aislamiento & purificación , Proteínas Proto-Oncogénicas p21(ras) , Proteínas ras/genética , Proteínas ras/aislamiento & purificaciónRESUMEN
The chromosomal translocation t(8;21) often found in acute myeloid leukemia generates an oncogenic fusion protein AML1-ETO. This chimeric oncoprotein disrupts wild-type AML1 function and dysregulates genes important for normal myelopoiesis. Monoclonal antibodies that can capture and detect the AML1-ETO fusion protein would help with early diagnosis and treatment prognosis of acute myeloid leukemia. We report the development of murine monoclonal antibodies (MAbs) that specifically bind epitopes encoded by either AML1 or ETO. Since alignment to the human ETO protein indicated almost 100% homology to the mouse ortholog, a strategy was needed to instruct humoral immunity in mice to focus and respond to self-epitopes. Our strategy to develop capture/detector reagents involved producing MAbs that would bind to epitopes within the non-fused myelopic protein (i.e., either AML1 or ETO). This included a process to select antibodies for their ability to also recognize the translocated chromosomal AML1-ETO fusion protein and to identify complementary capture/detector antibody pairs. Construction of a peptide hapten-carrier complex and use of a rapid immunization protocol resulted in IgM-IgG ETO specific MAbs. These MAbs bound specifically to a recombinant form of AML1-ETO fusion protein expressed in HEK and to an endogenous AML1-ETO form of the fusion protein expressed in Kasumi-1. We report the development of murine hybridoma MAbs derived from immunizations with a peptide "self-epitope." Our findings provide a potential strategy to instruct humoral immunity in mice to focus and respond to self-epitopes. This strategy has been validated with the oncogenic fusion protein AML1-ETO involved in acute myeloid leukemia.