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1.
Cell ; 145(4): 596-606, 2011 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-21565616

RESUMO

Under fasting conditions, metazoans maintain energy balance by shifting from glucose to fat burning. In the fasted state, SIRT1 promotes catabolic gene expression by deacetylating the forkhead factor FOXO in response to stress and nutrient deprivation. The mechanisms by which hormonal signals regulate FOXO deacetylation remain unclear, however. We identified a hormone-dependent module, consisting of the Ser/Thr kinase SIK3 and the class IIa deacetylase HDAC4, which regulates FOXO activity in Drosophila. During feeding, HDAC4 is phosphorylated and sequestered in the cytoplasm by SIK3, whose activity is upregulated in response to insulin. SIK3 is inactivated during fasting, leading to the dephosphorylation and nuclear translocation of HDAC4 and to FOXO deacetylation. SIK3 mutant flies are starvation sensitive, reflecting FOXO-dependent increases in lipolysis that deplete triglyceride stores; reducing HDAC4 expression restored lipid accumulation. Our results reveal a hormone-regulated pathway that functions in parallel with the nutrient-sensing SIRT1 pathway to maintain energy balance.


Assuntos
Drosophila melanogaster/metabolismo , Metabolismo Energético , Insulina/metabolismo , Transdução de Sinais , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Ingestão de Alimentos , Fatores de Transcrição Forkhead/metabolismo , Histona Desacetilases/metabolismo , Lipase/metabolismo , Metabolismo dos Lipídeos , Camundongos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Triglicerídeos/metabolismo
2.
Cell ; 140(1): 49-61, 2010 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-20079333

RESUMO

Tumor cells display progressive changes in metabolism that correlate with malignancy, including development of a lipogenic phenotype. How stored fats are liberated and remodeled to support cancer pathogenesis, however, remains unknown. Here, we show that the enzyme monoacylglycerol lipase (MAGL) is highly expressed in aggressive human cancer cells and primary tumors, where it regulates a fatty acid network enriched in oncogenic signaling lipids that promotes migration, invasion, survival, and in vivo tumor growth. Overexpression of MAGL in nonaggressive cancer cells recapitulates this fatty acid network and increases their pathogenicity-phenotypes that are reversed by an MAGL inhibitor. Impairments in MAGL-dependent tumor growth are rescued by a high-fat diet, indicating that exogenous sources of fatty acids can contribute to malignancy in cancers lacking MAGL activity. Together, these findings reveal how cancer cells can co-opt a lipolytic enzyme to translate their lipogenic state into an array of protumorigenic signals. PAPERFLICK:


Assuntos
Ácidos Graxos/metabolismo , Monoacilglicerol Lipases/metabolismo , Neoplasias Ovarianas/metabolismo , Animais , Linhagem Celular , Feminino , Humanos , Camundongos , Monoacilglicerol Lipases/genética , Monoglicerídeos/metabolismo , Transplante de Neoplasias , Transplante Heterólogo , Células Tumorais Cultivadas
3.
J Proteome Res ; 14(9): 3670-9, 2015 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-26151158

RESUMO

Tumor types can be defined cytologically by their regions of chromosomal amplification, which often results in the high expression of both mRNA and proteins of certain genes contained within the amplicon. An important strategy for defining therapeutically relevant targets in these situations is to ascertain which genes are amplified at the protein level and, concomitantly, are key drivers for tumor growth or maintenance. Furthermore, so-called passenger genes that are amplified with driver genes and a manifest on the cell surface can be attractive targets for an antibody-drug conjugate approach (ADC). We employed a tandem mass spectrometry proteomics approach using tumor cell lines to identify the cell surface proteins whose expression correlates with the 11q13 amplicon. The 11q13 amplicon is one of the most frequently amplified chromosomal regions in human cancer, being present in 45% of head and neck and oral squamous cell carcinoma (OSCC) and 13-21% of breast and liver carcinomas. Using a panel of tumor cell lines with defined 11q13 genomic amplification, we identified the membrane proteins that are differentially expressed in an 11q13 amplified cell line panel using membrane-enriched proteomic profiling. We found that DSG3, CD109, and CD14 were differentially overexpressed in head and neck and breast tumor cells with 11q13 amplification. The level of protein expression of each gene was confirmed by Western blot and FACS analysis. Because proteins with high cell surface expression on selected tumor cells could be potential antibody drug conjugate targets, we tested DSG3 and CD109 in antibody piggyback assays and validated that DSG3 and CD109 expression was sufficient to induce antibody internalization and cell killing in 11q13-amplified cell lines. Our results suggest that proteomic profiling using genetically stratified tumors can identify candidate antibody drug conjugate targets. Data are available via ProteomeXchange with the identifier PXD002486.


