562 results on '"Singer, A."'
Search Results
2. The glycerophosphocholine acyltransferase Gpc1 contributes to phosphatidylcholine biosynthesis, long-term viability, and embedded hyphal growth in Candida albicans
- Author
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King, William R., Singer, Justin, Warman, Mitchell, Wilson, Duncan, Hube, Bernard, Lager, Ida, and Patton-Vogt, Jana
- Published
- 2024
- Full Text
- View/download PDF
3. Sex hormones regulate metainflammation in diet-induced obesity in mice
- Author
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Varghese, Mita, Griffin, Cameron, Abrishami, Simin, Eter, Leila, Lanzetta, Nicholas, Hak, Layla, Clemente, Jeremy, Agarwal, Devyani, Lerner, Arianna, Westerhoff, Maria, Patel, Ravi, Bowers, Emily, Islam, Mohammed, Subbaiah, Perla, and Singer, Kanakadurga
- Published
- 2021
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4. The glycerophosphocholine acyltransferase Gpc1 contributes to phosphatidylcholine biosynthesis, long-term viability, and embedded hyphal growth in Candida albicans
- Author
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King, William R., primary, Singer, Justin, additional, Warman, Mitchell, additional, Wilson, Duncan, additional, Hube, Bernard, additional, Lager, Ida, additional, and Patton-Vogt, Jana, additional
- Published
- 2023
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5. Galactose 6-O-Sulfotransferases Are Not Required for the Generation of Siglec-F Ligands in Leukocytes or Lung Tissue*
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Patnode, Michael L, Cheng, Chu-Wen, Chou, Chi-Chi, Singer, Mark S, Elin, Matilda S, Uchimura, Kenji, Crocker, Paul R, Khoo, Kay-Hooi, and Rosen, Steven D
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Biomedical and Clinical Sciences ,Medicinal and Biomolecular Chemistry ,Chemical Sciences ,Lung ,Rare Diseases ,1.1 Normal biological development and functioning ,Underpinning research ,Respiratory ,Inflammatory and immune system ,Animals ,Antigens ,Differentiation ,Myelomonocytic ,Bronchoalveolar Lavage Fluid ,Cell Membrane ,Eosinophils ,Epithelial Cells ,Flow Cytometry ,Galactose ,Leukocytes ,Ligands ,Macrophages ,Alveolar ,Mass Spectrometry ,Mice ,Mice ,Knockout ,Microscopy ,Fluorescence ,Mucins ,Nippostrongylus ,Polysaccharides ,Sialic Acid Binding Immunoglobulin-like Lectins ,Strongylida Infections ,Sulfotransferases ,Galactose-6-O-Sulfate ,Lectin ,Siglec-F ,Sulfotransferase ,Biological Sciences ,Medical and Health Sciences ,Biochemistry & Molecular Biology ,Biological sciences ,Biomedical and clinical sciences ,Chemical sciences - Abstract
Eosinophil accumulation is a characteristic feature of the immune response to parasitic worms and allergens. The cell surface carbohydrate-binding receptor Siglec-F is highly expressed on eosinophils and negatively regulates their accumulation during inflammation. Although endogenous ligands for Siglec-F have yet to be biochemically defined, binding studies using glycan arrays have implicated galactose 6-O-sulfate (Gal6S) as a partial recognition determinant for this receptor. Only two sulfotransferases are known to generate Gal6S, namely keratan sulfate galactose 6-O-sulfotransferase (KSGal6ST) and chondroitin 6-O-sulfotransferase 1 (C6ST-1). Here we use mice deficient in both KSGal6ST and C6ST-1 to determine whether these sulfotransferases are required for the generation of endogenous Siglec-F ligands. First, we characterize ligand expression on leukocyte populations and find that ligands are predominantly expressed on cell types also expressing Siglec-F, namely eosinophils, neutrophils, and alveolar macrophages. We also detect Siglec-F ligand activity in bronchoalveolar lavage fluid fractions containing polymeric secreted mucins, including MUC5B. Consistent with these observations, ligands in the lung increase dramatically during infection with the parasitic nematode, Nippostrongylus brasiliensis, which is known to induce eosinophil accumulation and mucus production. Surprisingly, Gal6S is undetectable in sialylated glycans from eosinophils and BAL fluid analyzed by mass spectrometry. Furthermore, none of the ligands we describe are diminished in mice lacking KSGal6ST and C6ST-1, indicating that neither of the known galactose 6-O-sulfotransferases is required for ligand synthesis. These results establish that ligands for Siglec-F are present on several cell types that are relevant during allergic lung inflammation and argue against the widely held view that Gal6S is critical for glycan recognition by this receptor.
- Published
- 2013
6. Abstract 1651 Enhanced transport of glycerophosphocholine, a common lipid metabolite, inhibits growth of Saccharomyces cerevisiae and Schizosaccharomyces pombe
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Singer, Justin, Hrach, Victoria, Schwer, Beate, Shuman, Stewart, and Patton-Vogt, Jana
- Published
- 2024
- Full Text
- View/download PDF
7. mRNA on the Move: The Road to Its Biological Destiny
- Author
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Eliscovich, Carolina, Buxbaum, Adina R., Katz, Zachary B., and Singer, Robert H.
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- 2013
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8. The intrinsic kinase activity of BRD4 spans its BD2-B-BID domains
- Author
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Weissman, Jocelyn D., primary, Singh, Amit K., additional, Devaiah, Ballachanda N., additional, Schuck, Peter, additional, LaRue, Ross C., additional, and Singer, Dinah S., additional
- Published
- 2021
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9. Structural and Biochemical Characterization of the Type II Fructose-1,6-bisphosphatase GlpX from Escherichia coli
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Brown, Greg, Singer, Alexander, Lunin, Vladimir V., Proudfoot, Michael, Skarina, Tatiana, Flick, Robert, Kochinyan, Samvel, Sanishvili, Ruslan, Joachimiak, Andrzej, Edwards, Aled M., Savchenko, Alexei, and Yakunin, Alexander F.
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- 2009
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10. Transcriptional burst fraction and size dynamics during lens fiber cell differentiation and detailed insights into the denucleation process
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Melissa Lopez-Jones, Robert A. Coleman, Robert H. Singer, Peng Guo, Adrien Senecal, Saima Limi, Ales Cvekl, Christina Polumbo, and Arthur I. Skoultchi
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0301 basic medicine ,Transcription, Genetic ,Organogenesis ,Cellular differentiation ,Biochemistry ,Chromatin remodeling ,Mice ,03 medical and health sciences ,Lens, Crystalline ,Gene expression ,Transcriptional regulation ,Animals ,Gene Regulation ,Molecular Biology ,Cell Nucleus ,Transcriptional bursting ,Chemistry ,Gene Expression Regulation, Developmental ,Cell Differentiation ,Cell Biology ,Chromatin Assembly and Disassembly ,Embryo, Mammalian ,Chromatin ,Cell biology ,030104 developmental biology ,Animals, Newborn ,Fiber cell ,Female ,Lens fiber cell differentiation - Abstract
Genes are transcribed in irregular pulses of activity termed transcriptional bursts. Cellular differentiation requires coordinated gene expression; however, it is unknown whether the burst fraction (i.e. the number of active phases of transcription) or size/intensity (the number of RNA molecules produced within a burst) changes during cell differentiation. In the ocular lens, the positions of lens fiber cells correlate precisely with their differentiation status, and the most advanced cells degrade their nuclei. Here, we examined the transcriptional parameters of the β-actin and lens differentiation–specific α-, β-, and γ-crystallin genes by RNA fluorescent in situ hybridization (FISH) in the lenses of embryonic day (E) E12.5, E14.5, and E16.5 mouse embryos and newborns. We found that cellular differentiation dramatically alters the burst fraction in synchronized waves across the lens fiber cell compartment with less dramatic changes in burst intensity. Surprisingly, we observed nascent transcription of multiple genes in nuclei just before nuclear destruction. Nuclear condensation was accompanied by transfer of nuclear proteins, including histone and nonhistone proteins, to the cytoplasm. Although lens-specific deletion of the chromatin remodeler SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 (Smarca5/Snf2h) interfered with denucleation, persisting nuclei remained transcriptionally competent and exhibited changes in both burst intensity and fraction depending on the gene examined. Our results uncover the mechanisms of nascent transcriptional control during differentiation and chromatin remodeling, confirm the burst fraction as the major factor adjusting gene expression levels, and reveal transcriptional competence of fiber cell nuclei even as they approach disintegration.
