Noemi Kedei

National Cancer Institute (USA), Maryland, United States

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Publications (41)193.07 Total impact

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    ABSTRACT: Precise and accurate quantification of protein expression levels in a complex biological setting is challenging. Here, we describe a method for absolute quantitation of endogenous proteins in cell lysates using an automated capillary immunoassay system (the size-based Simple Western system, ProteinSimple, CA). The method was able to accurately measure the absolute amounts of target proteins at picogram or sub-picogram levels per nanogram of cell lysates. The measurements were independent of the cell matrix or the cell lysis buffer and were not affected by different antibody affinities for their specific epitopes. We then applied this method to quantitate absolute levels of expression of protein kinase C (PKC) isoforms in LNCaP and U937 cells, two cell lines used extensively for probing the downstream biological responses to PKC targeted ligands. Our absolute quantitation confirmed the predominance of PKCδ in both cells, supporting the important functional role of this PKC isoform in these cell lines. The method described here provides an approach to accurately quantitate levels of protein expression and correlate protein level with function. In addition to enhanced accuracy relative to conventional western analysis, it circumvents the distortions inherent in comparison of signal intensities from different antibodies with different affinities.
    Analytical Biochemistry 07/2013; · 2.58 Impact Factor
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    ABSTRACT: The bryostatins are a group of 20 macrolides isolated by Pettit and co-workers from the marine organism Bugula neritina. Bryostatin 1, the flagship member of the family, has been the subject of intense chemical and biological investigations due to its remarkably diverse biological activities, including promising indications as therapy for cancer, Alzheimer's disease, and HIV. Other bryostatins, however, have attracted far less attention, most probably due to their relatively low natural abundance and associated scarcity of supply. Among all macrolides in this family, bryostatin 7 is biologically the most potent protein kinase C (PKC) ligand (in terms of binding affinity) and also the first bryostatin to be synthesized in the laboratory. Nonetheless, almost no biological studies have been carried out on this agent. We describe herein the total synthesis of bryostatin 7 based on our pyran annulation technology, which allows for the first detailed biological characterizations of bryostatin 7 with side-by-side comparisons to bryostatin 1. The results suggest that the more easily synthesized and less lipophilic bryostatin 7 may be an effective surrogate for bryostatin 1.
    ACS Chemical Biology 02/2013; · 5.44 Impact Factor
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    ABSTRACT: PKCθ is predominantly expressed in T-cells and critically involved in immunity. Design of PKCθ selective molecules to manage autoimmune disorders by targeting its activator binding C1 domain requires the knowledge of its structure and the activator binding residues. The C1 domain consists of twin C1 domains, C1A and C1B, of which C1B plays the critical role in the membrane translocation and activation of PKCq. Here, we determined the crystal structure of the PKCθC1B to 1.63Å resolution, which showed that the Trp-253 at the rim of the activator binding pocket was oriented towards the membrane whereas in PKCdC1B, the homologous tryptophan was oriented away from the membrane. This particular orientation of Trp-253 abolishes the possible p-stacking interactions between Trp-253 and His-270 and the cation-p interactions between Trp-253 and Arg-272, which are present between the homologous residues in PKCdC1B. To further probe the structural constraints on activator binding, five residues lining the activator binding site were mutated (Y239A, T243A, W253G, L255G and Q258G) and the binding affinities of the PKCθC1B mutants were measured. These mutants showed reduced binding affinities for phorbol ester (PDBu) and diacylglycerol (DOG). All the five full length PKCθ mutants exhibited reduced phorbol ester-induced membrane translocation compared to the wild type. These results provide insights into the PKCq activator binding domain, which will aid in future design of PKCθ selective molecules.
    Biochemical Journal 01/2013; · 4.65 Impact Factor
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    ABSTRACT: Ingenol-3-angelate (I3A) is a non-tumor promoting phorbol ester-like compound identified in the sap of Euphoria peplus. Similar to tumor promoting phorbol esters, I3A is a diacylglycerol (DAG) analogue that binds with high affinity to the C1 domains of PKCs, recruits PKCs to cellular membranes and promotes enzyme activation. Numerous anti-cancer activities have been attributed to I3A and ascribed to I3A's effects on PKCs. We show here that I3A also binds to and activates members of the RasGRP family of Ras activators leading to robust elevation of Ras-GTP and engagement of the Raf-Mek-Erk kinase cascade. In response to I3A, recombinant proteins consisting of GFP fused separately to full-length RasGRP1 and RasGRP3 were rapidly recruited to cell membranes, consistent with direct binding of the compound to RasGRP's C1 domain. In the case of RasGRP3, IA3 treatment led to positive regulatory phosphorylation on T133 and activation of the candidate regulatory kinase PKCδ. I3A treatment of select B non-Hodgkin's lymphoma cell lines resulted in quantitative and qualitative changes in Bcl-2 family member proteins and induction of apoptosis, as previously demonstrated with the DAG analogue bryostatin 1 and its synthetic analogue pico. Our results offer further insights into the anticancer properties of I3A, support the idea that RasGRPs represent potential cancer therapeutic targets along with PKC, and expand the known range of ligands for RasGRP regulation.