Assuntos
Carcinoma de Células Escamosas/genética , Cromossomos Humanos Par 11 , Neoplasias de Cabeça e Pescoço/genética , Proteínas de Membrana/genética , Proteômica , Linhagem Celular Tumoral , Humanos , Proteínas de Membrana/química , Espectrometria de Massas em Tandem
4.
Proc Natl Acad Sci U S A ; 108(21): 8815-20, 2011 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-21555588

RESUMO

Filamentous cyanobacteria of the genus Lyngbya are important contributors to coral reef ecosystems, occasionally forming dominant cover and impacting the health of many other co-occurring organisms. Moreover, they are extraordinarily rich sources of bioactive secondary metabolites, with 35% of all reported cyanobacterial natural products deriving from this single pantropical genus. However, the true natural product potential and life strategies of Lyngbya strains are poorly understood because of phylogenetic ambiguity, lack of genomic information, and their close associations with heterotrophic bacteria and other cyanobacteria. To gauge the natural product potential of Lyngbya and gain insights into potential microbial interactions, we sequenced the genome of Lyngbya majuscula 3L, a Caribbean strain that produces the tubulin polymerization inhibitor curacin A and the molluscicide barbamide, using a combination of Sanger and 454 sequencing approaches. Whereas ∼ 293,000 nucleotides of the draft genome are putatively dedicated to secondary metabolism, this is far too few to encode a large suite of Lyngbya metabolites, suggesting Lyngbya metabolites are strain specific and may be useful in species delineation. Our analysis revealed a complex gene regulatory network, including a large number of sigma factors and other regulatory proteins, indicating an enhanced ability for environmental adaptation or microbial associations. Although Lyngbya species are reported to fix nitrogen, nitrogenase genes were not found in the genome or by PCR of genomic DNA. Subsequent growth experiments confirmed that L. majuscula 3L is unable to fix atmospheric nitrogen. These unanticipated life history characteristics challenge current views of the genus Lyngbya.


Assuntos
Cianobactérias/genética , Cianobactérias/fisiologia , Redes Reguladoras de Genes , Genoma Bacteriano/genética , Ciclopropanos , Ecologia , Genes Bacterianos/fisiologia , Biologia Marinha , Fixação de Nitrogênio/genética , Análise de Sequência de DNA , Tiazóis
5.
iScience ; 27(3): 109006, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38361610

RESUMO

Many vaccines, including those using recombinant antigen subunits, rely on adjuvant(s) to enhance the efficacy of the host immune responses. Among the few adjuvants clinically approved, QS-21, a saponin-based immunomodulatory molecule isolated from the tree bark of Quillaja saponaria (QS) is used in complex formulations in approved effective vaccines. High demand of the QS raw material as well as manufacturing scalability limitation has been barriers here. We report for the first-time successful plant cell culture production of QS-21 having structural, chemical, and biologic, properties similar to the bark extracted product. These data ensure QS-21 and related saponins are broadly available and accessible to drug developers.