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- 2018
11. TLR4, TRIF, and MyD88 are essential for myelopoiesis and CD11c+ adipose tissue macrophage production in obese mice
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Mita Varghese, Kaitlin McKernan, Kanakadurga Singer, Nico Lanzetta, Leila Eter, Jamie Lane, Simin Abrishami, Carey N. Lumeng, Lindsey A. Muir, and Cameron Griffin
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0301 basic medicine ,Myeloid ,Chemistry ,Adipose tissue macrophages ,Macrophage polarization ,Adipose tissue ,hemic and immune systems ,030209 endocrinology & metabolism ,Cell Biology ,Biochemistry ,Cell biology ,03 medical and health sciences ,Haematopoiesis ,030104 developmental biology ,0302 clinical medicine ,medicine.anatomical_structure ,TRIF ,medicine ,lipids (amino acids, peptides, and proteins) ,Myelopoiesis ,Molecular Biology ,Macrophage proliferation - Abstract
Obesity-induced chronic inflammation is associated with metabolic disease. Results from mouse models utilizing a high-fat diet (HFD) have indicated that an increase in activated macrophages, including CD11c+ adipose tissue macrophages (ATMs), contributes to insulin resistance. Obesity primes myeloid cell production from hematopoietic stem cells (HSCs) and Toll-like receptor 4 (TLR4), and the downstream TIR domain-containing adapter protein-inducing interferon-β (TRIF)- and MyD88-mediated pathways regulate production of similar myeloid cells after lipopolysaccharide stimulation. However, the role of these pathways in HFD-induced myelopoiesis is unknown. We hypothesized that saturated fatty acids and HFD alter myelopoiesis by activating TLR4 pathways in HSCs, differentially producing pro-inflammatory CD11c+ myeloid cells that contribute to obesity-induced metabolic disease. Results from reciprocal bone marrow transplants (BMTs) with Tlr4-/- and WT mice indicated that TLR4 is required for HFD-induced myelopoiesis and production of CD11c+ ATMs. Experiments with homozygous knockouts of Irakm (encoding a suppressor of MyD88 inactivation) and Trif in competitive BMTs revealed that MyD88 is required for HFD expansion of granulocyte macrophage progenitors and that Trif is required for pregranulocyte macrophage progenitor expansion. A comparison of WT, Tlr4-/-, Myd88-/-, and Trif-/- mice on HFD demonstrated that TLR4 plays a role in the production of CD11c+ ATMs, and both Myd88-/- and Trif-/- mice produced fewer ATMs than WT mice. Moreover, HFD-induced TLR4 activation inhibited macrophage proliferation, leading to greater accumulation of recruited CD11c+ ATMs. Our results indicate that HFD potentiates TLR4 and both its MyD88- and TRIF-mediated downstream pathways within progenitors and adipose tissue and leads to macrophage polarization.
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- 2018
12. Sex hormones regulate metainflammation in diet-induced obesity in mice
- Author
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Devyani Agarwal, Leila Eter, Kanakadurga Singer, Mita Varghese, Ravi Patel, Simin Abrishami, Maria Westerhoff, Perla Subbaiah, Jeremy Clemente, Cameron Griffin, Mohammed N. Islam, Layla Hak, Nicholas Lanzetta, Emily Bowers, and Arianna Lerner
- Subjects
Male ,sex differences ,obesity ,HFD, high-fat diet ,Myeloid ,ER, estrogen receptor ,Adipose tissue ,GX, gonadectomy ,Biochemistry ,ATM, adipose tissue macrophage ,Mice ,Sex hormone-binding globulin ,CFU, colony-forming unit ,OVX, ovariectomy ,CLS, crown-like structure ,Gonadal Steroid Hormones ,Sex Characteristics ,TG, triglyceride ,biology ,ND, normal diet ,medicine.anatomical_structure ,Adipose Tissue ,GTT, glucose tolerance test ,Female ,Myelopoiesis ,Research Article ,medicine.medical_specialty ,Normal diet ,Adipose tissue macrophages ,macrophage ,M, macrophage ,myelopoiesis ,Diet, High-Fat ,Proinflammatory cytokine ,ERα−/−, estrogen receptor–deficient alpha ,GWAT, gonadal white adipose tissue ,HSC, hematopoietic stem cell ,Internal medicine ,medicine ,Animals ,Molecular Biology ,ITT, insulin tolerance test ,Inflammation ,business.industry ,Macrophages ,androgens ,CAS, castration ,Cell Biology ,BMT, bone marrow transplantation ,G, granulocyte ,Endocrinology ,BM, bone marrow ,biology.protein ,AR, androgen receptor ,business ,metabolism ,Hormone - Abstract
Men have a statistically higher risk of metabolic and cardiovascular disease than premenopausal women, but the mechanisms mediating these differences are elusive. Chronic inflammation during obesity contributes to disease risk and is significantly more robust in males. Prior work demonstrated that compared with obese males, obese females have reduced proinflammatory adipose tissue macrophages (ATMs). Given the paucity of data on how sex hormones contribute to macrophage responses in obesity, we sought to understand the role of sex hormones in promoting obesity-induced myeloid inflammation. We used gonadectomy, estrogen receptor–deficient alpha chimeras, and androgen-insensitive mice to model sex hormone deficiency. These models were evaluated in diet-induced obesity conditions (high-fat diet [HFD]) and in vitro myeloid assays. We found that ovariectomy increased weight gain and adiposity. Ovariectomized females had increased ATMs and bone marrow myeloid colonies compared with sham-gonadectomized females. In addition, castrated males exposed to HFD had improved glucose tolerance, insulin sensitivity, and adiposity with fewer Ly6chi monocytes and bone marrow myeloid colonies compared with sham-gonadectomized males, although local adipose inflammation was enhanced. Similar findings were observed in androgen-insensitive mice; however, these mice had fewer CD11c+ ATMs, implying a developmental role for androgens in myelopoiesis and adipose inflammation. We concluded that gonadectomy results in convergence of metabolic and inflammatory responses to HFD between the sexes, and that myeloid estrogen receptor alpha contributes minimally to diet-induced inflammatory responses, whereas loss of androgen-receptor signaling improves metabolic and inflammatory outcomes. These studies demonstrate that sex hormones play a critical role in sex differences in obesity, metabolic dysfunction, and myeloid inflammation.
