RESUMO
Protein posttranslational modifications (PTMs) play a central role in the DNA damage response. In particular, protein phosphorylation and ubiquitination have been shown to be essential in the signaling cascade that coordinates break repair with cell cycle progression. Here, we performed whole-cell quantitative proteomics to identify global changes in protein ubiquitination that are induced by DNA double-strand breaks. In total, we quantified more than 9,400 ubiquitin sites and found that the relative abundance of â¼10% of these sites was altered in response to DNA double-strand breaks. Interestingly, a large proportion of ribosomal proteins, including those from the 40S as well as the 60S subunit, were ubiquitinated in response to DNA damage. In parallel, we discovered that DNA damage leads to the inhibition of ribosome function. Taken together, these data uncover the ribosome as a major target of the DNA damage response.
Assuntos
Quebras de DNA de Cadeia Dupla , Doxorrubicina/farmacologia , Biossíntese de Proteínas/fisiologia , Processamento de Proteína Pós-Traducional , Proteínas Ribossômicas/metabolismo , Ubiquitinação/fisiologia , Linhagem Celular Tumoral , Inibidores de Cisteína Proteinase/metabolismo , Fase G2/fisiologia , Humanos , Leupeptinas/metabolismo , Espectrometria de Massas , Proteínas Nucleares/metabolismo , Nucleofosmina , Fosfoproteínas/metabolismo , Fosforilação , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo , Proteínas Ubiquitinadas/metabolismo , NucleolinaRESUMO
Inhibition of the microtubule (MT) motor protein Eg5 results in a mitotic arrest due to the formation of monopolar spindles, making Eg5 an attractive target for anti-cancer therapies. However, Eg5-independent pathways for bipolar spindle formation exist, which might promote resistance to treatment with Eg5 inhibitors. To identify essential components for Eg5-independent bipolar spindle formation, we performed a genome-wide siRNA screen in Eg5-independent cells (EICs). We find that the kinase Aurora A and two kinesins, MCAK and Kif18b, are essential for bipolar spindle assembly in EICs and in cells with reduced Eg5 activity. Aurora A promotes bipolar spindle assembly by phosphorylating Kif15, hereby promoting Kif15 localization to the spindle. In turn, MCAK and Kif18b promote bipolar spindle assembly by destabilizing the astral MTs. One attractive way to interpret our data is that, in the absence of MCAK and Kif18b, excessive astral MTs generate inward pushing forces on centrosomes at the cortex that inhibit centrosome separation. Together, these data suggest a novel function for astral MTs in force generation on spindle poles and how proteins involved in regulating microtubule length can contribute to bipolar spindle assembly.
Assuntos
Aurora Quinase A/metabolismo , Cinesinas/metabolismo , Microtúbulos , Fuso Acromático , Estudo de Associação Genômica Ampla , Células HeLa , Humanos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Mitose , RNA Interferente Pequeno/genética , Fuso Acromático/metabolismoRESUMO
Affinity purification coupled with mass spectrometry (AP-MS) is a widely used approach for the identification of protein-protein interactions. However, for any given protein of interest, determining which of the identified polypeptides represent bona fide interactors versus those that are background contaminants (for example, proteins that interact with the solid-phase support, affinity reagent or epitope tag) is a challenging task. The standard approach is to identify nonspecific interactions using one or more negative-control purifications, but many small-scale AP-MS studies do not capture a complete, accurate background protein set when available controls are limited. Fortunately, negative controls are largely bait independent. Hence, aggregating negative controls from multiple AP-MS studies can increase coverage and improve the characterization of background associated with a given experimental protocol. Here we present the contaminant repository for affinity purification (the CRAPome) and describe its use for scoring protein-protein interactions. The repository (currently available for Homo sapiens and Saccharomyces cerevisiae) and computational tools are freely accessible at http://www.crapome.org/.