6.
Nat Chem Biol ; 7(7): 469-78, 2011 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-21572424

RESUMO

Serine hydrolases are a diverse enzyme class representing ∼1% of all human proteins. The biological functions of most serine hydrolases remain poorly characterized owing to a lack of selective inhibitors to probe their activity in living systems. Here we show that a substantial number of serine hydrolases can be irreversibly inactivated by 1,2,3-triazole ureas, which show negligible cross-reactivity with other protein classes. Rapid lead optimization by click chemistry-enabled synthesis and competitive activity-based profiling identified 1,2,3-triazole ureas that selectively inhibit enzymes from diverse branches of the serine hydrolase class, including peptidases (acyl-peptide hydrolase, or APEH), lipases (platelet-activating factor acetylhydrolase-2, or PAFAH2) and uncharacterized hydrolases (α,ß-hydrolase-11, or ABHD11), with exceptional potency in cells (sub-nanomolar) and mice (<1 mg kg(-1)). We show that APEH inhibition leads to accumulation of N-acetylated proteins and promotes proliferation in T cells. These data indicate 1,2,3-triazole ureas are a pharmacologically privileged chemotype for serine hydrolase inhibition, combining broad activity across the serine hydrolase class with tunable selectivity for individual enzymes.


Assuntos
Química Click/métodos , Serina Proteases/metabolismo , Inibidores de Serina Proteinase/síntese química , Triazóis/síntese química , Ureia/análogos & derivados , Animais , Encéfalo/enzimologia , Linhagem Celular Tumoral , Humanos , Camundongos , Estrutura Molecular , Miocárdio/enzimologia , Serina Proteases/genética , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologia , Transfecção , Triazóis/química , Triazóis/farmacologia , Ureia/farmacologia
7.
Proc Natl Acad Sci U S A ; 107(49): 20941-6, 2010 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-21084632

RESUMO

Serine hydrolases (SHs) are one of the largest and most diverse enzyme classes in mammals. They play fundamental roles in virtually all physiological processes and are targeted by drugs to treat diseases such as diabetes, obesity, and neurodegenerative disorders. Despite this, we lack biological understanding for most of the 110+ predicted mammalian metabolic SHs, in large part because of a dearth of assays to assess their biochemical activities and a lack of selective inhibitors to probe their function in living systems. We show here that the vast majority (> 80%) of mammalian metabolic SHs can be labeled in proteomes by a single, active site-directed fluorophosphonate probe. We exploit this universal activity-based assay in a library-versus-library format to screen 70+ SHs against 140+ structurally diverse carbamates. Lead inhibitors were discovered for ∼40% of the screened enzymes, including many poorly characterized SHs. Global profiles identified carbamate inhibitors that discriminate among highly sequence-related SHs and, conversely, enzymes that share inhibitor sensitivity profiles despite lacking sequence homology. These findings indicate that sequence relatedness is not a strong predictor of shared pharmacology within the SH superfamily. Finally, we show that lead carbamate inhibitors can be optimized into pharmacological probes that inactivate individual SHs with high specificity in vivo.


Assuntos
Carbamatos/farmacologia , Descoberta de Drogas/métodos , Inibidores Enzimáticos/química , Hidrolases/antagonistas & inibidores , Serina , Bibliotecas de Moléculas Pequenas/farmacologia , Carbamatos/uso terapêutico , Domínio Catalítico , Avaliação Pré-Clínica de Medicamentos , Fluoretos , Humanos , Sondas Moleculares , Fosfatos , Ligação Proteica , Proteoma , Especificidade por Substrato
8.
Int J Cancer ; 131(8): 1744-54, 2012 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-22275185

