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1.  Comparative Proteomic Analysis of a Cytosolic Fraction from β3 Integrin-deficient Cells 
Integrins are heterodimeric transmembrane receptors involved in sensing and transmitting informational cues from the extracellular environment to the cell. This study explored changes to a sub-proteome in response to elimination of the β3 integrin using a knockout murine model. Cleavable isotope-coded affinity tagging (cICAT) in combination with sub-cellular fractionation, multiple dimensions of separation and tandem mass spectrometry (MS/MS) were used to characterize differentially expressed proteins between β3 integrin−/− (β3−/−) mouse embryonic fibroblasts and isogenic wild-type (WT) controls. From a cytosolic protein fraction, 48 proteins were identified in which expression differed by >1.5-fold. Predominant ontological groups included actin-binding/cytoskeletal proteins and protease/protease inhibitors. Interestingly, β3 integrin expression was inversely correlated with expression of cathepsin B, a lysosomal cysteine protease, as its expression was greater by over 3.5-fold in the β3−/− cells. This inverse correlation was also observed in stable heterologous cells transfected with β3 integrin, where the intracellular expression and activity of cathepsin B was lower compared to control cells. Our data suggests that the composition of the cellular proteome is influenced by integrin expression patterns and reveals a strong functional relationship between β3 integrin and cathepsin B.
PMCID: PMC3627548  PMID: 22210044
Cathepsin B; integrin; isotope-coded affinity tags; protease; cancer
2.  Structural determinants of limited proteolysis 
Journal of proteome research  2011;10(8):3642-3651.
Limited or regulatory proteolysis plays a critical role in many important biological pathways like blood coagulation, cell proliferation, and apoptosis. A better understanding of mechanisms that control this process is required for discovering new proteolytic events and for developing inhibitors with potential therapeutic value. Two features that determine the susceptibility of peptide bonds to proteolysis are the sequence in the vicinity of the scissile bond and the structural context in which the bond is displayed. In this study we assessed statistical significance and predictive power of individual structural descriptors and combination thereof for the identification of cleavage sites. The analysis was performed on a dataset of >200 proteolytic events documented in CutDB for a variety of mammalian regulatory proteases and their physiological substrates with known 3D structures. The results confirmed the significance and provided a ranking within three main categories of structural features: exposure > flexibility > local interactions. Among secondary structure elements, the largest frequency of proteolytic cleavage was confirmed for loops and lower but significant frequency for helices. Limited proteolysis has lower albeit appreciable frequency of occurrence in certain types of β-strands, which is in contrast with some previous reports. Descriptors deduced directly from the amino acid sequence displayed only marginal predictive capabilities. Homology-based structural models showed a predictive performance comparable to protein substrates with experimentally established structures. Overall, this study provided a foundation for accurate automated prediction of segments of protein structure susceptible to proteolytic processing and, potentially, other post-translational modifications.
PMCID: PMC3164237  PMID: 21682278
proteolysis; proteolytic processing; limited proteolysis; regulatory proteolysis; protease; cleavage site; cleavage site prediction
3.  Proteins and an Inflammatory Network Expressed in Colon Tumors 
Journal of proteome research  2011;10(5):2129-2139.
The adenomatous polyposis coli (APC) protein is crucial to homeostasis of normal intestinal epithelia because it suppresses the β-catenin/TCF pathway. Consequently, loss or mutation of the APC gene causes colorectal tumors in humans and mice. Here, we describe our use of Multidimensional Protein Identification Technology (MudPIT) to compare protein expression in colon tumors to that of adjacent healthy colon tissue from ApcMin/+ mice. Twenty-seven proteins were found to be up-regulated in colon tumors and twenty-five down-regulated. As an extension of the proteomic analysis, the differentially expressed proteins were used as “seeds” to search for co-expressed genes. This approach revealed a co-expression network of 45 genes that is up-regulated in colon tumors. Members of the network include the antibacterial peptide cathelicidin (CAMP), Toll-like receptors (TLRs), IL-8, and triggering receptor expressed on myeloid cells 1 (TREM1). The co-expression network is associated with innate immunity and inflammation, and there is significant concordance between its connectivity in humans versus mice (Friedman: p value = 0.0056). This study provides new insights into the proteins and networks that are likely to drive the onset and progression of colon cancer.