Assuntos
Cromatografia de Afinidade/métodos , Espectrometria de Massas/métodos , Mapeamento de Interação de Proteínas/métodos , Proteínas/análise , Proteômica/métodos , Bases de Dados Factuais , HumanosRESUMO
Non-ionic surfactants such as Polysorbate 20/ 80 (PS20/ PS80), are commonly used in protein drug formulations to increase protein stability by protecting against interfacial stress and surface absorption. Polysorbate is susceptible to degradation which can impact product stability, leading to the formation of sub-visible and/or visible particles in the drug product during its shelf-life, affecting patient safety and efficacy. Therefore, it is important to monitor polysorbate concentration in drug product formulations of biotherapeutic drugs. The common method for measuring polysorbate concentration in drug product formulations uses mixed mode ion exchange reversed phase HPLC (MAX) coupled to evaporative light scattering detection (ELSD). However, high protein concentration can adversely impact method performance due to high sample viscosity, gel formation, column clogging, interfering peaks and loss of accuracy. To overcome this, a new method was developed based on EDTA mediated ethanol protein precipitation (EDTA/EtOH). This method was successfully implemented for the analysis of polysorbate in antibody formulations with wide range of protein concentration (10-250â¯mg/mL).
Assuntos
Precipitação Química , Ácido Edético , Etanol , Polissorbatos , Tensoativos , Polissorbatos/química , Polissorbatos/análise , Ácido Edético/química , Etanol/química , Tensoativos/química , Cromatografia Líquida de Alta Pressão/métodos , Proteínas/análise , Proteínas/química , Química Farmacêutica/métodos , Estabilidade Proteica , Produtos Biológicos/análise , Produtos Biológicos/químicaRESUMO
The forkhead transcription factor FoxM1 controls expression of a large number of genes that are specifically expressed during the G(2) phase of the cell cycle. Throughout most of the cell cycle, FoxM1 activity is restrained by an autoinhibitory mechanism, involving a repressor domain present in the N-terminal part of the protein. Activation of FoxM1 in G(2) is achieved by Cyclin A/Cyclin-dependent kinase (Cdk)-mediated phosphorylation, which alleviates autoinhibition by the N-terminal repressor domain. Here, we show that FoxM1 interacts with B55α, a regulatory subunit of protein phosphatase 2A (PP2A). B55α binds the catalytic subunit of PP2A, and this promotes dephosphorylation and inactivation of FoxM1. Indeed, we find that overexpression of B55α results in decreased FoxM1 activity. Inversely, depletion of B55α results in premature activation of FoxM1. The activation of FoxM1 that is observed upon depletion of B55α is fully dependent on Cyclin A/Cdk-mediated phosphorylation of FoxM1. Taken together, these data demonstrate that B55α acts to antagonize Cyclin A/Cdk-dependent activation of FoxM1, to ensure that FoxM1 activity is restricted to the G(2) phase of the cell cycle.