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- 2021
13. Critical Role of Vimentin Phosphorylation at Ser-56 by p21-activated Kinase in Vimentin Cytoskeleton Signaling
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Li, Qing-Fen, Spinelli, Amy M., Wang, Ruping, Anfinogenova, Yana, Singer, Harold A., and Tang, Dale D.
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- 2006
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14. Binding of DEAD-box helicase Dhh1 to the 5′-untranslated region of ASH1 mRNA represses localized translation of ASH1 in yeast cells
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Shaoyin Chen, Xiuhua Meng, Robert H. Singer, Wei Gu, Linjie Zhu, Jianmin Luo, Qianjun Zhang, and Delin Li
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0301 basic medicine ,Five prime untranslated region ,Nonsense-mediated decay ,RNA-binding protein ,Cell Biology ,Biology ,Biochemistry ,Molecular biology ,mRNA surveillance ,Messenger RNP ,03 medical and health sciences ,030104 developmental biology ,Polysome ,P-bodies ,MRNA transport ,Molecular Biology - Abstract
Local translation of specific mRNAs is regulated by dynamic changes in their subcellular localization, and these changes are due to complex mechanisms controlling cytoplasmic mRNA transport. The budding yeast Saccharomyces cerevisiae is well suited to studying these mechanisms because many of its transcripts are transported from the mother cell to the budding daughter cell. Here, we investigated the translational control of ASH1 mRNA after transport and localization. We show that although ASH1 transcripts were translated after they reached the bud tip, some mRNAs were bound by the RNA-binding protein Puf6 and were non-polysomal. We also found that the DEAD-box helicase Dhh1 complexed with the untranslated ASH1 mRNA and Puf6. Loss of Dhh1 affected local translation of ASH1 mRNA and resulted in delocalization of ASH1 transcript in the bud. Forcibly shifting the non-polysomal ASH1 mRNA into polysomes was associated with Dhh1 dissociation. We further demonstrated that Dhh1 is not recruited to ASH1 mRNA co-transcriptionally, suggesting that it could bind to ASH1 mRNA within the cytoplasm. Of note, Dhh1 bound to the 5′-UTR of ASH1 mRNA and inhibited its translation in vitro. These results suggest that after localization to the bud tip, a portion of the localized ASH1 mRNA becomes translationally inactive because of binding of Dhh1 and Puf6 to the 5′- and 3′-UTRs of ASH1 mRNA.
- Published
- 2017
15. Structural and Biochemical Analyses Reveal the Mechanism of Glutathione S-Transferase Pi 1 Inhibition by the Anti-cancer Compound Piperlongumine
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Scott B. Ficarro, Kenneth D. Westover, Lianbo Li, William D. Singer, Yan Liu, Wayne Harshbarger, Durga Udayakumar, Jarrod A. Marto, John C. Hunter, Sudershan R. Gondi, and Deepak Gurbani
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0301 basic medicine ,Protein Conformation ,Stereochemistry ,Crystallography, X-Ray ,urologic and male genital diseases ,Biochemistry ,Mass Spectrometry ,03 medical and health sciences ,chemistry.chemical_compound ,Tumor Cells, Cultured ,Humans ,Structure–activity relationship ,neoplasms ,Molecular Biology ,Piperlongumine ,Cell Proliferation ,chemistry.chemical_classification ,Reactive oxygen species ,biology ,Active site ,Dioxolanes ,Cell Biology ,Glutathione ,Pancreatic Neoplasms ,030104 developmental biology ,Glutathione S-Transferase pi ,chemistry ,Covalent bond ,Protein Structure and Folding ,Cancer cell ,biology.protein ,Protein Binding - Abstract
Glutathione S-transferase pi 1 (GSTP1) is frequently overexpressed in cancerous tumors and is a putative target of the plant compound piperlongumine (PL), which contains two reactive olefins and inhibits proliferation in cancer cells but not normal cells. PL exposure of cancer cells results in increased reactive oxygen species and decreased GSH. These data in tandem with other information led to the conclusion that PL inhibits GSTP1, which forms covalent bonds between GSH and various electrophilic compounds, through covalent adduct formation at the C7-C8 olefin of PL, whereas the C2-C3 olefin of PL was postulated to react with GSH. However, direct evidence for this mechanism has been lacking. To investigate, we solved the X-ray crystal structure of GSTP1 bound to PL and GSH at 1.1 Å resolution to rationalize previously reported structure activity relationship studies. Surprisingly, the structure showed that a hydrolysis product of PL (hPL) was conjugated to glutathione at the C7-C8 olefin, and this complex was bound to the active site of GSTP1; no covalent bond formation between hPL and GSTP1 was observed. Mass spectrometry (MS) analysis of the reactions between PL and GSTP1 confirmed that PL does not label GSTP1. Moreover, MS data also indicated that nucleophilic attack on PL at the C2-C3 olefin led to PL hydrolysis. Although hPL inhibits GSTP1 enzymatic activity in vitro, treatment of cells susceptible to PL with hPL did not have significant anti-proliferative effects, suggesting that hPL is not membrane-permeable. Altogether, our data suggest a model wherein PL is a prodrug whose intracellular hydrolysis initiates the formation of the hPL-GSH conjugate, which blocks the active site of and inhibits GSTP1 and thereby cancer cell proliferation.
- Published
- 2017
16. CD6 Receptor Regulates Intestinal Ischemia/Reperfusion-induced Injury by Modulating Natural IgM-producing B1a Cell Self-renewal
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Feng Lin, John J. Fung, Gospel Enyindah-Asonye, Neetu Gupta, Yan Li, Nora G. Singer, and Wei Xin
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Antigens, Differentiation, T-Lymphocyte ,0301 basic medicine ,Immunology ,Population ,Inflammation ,Biology ,Biochemistry ,Pathogenesis ,Mice ,03 medical and health sciences ,Peritoneal cavity ,Antigens, CD ,medicine ,Animals ,Intestinal Mucosa ,education ,Receptor ,Molecular Biology ,Mice, Knockout ,B-Lymphocytes ,education.field_of_study ,Innate immune system ,Cell Biology ,B-1 cell ,Disease Models, Animal ,Intestinal Diseases ,030104 developmental biology ,medicine.anatomical_structure ,Immunoglobulin M ,Reperfusion Injury ,Bone marrow ,medicine.symptom - Abstract
Intestinal ischemia/reperfusion (I/R) injury is a relatively common pathological condition that can lead to multi-organ failure and mortality. Regulatory mechanism for this disease is poorly understood, although it is established that circulating pathogenic natural IgM, which is primarily produced by B1a cells outside of the peritoneal cavity, are integrally involved. CD6 was originally identified as a marker for T cells and was later found to be present on some subsets of B cells in humans; however, whether CD6 plays any role in intestinal I/R-induced injury and, if so, the underlying mechanisms, remain unknown. Here we report that CD6−/− mice were significantly protected from intestinal inflammation and mucosal damage compared with WT mice in a model of intestinal I/R-induced injury. Mechanistically, we found that CD6 was selectively expressed on B1 cells outside of the bone marrow and peritoneal cavity and that pathogenic natural IgM titers were reduced in the CD6−/− mice in association with significantly decreased B1a cell population. Our results reveal an unexpected role of CD6 in the pathogenesis of intestinal IR-induced injury by regulating the self-renewal of B1a cells.