PMCID: PMC3090457  PMID: 21366352
proteomics; network; MudPIT; mass spectrometry; transcriptomics; colon cancer; inflammation; ApcMin/+
4.  Polymer Particle Shape Independently Influences Binding and Internalization by Macrophages 
The interaction of macrophages with micro and nano particles (MNPs) is important because these cells clear particles from the circulation, and because they are potential therapeutic targets in inflammatory conditions, atherosclerosis and cancer. Therefore, an understanding of the features of MNPs that influence their interaction with macrophages may allow optimization of their properties for enhanced drug delivery. In this study, we show that particle shape impacts phagocytosis by macrophages, and more importantly, that particle shape and size separately impact attachment and internalization. The study provides methodology for further exploring how particle shape can be controlled to achieve desired attachment and internalization. The results of the study also give mechanistic guidance on how particle shape can be manipulated to design drug carriers to evade macrophages, or alternatively to target macrophages.
PMCID: PMC2975856  PMID: 20691741
5.  Extracellular and Intracellular Mechanisms Mediating Metastatic Activity of Exogenous Osteopontin 
Cancer  2009;115(8):1753-1764.
Osteopontin affects several steps of the metastatic cascade. Despite direct correlation with metastasis in experimental systems and in patient studies, the extracellular and intracellular basis for these observations remains unsolved. We used human melanoma and sarcoma cell lines to evaluate the effects of soluble osteopontin on metastasis.
Exogenous osteopontin or negative controls, including a site-directed mutant osteopontin, were used in functional assays in vitro, ex vivo, and in vivo designed to test extracellular and intracellular mechanisms involved in experimental metastasis.
In the extracellular environment, we confirm that soluble osteopontin is required for its pro-metastatic effects; this phenomenon is specific, RGD-dependent, and evident in experimental models of metastasis. In the intracellular environment, osteopontin initially induces rapid Tyr-418 dephosphorylation of c-Src, with decreases in actin stress fibers and increased binding to the vascular endothelium. This heretofore undescribed Tyr dephosphorylation is followed by a tandem c-Src phosphorylation after tumor cell attachment to the metastatic site.
Our results reveal a complex molecular interaction as well as a dual role for osteopontin in metastasis that is dependent on whether tumor cells are in circulation or attached. Such context-dependent functional insights may contribute to anti-metastasis strategies.
PMCID: PMC2743165  PMID: 19224553
c-Src; endothelium; metastasis; osteopontin
6.  Glutamine-fueled mitochondrial metabolism is decoupled from glycolysis in melanoma 
Pigment cell & melanoma research  2012;25(6):732-739.
In this perspective, we revise the historic notion that cancer is a disease of mitochondria. We summarize recent findings on the function and rewiring of central carbon metabolism in melanoma. Metabolic profiling studies using stable isotope tracers show that glycolysis is decoupled from the tricarboxylic acid (TCA) cycle. This decoupling is not ‘dysfunction’ but rather an alternate wiring required by tumor cells to remain metabolically versatile. In large part, this requirement is met by glutamine feeding the TCA cycle as an alternative source of carbon. Glutamine is also used in non-conventional ways, like traveling in reverse through the TCA flux to feed fatty acid biosynthesis. The biosynthetic networks linked with non-essential amino acids alanine, serine, arginine, and proline are also significantly impacted by the use of glutamine as an alternate carbon source.
PMCID: PMC3639292  PMID: 22846158
metabolism; mitochondria; glutamine; systems biology; NMR
7.  Platelet Mimetic Particles for Targeting Thrombi in Flowing Blood 
PMCID: PMC3483800  PMID: 22641451
platelets; targeted drug delivery; polymeric particles; layer-by-layer technique; GPIb; VWF domain
8.  Reverse TCA cycle flux through isocitrate dehydrogenases 1 and 2 is required for lipogenesis in hypoxic melanoma cells 
Pigment cell & melanoma research  2012;25(3):375-383.
