RESUMO
BACKGROUND: The global warming potential of inhaled medication depends on the applied inhaler. Pressurised metered dose inhalers (pMDI) contain green-house gases (GHG) and are associated with a 10 to 40 times higher CO2-footprint than GHG-free dry-powder inhalers (DPI). AIM: Feasibility and relevance of prescription conversion from pMDI to DPI were investigated in a pulmonology outpatient clinic regarding the CO2-footprint and the economic costs under real-world conditions. METHODS: Based on exemplary therapy regimens of different intensity for three patients, the annual CO2-footprint and daily therapy costs were investigated. The effect of converting from pMDI to DPI on CO2-footprint and economic costs were calculated on the basis of prescriptions during the first quarter of 2020 compared to the first quarter of 2021. RESULTS: Conversion of a pMDI-based inhalative therapy of exemplary asthma and COPD patients to a DPI-based therapy saved between 115 and 480 kg CO2 equivalents (CO2e) per year and patient depending on intensity of therapy and GHG used. A total of 184,297 and 164,165 defined daily doses (DDD) were prescribed by the clinic for 2,610 (January-March 2020) and 2,693 (January-March 2021) patients, respectively. The proportion of DPI prescribed increased from 49 to 78% of total inhaler prescriptions. The increase in prescriptions for single-agent inhaled corticosteroids from 19.8 to 74.1% of total inhaler prescriptions was particularly striking. Due to the conversion, emissions were reduced by 35,000 to 40,000 kg CO2e between January-March 2020 and January-March 2021 in our clinic. During the same period, there was no increase in costs compared to nationwide costs. The relation of prescribed DPI and pMDI in the same period did not change among the pulmonologists in Saxony nor nationwide in Germany. If all ambulant pulmonologists in Germany would prescribe 75% DPI, CO2-emissions could be reduced by 11,650 tonnes CO2e per quarter and 46,600 tonnes CO2e per year, respectively. CONCLUSION: The type of inhalers can be converted from pMDI to DPI in a real-world setting. Thereby, a significant reduction of GHG emissions is possible without increased costs.
Assuntos
Asma , Gases de Efeito Estufa , Doença Pulmonar Obstrutiva Crônica , Administração por Inalação , Asma/tratamento farmacológico , Broncodilatadores , Dióxido de Carbono/uso terapêutico , Inaladores de Pó Seco , Humanos , Inaladores Dosimetrados , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológicoRESUMO
The delivery of newly synthesized soluble lysosomal hydrolases to the endosomal system is essential for lysosome function and cell homeostasis. This process relies on the proper trafficking of the mannose 6-phosphate receptors (MPRs) between the trans-Golgi network (TGN), endosomes and the plasma membrane. Many transmembrane proteins regulating diverse biological processes ranging from virus production to the development of multicellular organisms also use these pathways. To explore how cell signaling modulates MPR trafficking, we used high-throughput RNA interference (RNAi) to target the human kinome and phosphatome. Using high-content image analysis, we identified 127 kinases and phosphatases belonging to different signaling networks that regulate MPR trafficking and/or the dynamic states of the subcellular compartments encountered by the MPRs. Our analysis maps the MPR trafficking pathways based on enzymes regulating phosphatidylinositol phosphate metabolism. Furthermore, it reveals how cell signaling controls the biogenesis of post-Golgi tubular carriers destined to enter the endosomal system through a SRC-dependent pathway regulating ARF1 and RAC1 signaling and myosin II activity.