- Published
- 2017
17. Assembly and Molecular Architecture of the Phosphoinositide 3-Kinase p85α Homodimer
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Robert H. Singer, Jonathan M. Backer, Brian T. Chait, Andrej Sali, Jaclyn LoPiccolo, Bin Wu, Seung Joong Kim, Haiyan Wu, Anne R. Bresnick, Yi Shi, and Michael Brenowitz
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Phosphoinositide 3-kinase ,Molecular model ,biology ,Stereochemistry ,Dimer ,Class Ia Phosphatidylinositol 3-Kinase ,Cell Biology ,Biochemistry ,DNA-binding protein ,In vitro ,Protein Structure, Tertiary ,chemistry.chemical_compound ,chemistry ,biology.protein ,Humans ,Molecule ,Protein Multimerization ,Protein Structure, Quaternary ,Receptor ,Molecular Biology ,Signal Transduction - Abstract
Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that are activated by growth factor and G-protein-coupled receptors and propagate intracellular signals for growth, survival, proliferation, and metabolism. p85α, a modular protein consisting of five domains, binds and inhibits the enzymatic activity of class IA PI3K catalytic subunits. Here, we describe the structural states of the p85α dimer, based on data from in vivo and in vitro solution characterization. Our in vitro assembly and structural analyses have been enabled by the creation of cysteine-free p85α that is functionally equivalent to native p85α. Analytical ultracentrifugation studies showed that p85α undergoes rapidly reversible monomer-dimer assembly that is highly exothermic in nature. In addition to the documented SH3-PR1 dimerization interaction, we identified a second intermolecular interaction mediated by cSH2 domains at the C-terminal end of the polypeptide. We have demonstrated in vivo concentration-dependent dimerization of p85α using fluorescence fluctuation spectroscopy. Finally, we have defined solution conditions under which the protein is predominantly monomeric or dimeric, providing the basis for small angle x-ray scattering and chemical cross-linking structural analysis of the discrete dimer. These experimental data have been used for the integrative structure determination of the p85α dimer. Our study provides new insight into the structure and assembly of the p85α homodimer and suggests that this protein is a highly dynamic molecule whose conformational flexibility allows it to transiently associate with multiple binding proteins.
- Published
- 2015
18. Double genetic disruption of lactate dehydrogenases A and B is required to ablate the “Warburg effect” restricting tumor growth to oxidative metabolism
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Ždralević, Maša, primary, Brand, Almut, additional, Di Ianni, Lorenza, additional, Dettmer, Katja, additional, Reinders, Jörg, additional, Singer, Katrin, additional, Peter, Katrin, additional, Schnell, Annette, additional, Bruss, Christina, additional, Decking, Sonja-Maria, additional, Koehl, Gudrun, additional, Felipe-Abrio, Blanca, additional, Durivault, Jérôme, additional, Bayer, Pascale, additional, Evangelista, Marie, additional, O'Brien, Thomas, additional, Oefner, Peter J., additional, Renner, Kathrin, additional, Pouysségur, Jacques, additional, and Kreutz, Marina, additional
- Published
- 2018
- Full Text
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19. Differences in Hematopoietic Stem Cells Contribute to Sexually Dimorphic Inflammatory Responses to High Fat Diet-induced Obesity
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Kanakadurga Singer, Lindsey A. Muir, Brian F. Zamarron, Phillip Wachowiak, Jennifer B. DelProposto, Gabriel Martinez-Santibanez, Chaghig Demirjian, Lynn M. Geletka, Taleen Mergian, Carey N. Lumeng, Kae Won Cho, and Nidhi Maley
- Subjects
Blood Glucose ,Male ,medicine.medical_specialty ,Adipose tissue macrophages ,Adipose tissue ,Inflammation ,Biology ,Diet, High-Fat ,Weight Gain ,Biochemistry ,Proinflammatory cytokine ,Colony-Forming Units Assay ,Mice ,Sex Factors ,Internal medicine ,medicine ,Animals ,Obesity ,Molecular Biology ,Cells, Cultured ,Myelopoiesis ,Cell Biology ,Glucose Tolerance Test ,Flow Cytometry ,Hematopoietic Stem Cells ,Immunohistochemistry ,Lipids ,Mice, Inbred C57BL ,Sexual dimorphism ,Haematopoiesis ,Metabolism ,Endocrinology ,Adipose Tissue ,Female ,medicine.symptom ,Stem cell ,Biomarkers - Abstract
Women of reproductive age are protected from metabolic disease relative to postmenopausal women and men. Most preclinical rodent studies are skewed toward the use of male mice to study obesity-induced metabolic dysfunction because of a similar protection observed in female mice. How sex differences in obesity-induced inflammatory responses contribute to these observations is unknown. We have compared and contrasted the effects of high fat diet-induced obesity on glucose metabolism and leukocyte activation in multiple depots in male and female C57Bl/6 mice. With both short term and long term high fat diet, male mice demonstrated increased weight gain and CD11c+ adipose tissue macrophage content compared with female mice despite similar degrees of adipocyte hypertrophy. Competitive bone marrow transplant studies demonstrated that obesity induced a preferential contribution of male hematopoietic cells to circulating leukocytes and adipose tissue macrophages compared with female cells independent of the sex of the recipient. Sex differences in macrophage and hematopoietic cell in vitro activation in response to obesogenic cues were observed to explain these results. In summary, this report demonstrates that male and female leukocytes and hematopoietic stem cells have cell-autonomous differences in their response to obesity that contribute to an amplified response in males compared with females. Background: Diet-induced obesity leads to a chronic low grade inflammation with production of activated macrophages associated with systemic sexually dimorphic metabolic dysfunction. Results: Males have enhanced myelopoiesis and a proinflammatory response to obesity compared with females. Conclusion: Sex differences in myelopoiesis result in dimorphic responses to obesity-induced inflammation. Significance: Given differences in inflammatory responses, targeted treatment strategies are probably required for males and females.