The TCA cycle is the central hub of oxidative metabolism, running in the classic forward direction to provide carbon for biosynthesis and reducing agents for generation of ATP. Our metabolic tracer studies in melanoma cells showed that in hypoxic conditions the TCA cycle is largely disconnected from glycolysis. By studying the TCA branch point metabolites, acetyl CoA and citrate, as well as the metabolic endpoints glutamine and fatty acids, we developed a comprehensive picture of the rewiring of the TCA cycle that occurs in hypoxia. Hypoxic tumor cells maintain proliferation by running the TCA cycle in reverse. The source of carbon for acetyl CoA, citrate, and fatty acids switches from glucose in normoxia to glutamine in hypoxia. This hypoxic flux from glutamine into fatty acids is mediated by reductive carboxylation. This reductive carboxylation is catalyzed by two isocitrate dehydrogenases, IDH1 and IDH2. Their combined action is necessary and sufficient to effect the reverse TCA flux and maintain cellular viability.
PMCID: PMC3329592  PMID: 22360810
9.  Optical detection of indocyanine green encapsulated biocompatible poly (lactic-co-glycolic) acid nanoparticles with photothermal optical coherence tomography 
Optics letters  2012;37(5):981-983.
We describe a functional imaging paradigm that uses photothermal optical coherence tomography (PT-OCT) to detect indocyanine green (ICG)-encapsulated biocompatible poly(lactic-co-glycolic) acid (PLGA) nanoparticles embedded in highly scattering tissue phantoms with high resolution and sensitivity. The ICG-loaded PLGA nanoparticles were fabricated using a modified emulsification solvent diffusion method. With a 20 kHz axial scan rate, PT-OCT based on spectral-domain interferometric configuration at 1310 nm was used to detect phase changes induced by a 808 nm photothermal excitation of ICG-encapsulated PLGA nanoparticles. An algorithm based on Fourier transform analysis of differential phase of the spectral interferogram was developed for detecting the depth resolved localized photothermal signal. Excellent contrast difference was observed with PT-OCT between phantoms containing different concentrations of ICG-encapsulated PLGA nanoparticles. This technique has the potential to provide simultaneous structural and molecular-targeted imaging with excellent signal-to-noise for various clinical applications.
PMCID: PMC3625629  PMID: 22378459
10.  Crystal and Solution Structures of a Prokaryotic M16B Peptidase: an Open and Shut Case 
Structure (London, England : 1993)  2009;17(11):1465-1475.
The M16 family of zinc peptidases comprises a pair of homologous domains that form two halves of a ‘‘clam-shell’’ surrounding the active site. The M16A and M16C subfamilies form one class (‘‘peptidasomes’’): they degrade 30–70 residue peptides, and adopt both open and closed conformations. The eukaryotic M16B subfamily forms a second class (‘‘processing proteases’’): they adopt a single partly-open conformation that enables them to cleave signal sequences from larger proteins. Here, we report the solution and crystal structures of a prokaryotic M16B peptidase, and demonstrate that it has features of both classes: thus, it forms stable ‘‘open’’ homodimers in solution that resemble the processing proteases; but the clam-shell closes upon binding substrate, a feature of the M16A/C peptidasomes. Moreover, clam-shell closure is required for proteolytic activity. We predict that other prokaryotic M16B family members will form dimeric peptidasomes, and propose a model for the evolution of the M16 family.
PMCID: PMC3615642  PMID: 19913481
11.  One-Step Fabrication of Polymeric Janus Nanoparticles for Drug Delivery 
With its unique structure of two compartments, Janus particles inadequate, including to be used as a drug delivery system to deliver multiple payloads with widely different solubility. Here we report on a fluidic nanoprecipitation system (FNPS), capable of fabricating biocompatible Janus polymeric nanoparticles comprised of the FDA-approved polymer poly(lactic-co-glycolic acid) (PLGA). The FNPS contains dual inlets, one for each half of the particle, that insert into the precipitation stream. The system provides a one-step approach for production of Janus polymeric particles with submicrometer diameters and is likely amenable to substantial scale-up. To the best of our knowledge, this is the first demonstration of biocompatible Janus nanoparticles that encapsulate a hydrophobic drug (paclitaxel) on one side and a hydrophilic drug (doxorubicin hydrochloride) on the other.