Assuntos
Membrana Celular/enzimologia , Endossomos/enzimologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Interferência de RNA , Receptor IGF Tipo 2/metabolismo , Rede trans-Golgi/enzimologia , Fator 1 de Ribosilação do ADP/genética , Fator 1 de Ribosilação do ADP/metabolismo , Análise por Conglomerados , Regulação Enzimológica da Expressão Gênica , Redes Reguladoras de Genes , Células HeLa , Humanos , Fosfatos de Fosfatidilinositol/metabolismo , Mapas de Interação de Proteínas , Transporte Proteico/genética , Receptor IGF Tipo 2/genética , Transdução de Sinais , Transfecção , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Class IB phosphoinositide 3-kinases γ (PI3Kγ) are second-messenger-generating enzymes downstream of signalling cascades triggered by G-protein-coupled receptors (GPCRs). PI3Kγ variants have one catalytic p110γ subunit that can form two different heterodimers by binding to one of a pair of non-catalytic subunits, p87 or p101. Growing experimental data argue for a different regulation of p87-p110γ and p101-p110γ allowing integration into distinct signalling pathways. Pharmacological tools enabling distinct modulation of the two variants are missing. The ability of an anti-p110γ monoclonal antibody [mAb(A)p110γ] to block PI3Kγ enzymatic activity attracted us to characterize this tool in detail using purified proteins. In order to get insight into the antibody-p110γ interface, hydrogen-deuterium exchange coupled to MS (HDX-MS) measurements were performed demonstrating binding of the monoclonal antibody to the C2 domain in p110γ, which was accompanied by conformational changes in the helical domain harbouring the Gßγ-binding site. We then studied the modulation of phospholipid vesicles association of PI3Kγ by the antibody. p87-p110γ showed a significantly reduced Gßγ-mediated phospholipid recruitment as compared with p101-p110γ. Concomitantly, in the presence of mAb(A)p110γ, Gßγ did not bind to p87-p110γ. These data correlated with the ability of the antibody to block Gßγ-stimulated lipid kinase activity of p87-p110γ 30-fold more potently than p101-p110γ. Our data argue for differential regulatory functions of the non-catalytic subunits and a specific Gßγ-dependent regulation of p101 in PI3Kγ activation. In this scenario, we consider the antibody as a valuable tool to dissect the distinct roles of the two PI3Kγ variants downstream of GPCRs.
Assuntos
Anticorpos Monoclonais Murinos/química , Classe Ib de Fosfatidilinositol 3-Quinase , Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Animais , Classe Ib de Fosfatidilinositol 3-Quinase/química , Classe Ib de Fosfatidilinositol 3-Quinase/genética , Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Medição da Troca de Deutério , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Células Sf9 , SpodopteraRESUMO
The initial step of bone digestion is the adhesion of osteoclasts onto bone surfaces and the assembly of podosomal belts that segregate the bone-facing ruffled membrane from other membrane domains. During bone digestion, membrane components of the ruffled border also need to be recycled after macropinocytosis of digested bone materials. How osteoclast polarity and membrane recycling are coordinated remains unknown. Here, we show that the Cdc42-guanine nucleotide exchange factor FGD6 coordinates these events through its Src-dependent interaction with different actin-based protein networks. At the plasma membrane, FGD6 couples cell adhesion and actin dynamics by regulating podosome formation through the assembly of complexes comprising the Cdc42-interactor IQGAP1, the Rho GTPase-activating protein ARHGAP10, and the integrin interactors Talin-1/2 or Filamin A. On endosomes and transcytotic vesicles, FGD6 regulates retromer-dependent membrane recycling through its interaction with the actin nucleation-promoting factor WASH. These results provide a mechanism by which a single Cdc42-exchange factor controlling different actin-based processes coordinates cell adhesion, cell polarity, and membrane recycling during bone degradation.
Assuntos
Endossomos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Membranas Intracelulares/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Animais , Osso e Ossos/metabolismo , Adesão Celular , Linhagem Celular , Polaridade Celular , Fatores de Troca do Nucleotídeo Guanina/genética , Camundongos , Ligação Proteica , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Numerous transport processes occur between the two mitochondrial (mt) membranes due to the diverse functions and metabolic processes of the mt organelle. The metabolite and ion transport through the mt outer membrane (OM) is widely assumed to be mediated by the porin pore, whereas in the mt inner membrane (IM) specific carriers are responsible for transport processes. Here, we provide evidence by means of Blue Native (BN)-PAGE analysis, co-immunoprecipitation, and tandem affinity purification that the two mt OM proteins Om14p and Om45p associate with the porin pore. Porin molecules seem to assemble independently to build the core unit. A subpopulation of these core units interacts with Om14p and Om45p. With preparative tandem affinity purification followed by MS analysis, we could identify interaction partners of this OM complex, which are mainly localized within the mt IM and function as carriers for diverse molecules. We propose a model for the role of the two OM proteins in addressing the porin pore to bind to specific channels in the mt IM to facilitate transport of metabolites.
