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1.
Pharmacotherapy ; 39(7): 778-782, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31077424

RESUMO

Therapeutic drug monitoring (TDM) of vancomycin is commonly performed using immunoassays. This case describes falsely elevated vancomycin serum concentrations, possibly secondary to endogenous protein interference. Vancomycin was prescribed for a patient with a suspected septic knee. A blood sample for TDM was inadvertently collected before the first dose. The reported concentration was 36.1 mg/L using the Roche Modular P analyzer and remained high over the next 48 hours and 8 months later in the absence of vancomycin therapy. Vancomycin was undetectable in the patient sample by liquid chromatography-tandem mass spectrometry. The sample was subsequently investigated for endogenous protein interference. The responsible interference was removed by polyethylene glycol precipitation and heat inactivation. Four alternative immunoassays with varying test principles measured vancomycin concentrations ranging from undetectable to 108 mg/L. A glucose-6-phosphate dehydrogenase detection method was common to the two immunoassays exhibiting the greatest interference. To our knowledge, this is the first report of falsely elevated vancomycin concentrations on the Roche Modular P analyzer. Immunoassays are generally robust in facilitating TDM but are susceptible to cross-reactivity. Assay interference should be considered and laboratory professionals contacted when vancomycin levels do not correlate with clinical expectations.

5.
Can J Gastroenterol Hepatol ; 2017: 1450970, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28491862

RESUMO

Background. Pediatric inflammatory bowel disease (IBD) is on the rise worldwide. Endoscopies are necessary for IBD assessment but are invasive, expensive, and inconvenient. Recently, fecal calprotectin (FCal) was proposed as a noninvasive and specific marker of gut inflammation. We evaluated the analytical performance of three FCal assays and their clinical performance in predicting relapse in pediatric IBD. Methods. This study used 40 pediatric IBD and 40 random non-IBD patients' fecal samples. Two automated ELISAs (Bühlmann and PhiCal® Calprotectin-EIA) and an EliA (Phadia 250 EliA-Calprotectin) were used to evaluate the analytical performance. The clinical performance was assessed by PhiCal Calprotectin-EIA, EliA-Calprotectin, and Bühlmann immunochromatographic point-of-care test (POCT). Results. All assays displayed acceptable analytical performance below and above the medical decision cut-off [imprecision (CV < 10% intra-assay; <15% interassay); linearity (overall mean % deviation < 16.5%)]. The agreement with PhiCal Calprotectin-EIA was 100% and 78.6% for Bühlmann (95% CI, 87.5-100; Kappa: 1) and EliA-Calprotectin (95% CI, 60.5-89.8; Kappa: 0.32), respectively, and 63.6% between Bühlmann and EliA-Calprotectin (95% CI, 46.6-77.8; Kappa: 0.16). All assays evaluated had similar clinical performance [AUC: 0.84 (EliA-Calprotectin); 0.83 (POCT and PhiCal Calprotectin-EIA)]. Conclusion. FCal levels determined using the same method and assay together with clinical history would be a noninvasive and useful tool in monitoring pediatric IBD.


Assuntos
Fezes/química , Doenças Inflamatórias Intestinais/diagnóstico , Complexo Antígeno L1 Leucocitário/análise , Adolescente , Biomarcadores/análise , Criança , Diagnóstico Diferencial , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoensaio/métodos , Masculino , Valor Preditivo dos Testes , Recidiva , Reprodutibilidade dos Testes , Estudos Retrospectivos
6.
Mol Syst Biol ; 13(3): 918, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28298427

RESUMO

G-protein-coupled receptors (GPCRs) are the largest family of integral membrane receptors with key roles in regulating signaling pathways targeted by therapeutics, but are difficult to study using existing proteomics technologies due to their complex biochemical features. To obtain a global view of GPCR-mediated signaling and to identify novel components of their pathways, we used a modified membrane yeast two-hybrid (MYTH) approach and identified interacting partners for 48 selected full-length human ligand-unoccupied GPCRs in their native membrane environment. The resulting GPCR interactome connects 686 proteins by 987 unique interactions, including 299 membrane proteins involved in a diverse range of cellular functions. To demonstrate the biological relevance of the GPCR interactome, we validated novel interactions of the GPR37, serotonin 5-HT4d, and adenosine ADORA2A receptors. Our data represent the first large-scale interactome mapping for human GPCRs and provide a valuable resource for the analysis of signaling pathways involving this druggable family of integral membrane proteins.


