RESUMO
Dormant hematopoietic stem cells (dHSCs) are atop the hematopoietic hierarchy. The molecular identity of dHSCs and the mechanisms regulating their maintenance or exit from dormancy remain uncertain. Here, we use single-cell RNA sequencing (RNA-seq) analysis to show that the transition from dormancy toward cell-cycle entry is a continuous developmental path associated with upregulation of biosynthetic processes rather than a stepwise progression. In addition, low Myc levels and high expression of a retinoic acid program are characteristic for dHSCs. To follow the behavior of dHSCs in situ, a Gprc5c-controlled reporter mouse was established. Treatment with all-trans retinoic acid antagonizes stress-induced activation of dHSCs by restricting protein translation and levels of reactive oxygen species (ROS) and Myc. Mice maintained on a vitamin A-free diet lose HSCs and show a disrupted re-entry into dormancy after exposure to inflammatory stress stimuli. Our results highlight the impact of dietary vitamin A on the regulation of cell-cycle-mediated stem cell plasticity. VIDEO ABSTRACT.
Assuntos
Células-Tronco Hematopoéticas/citologia , Transdução de Sinais , Tretinoína/farmacologia , Vitamina A/administração & dosagem , Animais , Vias Biossintéticas , Técnicas de Cultura de Células , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular , Dieta , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/efeitos dos fármacos , Camundongos , Poli I-C/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Análise de Célula Única , Estresse Fisiológico , Vitamina A/farmacologia , Vitaminas/administração & dosagem , Vitaminas/farmacologiaRESUMO
Peroxiredoxins are central to cellular redox homeostasis and signaling. They serve as peroxide scavengers, sensors, signal transducers, and chaperones, depending on conditions and context. Typical 2-Cys peroxiredoxins are known to switch between different oligomeric states, depending on redox state, pH, posttranslational modifications, and other factors. Quaternary states and their changes are closely connected to peroxiredoxin activity and function but so far have been studied, almost exclusively, outside the context of the living cell. Here we introduce the use of homo-FRET (Förster resonance energy transfer between identical fluorophores) fluorescence polarization to monitor dynamic changes in peroxiredoxin quaternary structure inside the crowded environment of living cells. Using the approach, we confirm peroxide- and thioredoxin-related quaternary transitions to take place in cellulo and observe that the relationship between dimer-decamer transitions and intersubunit disulfide bond formation is more complex than previously thought. Furthermore, we demonstrate the use of the approach to compare different peroxiredoxin isoforms and to identify mutations and small molecules affecting the oligomeric state inside cells. Mutagenesis experiments reveal that the dimer-decamer equilibrium is delicately balanced and can be shifted by single-atom structural changes. We show how to use this insight to improve the design of peroxiredoxin-based redox biosensors.
Assuntos
Peroxirredoxinas/química , Linhagem Celular , Transferência Ressonante de Energia de Fluorescência , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mutação , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Multimerização Proteica/efeitos dos fármacos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Genetically encoded probes based on the H2O2-sensing proteins OxyR and Orp1 have greatly increased the ability to detect elevated H2O2 levels in stimulated or stressed cells. However, these proteins are not sensitive enough to monitor metabolic H2O2 baseline levels. Using yeast as a platform for probe development, we developed two peroxiredoxin-based H2O2 probes, roGFP2-Tsa2ΔCR and roGFP2-Tsa2ΔCPΔCR, that afford such sensitivity. These probes are â¼50% oxidized under 'normal' unstressed conditions and are equally responsive to increases and decreases in H2O2. Hence, they permit fully dynamic, real-time measurement of basal H2O2 levels, with subcellular resolution, in living cells. We demonstrate that expression of these probes does not alter endogenous H2O2 homeostasis. The roGFP2-Tsa2ΔCR probe revealed real-time interplay between basal H2O2 levels and partial oxygen pressure. Furthermore, it exposed asymmetry in H2O2 trafficking between the cytosol and mitochondrial matrix and a strong correlation between matrix H2O2 levels and cellular growth rate.