Assuntos
Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Porinas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico/fisiologia , Proteínas Mitocondriais/genética , Porinas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Bone digestion occurs when osteoclasts adhere onto bone surfaces and polarize to form acidic, hydrolase-rich resorption lacunae. For this process, they condense their actin-rich podosomes in tight belts to establish sealing zones, which segregate their basal membranes from those facing resorption lacunae. This polarization process remains poorly understood. Here, we combined quantitative proteomics and gene silencing to identify new substrates of the Src tyrosine kinase, a key regulator of osteoclast function. We now report that a depletion of the ARF GTPase-activating protein GIT2, which localizes to sealing zones upon Src phosphorylation, or a lack of GTP hydrolysis on ARF6 impairs sealing zone formation and polarized membrane traffic. Surprisingly, the Rho guanine nucleotide exchange factors alpha and beta PIX, which usually coordinate ARF and Rho signaling, were found to be dispensable. We conclude that the Src-dependent localization of GIT2 is essential for down-regulating ARF6 activity at sealing zones, and thus for maintaining osteoclast polarity.
Assuntos
Fatores de Ribosilação do ADP/genética , Osso e Ossos/metabolismo , Osteoclastos/metabolismo , Quinases da Família src/metabolismo , Fator 6 de Ribosilação do ADP , Animais , Reabsorção Óssea , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Cromatografia Líquida , Regulação para Baixo , Proteínas Ativadoras de GTPase , Guanosina Trifosfato/metabolismo , Hidrólise , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Osteoclastos/enzimologia , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica , Espectrometria de Massas em TandemRESUMO
Glucose and cAMP-inducing agents such as 3-isobutyl-1-methylxanthine (IBMX) rapidly change the expression profile of insulin-producing pancreatic beta-cells mostly through post-transcriptional mechanisms. A thorough analysis of these changes, however, has not yet been performed. By combining two-dimensional differential gel electrophoresis and mass spectrometry, we identified 165 spots, corresponding to 78 proteins, whose levels significantly change after stimulation of the beta-cell model INS-1 cells with 25 mM glucose + 1 mM IBMX for 2 h. Changes in the expression of selected proteins were verified by one- and two-dimensional immunoblotting. Most of the identified proteins are novel targets of rapid regulation in beta-cells. The transcription inhibitor actinomycin D failed to block changes in two-thirds of the spots, supporting their post-transcriptional regulation. More spots changed in response to IBMX than to glucose alone conceivably because of phosphorylation. Fourteen mRNA- binding proteins responded to stimulation, thus representing the most prominent class of rapidly regulated proteins. Bioinformatics analysis indicated that the mRNA 5'- and 3'-untranslated regions of 22 regulated proteins contain potential binding sites for polypyrimidine tract-binding protein 1, which promotes mRNA stability and translation in stimulated beta-cells. Overall our findings support the idea that mRNA-binding proteins play a major role in rapid adaptive changes in insulin-producing cells following their stimulation with glucose and cAMP-elevating agents.