Assuntos
Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Receptores Acoplados a Proteínas-G/metabolismo , Membrana Celular/metabolismo , Humanos , Receptor A2A de Adenosina/metabolismo , Receptores 5-HT4 de Serotonina/metabolismo , Transdução de Sinais , Técnicas do Sistema de Duplo-Híbrido
8.
Genome Med ; 6(4): 32, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24944581

RESUMO

Target identification is a critical step in the lengthy and expensive process of drug development. Here, we describe a genome-wide screening platform that uses systematic overexpression of pooled human ORFs to understand drug mode-of-action and resistance mechanisms. We first calibrated our screen with the well-characterized drug methotrexate. We then identified new genes involved in the bioactivity of diverse drugs including antineoplastic agents and biologically active molecules. Finally, we focused on the transcription factor RHOXF2 whose overexpression conferred resistance to DNA damaging agents. This approach represents an orthogonal method for functional screening and, to our knowledge, has never been reported before.

9.
Biochem Biophys Res Commun ; 445(4): 746-56, 2014 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-24561123

RESUMO

G-protein coupled receptors (GPCRs) are involved in a variety of disease processes and comprise major drug targets. However, the complexity of integral membrane proteins such as GPCRs makes the identification of their interacting partners and subsequent drug development challenging. A comprehensive understanding of GPCR protein interaction networks is needed to design effective therapeutic strategies to inhibit these drug targets. Here, we developed a novel split-ubiquitin membrane yeast two-hybrid (MYTH) technology called CHIP-MYTH, which allows the unbiased characterization of interaction partners of full-length GPCRs in a drug-dependent manner. This was achieved by coupling DNA microarray technology to the MYTH approach, which allows a quantitative evaluation of interacting partners of a given integral membrane protein in the presence or absence of drug. As a proof of principle, we applied the CHIP-MYTH approach to the human ß2-adrenergic receptor (ß2AR), a target of interest in the treatment of asthma, chronic obstructive pulmonary disease (COPD), neurological disease, cardiovascular disease, and obesity. A CHIP-MYTH screen was performed in the presence or absence of salmeterol, a long-acting ß2AR-agonist. Our results suggest that ß2AR activation with salmeterol can induce the dissociation of heterotrimeric G-proteins, Gαßγ, into Gα and Gßγ subunits, which in turn activates downstream signaling cascades. Using CHIP-MYTH, we confirmed previously known and identified novel ß2AR interactors involved in GPCR-mediated signaling cascades. Several of these interactions were confirmed in mammalian cells using LUminescence-based Mammalian IntERactome (LUMIER) and co-immunoprecipitation assays. In summary, the CHIP-MYTH approach is ideal for conducting comprehensive protein-protein interactions (PPI) screenings of full-length GPCRs in the presence or absence of drugs, thus providing a valuable tool to further our understanding of GPCR-mediated signaling.


Assuntos
Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Albuterol/análogos & derivados , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteômica/métodos , Receptores Adrenérgicos beta 2/metabolismo , Albuterol/farmacologia , Animais , Células HEK293 , Humanos , Modelos Moleculares , Receptores Acoplados a Proteínas-G/metabolismo , Xinafoato de Salmeterol , Transdução de Sinais/efeitos dos fármacos , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/metabolismo
10.
Pediatr Pulmonol ; 49(6): 574-80, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23843366