Assuntos
Peróxido de Hidrogênio/análise , Peróxido de Hidrogênio/metabolismo , Sondas Moleculares/metabolismo , Peroxirredoxinas/metabolismo , Citosol/metabolismo , Homeostase , Mitocôndrias/metabolismo , Oxigênio/metabolismo , Pressão Parcial , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Fatores de TempoRESUMO
Pkh is the yeast ortholog of the mammalian 3-phosphoinositide-dependent protein kinase 1 (PDK1). Pkh phosphorylates the activation loop of Ypks, Tpks, Sch9 and also phosphorylates the eisosome components Lsp1 and Pil1, which play fundamental roles upstream of diverse signaling pathways, including the cell wall integrity and sphingosine/long-chain base (LCB) signaling pathways. In S. cerevisiae, two isoforms, ScPkh1 and ScPkh2, are required for cell viability, while only one ortholog exists in C. albicans, CaPkh2. In spite of the extensive information gathered on the role of Pkh in the LCB signaling, the yeast Pkh kinases are not known to bind lipids and previous studies did not identify PH domains in Pkh sequences. We now describe that the C-terminal region of CaPkh2 is required for its intrinsic kinase activity. In addition, we found that the C-terminal region of CaPkh2 enables its interaction with structural and signaling lipids. Our results further show that phosphatidylserine, phosphatidic acid, phosphatidylinositol (3,4 and 4,5)-biphosphates, and phosphatidylinositol (3,4,5)-trisphosphate inhibit Pkh activity, whereas sulfatide binds with high affinity but does not affect the intrinsic activity of CaPkh2. Interestingly, we identified that its human ortholog PDK1 also binds to sulfatide. We propose a mechanism by which lipids and dihydrosphingosine regulate CaPkh2 kinase activity by modulating the interaction of the C-terminal region with the kinase domain, while sulfatide-like lipids support localization CaPkh2 mediated by a C-terminal PH domain, without affecting kinase intrinsic activity.
Assuntos
Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Candida albicans/enzimologia , Proteínas Fúngicas/metabolismo , Metabolismo dos Lipídeos , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/genética , Sequência de Aminoácidos , Sítios de Ligação , Candida albicans/genética , Biologia Computacional , Células HEK293 , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ácidos Fosfatídicos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilserinas/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Relação Estrutura-Atividade , Sulfoglicoesfingolipídeos/metabolismo , TransfecçãoRESUMO
BACKGROUND: Pkh proteins are the PDK1 orthologs in S. cerevisiae. They have redundant and essential activity and are responsible for the phosphorylation of several members of the AGC family of protein kinases. Pkh proteins have been involved in several cellular functions, including cell wall integrity and endocytosis. However the global expression changes caused by their depletion are still unknown. RESULTS: A doxycycline-repressible tetO7 promoter driving the expression of PKH2 in cells carrying deletions of the PKH1 and PKH3 genes allowed us to progressively deplete cells from Pkh proteins when treated with doxycycline. Global gene expression analysis indicate that depletion of Pkh results in the up-regulation of genes involved in the accumulation of glycogen and also of those related to stress responses. Moreover, genes involved in the ion transport were quickly down-regulated when the levels of Pkh decreased. The reduction in the mRNA levels required for protein translation, however, was only observed after longer doxycycline treatment (24 h). We uncovered that Pkh is important for the proper transcriptional response to heat shock, and is mostly required for the effects driven by the transcription factors Hsf1 and Msn2/Msn4, but is not required for down-regulation of the mRNA coding for ribosomal proteins. CONCLUSIONS: By using the tetO7 promoter we elucidated for the first time the transcriptomic changes directly or indirectly caused by progressive depletion of Pkh. Furthermore, this system enabled the characterization of the transcriptional response triggered by heat shock in wild-type and Pkh-depleted cells, showing that about 40 % of the observed expression changes were, to some degree, dependent on Pkh.