- Published
- 2015
20. Unusual Spectral Properties of Bacteriophytochrome Agp2 Result from a Deprotonation of the Chromophore in the Red-absorbing Form Pr
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Peter Hildebrandt, Francisco Velazquez Escobar, Isabel Molina, Dorothee Ehmer, Benjamin Zienicke, Tilman Lamparter, René Glenz, Rolf Diller, and Patrick Singer
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Phytochrome ,Absorption spectroscopy ,Chemistry ,Resonance Raman spectroscopy ,Mutation, Missense ,Protonation ,Cell Biology ,Chromophore ,Spectrum Analysis, Raman ,Photochemistry ,Biochemistry ,Recombinant Proteins ,Protein Structure, Tertiary ,Deprotonation ,Ultraviolet visible spectroscopy ,Amino Acid Substitution ,Bacterial Proteins ,Agrobacterium tumefaciens ,Mutagenesis, Site-Directed ,Flash photolysis ,Spectrophotometry, Ultraviolet ,Molecular Biology ,Signal Transduction - Abstract
Phytochromes are widely distributed photoreceptors with a bilin chromophore that undergo a typical reversible photoconversion between the two spectrally different forms, Pr and Pfr. The phytochrome Agp2 from Agrobacterium tumefaciens belongs to the group of bathy phytochromes that have a Pfr ground state as a result of the Pr to Pfr dark conversion. Agp2 has untypical spectral properties in the Pr form reminiscent of a deprotonated chromophore as confirmed by resonance Raman spectroscopy. UV/visible absorption spectroscopy showed that the pKa is >11 in the Pfr form and ∼7.6 in the Pr form. Unlike other phytochromes, photoconversion thus results in a pKa shift of more than 3 units. The Pr/Pfr ratio after saturating irradiation with monochromatic light is strongly pH-dependent. This is partially due to a back-reaction of the deprotonated Pr chromophore at pH 9 after photoexcitation as found by flash photolysis. The chromophore protonation and dark conversion were affected by domain swapping and site-directed mutagenesis. A replacement of the PAS or GAF domain by the respective domain of the prototypical phytochrome Agp1 resulted in a protonated Pr chromophore; the GAF domain replacement afforded an inversion of the dark conversion. A reversion was also obtained with the triple mutant N12S/Q190L/H248Q, whereas each single point mutant is characterized by decelerated Pr to Pfr dark conversion. Background: Typical phytochromes include a protonated chromophore in the parent states (Pr and Pfr) that transiently deprotonates during photoconversion. Results: In Agp2, the pKa of the chromophore is lowered from >11 to 7.6 during the conversion from Pfr to Pr. Conclusion: Chromophore protonation affects light-induced and thermal Pr to Pfr conversion. Significance: Agp2 can act as integrated light and pH sensor.
- Published
- 2013
21. Transcriptional burst fraction and size dynamics during lens fiber cell differentiation and detailed insights into the denucleation process
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Limi, Saima, primary, Senecal, Adrien, additional, Coleman, Robert, additional, Lopez-Jones, Melissa, additional, Guo, Peng, additional, Polumbo, Christina, additional, Singer, Robert H., additional, Skoultchi, Arthur I., additional, and Cvekl, Ales, additional
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- 2018
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22. DNA methylation regulates the neonatal CD4+ T-cell response to pneumonia in mice
- Author
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McGrath-Morrow, Sharon A., primary, Ndeh, Roland, additional, Helmin, Kathryn A., additional, Chen, Shang-Yang, additional, Anekalla, Kishore R., additional, Abdala-Valencia, Hiam, additional, D'Alessio, Franco R., additional, Collaco, J. Michael, additional, and Singer, Benjamin D., additional
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- 2018
- Full Text
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23. TLR4, TRIF, and MyD88 are essential for myelopoiesis and CD11c+ adipose tissue macrophage production in obese mice
- Author
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Griffin, Cameron, primary, Eter, Leila, additional, Lanzetta, Nico, additional, Abrishami, Simin, additional, Varghese, Mita, additional, McKernan, Kaitlin, additional, Muir, Lindsey, additional, Lane, Jamie, additional, Lumeng, Carey N., additional, and Singer, Kanakadurga, additional
- Published
- 2018
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24. Discovery of a highly selective chemical inhibitor of matrix metalloproteinase-9 (MMP-9) that allosterically inhibits zymogen activation
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Scannevin, Robert H., primary, Alexander, Richard, additional, Haarlander, Tara Mezzasalma, additional, Burke, Sharon L., additional, Singer, Monica, additional, Huo, Cuifen, additional, Zhang, Yue-Mei, additional, Maguire, Diane, additional, Spurlino, John, additional, Deckman, Ingrid, additional, Carroll, Karen I., additional, Lewandowski, Frank, additional, Devine, Eric, additional, Dzordzorme, Keli, additional, Tounge, Brett, additional, Milligan, Cindy, additional, Bayoumy, Shariff, additional, Williams, Robyn, additional, Schalk-Hihi, Celine, additional, Leonard, Kristi, additional, Jackson, Paul, additional, Todd, Matthew, additional, Kuo, Lawrence C., additional, and Rhodes, Kenneth J., additional
- Published
- 2017
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25. mRNA on the Move: The Road to Its Biological Destiny
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Zachary Katz, Carolina Eliscovich, Adina R. Buxbaum, and Robert H. Singer
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Cytoplasm ,Messenger RNA ,Models, Genetic ,Three prime untranslated region ,RNA-Binding Proteins ,Minireviews ,RNA-binding protein ,Cell Biology ,Biology ,Biochemistry ,RNA Transport ,mRNA surveillance ,Cell biology ,Microscopy, Fluorescence ,Live cell imaging ,eIF4A ,P-bodies ,Gene expression ,RNA Precursors ,Animals ,Humans ,RNA, Messenger ,Molecular Biology ,Neuroscience ,In Situ Hybridization - Abstract
Cells have evolved to regulate the asymmetric distribution of specific mRNA targets to institute spatial and temporal control over gene expression. Over the last few decades, evidence has mounted as to the importance of localization elements in the mRNA sequence and their respective RNA-binding proteins. Live imaging methodologies have shown mechanistic details of this phenomenon. In this minireview, we focus on the advanced biochemical and cell imaging techniques used to tweeze out the finer aspects of mechanisms of mRNA movement.
- Published
- 2013
26. Cross-talk Among RNA Polymerase II Kinases Modulates C-terminal Domain Phosphorylation
- Author
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Ballachanda N. Devaiah and Dinah S. Singer
- Subjects
genetic structures ,Transcription, Genetic ,viruses ,genetic processes ,Cell Cycle Proteins ,RNA polymerase II ,Models, Biological ,environment and public health ,Biochemistry ,Cell Line ,Cyclin-dependent kinase ,Serine ,Animals ,Humans ,Gene Regulation ,Phosphorylation ,Kinase activity ,Molecular Biology ,TATA-Binding Protein Associated Factors ,biology ,General transcription factor ,Kinase ,Nuclear Proteins ,Cell Biology ,Cyclin-Dependent Kinase 9 ,Molecular biology ,Cyclin-Dependent Kinases ,Protein Structure, Tertiary ,Cell biology ,enzymes and coenzymes (carbohydrates) ,health occupations ,biology.protein ,Drosophila ,Transcription Factor TFIID ,Cyclin-dependent kinase 9 ,RNA Polymerase II ,CTD ,Cyclin-dependent kinase 7 ,Cyclin-Dependent Kinase-Activating Kinase ,DNA Damage ,HeLa Cells ,Transcription Factors - Abstract
The RNA polymerase II (Pol II) C-terminal domain (CTD) serves as a docking site for numerous proteins, bridging various nuclear processes to transcription. The recruitment of these proteins is mediated by CTD phospho-epitopes generated during transcription. The mechanisms regulating the kinases that establish these phosphorylation patterns on the CTD are not known. We report that three CTD kinases, CDK7, CDK9, and BRD4, engage in cross-talk, modulating their subsequent CTD phosphorylation. BRD4 phosphorylates PTEFb/CDK9 at either Thr-29 or Thr-186, depending on its relative abundance, which represses or activates CDK9 CTD kinase activity, respectively. Conversely, CDK9 phosphorylates BRD4 enhancing its CTD kinase activity. The CTD Ser-5 kinase CDK7 also interacts with and phosphorylates BRD4, potently inhibiting BRD4 kinase activity. Additionally, the three kinases regulate each other indirectly through the general transcription factor TAF7. An inhibitor of CDK9 and CDK7 CTD kinase activities, TAF7 also binds to BRD4 and inhibits its kinase activity. Each of these kinases phosphorylates TAF7, affecting its subsequent ability to inhibit the other two. Thus, a complex regulatory network governs Pol II CTD kinases.