RESUMO

BACKGROUND: Aerosolized liposomal Amphotericin B may reduce the incidence of invasive pulmonary Aspergillosis in adults with chemotherapy-induced prolonged neutropenia with less nephrotoxicity. The breath-actuated AeroEclipse® BAN nebulizer is very efficient and minimizes environmental drug contamination since no aerosol is produced, unless the patient is inspiring through the device. Our aim is to develop an appropriate delivery system suitable for children that does not disrupt the liposomes due to the shear forces in nebulization. METHODS: This is an in vitro experimental study in vitro. Six ml of 4 mg/ml liposomal Amphotericin B solution (AmBisome®; Astellas Pharma Inc., Markham, Ontario, CA) was nebulized with the breath-actuated nebulizer (AeroEclipse®; Trudell Medical International, Canada) and captured by the glass liquid impinger. Sodium dodecyl sulfate was used as detergent to disrupt the liposomes in control samples. Gel filtration, electron microscopy, and high performance liquid chromatography (HPLC) were used to compare the size and shape of the liposomes, and amount of the drug before and after nebulization. The aerosol particle size was obtained by the laser diffraction. RESULTS: After nebulization, 97.5% of amphotericin B was captured by the liquid impinger and detected by HPLC. Gel filtration and electron microscopy demonstrated that the drug remained in its liposomal configuration after nebulization. The mass median diameter (MMD) was 3.7 µm and 66% of aerosol particles were less than 5 µm in diameter. CONCLUSIONS: We demonstrated that liposomal Amphotericin B can be nebulized successfully without disrupting the liposomes and minimize drug loss by using the breath-actuated nebulizer.


Assuntos
Anfotericina B/administração & dosagem , Antifúngicos/administração & dosagem , Hospedeiro Imunocomprometido/efeitos dos fármacos , Aspergilose Pulmonar Invasiva/prevenção & controle , Administração por Inalação , Aerossóis , Cromatografia Líquida de Alta Pressão , Humanos , Microscopia Eletrônica , Nebulizadores e Vaporizadores
11.
PLoS One ; 8(6): e67608, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23840749

RESUMO

The mu-opioid receptor (MOR) is the G-protein coupled receptor primarily responsible for mediating the analgesic and rewarding properties of opioid agonist drugs such as morphine, fentanyl, and heroin. We have utilized a combination of traditional and modified membrane yeast two-hybrid screening methods to identify a cohort of novel MOR interacting proteins (MORIPs). The interaction between the MOR and a subset of MORIPs was validated in pulldown, co-immunoprecipitation, and co-localization studies using HEK293 cells stably expressing the MOR as well as rodent brain. Additionally, a subset of MORIPs was found capable of interaction with the delta and kappa opioid receptors, suggesting that they may represent general opioid receptor interacting proteins (ORIPS). Expression of several MORIPs was altered in specific mouse brain regions after chronic treatment with morphine, suggesting that these proteins may play a role in response to opioid agonist drugs. Based on the known function of these newly identified MORIPs, the interactions forming the MOR signalplex are hypothesized to be important for MOR signaling and intracellular trafficking. Understanding the molecular complexity of MOR/MORIP interactions provides a conceptual framework for defining the cellular mechanisms of MOR signaling in brain and may be critical for determining the physiological basis of opioid tolerance and addiction.


Assuntos
Analgésicos Opioides/metabolismo , Receptores Opioides mu/metabolismo , Leveduras/metabolismo , Analgésicos Opioides/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Linhagem Celular , Feminino , Células HEK293 , Humanos , Imunoprecipitação/métodos , Camundongos , Camundongos Endogâmicos C57BL , Morfina/farmacologia , Proteínas Nucleares/metabolismo , Receptores Opioides delta/metabolismo , Receptores Opioides kappa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos
12.
G3 (Bethesda) ; 3(8): 1375-87, 2013 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-23797109