PMCID: PMC3582348  PMID: 22251479
12.  Risk modification of colorectal adenoma by CYP7A1 polymorphisms and the role of bile acid metabolism in carcinogenesis 
Cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in the conversion of cholesterol to bile acids, is a postulated gene modifier of colorectal cancer risk and target for the therapeutic bile acid, ursodeoxycholic acid (UDCA). We investigated associations between CYP7A1 polymorphisms and fecal bile acids, colorectal adenoma (CRA), and UDCA efficacy for CRA prevention. Seven tagging, single-nucleotide polymorphisms (SNPs) in CYP7A1 were measured in 703 (355 UDCA, 348 placebo) participants of a phase III chemoprevention trial, of which 495 had known baseline fecal bile acid concentrations. In the placebo arm, participants with two minor Grs8192871 alleles (tag for a low-activity promoter polymorphism at -204) had lower odds of high secondary bile acids (OR, 0.26; 95% CI, 0.10–0.69), and CRA at 3 years' follow-up (OR, 0.41; 95% CI, 0.19–0.89), than AA carriers. Haplotype construction from the six polymorphic SNPs showed participants with the third-most common haplotype (Crs10957057Crs8192879Grs8192877Trs11786580Ars8192871Grs13251096) had higher odds of high primary bile acids (OR, 2.34; 95% CI, 1.12–4.89) and CRA (OR, 1.89; 95% CI, 1.00–3.57) than those with the most common CTACAG haplotype. Furthermore, three SNPs (rs8192877, rs8192871, and rs13251096) each modified UDCA efficacy for CRA prevention, and CCGTAG-haplotype carriers experienced 71% lower odds of CRA recurrence with UDCA treatment, an effect not present for other haplotypes (test for UDCA-by-haplotype interaction, P=0.020). Our findings support CYP7A1 polymorphisms as determinants of fecal bile acids and risk factors for CRA. Further, UDCA efficacy for CRA prevention may be modified by genetic variation in CYP7A1, limiting treatment benefit to a subgroup of the population.
PMCID: PMC3400261  PMID: 22058145
CYP7A1; colorectal adenoma; chemoprevention; bile acids
Nano LIFE  2010;1(3-4):207-214.
Macrophage foam cells are key components of atherosclerotic plaque and play an important role in the progression of atherosclerosis leading to plaque rupture and thrombosis. Foam cells are emerging as attractive targets for therapeutic intervention and for imaging the progression of disease. Therefore, designing nanoparticles (NPs) targeted to macrophage foam cells in plaque is of considerable therapeutic significance. Here we report the construction of an oligonucleotide functionalized NP system with high affinity for foam cells. Nanoparticles functionalized with a 23-mer poly-Guanine (polyG) oligonucleotide are specifically recognized by the scavenger receptors on lipid-laden foam cells in vitro and ex vivo. The enhanced uptake of polyG-functionalized NPs by foam cells is inhibited in the presence of acetylated-LDL, a known ligand of scavenger receptors. Since polyG oligonucleotides are stable in serum and are unlikely to induce an immune response, their use for scavenger receptor-mediated targeting of macrophage foam cells provides a strategy for targeting atherosclerotic lesions.
PMCID: PMC3484886  PMID: 23125876
Atherosclerosis; Nanoparticles; polyG; foam cells; scavenger receptors
14.  Functional Specialization in Proline Biosynthesis of Melanoma 
PLoS ONE  2012;7(9):e45190.
Proline metabolism is linked to hyperprolinemia, schizophrenia, cutis laxa, and cancer. In the latter case, tumor cells tend to rely on proline biosynthesis rather than salvage. Proline is synthesized from either glutamate or ornithine; both are converted to pyrroline-5-carboxylate (P5C), and then to proline via pyrroline-5-carboxylate reductases (PYCRs). Here, the role of three isozymic versions of PYCR was addressed in human melanoma cells by tracking the fate of 13C-labeled precursors. Based on these studies we conclude that PYCR1 and PYCR2, which are localized in the mitochondria, are primarily involved in conversion of glutamate to proline. PYCRL, localized in the cytosol, is exclusively linked to the conversion of ornithine to proline. This analysis provides the first clarification of the role of PYCRs to proline biosynthesis.
PMCID: PMC3443215  PMID: 23024808
15.  High Throughput Substrate Phage Display for Protease Profiling 
The interplay between a protease and its substrates is controlled at many different levels, including coexpression, colocalization, binding driven by ancillary contacts, and the presence of natural inhibitors. Here we focus on the most basic parameter that guides substrate recognition by a protease, the recognition specificity at the catalytic cleft. An understanding of this substrate specificity can be used to predict the putative substrates of a protease, to design protease activated imaging agents, and to initiate the design of active site inhibitors. Our group has characterized protease specificities of several matrix metalloproteinases using substrate phage display. Recently, we have adapted this method to a semiautomated platform that includes several high-throughput steps. The semiautomated platform allows one to obtain an order of magnitude more data, thus permitting precise comparisons among related proteases to define their functional distinctions.