RESUMO

The recepteur d'origine nantais (RON) receptor tyrosine kinase is overexpressed and stimulates invasive growth in pancreatic cancer cells, yet the mechanisms that underlie RON-mediated phenotypes remain poorly characterized. To better understand RON function in pancreatic cancer cells, we sought to identify novel RON interactants using multidimensional protein identification analysis. These studies revealed plectin, a large protein of the spectrin superfamily, to be a novel RON interactant. Plectin is a multifunctional protein that complexes with integrin-ß4 (ITGB4) to form hemidesmosomes, serves as a scaffolding platform crucial to the function of numerous protein signaling pathways and was recently described as an overexpressed protein in pancreatic cancer (Bausch D et al., Clin Cancer Res 2010; Kelly et al., PLoS Med 2008;5:e85). In this study, we demonstrate that on exposure to its ligand, macrophage-stimulating protein, RON binds to plectin and ITGB4, which results in disruption of the plectin-ITGB4 interaction and enhanced cell migration, a phenotype that can be recapitulated by small hairpin ribosomal nucleic acid (shRNA)-mediated suppression of plectin expression. We demonstrate that disruption of plectin-ITGB4 is dependent on RON and phosphoinositide-3 (PI3) kinase, but not mitogen-activated protein kinase (MEK), activity. Thus, in pancreatic cancer cells, plectin and ITGB4 form hemidesmosomes which serve to anchor cells to the extracellular matrix (ECM) and restrain migration. The current study defines a novel interaction between RON and plectin, provides new insight into RON-mediated migration and further supports efforts to target RON kinase activity in pancreatic cancer.


Assuntos
Movimento Celular , Hemidesmossomos/metabolismo , Integrina beta4/metabolismo , Neoplasias Pancreáticas/patologia , Plectina/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Western Blotting , Proliferação de Células , Células Cultivadas , Cromatografia Líquida , Imunofluorescência , Humanos , Rim/citologia , Rim/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Neoplasias Pancreáticas/metabolismo , Fosforilação , Transdução de Sinais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Cicatrização
9.
Proteomics ; 11(12): 2377-88, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21584940

RESUMO

Proteases are critical in many physiological processes and the human genome encodes for 566 predicted proteolytic enzymes. Therefore, there is great interest in identifying and characterizing physiologic protease-substrate relationships. The coagulation cascade is a well-described network of serine proteases. However, new interactions of the coagulation cascade with other biological pathways have been discovered only recently. Therefore, we hypothesized that a non-biased protease degradomics analysis of the physiologic coagulation reaction would identify new interactions between the coagulation cascade and other pathways. We used the recently described PROTOMAP technique to profile the complete coagulation degradome. This analysis detected virtually all of the proteins of the coagulation cascade and identified a majority of the expected proteolytic events, suggesting significant coverage of the coagulation degradome. Multiple potential new proteolytic cleavages were detected, including two of transmembrane proteins that may be shed from the surface of blood cells. In addition, this analysis was able to identify several new potentially secreted proteins. A significant majority of the newly identified events were of proteins involved in innate immunity (complement and inflammation). This highlights potential new areas of crosstalk between these linked systems. Future studies will elucidate the details and functional consequences of these proteolytic events during coagulation.


Assuntos
Proteínas do Sistema Complemento/metabolismo , Proteínas de Membrana/metabolismo , Fragmentos de Peptídeos/análise , Peptídeo Hidrolases/metabolismo , Mapeamento de Peptídeos/métodos , Plasma/enzimologia , Proteômica/métodos , Coagulação Sanguínea/genética , Western Blotting , Cromatografia Líquida , Proteínas do Sistema Complemento/genética , Eletroforese em Gel de Poliacrilamida , Humanos , Espectrometria de Massas , Proteínas de Membrana/genética , Fragmentos de Peptídeos/química , Peptídeo Hidrolases/genética , Plasma/química , Especificidade por Substrato , Tripsina/metabolismo
10.
Carcinogenesis ; 32(8): 1151-6, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21565828

RESUMO

The RON receptor tyrosine kinase (RTK) is overexpressed in the majority of pancreatic cancers, yet its role in pancreatic cancer cell biology remains to be clarified. Recent work in childhood sarcoma identified RON as a mediator of resistance to insulin-like growth factor receptor (IGF1-R)-directed therapy. To better understand RON function in pancreatic cancer cells, we sought to identify novel RON interactants. Using multidimensional protein identification analysis, IGF-1R was identified and confirmed to interact with RON in pancreatic cancer cell lines. IGF-1 induces rapid phosphorylation of RON, but RON signaling did not activate IGF-1R indicating unidirectional signaling between these RTKs. We next demonstrate that IGF-1 induces pancreatic cancer cell migration that is RON dependent, as inhibition of RON signaling by either shRNA-mediated RON knockdown or by a RON kinase inhibitor abrogated IGF-1 induced wound closure in a scratch assay. In pancreatic cancer cells, unlike childhood sarcoma, STAT-3, rather than RPS6, is activated in response to IGF-1, in a RON-dependent manner. The current study defines a novel interaction between RON and IGF-1R and taken together, these two studies demonstrate that RON is an important mediator of IGF1-R signaling and that this finding is consistent in both human epithelial and mesenchymal cancers. These findings demand additional investigation to determine if IGF-1R independent RON activation is associated with resistance to IGF-1R-directed therapies in vivo and to identify suitable biomarkers of activated RON signaling.