Assuntos
1-Metil-3-Isobutilxantina/farmacologia , Glucose/farmacologia , Insulinoma/metabolismo , Insulinoma/patologia , Proteínas de Ligação a RNA/metabolismo , Motivos de Aminoácidos , Animais , Sítios de Ligação , Western Blotting , Biologia Computacional , Sequência Conservada , Eletroforese em Gel Bidimensional , Ribonucleoproteínas Nucleares Heterogêneas , Humanos , Espectrometria de Massas , Camundongos , Proteínas Musculares/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas , Proteômica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Ratos , Reprodutibilidade dos Testes , Vesículas Secretórias/efeitos dos fármacos , Vesículas Secretórias/metabolismo , Regiões não Traduzidas/genéticaRESUMO
Receptor-regulated class I phosphoinositide 3-kinases (PI3K) phosphorylate the membrane lipid phosphatidylinositol (PtdIns)-4,5-P2 to PtdIns-3,4,5-P3. This, in turn, recruits and activates cytosolic effectors with PtdIns-3,4,5-P3-binding pleckstrin homology (PH) domains, thereby controlling important cellular functions such as proliferation, survival, or chemotaxis. The class IB p110 gamma/p101 PI3K gamma is activated by G beta gamma on stimulation of G protein-coupled receptors. It is currently unknown whether in living cells G beta gamma acts as a membrane anchor or an allosteric activator of PI3K gamma, and which role its noncatalytic p101 subunit plays in its activation by G beta gamma. Using GFP-tagged PI3K gamma subunits expressed in HEK cells, we show that G beta gamma recruits the enzyme from the cytosol to the membrane by interaction with its p101 subunit. Accordingly, p101 was found to be required for G protein-mediated activation of PI3K gamma in living cells, as assessed by use of GFP-tagged PtdIns-3,4,5-P3-binding PH domains. Furthermore, membrane-targeted p110 gamma displayed basal enzymatic activity, but was further stimulated by G beta gamma, even in the absence of p101. Therefore, we conclude that in vivo, G beta gamma activates PI3K gamma by a mechanism assigning specific roles for both PI3K gamma subunits, i.e., membrane recruitment is mediated via the noncatalytic p101 subunit, and direct stimulation of G beta gamma with p110 gamma contributes to activation of PI3K gamma.
Assuntos
Membrana Celular/metabolismo , Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Proteínas Heterotriméricas de Ligação ao GTP/fisiologia , Proteínas de Membrana , Fosfatidilinositol 3-Quinases/metabolismo , Sítio Alostérico , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Linhagem Celular , Cromatografia em Gel , Dimerização , Relação Dose-Resposta a Droga , Transferência de Energia , Proteínas de Fluorescência Verde , Humanos , Immunoblotting , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Modelos Biológicos , Fosforilação , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Fluorescência , TransfecçãoRESUMO
Clathrin/adaptor protein-1-coated carriers connect the secretory and the endocytic pathways. Carrier biogenesis relies on distinct protein networks changing membrane shape at the trans-Golgi network, each regulating coat assembly, F-actin-based mechanical forces, or the biophysical properties of lipid bilayers. How these different hubs are spatiotemporally coordinated remains largely unknown. Using in vitro reconstitution systems, quantitative proteomics, and lipidomics, as well as in vivo cell-based assays, we characterize the protein networks controlling membrane lipid composition, membrane shape, and carrier scission. These include PIP5K1A and phospholipase C-beta 3 controlling the conversion of PI[4]P into diacylglycerol. PIP5K1A binding to RAC1 provides a link to F-actin-based mechanical forces needed to tubulate membranes. Tubular membranes then recruit the BAR-domain-containing arfaptin-1/2 guiding carrier scission. These findings provide a framework for synchronizing the chemical/biophysical properties of lipid bilayers, F-actin-based mechanical forces, and the activity of proteins sensing membrane shape during clathrin/adaptor protein-1-coated carrier biogenesis.