Assuntos
Ciclina A/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Proteína Fosfatase 2/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Proteína Forkhead Box M1 , Fatores de Transcrição Forkhead/química , Fatores de Transcrição Forkhead/deficiência , Fatores de Transcrição Forkhead/genética , Humanos , Dados de Sequência Molecular , Peptídeos/química , Fosforilação , Ligação Proteica , Transcrição GênicaRESUMO
Activation of the DNA-damage checkpoint culminates in the inhibition of cyclin-dependent kinase (Cdk) complexes to prevent cell-cycle progression. We have shown recently that Cdk activity is required for activation of the Forkhead transcription factor FoxM1, an important regulator of gene expression in the G2 phase of the cell cycle. Here, we show that FoxM1 is transcriptionally active during a DNA-damage-induced G2 arrest and is essential for checkpoint recovery. Paradoxically, Cdk activity, although reduced after checkpoint activation, is required to maintain FoxM1-dependent transcription during the arrest and for expression of pro-mitotic targets such as cyclin A, cyclin B and Plk1. Indeed, we find that cells need to retain sufficient levels of Cdk activity during the DNA-damage response to maintain cellular competence to recover from a DNA-damaging insult.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Dano ao DNA , Fatores de Transcrição Forkhead/genética , Fase G2 , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/antagonistas & inibidores , Proteína Forkhead Box M1 , Fatores de Transcrição Forkhead/metabolismo , Humanos , Transcrição GênicaRESUMO
To elucidate the molecular mechanisms underlying pathogen-associated molecular pattern (PAMP)-induced defense responses in potato (Solanum tuberosum), the role of the signaling compounds salicylic acid (SA) and jasmonic acid (JA) was analyzed. Pep-13, a PAMP from Phytophthora, induces the accumulation of SA, JA and hydrogen peroxide, as well as the activation of defense genes and hypersensitive-like cell death. We have previously shown that SA is required for Pep-13-induced defense responses. To assess the importance of JA, RNA interference constructs targeted at the JA biosynthetic genes, allene oxide cyclase and 12-oxophytodienoic acid reductase, were expressed in transgenic potato plants. In addition, expression of the F-box protein COI1 was reduced by RNA interference. Plants expressing the RNA interference constructs failed to accumulate the respective transcripts in response to wounding or Pep-13 treatment, neither did they contain significant amounts of JA after elicitation. In response to infiltration of Pep-13, the transgenic plants exhibited a highly reduced accumulation of reactive oxygen species as well as reduced hypersensitive cell death. The ability of the JA-deficient plants to accumulate SA suggests that SA accumulation is independent or upstream of JA accumulation. These data show that PAMP responses in potato require both SA and JA and that, in contrast to Arabidopsis, these compounds act in the same signal transduction pathway. Despite their inability to fully respond to PAMP treatment, the transgenic RNA interference plants are not altered in their basal defense against Phytophthora infestans.
Assuntos
Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Doenças das Plantas/genética , Reguladores de Crescimento de Plantas/metabolismo , Ácido Salicílico/metabolismo , Solanum tuberosum/metabolismo , Morte Celular , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/metabolismo , Phytophthora infestans/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Solanum tuberosum/genéticaRESUMO
Being able to rapidly and sensitively detect specific enzymatic products is important when screening biological samples for enzymatic activity. We present a simple method for assaying protease activity in the presence of protease inhibitors (PIs) by measuring tryptic peptide accumulation on copolymer pMALDI target chips using a dual fluorescence/MALDI-TOF-MS read-out. The small platform of the chip accommodates microliter amounts of sample and allows for rapid protein digestion. Fluorescamine labeling of tryptic peptides is used to indicate the proteolytic activity and is shown to be an affordable, simple process, yielding a strong fluorescence signal with a low background. Subsequent MALDI-TOF-MS analysis, performed in the same sample well, or in a parallel well without adding fluorescamine, detects the specific tryptic peptides and provides confidence in the assay. The dual read-out method was applied to screen the inhibition activity of plant PIs, components of plant defense against herbivores and pathogens. Extracts of PIs from Solanum nigrum and trypsin were applied together to a pMALDI chip on which a suitable substrate was adsorbed. The fluorescence and MALDI-TOF-MS signal decrease were associated with the inhibitory effect of the PIs on trypsin. The developed platform can be modified to screen novel protease inhibitors, namely, those potentially useful for treating or preventing infection by viruses, including HIV and hepatitis C.