    PLoS ONE 01/2013; 8(8):e72331. · 3.73 Impact Factor
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    ABSTRACT: Mice expressing a germline mutation in the phospholipase C-γ1-binding site of linker for activation of T cells (LAT) show progressive lymphoproliferation and ultimately die at 4-6 mo age. The hyperactivated T cells in these mice show defective TCR-induced calcium flux but enhanced Ras/ERK activation, which is critical for disease progression. Despite the loss of LAT-dependent phospholipase C-γ1 binding and activation, genetic analysis revealed RasGRP1, and not Sos1 or Sos2, to be the major Ras guanine exchange factor responsible for ERK activation and the lymphoproliferative phenotype in these mice. Analysis of isolated CD4(+) T cells from LAT-Y136F mice showed altered proximal TCR-dependent kinase signaling, which activated a Zap70- and LAT-independent pathway. Moreover, LAT-Y136F T cells showed ERK activation that was dependent on Lck and/or Fyn, protein kinase C-θ, and RasGRP1. These data demonstrate a novel route to Ras activation in vivo in a pathological setting.
    The Journal of Immunology 12/2012; · 5.52 Impact Factor
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    ABSTRACT: Bryostatin 1, like the phorbol esters, binds to and activates protein kinase C (PKC) but paradoxically antagonizes many but not all phorbol ester responses. Previously, we have compared patterns of biological response to bryostatin 1, phorbol ester, and the bryostatin 1 derivative Merle 23 in two human cancer cell lines, LNCaP and U937. Bryostatin 1 fails to induce a typical phorbol ester biological response in either cell line, whereas Merle 23 resembles phorbol ester in the U937 cells and bryostatin 1 in the LNCaP cells. Here, we have compared the pattern of their transcriptional response in both cell lines. We examined by qPCR the transcriptional response as a function of dose and time for a series of genes regulated by PKCs. In both cell lines bryostatin 1 differed primarily from phorbol ester in having a shorter duration of transcriptional modulation. This was not due to bryostatin 1 instability, since bryostatin 1 suppressed the phorbol ester response. In both cell lines Merle 23 induced a pattern of transcription largely like that of phorbol ester although with a modest reduction at later times in the LNCaP cells, suggesting that the difference in biological response of the two cell lines to Merle 23 lies downstream of this transcriptional regulation. For a series of bryostatins and analogues which ranged from bryostatin 1-like to phorbol ester-like in activity on the U937 cells, the duration of transcriptional response correlated with the pattern of biological activity, suggesting that this may provide a robust platform for structure activity analysis.
    Biochemical pharmacology 11/2012; · 4.25 Impact Factor
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    ABSTRACT: The role of the C(8) gem-dimethyl group in the A-ring of bryostatin 1 has been examined through chemical synthesis and biological evaluation of a new analogue. Assays for biological function using U937, K562, and MV4-11 cells as well as the profiles for downregulation of PKC isozymes revealed that the presence of this group is not a critical determinant for the unique pattern of biological activity of bryostatin.
    Bioorganic & medicinal chemistry letters 04/2012; 22(12):4084-8. · 2.65 Impact Factor
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    ABSTRACT: C1 domains, the recognition motif of the second messenger diacylglycerol and of the phorbol esters, are classified as typical (ligand-responsive) or atypical (not ligand-responsive). The C1 domain of Vav1, a guanine nucleotide exchange factor, plays a critical role in regulation of Vav activity through stabilization of the Dbl homology domain, which is responsible for exchange activity of Vav. Although the C1 domain of Vav1 is classified as atypical, it retains a binding pocket geometry homologous to that of the typical C1 domains of PKCs. This study clarifies the basis for its failure to bind ligands. Substituting Vav1-specific residues into the C1b domain of PKCδ, we identified five crucial residues (Glu(9), Glu(10), Thr(11), Thr(24), and Tyr(26)) along the rim of the binding cleft that weaken binding potency in a cumulative fashion. Reciprocally, replacing these incompatible residues in the Vav1 C1 domain with the corresponding residues from PKCδ C1b (δC1b) conferred high potency for phorbol ester binding. Computer modeling predicts that these unique residues in Vav1 increase the hydrophilicity of the rim of the binding pocket, impairing membrane association and thereby preventing formation of the ternary C1-ligand-membrane binding complex. The initial design of diacylglycerol-lactones to exploit these Vav1 unique residues showed enhanced selectivity for C1 domains incorporating these residues, suggesting a strategy for the development of ligands targeting Vav1.