Assuntos
Actinas/metabolismo , Complexo 1 de Proteínas Adaptadoras/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Metabolismo dos Lipídeos , Animais , Fenômenos Biomecânicos , Proteínas de Transporte/metabolismo , Clatrina/metabolismo , Diglicerídeos/biossíntese , Células HeLa , Humanos , Camundongos , Fosfatos de Fosfatidilinositol/metabolismo , Fosfolipase C beta/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Polimerização , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Bone resorption in vertebrates relies on the ability of osteoclasts to assemble F-actin-rich podosomes that condense into podosomal belts, forming sealing zones. Sealing zones segregate bone-facing ruffled membranes from other membrane domains, and disassemble when osteoclasts migrate to new areas. How podosome/sealing zone dynamics is regulated remains unknown. We illustrate the essential role of the membrane scaffolding F-BAR-Proline-Serine-Threonine Phosphatase Interacting Proteins (PSTPIP) 1 and 2 in this process. Whereas PSTPIP2 regulates podosome assembly, PSTPIP1 regulates their disassembly. PSTPIP1 recruits, through its F-BAR domain, the protein tyrosine phosphatase non-receptor type 6 (PTPN6) that de-phosphophorylates the phosphatidylinositol 5-phosphatases SHIP1/2 bound to the SH3 domain of PSTPIP1. Depletion of any component of this complex prevents sealing zone disassembly and increases osteoclast activity. Thus, our results illustrate the importance of BAR domain proteins in podosome structure and dynamics, and identify a new PSTPIP1/PTPN6/SHIP1/2-dependent negative feedback mechanism that counterbalances Src and PI(3,4,5)P3 signalling to control osteoclast cell polarity and activity during bone resorption.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Proteínas do Citoesqueleto/metabolismo , Osteoclastos/metabolismo , Osteoclastos/patologia , Podossomos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/genética , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Camundongos , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Domínios Proteicos , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Proteômica , Células RAW 264.7 , Interferência de RNARESUMO
The Saccharomyces cerevisiae kinase Sat4p has been originally identified as a protein involved in salt tolerance and stabilization of plasma membrane transporters, implicating a cytoplasmic localization. Our study revealed an additional mitochondrial (mt) localization, suggesting a dual function for Sat4p. While no mt related phenotype was observed in the absence of Sat4p, its overexpression resulted in significant changes of a specific mitochondrial subproteome. As shown by a comparative two dimensional difference gel electrophoresis (2D-DIGE) approach combined with mass spectrometry, particularly two groups of proteins were affected: the iron-sulfur containing aconitase-type proteins (Aco1p, Lys4p) and the lipoamide-containing subproteome (Lat1p, Kgd2p and Gcv3p). The lipoylation sites of all three proteins could be assigned by nanoLC-MS/MS to Lys75 (Lat1p), Lys114 (Kgd2p) and Lys102 (Gcv3p), respectively. Sat4p overexpression resulted in accumulation of the delipoylated protein variants and in reduced levels of aconitase-type proteins, accompanied by a decrease in the activities of the respective enzyme complexes. We propose a regulatory role of Sat4p in the late steps of the maturation of a specific subset of mitochondrial iron-sulfur cluster proteins, including Aco1p and lipoate synthase Lip5p. Impairment of the latter enzyme may account for the observed lipoylation defects.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitocôndrias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Citoplasma/metabolismo , Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , Proteoma , Proteômica/métodos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The activity of yeast pyruvate dehydrogenase complex is regulated by reversible phosphorylation. Recently we identified two enzymes that are involved in the phosphorylation (Pkp1p) and dephosphorylation (Ppp1p) of Pda1p, the alpha-subunit of the pyruvate dehydrogenase complex. Here we provide evidence that two additional mitochondrial proteins, Pkp2p (Ygl059wp) and Ppp2p (Ycr079wp), are engaged in the regulation of this complex by affecting the phosphorylation state of Pda1p. Our data indicate complementary activities of the kinases and a redundant function for the phosphatases. Both proteins are associated with the complex. We propose a model for the role of the regulatory enzymes and the phosphorylation state of Pda1p in the assembly process of the pyruvate dehydrogenase complex.