PMCID: PMC3372406  PMID: 19377968
Substrate phage display; Substrate; Protease; Specificity; Proteolysis; Filamentous phage; M13 coat protein; 3 gene protein
16.  Synthesis of Novel β-Lactone Inhibitors of Fatty Acid Synthase 
Journal of medicinal chemistry  2008;51(17):5285-5296.
Fatty acid synthase (FAS) is necessary for growth and survival of tumor cells and is a promising drug target for oncology. Here, we report on the syntheses and activity of novel inhibitors of the thioesterase domain of FAS. Using the structure of orlistat as a starting point, which contains a β-lactone as the central pharmacophore, 28 novel congeners were synthesized and examined. Structural features such as the length of the α- and β-alkyl chains, their chemical composition, and amino ester substitutions were altered and the resulting compounds explored for inhibitory activity toward the thioesterase domain of FAS. Nineteen congeners show improved potency for FAS in biochemical assays relative to orlistat. Three of that subset, including the natural product valilactone, also display an increased potency in inducing tumor cell death and improved solubility compared to orlistat. These findings support the idea that an orlistat congener can be optimized for use in a preclinical drug design and for clinical drug development.
PMCID: PMC3172131  PMID: 18710210
17.  Mitochondrial p32 Protein Is a Critical Regulator of Tumor Metabolism via Maintenance of Oxidative Phosphorylation ▿  
Molecular and Cellular Biology  2010;30(6):1303-1318.
p32/gC1qR/C1QBP/HABP1 is a mitochondrial/cell surface protein overexpressed in certain cancer cells. Here we show that knocking down p32 expression in human cancer cells strongly shifts their metabolism from oxidative phosphorylation (OXPHOS) to glycolysis. The p32 knockdown cells exhibited reduced synthesis of the mitochondrial-DNA-encoded OXPHOS polypeptides and were less tumorigenic in vivo. Expression of exogenous p32 in the knockdown cells restored the wild-type cellular phenotype and tumorigenicity. Increased glucose consumption and lactate production, known as the Warburg effect, are almost universal hallmarks of solid tumors and are thought to favor tumor growth. However, here we show that a protein regularly overexpressed in some cancers is capable of promoting OXPHOS. Our results indicate that high levels of glycolysis, in the absence of adequate OXPHOS, may not be as beneficial for tumor growth as generally thought and suggest that tumor cells use p32 to regulate the balance between OXPHOS and glycolysis.
PMCID: PMC2832503  PMID: 20100866
18.  Fabrication of PLGA nanoparticles with a fluidic nanoprecipitation system 
Particle size is a key feature in determining performance of nanoparticles as drug carriers because it influences circulating half-life, cellular uptake and biodistribution. Because the size of particles has such a major impact on their performance, the uniformity of the particle population is also a significant factor. Particles comprised of the polymer poly(lactic-co-glycolic acid) (PLGA) are widely studied as therapeutic delivery vehicles because they are biodegradable and biocompatible. In fact, microparticles comprised of PLGA are already approved for drug delivery. Unfortunately, PLGA nanoparticles prepared by conventional methods usually lack uniformity. We developed a novel Fluidic NanoPrecipitation System (FNPS) to fabricate highly uniform PLGA particles. Several parameters can be fine-tuned to generate particles of various sizes.
PMCID: PMC2929212  PMID: 20707919
19.  Mass Spectrometry-Based Label-Free Quantitative Proteomics 
In order to study the differential protein expression in complex biological samples, strategies for rapid, highly reproducible and accurate quantification are necessary. Isotope labeling and fluorescent labeling techniques have been widely used in quantitative proteomics research. However, researchers are increasingly turning to label-free shotgun proteomics techniques for faster, cleaner, and simpler results. Mass spectrometry-based label-free quantitative proteomics falls into two general categories. In the first are the measurements of changes in chromatographic ion intensity such as peptide peak areas or peak heights. The second is based on the spectral counting of identified proteins. In this paper, we will discuss the technologies of these label-free quantitative methods, statistics, available computational software, and their applications in complex proteomics studies.