Assuntos
Peptídeo Hidrolases/análise , Peptídeo Hidrolases/metabolismo , Análise Serial de Proteínas/métodos , Solanum nigrum/enzimologia , Fluorescamina/química , Peptídeos/metabolismo , Proteínas de Plantas/análise , Proteínas de Plantas/metabolismo , Inibidores de Proteases/análise , Inibidores de Proteases/metabolismo , Análise Serial de Proteínas/instrumentação , Ribulose-Bifosfato Carboxilase/metabolismo , Soroalbumina Bovina/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/instrumentação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Tripsina/metabolismoRESUMO
The importance of the signaling compound salicylic acid for basal defense of potato (Solanum tuberosum L. cv. Désirée) against Phytophthora infestans, the causal agent of late blight disease, was assessed using transgenic NahG potato plants which are unable to accumulate salicylic acid. Although the size of lesions caused by P. infestans was not significantly different in wild-type and transgenic NahG plants, real-time polymerase chain reaction analyses revealed a drastic enhancement of pathogen growth in potato plants depleted of salicylic acid. Increased susceptibility of NahG plants correlated with compromised callose formation and reduced early defense gene expression. NahG plants pretreated with the salicylic acid analog 2,6-dichloro-isonicotinic acid allowed pathogen growth to a similar extent as did wild-type plants, indicating that salicylic acid is an important compound required for basal defense of potato against P. infestans.
Assuntos
Phytophthora/fisiologia , Doenças das Plantas/microbiologia , Ácido Salicílico/metabolismo , Solanum tuberosum/metabolismo , Solanum tuberosum/microbiologia , Biomassa , Regulação da Expressão Gênica de Plantas , Ácidos Isonicotínicos/farmacologia , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Folhas de Planta/microbiologia , Folhas de Planta/ultraestrutura , Plantas Geneticamente Modificadas , Solanum tuberosum/efeitos dos fármacos , Solanum tuberosum/genéticaRESUMO
How cells recover from a DNA damage-induced arrest is currently poorly understood. We performed large-scale quantitative phosphoproteomics to identify changes in protein phosphorylation that occurred during recovery from arrest in the G2 phase of the cell cycle caused by DNA damage. We identified 154 proteins that were differentially phosphorylated, and systematic depletion of each of these differentially phosphorylated proteins by small interfering RNA (siRNA) identified at least 10 potential regulators of recovery. Astrin, a protein associated with the mitotic spindle, was among the potential regulators of recovery. We found that astrin controlled the abundance of the cell cycle regulator p53 during DNA damage-induced arrest. Cells in which astrin was depleted had decreased murine double minute 2 (MDM2) abundance and increased p53 at the later stages of the DNA damage response. Astrin was required for continued expression of genes encoding proteins that promote cell cycle progression in arrested cells. Thus, by controlling p53 abundance in cells recovering from DNA damage, astrin maintains the cells in a state competent to resume the cell cycle.
Assuntos
Dano ao DNA , Pontos de Checagem da Fase G2 do Ciclo Celular , Fosfoproteínas/metabolismo , Linhagem Celular Tumoral , Humanos , Fosfoproteínas/genética , Fosforilação , Proteômica , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/genética , Fuso Acromático/genética , Fuso Acromático/metabolismoRESUMO
Cells are constantly challenged by DNA damage and protect their genome integrity by activation of an evolutionary conserved DNA damage response pathway (DDR). A central core of DDR is composed of a spatiotemporally ordered net of post-translational modifications, among which protein phosphorylation plays a major role. Activation of checkpoint kinases ATM/ATR and Chk1/2 leads to a temporal arrest in cell cycle progression (checkpoint) and allows time for DNA repair. Following DNA repair, cells re-enter the cell cycle by checkpoint recovery. Wip1 phosphatase (also called PPM1D) dephosphorylates multiple proteins involved in DDR and is essential for timely termination of the DDR. Here we have investigated how Wip1 is regulated in the context of the cell cycle. We found that Wip1 activity is downregulated by several mechanisms during mitosis. Wip1 protein abundance increases from G(1) phase to G(2) and declines in mitosis. Decreased abundance of Wip1 during mitosis is caused by proteasomal degradation. In addition, Wip1 is phosphorylated at multiple residues during mitosis, and this leads to inhibition of its enzymatic activity. Importantly, ectopic expression of Wip1 reduced γH2AX staining in mitotic cells and decreased the number of 53BP1 nuclear bodies in G(1) cells. We propose that the combined decrease and inhibition of Wip1 in mitosis decreases the threshold necessary for DDR activation and enables cells to react adequately even to modest levels of DNA damage encountered during unperturbed mitotic progression.