RESUMO

The application of new proteomics and genomics technologies support a view in which few drugs act solely by inhibiting a single cellular target. Indeed, drug activity is modulated by complex, often incompletely understood cellular mechanisms. Therefore, efforts to decipher mode of action through genetic perturbation such as RNAi typically yields "hits" that fall into several categories. Of particular interest to the present study, we aimed to characterize secondary activities of drugs on cells. Inhibiting a known target can result in clinically relevant synthetic phenotypes. In one scenario, drug perturbation could, for example, improperly activate a protein that normally inhibits a particular kinase. In other cases, additional, lower affinity targets can be inhibited as in the example of inhibition of c-Kit observed in Bcr-Abl-positive cells treated with Gleevec. Drug transport and metabolism also play an important role in the way any chemicals act within the cells. Finally, RNAi per se can also affect cell fitness by more general off-target effects, e.g., via the modulation of apoptosis or DNA damage repair. Regardless of the root cause of these unwanted effects, understanding the scope of a drug's activity and polypharmacology is essential for better understanding its mechanism(s) of action, and such information can guide development of improved therapies. We describe a rapid, cost-effective approach to characterize primary and secondary effects of small-molecules by using small-scale libraries of virally integrated short hairpin RNAs. We demonstrate this principle using a "minipool" composed of shRNAs that target the genes encoding the reported protein targets of approved drugs. Among the 28 known reported drug-target pairs, we successfully identify 40% of the targets described in the literature and uncover several unanticipated drug-target interactions based on drug-induced synthetic lethality. We provide a detailed protocol for performing such screens and for analyzing the data. This cost-effective approach to mammalian knockdown screens, combined with the increasing maturation of RNAi technology will expand the accessibility of similar approaches in academic settings.


Assuntos
Antineoplásicos/farmacologia , Benzamidas/farmacologia , Proliferação de Células/efeitos dos fármacos , Piperazinas/farmacologia , Pirimidinas/farmacologia , RNA Interferente Pequeno/genética , Linhagem Celular Tumoral , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mesilato de Imatinib , Lentivirus/genética , Miniaturização , Proteínas/antagonistas & inibidores , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo
13.
Cell Rep ; 2(4): 951-63, 2012 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-23084749

RESUMO

The pentaspan membrane glycoprotein CD133 marks lineage-specific cancer progenitor cells and is associated with poor prognosis in a number of tumor types. Despite its utility as a cancer progenitor cell marker, CD133 protein regulation and molecular function remain poorly understood. We find that the deacetylase HDAC6 physically associates with CD133 to negatively regulate CD133 trafficking down the endosomal-lysosomal pathway for degradation. We further demonstrate that CD133, HDAC6, and the central molecule of the canonical Wnt signaling pathway, ß-catenin, can physically associate as a ternary complex. This association stabilizes ß-catenin via HDAC6 deacetylase activity, which leads to activation of ß-catenin signaling targets. Downregulation of either CD133 or HDAC6 results in increased ß-catenin acetylation and degradation, which correlates with decreased proliferation in vitro and tumor xenograft growth in vivo. Given that CD133 marks progenitor cells in a wide range of cancers, targeting CD133 may be a means to treat multiple cancer types.


Assuntos
Antígenos CD/metabolismo , Glicoproteínas/metabolismo , Histona Desacetilases/metabolismo , Peptídeos/metabolismo , beta Catenina/metabolismo , Antígeno AC133 , Acetilação , Animais , Antígenos CD/genética , Células CACO-2 , Diferenciação Celular , Linhagem Celular Tumoral , Regulação para Baixo , Endossomos/metabolismo , Transição Epitelial-Mesenquimal , Feminino , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/genética , Células HEK293 , Células HT29 , Desacetilase 6 de Histona , Histona Desacetilases/química , Histona Desacetilases/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Neoplasias/metabolismo , Neoplasias/patologia , Peptídeos/antagonistas & inibidores , Peptídeos/genética , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Transplante Heterólogo , Proteínas Wnt/metabolismo
14.
Methods Mol Biol ; 910: 55-69, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22821592

RESUMO

With the advent of next-generation sequencing (NGS) technology, methods previously developed for microarrays have been adapted for use by NGS. Here we describe in detail a protocol for Barcode analysis by sequencing (Bar-seq) to assess pooled competitive growth of individually barcoded yeast deletion mutants. This protocol has been optimized on two sequencing platforms: Illumina's Genome Analyzer IIx/HiSeq2000 and Life Technologies SOLiD3/5500. In addition, we provide guidelines for assessment of human knockdown cells using short-hairpin RNAs (shRNA) and an Illumina sequencing readout.