Assuntos
Proteínas Quinases Dependentes de 3-Fosfoinositídeo/genética , Regulação Fúngica da Expressão Gênica , Saccharomyces cerevisiae/genética , Estresse Fisiológico/genética , Transcrição Gênica , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Análise por Conglomerados , Fermentação , Perfilação da Expressão Gênica , Genes Letais , Glicogênio/metabolismo , Resposta ao Choque Térmico/genética , Íons , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The group of AGC protein kinases includes more than 60 protein kinases in the human genome, classified into 14 families: PDK1, AKT/PKB, SGK, PKA, PKG, PKC, PKN/PRK, RSK, NDR, MAST, YANK, DMPK, GRK and SGK494. This group is also widely represented in other eukaryotes, including causative organisms of human infectious diseases. AGC kinases are involved in diverse cellular functions and are potential targets for the treatment of human diseases such as cancer, diabetes, obesity, neurological disorders, inflammation and viral infections. Small molecule inhibitors of AGC kinases may also have potential as novel therapeutic approaches against infectious organisms. Fundamental in the regulation of many AGC kinases is a regulatory site termed the "PIF-pocket" that serves as a docking site for substrates of PDK1. This site is also essential to the mechanism of activation of AGC kinases by phosphorylation and is involved in the allosteric regulation of N-terminal domains of several AGC kinases, such as PKN/PRKs and atypical PKCs. In addition, the C-terminal tail and its interaction with the PIF-pocket are involved in the dimerization of the DMPK family of kinases and may explain the molecular mechanism of allosteric activation of GRKs by GPCR substrates. In this review, we briefly introduce the AGC kinases and their known roles in physiology and disease and the discovery of the PIF-pocket as a regulatory site in AGC kinases. Finally, we summarize the current status and future therapeutic potential of small molecules directed to the PIF-pocket; these molecules can allosterically activate or inhibit the kinase as well as act as substrate-selective inhibitors. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).
Assuntos
Modelos Moleculares , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Regulação Alostérica , Candida albicans/efeitos dos fármacos , Candida albicans/enzimologia , Candidíase/tratamento farmacológico , Candidíase/microbiologia , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/classificaçãoRESUMO
The activation of at least 23 different mammalian kinases requires the phosphorylation of their hydrophobic motifs by the kinase PDK1. A linker connects the phosphoinositide-binding PH domain to the catalytic domain, which contains a docking site for substrates called the PIF pocket. Here, we used a chemical biology approach to show that PDK1 existed in equilibrium between at least three distinct conformations with differing substrate specificities. The inositol polyphosphate derivative HYG8 bound to the PH domain and disrupted PDK1 dimerization by stabilizing a monomeric conformation in which the PH domain associated with the catalytic domain and the PIF pocket was accessible. In the absence of lipids, HYG8 potently inhibited the phosphorylation of Akt (also termed PKB) but did not affect the intrinsic activity of PDK1 or the phosphorylation of SGK, which requires docking to the PIF pocket. In contrast, the small-molecule valsartan bound to the PIF pocket and stabilized a second distinct monomeric conformation. Our study reveals dynamic conformations of full-length PDK1 in which the location of the linker and the PH domain relative to the catalytic domain determines the selective phosphorylation of PDK1 substrates. The study further suggests new approaches for the design of drugs to selectively modulate signaling downstream of PDK1.
Assuntos
Mamíferos , Polifosfatos , Animais , Especificidade por Substrato , Fosforilação , Domínio Catalítico , DimerizaçãoRESUMO
Aims: Whether H2O2 contributes to the glucose-dependent stimulation of insulin secretion (GSIS) by pancreatic ß cells is highly controversial. We used two H2O2-sensitive probes, roGFP2-Orp1 (reduction/oxidation-sensitive enhanced green fluorescent protein fused to oxidant receptor peroxidase 1) and HyPer (hydrogen peroxide sensor) with its pH-control SypHer, to test the acute effects of glucose, monomethyl succinate, leucine with glutamine, and α-ketoisocaproate on ß cell cytosolic and mitochondrial H2O2 concentrations. We then tested the effects of low H2O2 and menadione concentrations on insulin secretion. Results: RoGFP2-Orp1 was more sensitive than HyPer to H2O2 (response at 2-5 vs. 10 µM) and less pH-sensitive. Under control conditions, stimulation with glucose reduced mitochondrial roGFP2-Orp1 oxidation without affecting cytosolic roGFP2-Orp1 and HyPer fluorescence ratios, except for the pH-dependent effects on HyPer. However, stimulation with glucose decreased the oxidation of both cytosolic probes by 15 µM exogenous H2O2. The glucose effects were not affected by overexpression of catalase, mitochondrial catalase, or superoxide dismutase 1 and 2. They followed the increase in NAD(P)H autofluorescence, were maximal at 5 mM glucose in the cytosol and 10 mM glucose in the mitochondria, and were partly mimicked by the other nutrients. Exogenous H2O2 (1-15 µM) did not affect insulin secretion. By contrast, menadione (1-5 µM) did not increase basal insulin secretion but reduced the stimulation of insulin secretion by 20 mM glucose. Innovation: Subcellular changes in ß cell H2O2 levels are better monitored with roGFP2-Orp1 than HyPer/SypHer. Nutrients acutely lower mitochondrial H2O2 levels in ß cells and promote degradation of exogenously supplied H2O2 in both cytosolic and mitochondrial compartments. Conclusion: The GSIS occurs independently of a detectable increase in ß cell cytosolic or mitochondrial H2O2 levels.