Assuntos
Dano ao DNA , Regulação da Expressão Gênica/fisiologia , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Mitose/fisiologia , Fosfoproteínas Fosfatases/metabolismo , Transdução de Sinais/fisiologia , Linhagem Celular Tumoral , Primers do DNA/genética , Imunofluorescência , Humanos , Espectrometria de Massas , Fosforilação , Proteína Fosfatase 2C , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , TransfecçãoRESUMO
Metastatic breast cancer is the major cause of cancer-related death among women in the Western world. Invasive carcinoma cells are able to counteract apoptotic signals in the absence of anchorage, enabling cell survival during invasion and dissemination. Although loss of E-cadherin is a cardinal event in the development and progression of invasive lobular carcinoma (ILC), little is known about the underlying mechanisms that govern these processes. Using a mouse model of human ILC, we show here that cytosolic p120-catenin (p120) regulates tumor growth upon loss of E-cadherin through the induction of anoikis resistance. p120 conferred anchorage independence by indirect activation of Rho/Rock signaling through interaction and inhibition of myosin phosphatase Rho-interacting protein (Mrip), an antagonist of Rho/Rock function. Consistent with these data, primary human ILC samples expressed hallmarks of active Rock signaling, and Rock controlled the anoikis resistance of human ILC cells. Thus, we have linked loss of E-cadherin - an initiating event in ILC development - to Rho/Rock-mediated control of anchorage-independent survival. Because activation of Rho and Rock are strongly linked to cancer progression and are susceptible to pharmacological inhibition, these insights may have clinical implications for the development of tailor-made intervention strategies to better treat invasive and metastatic lobular breast cancer.
Assuntos
Carcinoma Lobular/metabolismo , Cateninas/fisiologia , Regulação Neoplásica da Expressão Gênica , Regulação da Expressão Gênica , Quinases Associadas a rho/metabolismo , Animais , Anoikis , Caderinas/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Progressão da Doença , Feminino , Humanos , Camundongos , Metástase Neoplásica , Transporte Proteico , delta CateninaRESUMO
Natural variation of plant pathogen resistance is often quantitative. This type of resistance can be genetically dissected in quantitative resistance loci (QRL). To unravel the molecular basis of QRL in potato (Solanum tuberosum), we employed the model plant Arabidopsis thaliana for functional analysis of natural variants of potato allene oxide synthase 2 (StAOS2). StAOS2 is a candidate gene for QRL on potato chromosome XI against the oömycete Phytophthora infestans causing late blight, and the bacterium Erwinia carotovora ssp. atroseptica causing stem black leg and tuber soft rot, both devastating diseases in potato cultivation. StAOS2 encodes a cytochrome P450 enzyme that is essential for biosynthesis of the defense signaling molecule jasmonic acid. Allele non-specific dsRNAi-mediated silencing of StAOS2 in potato drastically reduced jasmonic acid production and compromised quantitative late blight resistance. Five natural StAOS2 alleles were expressed in the null Arabidopsis aos mutant under control of the Arabidopsis AOS promoter and tested for differential complementation phenotypes. The aos mutant phenotypes evaluated were lack of jasmonates, male sterility and susceptibility to Erwinia carotovora ssp. carotovora. StAOS2 alleles that were associated with increased disease resistance in potato complemented all aos mutant phenotypes better than StAOS2 alleles associated with increased susceptibility. First structure models of 'quantitative resistant' versus 'quantitative susceptible' StAOS2 alleles suggested potential mechanisms for their differential activity. Our results demonstrate how a candidate gene approach in combination with using the homologous Arabidopsis mutant as functional reporter can help to dissect the molecular basis of complex traits in non model crop plants.