- Published
- 2012
27. Biochemical and Structural Studies of Uncharacterized Protein PA0743 from Pseudomonas aeruginosa Revealed NAD+-dependent l-Serine Dehydrogenase
- Author
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Claudio F. Gonzalez, Elena Evdokimova, Kemin Tan, Alexei Savchenko, Alexander F. Yakunin, Greg Brown, Alex U. Singer, Robert Flick, and Anatoli Tchigvintsev
- Subjects
Rossmann fold ,Stereochemistry ,Dehydrogenase ,Crystallography, X-Ray ,Biochemistry ,Catalysis ,Cofactor ,Substrate Specificity ,Enzyme catalysis ,Bacterial Proteins ,Oxidoreductase ,Serine ,Phosphoglycerate dehydrogenase ,Molecular Biology ,chemistry.chemical_classification ,biology ,Active site ,Cell Biology ,NAD ,Protein Structure, Tertiary ,chemistry ,Pseudomonas aeruginosa ,Mutagenesis, Site-Directed ,Enzymology ,biology.protein ,Oxidoreductases ,Branched-chain alpha-keto acid dehydrogenase complex ,Oxidation-Reduction - Abstract
The β-hydroxyacid dehydrogenases form a large family of ubiquitous enzymes that catalyze oxidation of various β-hydroxy acid substrates to corresponding semialdehydes. Several known enzymes include β-hydroxyisobutyrate dehydrogenase, 6-phosphogluconate dehydrogenase, 2-(hydroxymethyl)glutarate dehydrogenase, and phenylserine dehydrogenase, but the vast majority of β-hydroxyacid dehydrogenases remain uncharacterized. Here, we demonstrate that the predicted β-hydroxyisobutyrate dehydrogenase PA0743 from Pseudomonas aeruginosa catalyzes an NAD(+)-dependent oxidation of l-serine and methyl-l-serine but exhibits low activity against β-hydroxyisobutyrate. Two crystal structures of PA0743 were solved at 2.2-2.3-Å resolution and revealed an N-terminal Rossmann fold domain connected by a long α-helix to the C-terminal all-α domain. The PA0743 apostructure showed the presence of additional density modeled as HEPES bound in the interdomain cleft close to the predicted catalytic Lys-171, revealing the molecular details of the PA0743 substrate-binding site. The structure of the PA0743-NAD(+) complex demonstrated that the opposite side of the enzyme active site accommodates the cofactor, which is also bound near Lys-171. Site-directed mutagenesis of PA0743 emphasized the critical role of four amino acid residues in catalysis including the primary catalytic residue Lys-171. Our results provide further insight into the molecular mechanisms of substrate selectivity and activity of β-hydroxyacid dehydrogenases.
- Published
- 2012
28. Crystal Structure of Hexokinase KlHxk1 of Kluyveromyces lactis
- Author
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Dmitri I. Svergun, Albrecht Otto, Thomas M. Kriegel, Antje Keim, Eva-Christina Müller, Karina Kettner, Daniela Volke, Ralf Hoffmann, Norbert Sträter, David Singer, and E. Bartholomeus Kuettner
- Subjects
Kluyveromyces lactis ,Hexokinase ,Molecular model ,Stereochemistry ,Protein subunit ,Dimer ,Cell Biology ,Biology ,biology.organism_classification ,Biochemistry ,Yeast ,chemistry.chemical_compound ,Crystallography ,Protein structure ,chemistry ,Protein phosphorylation ,Molecular Biology - Abstract
Crystal structures of the unique hexokinase KlHxk1 of the yeast Kluyveromyces lactis were determined using eight independent crystal forms. In five crystal forms, a symmetrical ring-shaped homodimer was observed, corresponding to the physiological dimer existing in solution as shown by small-angle x-ray scattering. The dimer has a head-to-tail arrangement such that the small domain of one subunit interacts with the large domain of the other subunit. Dimer formation requires favorable interactions of the 15 N-terminal amino acids that are part of the large domain with amino acids of the small domain of the opposite subunit, respectively. The head-to-tail arrangement involving both domains of the two KlHxk1 subunits is appropriate to explain the reduced activity of the homodimer as compared with the monomeric enzyme and the influence of substrates and products on dimer formation and dissociation. In particular, the structure of the symmetrical KlHxk1 dimer serves to explain why phosphorylation of conserved residue Ser-15 may cause electrostatic repulsions with nearby negatively charged residues of the adjacent subunit, thereby inducing a dissociation of the homologous dimeric hexokinases KlHxk1 and ScHxk2. Two complex structures of KlHxk1 with bound glucose provide a molecular model of substrate binding to the open conformation and the subsequent classical domain closure motion of yeast hexokinases. The entirety of the novel data extends the current concept of glucose signaling in yeast and complements the induced-fit model by integrating the events of N-terminal phosphorylation and dissociation of homodimeric yeast hexokinases.
- Published
- 2010
29. Structure and Activity of the Metal-independent Fructose-1,6-bisphosphatase YK23 from Saccharomyces cerevisiae
- Author
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Hong Cui, Aiping Dong, Ekaterina Kuznetsova, Linda Xu, Alexander F. Yakunin, Alexei Savchenko, Greg Brown, Robert Flick, Andrzej Joachimiak, Alex U. Singer, and Marianne E. Cuff
- Subjects
Models, Molecular ,Fructose 1,6-bisphosphate ,Protein Conformation ,Molecular Sequence Data ,Saccharomyces cerevisiae ,Fructose 1,6-bisphosphatase ,Crystallography, X-Ray ,Biochemistry ,Substrate Specificity ,Phosphoglycerate mutase ,chemistry.chemical_compound ,Hydrolase ,Fructosediphosphates ,Magnesium ,Amino Acid Sequence ,Cloning, Molecular ,Molecular Biology ,Alanine ,Manganese ,Sequence Homology, Amino Acid ,biology ,Hydrolysis ,Gene Amplification ,Active site ,Cell Biology ,biology.organism_classification ,Enzyme structure ,Fructose-Bisphosphatase ,Kinetics ,Gene Expression Regulation ,chemistry ,Mutagenesis, Site-Directed ,Enzymology ,biology.protein ,Sequence Alignment - Abstract
Fructose-1,6-bisphosphatase (FBPase), a key enzyme of gluconeogenesis and photosynthetic CO(2) fixation, catalyzes the hydrolysis of fructose 1,6-bisphosphate (FBP) to produce fructose 6-phosphate, an important precursor in various biosynthetic pathways. All known FBPases are metal-dependent enzymes, which are classified into five different classes based on their amino acid sequences. Eukaryotes are known to contain only the type-I FBPases, whereas all five types exist in various combinations in prokaryotes. Here we demonstrate that the uncharacterized protein YK23 from Saccharomyces cerevisiae efficiently hydrolyzes FBP in a metal-independent reaction. YK23 is a member of the histidine phosphatase (phosphoglyceromutase) superfamily with homologues found in all organisms. The crystal structure of the YK23 apo-form was solved at 1.75-A resolution and revealed the core domain with the alpha/beta/alpha-fold covered by two small cap domains. Two liganded structures of this protein show the presence of two phosphate molecules (an inhibitor) or FBP (a substrate) bound to the active site. FBP is bound in its linear, open conformation with the cleavable C1-phosphate positioned deep in the active site. Alanine replacement mutagenesis of YK23 identified six conserved residues absolutely required for activity and suggested that His(13) and Glu(99) are the primary catalytic residues. Thus, YK23 represents the first family of metal-independent FBPases and a second FBPase family in eukaryotes.