Assuntos
Movimento Celular/fisiologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais , Western Blotting , Adesão Celular , Humanos , Imunoprecipitação , Neoplasias Pancreáticas/genética , RNA Interferente Pequeno/genética , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Células Tumorais Cultivadas , Cicatrização
11.
Mol Cell Proteomics ; 8(5): 1082-93, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19136719

RESUMO

Activity-based protein profiling is a powerful method to display enzyme activities in proteomes and provides crucial information on enzyme activity rather than protein or transcript abundance. We applied activity-based protein profiling using fluorophosphonate-based probes to display the activities of Ser hydrolases in the model plant Arabidopsis thaliana. Multidimensional protein identification technology and in-gel analysis of fluorophosphonate-labeled leaf extracts revealed over 50 Ser hydrolases, including dozens of proteases, esterases, and lipases, representing over 10 different enzyme families. Except for some well characterized Ser hydrolases like subtilases TPP2 and ARA12, prolyl oligopeptidase acylamino acid-releasing enzyme, serine carboxypeptidase-like SNG1 and BRS1, carboxylesterase-like CXE12, methylesterases MES2 and MES3, and S-formylglutathione hydrolase, the majority of these serine hydrolases have not been described before. We studied transiently expressed SNG1 and investigated plants infected with the fungal pathogen Botrytis cinerea. Besides the down-regulation of several Arabidopsis Ser hydrolase activities during Botrytis infection, we detected the activities of Botrytis-derived cutinases and lipases, which are thought to contribute to pathogenicity.


Assuntos
Arabidopsis/enzimologia , Arabidopsis/microbiologia , Botrytis/fisiologia , Serina Endopeptidases/metabolismo , Aciltransferases/metabolismo , Agrobacterium tumefaciens , Sequência de Aminoácidos , Proteínas de Arabidopsis/metabolismo , Interações Hospedeiro-Patógeno , Dados de Sequência Molecular , Organofosfonatos/metabolismo , Peptídeos/química , Folhas de Planta/enzimologia , Folhas de Planta/microbiologia , Serina Endopeptidases/análise , Extratos de Tecidos , Nicotiana/metabolismo
12.
mBio ; 11(3)2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32518184

RESUMO

Antisense transcription is widespread in bacteria. By base pairing with overlapping sense RNAs, antisense RNAs (asRNA) can form double-stranded RNAs (dsRNA), which are cleaved by RNase III, a dsRNA endoribonuclease. The ectopic expression of plant Tombusvirus p19 in Escherichia coli stabilizes ∼21-nucleotide (nt) dsRNA RNase III decay intermediates, which enabled us to characterize otherwise highly unstable asRNA by deep sequencing of p19-captured dsRNA. RNase III-produced small dsRNA were formed at most bacterial genes in the bacterial genome and in a plasmid. We classified the types of asRNA in genomic clusters producing the most abundant p19-captured dsRNA and confirmed RNase III regulation of asRNA and sense RNA decay at three type I toxin-antitoxin loci and at a coding gene, rsd Furthermore, we provide potential evidence for the RNase III-dependent regulation of CspD protein by asRNA. The analysis of p19-captured dsRNA revealed an RNase III sequence preference for AU-rich sequences 3 nucleotides on either side of the cleavage sites and for GC-rich sequences in the 2-nt overhangs. Unexpectedly, GC-rich sequences were enriched in the middle section of p19-captured dsRNA, suggesting some unexpected sequence bias in p19 protein binding. Nonetheless, the ectopic expression of p19 is a sensitive method for identifying antisense transcripts and RNase III cleavage sites in dsRNA formed by overlapping sense and antisense transcripts in bacteria.