Assuntos
Citosol/efeitos dos fármacos , Glucose/farmacologia , Peróxido de Hidrogênio/antagonistas & inibidores , Células Secretoras de Insulina/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Animais , Citosol/metabolismo , Glucose/metabolismo , Peróxido de Hidrogênio/metabolismo , Células Secretoras de Insulina/metabolismo , Masculino , Mitocôndrias/metabolismo , Oxirredução , Ratos , Ratos WistarRESUMO
Mitochondrially generated oxidants are believed to play important roles in both physiology and pathophysiology. Therefore, it is of significant interest to better understand the metabolic conditions leading to enhanced mitochondrial oxidant generation. Here, we investigate the influence of oxygen and glucose availability on the redox state of peroxiredoxin-based redox probes, expressed in the cytosol and mitochondrial matrix of yeast cells. We observe that the redox state of peroxiredoxin probes reflects the balance between dioxygen-dependent peroxide generation and glucose-dependent generation of reducing equivalents. The oxidative pentose phosphate pathway appears to be the dominant source of NADPH in the system under study.
RESUMO
There is a current and pressing need for improved cancer therapies. The use of small molecule kinase inhibitors and their application in combinatorial regimens represent an approach to personalized targeted cancer therapy. A number of AGC kinases, including atypical Protein Kinase C enzymes (PKCs), are validated drug targets for cancer treatment. Most drug development programs for protein kinases focus on the development of drugs that bind at the ATP-binding site. Alternatively, allosteric drugs have great potential for the development of future innovative drugs. However, the rational development of allosteric drugs poses important challenges because the compounds not only must bind to a given site but also must stabilize forms of the protein with a desired effect at a distant site. Here we describe the development of a new class of compounds targeting a regulatory site (PIF-pocket) present in the kinase domain and provide biochemical and crystallographic data showing that these compounds allosterically inhibit the activity of atypical PKCs. PS432, a representative compound, decreased the rate of proliferation of non-small cell lung cancer cells more potently than aurothiomalate, an atypical PKCι inhibitor currently under evaluation in clinical trials, and significantly reduced tumor growth without side effects in a mouse xenograft model. The druglike chemical class provides ample possibilities for the synthesis of derivative compounds, with the potential to allosterically modulate the activity of atypical PKCs and other kinases.
Assuntos
Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Regulação Alostérica , Animais , Linhagem Celular Tumoral , Feminino , Xenoenxertos , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos NusRESUMO
SCOPE: Many phytochemicals with beneficial pharmacological properties contain electrophilic sites, e.g. α,ß-unsaturated carbonyl (enone) groups. There is increasing evidence that many biological effects of electrophilic compounds depend on covalent conjugation to reactive protein thiols. For example, the reaction of electrophiles with cysteinyl residues of the sensor protein Keap1 activates the cell-protective Nrf2 response. Thus it is of interest to identify more generally the proteins to which small molecule electrophiles bind covalently. METHODS AND RESULTS: Here we use a Click chemistry approach to identify target proteins of the chemopreventive phytochemical xanthohumol (XN), an enone-containing chalcone from hops (Humulus lupulus L.). Using an alkynylated analog of XN (XN-alkyne), we purified covalent protein-electrophile conjugates from cell lysates. We confirm the previously described conjugation of XN to Keap1. One of the newly identified candidate target proteins is glucose-6-phosphate dehydrogenase (G6PDH). We confirm that XN attenuates intracellular G6PDH activity at low micromolar concentrations. CONCLUSION: We find support for the notion that XN modulates multiple pathways and processes by covalent modification of proteins with reactive cysteines.