- Published
- 2010
30. Calcium/Calmodulin-dependent Protein Kinase II Delta 6 (CaMKIIδ6) and RhoA Involvement in Thrombin-induced Endothelial Barrier Dysfunction
- Author
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Harold A. Singer, Roman Ginnan, Zhen Wang, Peter A. Vincent, Iskandar F. Abdullaev, and Mohamed Trebak
- Subjects
Male ,RHOA ,Endothelium ,Models, Biological ,Biochemistry ,Gene Expression Regulation, Enzymologic ,Rats, Sprague-Dawley ,Thrombin ,Ca2+/calmodulin-dependent protein kinase ,medicine ,Animals ,Humans ,Protein Isoforms ,Extracellular Signal-Regulated MAP Kinases ,Molecular Biology ,Rho-associated protein kinase ,biology ,Autophosphorylation ,Endothelial Cells ,Cell Biology ,Rats ,Cell biology ,medicine.anatomical_structure ,biology.protein ,Calcium ,Human umbilical vein endothelial cell ,Endothelium, Vascular ,Signal transduction ,Calcium-Calmodulin-Dependent Protein Kinase Type 2 ,rhoA GTP-Binding Protein ,Signal Transduction ,medicine.drug - Abstract
Multiple Ca(2+) release and entry mechanisms and potential cytoskeletal targets have been implicated in vascular endothelial barrier dysfunction; however, the immediate downstream effectors of Ca(2+) signals in the regulation of endothelial permeability still remain unclear. In the present study, we evaluated the contribution of multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) as a mediator of thrombin-stimulated increases in human umbilical vein endothelial cell (HUVEC) monolayer permeability. For the first time, we identified the CaMKIIdelta(6) isoform as the predominant CaMKII isoform expressed in endothelium. As little as 2.5 nM thrombin maximally increased CaMKIIdelta(6) activation assessed by Thr(287) autophosphorylation. Electroporation of siRNA targeting endogenous CaMKIIdelta (siCaMKIIdelta) suppressed expression of the kinase by >80% and significantly inhibited 2.5 nM thrombin-induced increases in monolayer permeability assessed by electrical cell-substrate impedance sensing (ECIS). siCaMKIIdelta inhibited 2.5 nM thrombin-induced activation of RhoA, but had no effect on thrombin-induced ERK1/2 activation. Although Rho kinase inhibition strongly suppressed thrombin-induced HUVEC hyperpermeability, inhibiting ERK1/2 activation had no effect. In contrast to previous reports, these results indicate that thrombin-induced ERK1/2 activation in endothelial cells is not mediated by CaMKII and is not involved in endothelial barrier hyperpermeability. Instead, CaMKIIdelta(6) mediates thrombin-induced HUVEC barrier dysfunction through RhoA/Rho kinase as downstream intermediates. Moreover, the relative contribution of the CaMKIIdelta(6)/RhoA pathway(s) diminished with increasing thrombin stimulation, indicating recruitment of alternative signaling pathways mediating endothelial barrier dysfunction, dependent upon thrombin concentration.
- Published
- 2010
31. ADP-dependent 6-Phosphofructokinase from Pyrococcus horikoshii OT3
- Author
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Alexander F. Yakunin, Zongchao Jia, Andrés Caniuguir, Greg Brown, Alexei Savchenko, Felipe Merino, Tatiana Skarina, Alex U. Singer, Andrew Wong, Mark A. Currie, and Victoria Guixé
- Subjects
Methanococcus ,Conformational change ,biology ,Glucokinase ,Substrate (chemistry) ,Cell Biology ,biology.organism_classification ,Biochemistry ,Pyrococcus horikoshii ,Transferase ,Ribokinase ,Molecular Biology ,Phosphofructokinase - Abstract
Some hyperthermophilic archaea use a modified glycolytic pathway that employs an ADP-dependent glucokinase (ADP-GK) and an ADP-dependent phosphofructokinase (ADP-PFK) or, in the case of Methanococcus jannaschii, a bifunctional ADP-dependent glucophosphofructokinase (ADP-GK/PFK). The crystal structures of three ADP-GKs have been determined. However, there is no structural information available for ADP-PFKs or the ADP-GK/PFK. Here, we present the first crystal structure of an ADP-PFK from Pyrococcus horikoshii OT3 (PhPFK) in both apo- and AMP-bound forms determined to 2.0-A and 1.9-A resolution, respectively, along with biochemical characterization of the enzyme. The overall structure of PhPFK maintains a similar large and small alpha/beta domain structure seen in the ADP-GK structures. A large conformational change accompanies binding of phosphoryl donor, acceptor, or both, in all members of the ribokinase superfamily characterized thus far, which is believed to be critical to enzyme function. Surprisingly, no such conformational change was observed in the AMP-bound PhPFK structure compared with the apo structure. Through comprehensive site-directed mutagenesis of the substrate binding pocket we identified residues that were critical for both substrate recognition and the phosphotransfer reaction. The catalytic residues and many of the substrate binding residues are conserved between PhPFK and ADP-GKs; however, four key residues differ in the sugar-binding pocket, which we have shown determine the sugar-binding specificity. Using these results we were able to engineer a mutant PhPFK that mimics the ADP-GK/PFK and is able to phosphorylate both fructose 6-phosphate and glucose.