Assuntos
Escherichia coli/genética , RNA Antissenso/genética , RNA de Cadeia Dupla/metabolismo , Ribonuclease III/metabolismo , Tombusvirus/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Estabilidade de RNA , RNA Antissenso/classificação , RNA de Cadeia Dupla/genética
13.
Bioorg Med Chem Lett ; 18(22): 5838-41, 2008 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-18657971

RESUMO

The endocannabinoid 2-arachidonoylglycerol (2-AG) has been implicated as a key retrograde mediator in the nervous system based on pharmacological studies using inhibitors of the 2-AG biosynthetic enzymes diacyglycerol lipase alpha and beta (DAGL-alpha/beta). Here, we show by competitive activity-based protein profiling that the DAGL-alpha/beta inhibitors, tetrahydrolipstatin (THL) and RHC80267, block several brain serine hydrolases with potencies equal to or greater than their inhibitory activity against DAGL enzymes. Interestingly, a minimal overlap in target profiles was observed for THL and RHC80267, suggesting that pharmacological effects observed with both agents may be viewed as good initial evidence for DAGL-dependent events.


Assuntos
Ácidos Araquidônicos/antagonistas & inibidores , Ácidos Araquidônicos/biossíntese , Encéfalo/enzimologia , Moduladores de Receptores de Canabinoides/antagonistas & inibidores , Moduladores de Receptores de Canabinoides/biossíntese , Cicloexanonas/farmacologia , Endocanabinoides , Glicerídeos/antagonistas & inibidores , Glicerídeos/biossíntese , Lactonas/farmacologia , Lipase/antagonistas & inibidores , Inibidores de Serina Proteinase/farmacologia , Humanos , Isoenzimas/antagonistas & inibidores , Estrutura Molecular , Orlistate , Receptor CB1 de Canabinoide/metabolismo
14.
Science ; 361(6404): 769-773, 2018 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-30072576

RESUMO

Proteins circulating in the blood are critical for age-related disease processes; however, the serum proteome has remained largely unexplored. To this end, 4137 proteins covering most predicted extracellular proteins were measured in the serum of 5457 Icelanders over 65 years of age. Pairwise correlation between proteins as they varied across individuals revealed 27 different network modules of serum proteins, many of which were associated with cardiovascular and metabolic disease states, as well as overall survival. The protein modules were controlled by cis- and trans-acting genetic variants, which in many cases were also associated with complex disease. This revealed co-regulated groups of circulating proteins that incorporated regulatory control between tissues and demonstrated close relationships to past, current, and future disease states.


Assuntos
Proteínas Sanguíneas/análise , Proteínas Sanguíneas/genética , Doenças Cardiovasculares/genética , Doenças Metabólicas/genética , Proteoma/análise , Proteoma/genética , Proteômica/métodos , Aptâmeros de Nucleotídeos , Predisposição Genética para Doença , Variação Genética , Humanos , Islândia , Redes e Vias Metabólicas
15.
J Med Chem ; 50(5): 1058-68, 2007 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-17279740

RESUMO

A study of the structure-activity relationships (SAR) of 2f (OL-135), a potent inhibitor of fatty acid amide hydrolase (FAAH), is detailed, targeting the 5-position of the oxazole. Examination of a series of substituted benzene derivatives (12-14) revealed that the optimal position for substitution was the meta-position with selected members approaching or exceeding the potency of 2f. Concurrent with these studies, the effect of substitution on the pyridine ring of 2f was also examined. A series of small, nonaromatic C5-substituents was also explored and revealed that the K(i) follows a well-defined correlation with the Hammett sigma(p) constant (rho = 3.01, R2 = 0.91) in which electron-withdrawing substituents enhance potency, leading to inhibitors with K(i)s as low as 400 pM (20n). Proteomic-wide screening of the inhibitors revealed that most are exquisitely selective for FAAH over all other mammalian proteases, reversing the 100-fold preference of 20a (C5 substituent = H) for the enzyme TGH.


