[Show abstract][Hide abstract] ABSTRACT: 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-β4 (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.
International Journal of Cancer 01/2012; 131(8):1744-54. · 6.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism 07/2011; 31(12):2267-81. · 5.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Proceedings of the National Academy of Sciences 05/2011; 108(21):8815-20. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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,β-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.
Nature Chemical Biology 01/2011; 7(7):469-78. · 12.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Proceedings of the National Academy of Sciences 11/2010; 107(49):20941-6. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
The Journal of Cell Biology 01/2010; 188(2):191-8. · 10.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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:
[Show abstract][Hide abstract] ABSTRACT: 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.
ACS Chemical Biology 09/2009; 4(11):948-57. · 5.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Medicinal Chemistry 04/2007; 50(5):1058-68. · 5.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Nature Chemical Biology 06/2006; 2(5):274-81. · 12.95 Impact Factor