    Journal of Biological Chemistry 02/2012; 287(16):13137-58. · 4.65 Impact Factor
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    ABSTRACT: N-methyl-substituted diacylglycerol-indololactones (DAG-indololactones) are newly synthesized effectors of protein kinase C (PKC) isoforms and exhibit substantial selectivity between RasGRP3 and PKCα. We present a comprehensive analysis of membrane interactions and biological activities of several DAG-indololactones. Translocation and binding activity assays underline significant variations between the PKC translocation characteristics affected by the ligands as compared to their binding activities. In parallel, the fluorescent properties of the ligands were employed for analysis of their membrane association profiles. Specifically, we found that a slight change in the linkage to the indole ring resulted in significant differences in membrane binding and association of the DAG-indololactones with lipid bilayers. Our analysis shows that seemingly small structural modifications of the hydrophobic regions of these biomimetic PKC effectors contribute to pronounced modulation of membrane interactions of the ligands.
    ChemBioChem 10/2011; 12(15):2331-40. · 3.74 Impact Factor
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    ABSTRACT: RasGRP3, an activator for H-Ras, R-Ras and Ras-associated protein-1/2, has emerged as an important mediator of signaling downstream from receptor coupled phosphoinositide turnover in B and T cells. Here, we report that RasGRP3 showed a high level of expression in multiple human melanoma cell lines as well as in a subset of human melanoma tissue samples. Suppression of endogenous RasGRP3 expression in these melanoma cell lines reduced Ras-GTP formation as well as c-Met expression and Akt phosphorylation downstream from hepatocyte growth factor (HGF) or epidermal growth factor (EGF) stimulation. RasGRP3 suppression also inhibited cell proliferation and reduced both colony formation in soft agar and xenograft tumor growth in immunodeficient mice, demonstrating the importance of RasGRP3 for the transformed phenotype of the melanoma cells. Reciprocally, overexpression of RasGRP3 in human primary melanocytes altered cellular morphology, markedly enhanced cell proliferation and rendered the cells tumorigenic in a mouse xenograft model. Suppression of RasGRP3 expression in these cells inhibited downstream RasGRP3 responses and suppressed cell growth, confirming the functional role of RasGRP3 in the altered behavior of these cells. The identification of the role of RasGRP3 in melanoma highlights its importance, as a Ras activator, in the phosphoinositide signaling pathway in human melanoma and provides a new potential therapeutic target.
    Oncogene 05/2011; 30(45):4590-600. · 7.36 Impact Factor
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    ABSTRACT: Phorbol 12-myristate 13-acetate (PMA) and bryostatin 1 are both potent protein kinase C (PKC) activators. In LNCaP human prostate cancer cells, PMA induces tumor necrosis factor alpha (TNFα) secretion and inhibits proliferation; bryostatin 1 does not, and indeed blocks the response to PMA. This difference has been attributed to bryostatin 1 not localizing PKCδ to the plasma membrane. Since phorbol ester lipophilicity influences PKCδ localization, we have examined in LNCaP cells a series of phorbol esters and related derivatives spanning some eight logs in lipophilicity (logP) to see if any behave like bryostatin 1. The compounds showed marked differences in their effects on proliferation and TNFα secretion. For example, maximal responses for TNFα secretion relative to PMA ranged from 97 % for octyl-indolactam V to 24 % for phorbol 12,13-dibenzoate. Dose-response curves ranged from monophasic for indolactam V to markedly biphasic for sapintoxin D. The divergent patterns of response, however, correlated neither to lipophilicity, to plasma membrane translocation of PKCδ, nor to the ability to interact with model membranes. In U937 human leukemia cells, a second system in which PMA and bryostatin 1 have divergent effects, viz. PMA but not bryostatin 1 inhibits proliferation and induces attachment, all the compounds acted like PMA for proliferation, but several induced a reduced level or a biphasic dose-response curve for attachment. We conclude that active phorbol esters are not all equivalent. Depending on the system, some might partially resemble bryostatin 1 in their behavior; this encourages the concept that bryostatin-like behavior may be obtained from other structural templates.