Assuntos
Modelos Biológicos , Fosfoproteínas Fosfatases/metabolismo , Proteínas Quinases/metabolismo , Complexo Piruvato Desidrogenase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Fosfoproteínas Fosfatases/genética , Fosforilação , Proteínas Quinases/genética , Complexo Piruvato Desidrogenase/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The AP-3 adaptor complex targets selected transmembrane proteins to lysosomes and lysosome-related organelles. We reconstituted its preferred interaction with liposomes containing the ADP ribosylation factor (ARF)-1 guanosine triphosphatase (GTPase), specific cargo tails, and phosphatidylinositol-3 phosphate, and then we performed a proteomic screen to identify new proteins supporting its sorting function. We identified approximately 30 proteins belonging to three networks regulating either AP-3 coat assembly or septin polymerization or Rab7-dependent lysosomal transport. RNA interference shows that, among these proteins, the ARF-1 exchange factor brefeldin A-inhibited exchange factor 1, the ARF-1 GTPase-activating protein 1, the Cdc42-interacting Cdc42 effector protein 4, an effector of septin-polymerizing GTPases, and the phosphatidylinositol-3 kinase IIIC3 are key components regulating the targeting of lysosomal membrane proteins to lysosomes in vivo. This analysis reveals that these proteins, together with AP-3, play an essential role in protein sorting at early endosomes, thereby regulating the integrity of these organelles.
Assuntos
Complexo 3 de Proteínas Adaptadoras/metabolismo , Proteínas de Membrana Lisossomal/metabolismo , Fator 1 de Ribosilação do ADP/metabolismo , Proteínas do Citoesqueleto/metabolismo , Células HeLa , Humanos , Lipossomos/química , Lipossomos/metabolismo , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Ligação Proteica , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteômica , RNA Interferente Pequeno/metabolismo , Termodinâmica , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
Coat components localize to specific membrane domains, where they sort selected transmembrane proteins. To study how clathrin coats are stabilized on such domains and to identify the protein networks involved, we combined proteomic screens and in vitro liposome-based assays that recapitulate the fidelity of protein sorting in vivo. Our study identifying approximately 40 proteins on AP-1A-coated liposomes revealed that AP-1A coat assembly triggers the concomitant recruitment of Rac1, its effectors, and the Wave/Scar complex as well as that of Rab11 and Rab14. The coordinated recruitment of these different machineries requires a mosaic of membrane components comprising the GTPase ADP-ribosylation factor 1, sorting signals in selected transmembrane proteins, and phosphatidylinositol 4-phosphate. These results demonstrate that the combinatorial use of low-affinity binding sites present on the same membrane domain accounts not only for a selective coat assembly but also for the coordinated assembly of selected machineries required for actin polymerization and subsequent membrane fusion.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Proteômica , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Fusão de Membrana , Dados de Sequência Molecular , Fosfatos de Fosfatidilinositol/farmacologia , Ligação Proteica , Suínos , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Osteoclasts, the bone-digesting cells, are polarized cells that secrete acid hydrolases into a resorption lacuna where bone degradation takes place. The molecular mechanisms underlying this process are poorly understood. To analyze the nature of acid hydrolases secreted by osteoclasts, we used the mouse myeloid Raw 264.7 cell line that differentiates in vitro into mature osteoclasts in the presence of the receptor activator of NF-kappaB ligand. Upon differentiation, we observed a strong increase in the secretion of mannose 6-phosphate-containing acid hydrolases. A proteomic analysis of the secreted proteins captured on a mannose 6-phosphate receptor affinity column revealed 58 different proteins belonging to several families of acid hydrolases of which 16 are clearly involved in bone homeostasis. Moreover these acid hydrolases were secreted as proproteins. The expression of most of the identified acid hydrolases is unchanged during osteoclastogenesis. Thus, our data strongly support the notion that the polarized secretion of acid hydrolases by osteoclasts results from a reorganization of key steps of membrane traffic along the lysosomal pathway rather than from a fusion of lysosomes with the membrane facing the resorption lacuna.