Assuntos
Genes/genética , Preparações Farmacêuticas/metabolismo , Análise de Sequência de DNA/métodos , Animais , Deleção de Genes , Técnicas de Inativação de Genes , Humanos , Mutação , RNA Interferente Pequeno/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética
15.
Nat Protoc ; 5(7): 1281-93, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20595957

RESUMO

The biological function of proteins may be predicted by identification of their interacting partners, and one of the major goals of the postgenomic era is the mapping of protein interaction networks. Membrane proteins are of particular interest because of their role in disease and because of their prevalence as major pharmaceutical targets. Unfortunately, because of their hydrophobic nature, they have long been difficult to study in a high-throughput format. A powerful technology recently developed to facilitate the characterization of membrane protein interactions is the membrane yeast two-hybrid (MYTH) assay. MYTH adapts the principle of split ubiquitin for use as a potent in vivo sensor of protein-protein interactions, allowing large-scale screening for interactors of full-length membrane proteins, from a range of organisms, using Saccharomyces cerevisiae as a host. In this article, we describe a protocol for MYTH bait generation, validation and library screening. The entire MYTH procedure can generally be completed in 4-6 weeks.


Assuntos
Proteínas de Membrana/análise , Mapeamento de Interação de Proteínas/métodos , Saccharomyces cerevisiae/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Proteínas de Membrana/metabolismo
16.
BMC Neurosci ; 11: 33, 2010 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-20214800

RESUMO

BACKGROUND: Opioid agonist drugs produce analgesia. However, long-term exposure to opioid agonists may lead to opioid dependence. The analgesic and addictive properties of opioid agonist drugs are mediated primarily via the mu-opioid receptor (MOR). Opioid agonists appear to alter neuronal morphology in key brain regions implicated in the development of opioid dependence. However, the precise role of the MOR in the development of these neuronal alterations remains elusive. We hypothesize that identifying and characterizing novel MOR interacting proteins (MORIPs) may help to elucidate the underlying mechanisms involved in the development of opioid dependence. RESULTS: GPR177, the mammalian ortholog of Drosophila Wntless/Evi/Sprinter, was identified as a MORIP in a modified split ubiquitin yeast two-hybrid screen. GPR177 is an evolutionarily conserved protein that plays a critical role in mediating Wnt protein secretion from Wnt producing cells. The MOR/GPR177 interaction was validated in pulldown, coimmunoprecipitation, and colocalization studies using mammalian tissue culture cells. The interaction was also observed in rodent brain, where MOR and GPR177 were coexpressed in close spatial proximity within striatal neurons. At the cellular level, morphine treatment caused a shift in the distribution of GPR177 from cytosol to the cell surface, leading to enhanced MOR/GPR177 complex formation at the cell periphery and the inhibition of Wnt protein secretion. CONCLUSIONS: It is known that chronic morphine treatment decreases dendritic arborization and hippocampal neurogenesis, and Wnt proteins are essential for these processes. We therefore propose that the morphine-mediated MOR/GPR177 interaction may result in decreased Wnt secretion in the CNS, resulting in atrophy of dendritic arbors and decreased neurogenesis. Our results demonstrate a previously unrecognized role for GPR177 in regulating cellular response to opioid drugs.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Opioides mu/metabolismo , Proteínas Wnt/metabolismo , Analgésicos Opioides/farmacologia , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Corpo Estriado/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Humanos , Camundongos , Modelos Neurológicos , Morfina/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Transtornos Relacionados ao Uso de Opioides/metabolismo , Células PC12 , Ratos , Receptores Opioides mu/antagonistas & inibidores , Proteínas Wnt/antagonistas & inibidores
17.
J Vis Exp ; (36)2010 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-20125081

RESUMO

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (C(ub)) and the N-terminal half of the ubiquitin moiety (N(ub)). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to C(ub) which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the N(ub) moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.