    ChemBioChem 05/2011; 12(8):1242-51. · 3.74 Impact Factor
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    ABSTRACT: Bryostatin 1 has attracted considerable attention both as a cancer chemotherapeutic agent and for its unique activity. Although it functions, like phorbol esters, as a potent protein kinase C (PKC) activator, it paradoxically antagonizes many phorbol ester responses in cells. Because of its complex structure, little is known of its structure-function relations. Merle 23 is a synthetic derivative, differing from bryostatin 1 at only four positions. However, in U-937 human leukemia cells, Merle 23 behaves like a phorbol ester and not like bryostatin 1. Here, we characterize the behavior of Merle 23 in the human prostate cancer cell line LNCaP. In this system, bryostatin 1 and phorbol ester have contrasting activities, with the phorbol ester but not bryostatin 1 blocking cell proliferation or tumor necrosis factor alpha secretion, among other responses. We show that Merle 23 displays a highly complex pattern of activity in this system. Depending on the specific biological response or mechanistic change, it was bryostatin-like, phorbol ester-like, intermediate in its behavior, or more effective than either. The pattern of response, moreover, varied depending on the conditions. We conclude that the newly emerging bryostatin derivatives such as Merle 23 provide powerful tools to dissect subsets of bryostatin mechanism and response.
    Biochemical pharmacology 03/2011; 81(11):1296-308. · 4.25 Impact Factor
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    ABSTRACT: Synthetic diacylglycerol-lactones (DAG-lactones) are effective modulators of critical cellular signaling pathways, downstream of the lipophilic second messenger diacylglycerol, that activate a host of protein kinase C (PKC) isozymes and other nonkinase proteins that share similar C1 membrane-targeting domains with PKC. A fundamental determinant of the biological activity of these amphiphilic molecules is the nature of their interactions with cellular membranes. This study examines the biological properties of charged DAG-lactones exhibiting different alkyl groups attached to the heterocyclic nitrogen of an α-pyridylalkylidene chain, and particularly the relationship between membrane interactions of the substituted DAG-lactones and their respective biological activities. Our results suggest that bilayer interface localization of the N-alkyl chain in the R(2) position of the DAG-lactones inhibits translocation of PKC isoenzymes onto the cellular membrane. However, the orientation of a branched alkyl chain at the bilayer surface facilitates PKC binding and translocation. This investigation emphasizes that bilayer localization of the aromatic side residues of positively charged DAG-lactone derivatives play a central role in determining biological activity, and that this factor contributes to the diversity of biological actions of these synthetic biomimetic ligands.
    ChemBioChem 09/2010; 11(14):2003-9. · 3.74 Impact Factor
  • ChemBioChem 09/2010; 11(14):1926. · 3.74 Impact Factor
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    ABSTRACT: RasGRP3 mediates the activation of the Ras signaling pathway that is present in many human cancers. Here, we explored the involvement of RasGRP3 in the formation and maintenance of the prostate cancer phenotype. RasGRP3 expression was elevated in multiple human prostate tumor tissue samples and in the human androgen-independent prostate cancer cell lines PC-3 and DU 145 compared with the androgen-dependent prostate cancer cell line LNCaP. Downregulation of endogenous RasGRP3 in PC-3 and DU 145 cells reduced Ras-GTP formation, inhibited cell proliferation, impeded cell migration, and induced apoptosis. Anchorage-independent growth of the PC-3 cells and tumor formation in mouse xenografts of both cell lines were likewise inhibited. Inhibition of RasGRP3 expression reduced AKT and extracellular signal-regulated kinase 1/2 phosphorylation and sensitized the cells to killing by carboplatin. Conversely, exogenous RasGRP3 elevated Ras-GTP, stimulated proliferation, and provided resistance to phorbol 12-myristate 13-acetate-induced apoptosis in LNCaP cells. RasGRP3-overexpressing LNCaP cells displayed a markedly enhanced rate of xenograft tumor formation in both male and female mice compared with the parental line. Suppression of RasGRP3 expression in these cells inhibited downstream RasGRP3 responses, caused the cells to resume the LNCaP morphology, and suppressed growth, confirming the functional role of RasGRP3 in the altered behavior of these cells. We conclude that RasGRP3 contributes to the malignant phenotype of the prostate cancer cells and may constitute a novel therapeutic target for human prostate cancer.