Assuntos
Osso e Ossos/metabolismo , Hidrolases/análise , Lisossomos/enzimologia , Osteoclastos/enzimologia , Animais , Proteínas de Transporte/metabolismo , Catepsina D/metabolismo , Diferenciação Celular , Cromatografia de Afinidade , Perfilação da Expressão Gênica , Glicoproteínas de Membrana/metabolismo , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Transporte Proteico , Proteômica , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-B , Receptor IGF Tipo 2/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Osteoclasts, the bone-digesting cells, are key players in bone remodeling. To identify proteins potentially involved in osteoclast function, we analyzed the patterns of protein expression during osteoclastogenesis by2-D DIGE. As a model system we used the mouse myeloid Raw 264.7 cell line that differentiates in vitro into osteoclasts upon treatment with specific growth factors. In 2-D DIGE, we identified 86 up- and 34 down-regulated proteins including known osteoclast differentiation markers as well as proteins regulating key cellular functions of osteoclasts such as energy production, cytoskeleton dynamics, and digestion of organic and inorganic bone matrix. Comparison of protein expression using 2-D DIGE techniques with mRNA expression analyzed by DNA microarrays revealed essentially two groups of genes. The first group comprises genes for which differences in both mRNA and protein expressions were found. A second group covers genes whose expression was not altered at the mRNA level but whose corresponding gene products exhibited different electrophoretic mobilities, thereby revealing potential changes in post-transcriptional processing and PTM. Thus, these combined approaches identify new potential therapeutic targets for treatment of bone diseases and provide complementary information on regulatory processes that might affect osteoclastogenesis.
Assuntos
Osteoclastos/citologia , Proteínas/metabolismo , RNA Mensageiro/metabolismo , Animais , Proteínas de Transporte/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Eletroforese em Gel Bidimensional , Regulação da Expressão Gênica , Glicoproteínas de Membrana/farmacologia , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Osteoclastos/metabolismo , Proteínas/isolamento & purificação , Ligante RANK , RNA Mensageiro/isolamento & purificação , Receptor Ativador de Fator Nuclear kappa-BRESUMO
This article describes the identification of the autophosphorylation sites of the G protein-sensitive class I phosphoinositide 3-kinase isoforms beta and gamma by mass spectrometry. Since discrimination and suppression effects prevented the immediate detection and sequencing of phosphopeptides in complex mixtures, a strategy was applied that involved (32)P-radiolabeling of the phosphoproteins, cleavage of the phosphoproteins with several proteases and/or cyanogen bromide, separation of the resulting peptide mixtures by micro-reversed-phase liquid chromatography, and mass spectrometric analysis of fractions containing phosphopeptides. As a result the primary autophosphorylation sites of phosphoinositide 3-kinase p110beta and p110gamma subunits could be unambiguously assigned to the C-terminal Ser 1070 and Ser 1101, respectively.
Assuntos
Fosfatidilinositol 3-Quinases/classificação , Fosfatidilinositol 3-Quinases/metabolismo , Fosfosserina/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Cromatografia Líquida , Isoenzimas/química , Isoenzimas/classificação , Isoenzimas/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosfatidilinositol 3-Quinases/química , Fosforilação , TripsinaRESUMO
Class I phosphoinositide 3-kinases (PI3Ks) are bifunctional enzymes possessing lipid kinase activity and the capacity to phosphorylate their catalytic and/or regulatory subunits. In this study, in vitro autophosphorylation of the G protein-sensitive p85-coupled class I(A) PI3K beta and p101-coupled class I(B) PI3K gamma was examined. Autophosphorylation sites of both PI3K isoforms were mapped to C-terminal serine residues of the catalytic p110 subunit (i.e. serine 1070 of p110 beta and serine 1101 of p110 gamma). Like other class I(A) PI3K isoforms, autophosphorylation of p110 beta resulted in down-regulated PI3K beta lipid kinase activity. However, no inhibitory effect of p110 gamma autophosphorylation on PI3K gamma lipid kinase activity was observed. Moreover, PI3K beta and PI3K gamma differed in the regulation of their autophosphorylation. Whereas p110 beta autophosphorylation was stimulated neither by G beta gamma complexes nor by a phosphotyrosyl peptide derived from the platelet-derived growth factor receptor, autophosphorylation of p110 gamma was significantly enhanced by G beta gamma in a time- and concentration-dependent manner. In summary, we show that autophosphorylation of both PI3K beta and PI3K gamma occurs in a C-terminal region of the catalytic p110 subunit but differs in its regulation and possible functional consequences, suggesting distinct roles of autophosphorylation of PI3K beta and PI3K gamma.