PMCID: PMC2775274  PMID: 19911078
20.  Matrix Metalloproteinase Proteolysis of the Myelin Basic Protein Isoforms Is a Source of Immunogenic Peptides in Autoimmune Multiple Sclerosis 
PLoS ONE  2009;4(3):e4952.
Matrix metalloproteinases (MMPs) play a significant role in the fragmentation of myelin basic protein (MBP) and demyelination leading to autoimmune multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The classic MBP isoforms are predominantly expressed in the oligodendrocytes of the CNS. The splice variants of the single MBP gene (Golli-MBP BG21 and J37) are widely expressed in the neurons and also in the immune cells. The relative contribution of the individual MMPs to the MBP cleavage is not known.
Methodology/Principal Findings
To elucidate which MMP plays the primary role in cleaving MBP, we determined the efficiency of MMP-2, MMP-8, MMP-9, MMP-10, MMP-12, MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP and MT6-MMP in the cleavage of the MBP, BG21 and J37 isoforms in the in vitro cleavage reactions followed by mass-spectroscopy analysis of the cleavage fragments. As a result, we identified the MMP cleavage sites and the sequence of the resulting fragments. We determined that MBP, BG21 and J37 are highly sensitive to redundant MMP proteolysis. MT6-MMP (initially called leukolysin), however, was superior over all of the other MMPs in cleaving the MBP isoforms. Using the mixed lymphocyte culture assay, we demonstrated that MT6-MMP proteolysis of the MBP isoforms readily generated, with a near quantitative yield, the immunogenic N-terminal 1–15 MBP peptide. This peptide selectively stimulated the proliferation of the PGPR7.5 T cell clone isolated from mice with EAE and specific for the 1–15 MBP fragment presented in the MHC H-2U context.
In sum, our biochemical observations led us to hypothesize that MT6-MMP, which is activated by furin and associated with the lipid rafts, plays an important role in MS pathology and that MT6-MMP is a novel and promising drug target in MS especially when compared with other individual MMPs.
PMCID: PMC2654159  PMID: 19300513
21.  PMAP: databases for analyzing proteolytic events and pathways 
Nucleic Acids Research  2008;37(Database issue):D611-D618.
The Proteolysis MAP (PMAP, is a user-friendly website intended to aid the scientific community in reasoning about proteolytic networks and pathways. PMAP is comprised of five databases, linked together in one environment. The foundation databases, ProteaseDB and SubstrateDB, are driven by an automated annotation pipeline that generates dynamic ‘Molecule Pages’, rich in molecular information. PMAP also contains two community annotated databases focused on function; CutDB has information on more than 5000 proteolytic events, and ProfileDB is dedicated to information of the substrate recognition specificity of proteases. Together, the content within these four databases will ultimately feed PathwayDB, which will be comprised of known pathways whose function can be dynamically modeled in a rule-based manner, and hypothetical pathways suggested by semi-automated culling of the literature. A Protease Toolkit is also available for the analysis of proteases and proteolysis. Here, we describe how the databases of PMAP can be used to foster understanding of proteolytic pathways, and equally as significant, to reason about proteolysis.
PMCID: PMC2686432  PMID: 18842634
22.  Central carbon metabolism in the progression of mammary carcinoma 
There is a growing belief that the metabolic program of breast tumor cells could be a therapeutic target. Yet, without detailed information on central carbon metabolism in breast tumors it is impossible to know which metabolic pathways to target, and how their inhibition might influence different stages of breast tumor progression. Here we perform the first comprehensive profiling of central metabolism in the MCF10 model of mammary carcinoma, where the steps of breast tumor progression (transformation, tumorigenicity and metastasis) can all be examined in the context of the same genetic background. The metabolism of [U-13C]-glucose by a series of progressively more aggressive MCF10 cell lines was tracked by 2D NMR and mass spectrometry. From this analysis the flux of carbon through distinct metabolic reactions was quantified by isotopomer modeling. The results indicate widespread changes to central metabolism upon cellular transformation including increased carbon flux through the pentose phosphate pathway (PPP), the TCA cycle, as well as increased synthesis of glutamate, glutathione and fatty acids (including elongation and desaturation). The de novo synthesis of glycine increased upon transformation as well as at each subsequent step of breast tumor cell progression. Interestingly, the major metabolic shift in metastatic cells is a large increase in the de novo synthesis of proline. This work provides the first comprehensive view of changes to central metabolism as a result of breast tumor progression.