    Cancer Research 09/2010; 70(20):7905-17. · 8.65 Impact Factor
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    Angewandte Chemie International Edition 06/2010; 49(27):4580-4. · 13.73 Impact Factor
  • Angewandte Chemie 05/2010; 122(27):4684 - 4688.
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    ABSTRACT: Protein kinase C (PKC) represents the most prominent of the families of signaling proteins integrating response to the ubiquitous lipophilic second messenger sn-1,2-diacylglycerol and to its ultrapotent analogs, the tumor-promoting phorbol esters. Response is mediated through twin conserved zinc finger structures, the C1 domains. The C1 domains function as hydrophobic switches, for which ligand binding completes a hydrophobic surface on the face of the C1 domain, driving membrane association of PKC and enzymatic activation. Since the lipid bilayer provides critical contacts for ligand binding, along with the C1 domain, membrane heterogeneity provides an important mechanism for diversity, as do the differential functions of the twin C1 domains. Consistent with such mechanistic diversity, PKC ligands can differ dramatically in biological consequences. Thus, whereas PKC ligands have provided the paradigm for tumor promoters, some PKC ligands in fact function as inhibitors of tumor promotion. Reflecting the central role of PKC in cellular signaling, PKC has emerged as a promising therapeutic target for cancer with several PKC ligands currently in clinical trials. KeywordsC1 domain-Diacylglycerol-Phorbol ester-Protein kinase C
    12/2009: pages 25-53;
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    ABSTRACT: Synthetic diacylglycerol lactones (DAG lactones) are effective modulators of critical cellular signaling pathways downstream of the lipophilic second messenger diacylglycerol that activate a host of protein kinase C (PKC) isozymes as well as other non-kinase proteins that share with PKC similar C1 membrane-targeting domains. A fundamental determinant of the biological activity of these amphiphilic molecules is the nature of their interactions with cellular membranes. This study characterizes the membrane interactions and bilayer anchoring of a series of DAG lactones in which the hydrophobic moiety is a 'molecular rod', namely a rigid 4-[2-(R-phenyl)ethynyl]benzoate moiety in the acyl position. Use of assays employing chromatic biomimetic vesicles and biophysical techniques revealed that the mode of membrane anchoring of the DAG lactone derivatives was markedly affected by the presence of the hydrophobic diphenyl rod and by the size of the functional unit at the terminus of the rod. Two primary mechanisms of interaction were observed: surface binding of the DAG lactones at the lipid/water interface and deep insertion of the ligands into the alkyl core of the lipid bilayer. These membrane-insertion properties could explain the different patterns of the PKC translocation from the cytosol to membranes that is induced by the molecular-rod DAG lactones. This investigation emphasizes that the side residues of DAG lactones, rather than simply conferring hydrophobicity, profoundly influence membrane interactions, and thus may further contribute to the diversity of biological actions of these synthetic biomimetic ligands.
    FEBS Journal 12/2009; 277(1):233-43. · 4.25 Impact Factor
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    ABSTRACT: The contribution of the A-ring C(7) acetate to the function of bryostatin 1 has been investigated through synthesis and biological evaluation of an analogue incorporating this feature into the bryopyran core structure. No enhanced binding affinity for protein kinase C (PKC) was observed, relative to previously characterized analogues lacking the C(7) acetate. Functional assays showed biological responses characteristic of those induced by the phorbol ester PMA and distinctly different from those observed with bryostatin 1.
    Organic Letters 06/2009; 11(11):2277-80. · 6.14 Impact Factor

Publication Stats

777 Citations
202 Downloads
2k Views
193.07 Total Impact Points

Institutions

  • 2004–2013
    • National Cancer Institute (USA)
      • • Laboratory of Cell Biology
      • • Laboratory of Cancer Biology and Genetics
      • • Center for Cancer Research
      Maryland, United States
  • 2009–2012
    • NCI-Frederick
      Maryland, United States
  • 2008–2012
    • University of Utah
      • Department of Chemistry
      Salt Lake City, UT, United States
  • 2001–2012
    • National Institutes of Health
      • • Laboratory of Cancer Biology and Genetics
      • • Center for Cancer Research
      Bethesda, MD, United States
  • 2008–2011
    • Ben-Gurion University of the Negev
      • Department of Chemistry
      Beersheba, Southern District, Israel