Assuntos
Isoenzimas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Catálise , Linhagem Celular , Primers do DNA , Humanos , Isoenzimas/química , Dados de Sequência Molecular , Fosfatidilinositol 3-Quinases/química , Fosforilação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Serina/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
We have previously shown that alkyl-substituted amino acid derivatives directly activate G(i/o) proteins. N-Dodecyl-N(alpha),N(epsilon)-(bis-l-lysinyl)-l-lysine amide (FUB132) is a new representative of this class of compounds with increased efficacy. Here, we characterized the molecular mechanism of action of this class of compounds. FUB132 and its predecessor FUB86 were selective receptomimetics for G(i/o) because they stimulated the guanine nucleotide exchange reaction of purified G(i/o) as documented by an increased rate of GDP release, GTP gamma S binding, and GTP hydrolysis. In contrast to the receptomimetic peptide mastoparan, stimulation of G proteins by lipoamines required the presence of neither G beta gamma-dimers nor lipids. On the contrary, G beta gamma-dimers suppressed the stimulatory effect of FUB132. The stimulation of G(i/o) by lipoamines and by mastoparan was not additive. A peptide derived from the C terminus of G alpha(o3), but not a corresponding G alpha(q)-derived peptide, quenched the FUB132-induced activation of G alpha(o). In membranes prepared from human embryonic kidney 293 cells that stably expressed the G(i/o)-coupled human A(1)-adenosine receptor, lipoamines impeded high-affinity agonist binding. In contrast, antagonist binding was not affected. We conclude that alkyl-substituted amines target a site, most likely at the C terminus of G alpha(i/o)-subunits, that is also contacted by receptors. However, because G beta gamma-dimers blunt rather than enhance their efficacy, their mechanism of action differs fundamentally from that of a receptor. Thus, despite their receptomimetic effect in vitro, alkyl-substituted amines and related polyamines are poor direct G protein activators in vivo. In the presence of G beta gamma, they rather antagonize G protein-coupled receptor signaling.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Lisina/farmacologia , Proteínas de Ligação ao GTP/antagonistas & inibidores , Células HL-60 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Lisina/análogos & derivados , Peptídeos , Ensaio Radioligante , Transdução de Sinais/efeitos dos fármacos , Venenos de Vespas/farmacologiaRESUMO
Migration of endothelial cells (EC) is a key event in angiogenesis that contributes to neovascularization in diabetic vasculopathy. Leptin induces angiogenesis and is elevated in obesity and hyperinsulinemia. The antidiabetic thiazolidinediones (TZD) inhibit leptin gene expression and vascular smooth muscle cell migration through activation of the peroxisome proliferator-activated receptor-gamma (PPARgamma). This study investigates the role of leptin in EC migration, the chemotactic signaling pathways involved, and the effects of the TZD-PPARgamma ligands troglitazone (TRO) and ciglitazone (CIG) on EC migration. We demonstrate that leptin induces EC migration. Because activation of two signaling pathways, the phosphatidylinositol-3 kinase (PI3K)-->Akt-->eNOS and the ERK1/2 MAPK pathway, is known to be involved in cell migration, we used the pharmacological inhibitors wortmannin and PD98059 to determine if chemotactic signaling by leptin involves Akt or ERK1/2, respectively. Both wortmannin and PD98059 significantly inhibited leptin-induced migration. Treatment with the TZD-PPARgamma-ligands TRO and CIG significantly inhibited the chemotactic response toward leptin. Both PPARgamma-ligands inhibited leptin-stimulated Akt and eNOS phosphorylation, but neither attenuated ERK 1/2 activation in response to leptin. The inhibition of Akt-phosphorylation was accompanied by a PPARgamma-ligand-mediated upregulation of PTEN, a phosphatase that functions as a negative regulator of PI3K-->Akt signaling. These experiments provide the first evidence that activation of Akt and ERK 1/2 are crucial events in leptin-mediated signal transduction leading to EC migration. Moreover, inhibition of leptin-directed migration by the PPARgamma-ligands TRO and CIG through inhibition of Akt underscores their potential in the prevention of diabetes-associated complications.