Assuntos
Química Click/métodos , Flavonoides/química , Flavonoides/metabolismo , Propiofenonas/química , Propiofenonas/metabolismo , Proteínas/metabolismo , Alcinos/química , Alcinos/metabolismo , Cisteamina/química , Flavonoides/farmacologia , Glucosefosfato Desidrogenase/antagonistas & inibidores , Glucosefosfato Desidrogenase/química , Glucosefosfato Desidrogenase/metabolismo , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/química , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Espectroscopia de Ressonância Magnética , Peso Molecular , Propiofenonas/farmacologia , Proteínas/química , Solubilidade , Espectrometria de Massas por Ionização por Electrospray , Compostos de Sulfidrila/químicaRESUMO
Hydrogen sulfide signaling involves persulfide formation at specific protein Cys residues. However, overcoming current methodological challenges in persulfide detection and elucidation of Cys regeneration mechanisms from persulfides are prerequisites for constructing a bona fide signaling model. We here establish a novel, highly specific protein persulfide detection protocol, ProPerDP, with which we quantify 1.52 ± 0.6 and 11.6 ± 6.9 µg/mg protein steady-state protein persulfide concentrations in human embryonic kidney 293 (HEK293) cells and mouse liver, respectively. Upon treatment with polysulfides, HEK293 and A549 cells exhibited increased protein persulfidation. Deletion of the sulfide-producing cystathionine-γ-lyase or cystathionine-ß-synthase enzymes in yeast diminished protein persulfide levels, thereby corroborating their involvement in protein persulfidation processes. We here establish that thioredoxin (Trx) and glutathione (GSH) systems can independently catalyze reductions of inorganic polysulfides and protein persulfides. Increased endogenous persulfide levels and protein persulfidation following polysulfide treatment in thioredoxin reductase-1 (TrxR1) or thioredoxin-related protein of 14 kDa (TRP14) knockdown HEK293 cells indicated that these enzymes constitute a potent regeneration system of Cys residues from persulfides in a cellular context. Furthermore, TrxR1-deficient cells were less viable upon treatment with toxic amounts of polysulfides compared to control cells. Emphasizing the dominant role of cytosolic disulfide reduction systems in maintaining sulfane sulfur homeostasis in vivo, protein persulfide levels were markedly elevated in mouse livers where hepatocytes lack both TrxR1 and glutathione reductase (TR/GR-null). The different persulfide patterns observed in wild-type, GR-null, and TR/GR-null livers suggest distinct roles for the Trx and GSH systems in regulating subsets of protein persulfides and thereby fine-tuning sulfide signaling pathways.
Assuntos
Glutationa/metabolismo , Sulfetos/metabolismo , Tiorredoxinas/metabolismo , Animais , Cistationina beta-Sintase/metabolismo , Cistationina gama-Liase/metabolismo , Glutationa Redutase/metabolismo , Células HEK293 , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Masculino , Camundongos , Ratos , Tiorredoxina Redutase 1/metabolismoRESUMO
The phosphoinositide-dependent protein kinase 1, PDK1, is a master kinase that phosphorylates the activation loop of up to 23 AGC kinases. S. cerevisiae has three PDK1 orthologues, Pkh1-3, which also phosphorylate AGC kinases (e.g., Ypk, Tpk, Pkc1, and Sch9). Pkh1 and 2 are redundant proteins involved in multiple essential cellular functions, including endocytosis and cell wall integrity. Based on similarities with the budding yeast, the Pkh of fungal infectious species was postulated as a novel target for antifungals. Here, we found that depletion of Pkh eventually induces oxidative stress and DNA double-strand breaks, leading to programmed cell death. This finding supports Pkh as an antifungal target since pharmacological inhibition of Pkh would lead to the death of yeast cells, the ultimate goal of antifungals. It was therefore of interest to further investigate the possibility to develop Pkh inhibitors with selectivity for Candida Pkh that would not inhibit the human ortholog. Here, we describe C. albicans Pkh2 biochemically, structurally and by using chemical probes in comparison to human PDK1. We found that a regulatory site on the C. albicans Pkh2 catalytic domain, the PIF-pocket, diverges from human PDK1. Indeed, we identified and characterized PS77, a new small allosteric inhibitor directed to the PIF-pocket, which has increased selectivity for C. albicans Pkh2. Together, our results describe novel features of the biology of Pkh and chemical biology approaches that support the validation of Pkh as a drug target for selective antifungals.