Assuntos
Amidoidrolases/antagonistas & inibidores , Ácidos Araquidônicos/metabolismo , Derivados de Benzeno/síntese química , Ácidos Oleicos/metabolismo , Oxazóis/síntese química , Alcamidas Poli-Insaturadas/metabolismo , Amidoidrolases/química , Animais , Derivados de Benzeno/química , Derivados de Benzeno/farmacologia , Células COS , Chlorocebus aethiops , Endocanabinoides , Humanos , Oxazóis/química , Oxazóis/farmacologia , Proteômica , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Relação Estrutura-Atividade
16.
J Cereb Blood Flow Metab ; 31(12): 2267-81, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21792245

RESUMO

The blood-brain barrier (BBB) is a multicellular vascular structure separating blood from the brain parenchyma that is composed of endothelial cells with tight intercellular junctions, surrounded by a basal lamina, astrocytes, and pericytes. Previous studies have generated detailed databases of the microvessel transcriptome; however, less information is available on the BBB at the protein level. In this study, we specifically focused on characterization of the membrane fraction of cells within the BBB to generate a more complete understanding of membrane transporters, tight junction proteins, and associated extracellular matrix proteins that are functional hallmarks of the BBB. We used Multidimensional Protein Identification Technology to identify a total of 1,143 proteins in mouse brain microvessels, of which 53% were determined to be membrane associated. Analyses of specific classes of BBB-associated proteins in the context of recent transcriptome reports provide a unique database to assess the relative contribution of genes at the level of both RNA and protein in the maintenance of normal BBB integrity.


Assuntos
Membrana Basal/metabolismo , Química Encefálica/genética , Capilares/metabolismo , Membrana Celular/metabolismo , Proteoma/genética , Animais , Biomarcadores , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/metabolismo , Cromatografia Líquida de Alta Pressão , Biologia Computacional , Células Endoteliais/fisiologia , Proteínas da Matriz Extracelular/metabolismo , Integrinas/metabolismo , Espectrometria de Massas , Camundongos , Proteoglicanas/metabolismo , Junções Íntimas/metabolismo
17.
Chem Biol ; 18(7): 846-56, 2011 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-21802006

RESUMO

Cancer cells couple heightened lipogenesis with lipolysis to produce fatty acid networks that support malignancy. Monoacylglycerol lipase (MAGL) plays a principal role in this process by converting monoglycerides, including the endocannabinoid 2-arachidonoylglycerol (2-AG), to free fatty acids. Here, we show that MAGL is elevated in androgen-independent versus androgen-dependent human prostate cancer cell lines, and that pharmacological or RNA-interference disruption of this enzyme impairs prostate cancer aggressiveness. These effects were partially reversed by treatment with fatty acids or a cannabinoid receptor-1 (CB1) antagonist, and fully reversed by cotreatment with both agents. We further show that MAGL is part of a gene signature correlated with epithelial-to-mesenchymal transition and the stem-like properties of cancer cells, supporting a role for this enzyme in protumorigenic metabolism that, for prostate cancer, involves the dual control of endocannabinoid and fatty acid pathways.


Assuntos
Moduladores de Receptores de Canabinoides/metabolismo , Endocanabinoides , Ácidos Graxos/metabolismo , Monoacilglicerol Lipases/metabolismo , Neoplasias da Próstata/enzimologia , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Monoacilglicerol Lipases/antagonistas & inibidores , Monoacilglicerol Lipases/genética , Células-Tronco Neoplásicas/enzimologia , Células-Tronco Neoplásicas/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Interferência de RNA , Transdução de Sinais
18.
J Cell Biol ; 188(2): 191-8, 2010 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-20100909