Electronic supplementary material
The online version of this article (doi:10.1007/s10549-007-9732-3) contains supplementary material, which is available to authorized users.
PMCID: PMC2440942  PMID: 17879159
Cellular metabolism; Flux modeling; Glucose; Glycolysis; Metastasis; Tumor progression
23.  Expression and purification of a two-component flaviviral proteinase resistant to autocleavage at the NS2B-NS3 junction region 
Regulated proteolysis of the polyprotein precursor of West Nile virus (WNV) by the essential NS2B-NS3(pro)tease, a promising drug target for WNV inhibitors, is required for the propagation of infectious virions. Structural and drug design studies, however, require pilot-scale quantities of a pure and catalytically active WNV protease that is resistant to self-proteolysis. Autolytic cleavage at the NS2B-NS3 boundary leads to individual, non-covalently associated, NS2B and NS3 domains, together with residual amounts of the intact NS2B-NS3, in the NS2B-NS3pro samples. We modified the cleavage site sequence of the NS2B-NS3 junction region and then developed expression and purification procedures to prepare a covalently linked, single-chain, NS2B-NS3pro K48A mutant construct. This construct exhibits high stability and functional activity and is thus well suited for the follow-up purification and structural and drug design studies.
PMCID: PMC1857357  PMID: 17189703
24.  Switching the Substrate Specificity of the Two-Component NS2B-NS3 Flavivirus Proteinase by Structure-Based Mutagenesis▿  
Journal of Virology  2007;81(9):4501-4509.
The flavivirus NS2B-NS3(pro)teinase is an essential element in the proteolytic processing of the viral precursor polyprotein and therefore a potential drug target. Recently, crystal structures and substrate preferences of NS2B-NS3pro from Dengue and West Nile viruses (DV and WNV) were determined. We established that the presence of Gly-Gly at the P1′-P2′ positions is optimal for cleavage by WNV NS3pro, whereas DV NS3pro tolerates well the presence of bulky residues at either P1′ or P2′. Structure-based modeling suggests that Arg76 and Pro131-Thr132 limit the P1′-P2′ subsites and restrict the cleavage preferences of the WNV enzyme. In turn, Leu76 and Lys131-Pro132 widen the specificity of DV NS3pro. Guided by these structural models, we expressed and purified mutant WNV NS2B-NS3pro and evaluated cleavage preferences by using positional scanning of the substrate peptides in which the P4-P1 and the P3′-P4′ positions were fixed and the P1′ and P2′ positions were each randomized. We established that WNV R76L and P131K-T132P mutants acquired DV-like cleavage preferences, whereas T52V had no significant effect. Our work is the first instance of engineering a viral proteinase with switched cleavage preferences and should provide valuable data for the design of optimized substrates and substrate-based selective inhibitors of flaviviral proteinases.
PMCID: PMC1900165  PMID: 17301157
25.  Genome-wide changes accompanying knockdown of fatty acid synthase in breast cancer 
BMC Genomics  2007;8:168.
The lipogenic enzyme fatty acid synthase (FAS) is up-regulated in a wide variety of cancers, and is considered a potential metabolic oncogene by virtue of its ability to enhance tumor cell survival. Inhibition of tumor FAS causes both cell cycle arrest and apoptosis, indicating FAS is a promising target for cancer treatment.
Here, we used gene expression profiling to conduct a global study of the cellular processes affected by siRNA mediated knockdown of FAS in MDA-MB-435 mammary carcinoma cells. The study identified 169 up-regulated genes (≥ 1.5 fold) and 110 down-regulated genes (≤ 0.67 fold) in response to knockdown of FAS. These genes regulate several aspects of tumor function, including metabolism, cell survival/proliferation, DNA replication/transcription, and protein degradation. Quantitative pathway analysis using Gene Set Enrichment Analysis software further revealed that the most pronounced effect of FAS knockdown was down-regulation in pathways that regulate lipid metabolism, glycolysis, the TCA cycle and oxidative phosphorylation. These changes were coupled with up-regulation in genes involved in cell cycle arrest and death receptor mediated apoptotic pathways.
Together these findings reveal a wide network of pathways that are influenced in response to FAS knockdown and provide new insight into the role of this enzyme in tumor cell survival and proliferation.
PMCID: PMC1913522  PMID: 17565694

Results 1-25 (26)