Assuntos
Proteínas de Membrana/análise , Mapeamento de Interação de Proteínas/métodos , Saccharomyces cerevisiae/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/metabolismo
18.
Sci Signal ; 2(102): ra84, 2009 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-20029029

RESUMO

Binding of epidermal growth factor (EGF) to its receptor leads to receptor dimerization, assembly of protein complexes, and activation of signaling networks that control key cellular responses. Despite their fundamental role in cell biology, little is known about protein complexes associated with the EGF receptor (EGFR) before growth factor stimulation. We used a modified membrane yeast two-hybrid system together with bioinformatics to identify 87 candidate proteins interacting with the ligand-unoccupied EGFR. Among them was histone deacetylase 6 (HDAC6), a cytoplasmic lysine deacetylase, which we found negatively regulated EGFR endocytosis and degradation by controlling the acetylation status of alpha-tubulin and, subsequently, receptor trafficking along microtubules. A negative feedback loop consisting of EGFR-mediated phosphorylation of HDAC6 Tyr(570) resulted in reduced deacetylase activity and increased acetylation of alpha-tubulin. This study illustrates the complexity of the EGFR-associated interactome and identifies protein acetylation as a previously unknown regulator of receptor endocytosis and degradation.


Assuntos
Receptores ErbB/metabolismo , Histona Desacetilases/metabolismo , Complexos Multiproteicos/metabolismo , Transdução de Sinais/fisiologia , Acetilação , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Biologia Computacional , Desacetilase 6 de Histona , Humanos , Imunoprecipitação , Espectrometria de Massas , Microscopia de Fluorescência , Dados de Sequência Molecular , RNA/genética , Transfecção , Tubulina (Proteína)/metabolismo , Técnicas do Sistema de Duplo-Híbrido
19.
Methods Mol Biol ; 548: 247-71, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19521829

RESUMO

Recent research has begun to elucidate the global network of cytosolic and membrane protein interactions. The resulting interactome map facilitates numerous biological studies, including those for cell signalling, protein trafficking and protein regulation. Due to the hydrophobic nature of membrane proteins such as tyrosine kinases, G-protein coupled receptors, membrane bound phosphatases and transporters it is notoriously difficult to study their relationship to signaling molecules, the cytoskeleton, or any other interacting partners. Although conventional yeast-two hybrid is a simple and robust technique that is effective in the identification of specific protein-protein interactions, it is limited in its use for membrane proteins. However, the split-ubiquitin membrane based yeast two-hybrid assay (MYTH) has been described as a tool that allows for the identification of membrane protein interactions. In the MYTH system, ubiquitin has been split into two halves, each of which is fused to a protein, at least one of which is membrane bound. Upon interaction of these two proteins, the two halves of ubiquitin are reconstituted and a transcription factor that is fused to the membrane protein is released. The transcription factor then enters the nucleus and activates transcription of reporter genes. Currently, large-scale MYTH screens using cDNA or gDNA libraries are performed to identify and map the binding partners of various membrane proteins. Thus, the MYTH system is proving to be a powerful tool for the elucidation of specific protein-protein interactions, contributing greatly to the mapping of the membrane protein interactome.


Assuntos
Proteínas de Membrana/análise , Mapeamento de Interação de Proteínas/métodos , Proteínas de Saccharomyces cerevisiae/análise , Técnicas do Sistema de Duplo-Híbrido , Sequência de Aminoácidos , Sequência de Bases , Biologia Computacional , Primers do DNA/genética , DNA Recombinante/genética , Vetores Genéticos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Complexos Multiproteicos/análise , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Plasmídeos/genética , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transformação Genética , Técnicas do Sistema de Duplo-Híbrido/estatística & dados numéricos , Ubiquitina/metabolismo
20.
Curr Opin Biotechnol ; 19(4): 316-23, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18619540

RESUMO

Given that protein-protein interactions (PPIs) regulate nearly every living process; the exploration of global and pathway-specific protein interaction networks is expected to have major implications in the understanding of diseases and for drug discovery. Consequently, the development and application of methodologies that address physical associations among proteins is of major importance in today's proteomics research. The most widely and successfully used methodology to assess PPIs is the yeast two-hybrid system (YTH). Here we present an overview on the current applications of YTH and variant technologies in yeast and mammalian systems. Two-hybrid-based methods will not only continue to have a dominant role in the assessment of protein interactomes but will also become important in the development of novel compounds that target protein interaction interfaces for therapeutic intervention.


Assuntos
Proteômica , Técnicas do Sistema de Duplo-Híbrido , Animais , Desenho de Drogas , Mamíferos
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