RESUMO

Accurate control of the number of centrosomes, the major microtubule-organizing centers of animal cells, is critical for the maintenance of genome integrity. Abnormalities in centrosome number can promote errors in spindle formation that lead to subsequent chromosome missegregation, and extra centrosomes are found in many cancers. Centrosomes are comprised of a pair of centrioles surrounded by amorphous pericentriolar material, and centrosome duplication is controlled by centriole replication. Polo-like kinase 4 (Plk4) plays a key role in initiating centriole duplication, and overexpression of Plk4 promotes centriole overduplication and the formation of extra centrosomes. Using chemical genetics, we show that kinase-active Plk4 is inherently unstable and targeted for degradation. Plk4 is shown to multiply self-phosphorylate within a 24-amino acid phosphodegron. Phosphorylation of multiple sites is required for Plk4 instability, indicating a requirement for a threshold level of Plk4 kinase activity to promote its own destruction. We propose that kinase-mediated, autoregulated instability of Plk4 self-limits Plk4 activity so as to prevent centrosome amplification.


Assuntos
Centríolos/metabolismo , Centrossomo/metabolismo , Duplicação Gênica , Proteínas Serina-Treonina Quinases/metabolismo , Aneuploidia , Animais , Domínio Catalítico/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Centríolos/genética , Estabilidade Enzimática/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Homeostase/genética , Humanos , Camundongos , Fosforilação , Fosfotransferases/genética , Fosfotransferases/metabolismo , Ligação Proteica/fisiologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Fuso Acromático/genética , Fuso Acromático/metabolismo
19.
ACS Chem Biol ; 4(11): 948-57, 2009 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-19785476

RESUMO

A significant gap exists between genetics-based investigations of polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) biosynthetic pathways and our understanding of their regulation, interaction, and activity in living systems. To help bridge this gap, here we present an orthogonal active site identification system (OASIS) for the proteomic identification and analysis of PKS/NRPS biosynthetic enzymes. OASIS probes target conserved features of PKS/NRPS active sites to provide activity-based enrichment of modular synthases, followed by analysis through multidimensional protein identification technology (MudPIT) LC-MS/MS analysis. When applied to the model bacterium Bacillus subtilis, this functional proteomics method detects and quantifies all four modular synthases in the organism. Furthermore, tandem application of multiple OASIS probes enhances identification of specific PKS/NRPS modules from complex proteomic mixtures. By expanding the dynamic range of proteomic analysis for PKS/NRPS enzymes, OASIS offers a valuable tool for strain comparison, culture condition optimization, and enzyme discovery.


Assuntos
Produtos Biológicos/análise , Peptídeo Sintases/análise , Proteômica/métodos , Sequência de Aminoácidos , Bacillus subtilis/enzimologia , Produtos Biológicos/metabolismo , Domínio Catalítico , Dados de Sequência Molecular , Peptídeo Sintases/química , Peptídeo Sintases/metabolismo
20.
Nat Chem Biol ; 2(5): 274-81, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16565715

RESUMO

Metalloproteases are a large, diverse class of enzymes involved in many physiological and disease processes. Metalloproteases are regulated by post-translational mechanisms that diminish the effectiveness of conventional genomic and proteomic methods for their functional characterization. Chemical probes directed at active sites offer a potential way to measure metalloprotease activities in biological systems; however, large variations in structure limit the scope of any single small-molecule probe aimed at profiling this enzyme class. Here, we address this problem by creating a library of metalloprotease-directed probes that show complementary target selectivity. These probes were applied as a 'cocktail' to proteomes and their labeling profiles were analyzed collectively using an advanced liquid chromatography-mass spectrometry platform. More than 20 metalloproteases were identified, including members from nearly all of the major branches of this enzyme class. These findings suggest that chemical proteomic methods can serve as a universal strategy to profile the activity of the metalloprotease superfamily in complex biological systems.


Assuntos
Metaloproteases/metabolismo , Técnicas de Sonda Molecular , Proteoma , Proteômica/métodos , Marcadores de Afinidade/química , Marcadores de Afinidade/metabolismo , Sítios de Ligação , Espectrometria de Massas , Metaloproteases/análise , Estrutura Molecular , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/fisiologia
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