- Published
- 2009
32. Structural and Biochemical Characterization of the Type II Fructose-1,6-bisphosphatase GlpX from Escherichia coli
- Author
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Aled M. Edwards, Ruslan Sanishvili, Alexei Savchenko, Alex U. Singer, Alexander F. Yakunin, Andrzej Joachimiak, Vladimir V. Lunin, Samvel Kochinyan, Michael Proudfoot, Greg Brown, Tatiana Skarina, and Robert Flick
- Subjects
Phosphatase ,Mutation, Missense ,Fructose 1,6-bisphosphatase ,Lithium ,Crystallography, X-Ray ,Biochemistry ,Catalysis ,Phosphates ,Substrate Specificity ,Evolution, Molecular ,chemistry.chemical_compound ,Escherichia coli ,Fructosediphosphates ,Molecular Biology ,Alanine ,chemistry.chemical_classification ,biology ,Escherichia coli Proteins ,Gluconeogenesis ,Active site ,Fructose ,Cell Biology ,Fructose-Bisphosphatase ,Protein Structure, Tertiary ,Amino acid ,Metabolic pathway ,Enzyme ,Amino Acid Substitution ,chemistry ,Mutagenesis ,Protein Structure and Folding ,biology.protein - Abstract
Gluconeogenesis is an important metabolic pathway, which produces glucose from noncarbohydrate precursors such as organic acids, fatty acids, amino acids, or glycerol. Fructose-1,6-bisphosphatase, a key enzyme of gluconeogenesis, is found in all organisms, and five different classes of these enzymes have been identified. Here we demonstrate that Escherichia coli has two class II fructose-1,6-bisphosphatases, GlpX and YggF, which show different catalytic properties. We present the first crystal structure of a class II fructose-1,6-bisphosphatase (GlpX) determined in a free state and in the complex with a substrate (fructose 1,6-bisphosphate) or inhibitor (phosphate). The crystal structure of the ligand-free GlpX revealed a compact, globular shape with two α/β-sandwich domains. The core fold of GlpX is structurally similar to that of Li+-sensitive phosphatases implying that they have a common evolutionary origin and catalytic mechanism. The structure of the GlpX complex with fructose 1,6-bisphosphate revealed that the active site is located between two domains and accommodates several conserved residues coordinating two metal ions and the substrate. The third metal ion is bound to phosphate 6 of the substrate. Inorganic phosphate strongly inhibited activity of both GlpX and YggF, and the crystal structure of the GlpX complex with phosphate demonstrated that the inhibitor molecule binds to the active site. Alanine replacement mutagenesis of GlpX identified 12 conserved residues important for activity and suggested that Thr90 is the primary catalytic residue. Our data provide insight into the molecular mechanisms of the substrate specificity and catalysis of GlpX and other class II fructose-1,6-bisphosphatases.
- Published
- 2009
33. Binding of DEAD-box helicase Dhh1 to the 5′-untranslated region of ASH1 mRNA represses localized translation of ASH1 in yeast cells
- Author
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Zhang, Qianjun, primary, Meng, Xiuhua, additional, Li, Delin, additional, Chen, Shaoyin, additional, Luo, Jianmin, additional, Zhu, Linjie, additional, Singer, Robert H., additional, and Gu, Wei, additional
- Published
- 2017
- Full Text
- View/download PDF
34. Structural and Biochemical Analyses Reveal the Mechanism of Glutathione S-Transferase Pi 1 Inhibition by the Anti-cancer Compound Piperlongumine
- Author
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Harshbarger, Wayne, primary, Gondi, Sudershan, additional, Ficarro, Scott B., additional, Hunter, John, additional, Udayakumar, Durga, additional, Gurbani, Deepak, additional, Singer, William D., additional, Liu, Yan, additional, Li, Lianbo, additional, Marto, Jarrod A., additional, and Westover, Kenneth D., additional
- Published
- 2017
- Full Text
- View/download PDF
35. CD6 Receptor Regulates Intestinal Ischemia/Reperfusion-induced Injury by Modulating Natural IgM-producing B1a Cell Self-renewal
- Author
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Enyindah-Asonye, Gospel, primary, Li, Yan, additional, Xin, Wei, additional, Singer, Nora G., additional, Gupta, Neetu, additional, Fung, John, additional, and Lin, Feng, additional
- Published
- 2017
- Full Text
- View/download PDF
36. The Cell Adhesion Receptor Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 Regulates Nucleocytoplasmic Trafficking of DNA Polymerase δ-Interacting Protein 38
- Author
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Mario M. Müller, Lothar Lucka, Bernhard B. Singer, Werner Reutter, Wolfgang Otto, Christoph Kannicht, Björn Öbrink, and Esther Klaile
- Subjects
Cytoplasm ,L1 ,Active Transport, Cell Nucleus ,CHO Cells ,Biology ,Biochemistry ,Cricetulus ,Antigens, CD ,Cricetinae ,Animals ,Humans ,Protein Isoforms ,Immunologic Capping ,Cell adhesion ,Receptor ,Molecular Biology ,Cell Nucleus ,Confluency ,Cell adhesion molecule ,Cell Membrane ,Antibodies, Monoclonal ,Membrane Proteins ,Nuclear Proteins ,Cell Biology ,Subcellular localization ,Rats ,Cell biology ,Neural cell adhesion molecule ,Cell Adhesion Molecules ,HeLa Cells ,Signal Transduction - Abstract
The homophilic cell-cell adhesion receptor CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1, CD66a) acts as a regulator of contact-dependent cell survival, differentiation, and growth. It is involved in the control of proliferation in hematopoietic and epithelial cells and can act as a tumor suppressor. In this study, we identify DNA polymerase delta-interacting protein 38 (PDIP38) as a novel binding partner for CEACAM1-L and CEACAM1-S. We show that PDIP38 can occur in the nucleus, in the cytoplasm and at the plasma membrane in NBT-II, IEC18, RBE, and HeLa cells and that the distribution in NBT-II cells is influenced by the confluency of the cells. We also demonstrate that the interaction of CEACAM1 and PDIP38 is of functional importance in NBT-II cells, which co-express the long and the short CEACAM1 isoform. In subconfluent, proliferating NBT-II cells, perturbation of CEACAM1 by antibody clustering induces increased binding to PDIP38 and results in rapid recruitment of PDIP38 to the plasma membrane. The same treatment of confluent, quiescent NBT-II cells leads to a different response, i.e. translocation of PDIP38 to the nucleus. Together, our data show that PDIP38 can shuttle between the cytoplasmic and the nuclear compartments and that its subcellular localization is regulated by CEACAM1, implicating that PDIP38 may constitute a novel downstream target of CEACAM1 signaling.
- Published
- 2007
37. Critical Role of Vimentin Phosphorylation at Ser-56 by p21-activated Kinase in Vimentin Cytoskeleton Signaling
- Author
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Ruping Wang, Yana Anfinogenova, Dale D. Tang, Harold A. Singer, Amy M. Spinelli, and Qing-Fen Li
- Subjects
Serotonin ,Myocytes, Smooth Muscle ,Vimentin ,macromolecular substances ,Protein Serine-Threonine Kinases ,environment and public health ,Biochemistry ,Article ,Gene Expression Regulation, Enzymologic ,Phosphoserine ,Dogs ,Serotonin Agents ,PAK1 ,Animals ,Myocyte ,Phosphorylation ,Cytoskeleton ,Molecular Biology ,Actin ,biology ,Kinase ,Cell Biology ,Smooth muscle contraction ,Actins ,Cell biology ,Trachea ,enzymes and coenzymes (carbohydrates) ,Crk-Associated Substrate Protein ,p21-Activated Kinases ,biology.protein ,Signal Transduction - Abstract
Phosphorylation and spatial reorganization of the vimentin network have been implicated in mediating smooth muscle contraction, cell migration, and cell mitosis. In this report, stimulation of cultured smooth muscle cells with 5-hydroxytryptamine (5-HT) induced PAK1 phosphorylation at Thr-423 (an indication of PAK activation). Treatment with PAK led to disassembly of wild type vimentin filaments, but not vimentin S56A (alanine substitution at serine-56) mutant filaments, as assessed by an in vitro filament assembly assay. Furthermore, stimulation with 5-HT resulted in the dissociation of Crk-associated substrate (CAS, an adapter protein associated with smooth muscle force development) from cytoskeletal vimentin. Expression of the vimentin S56A mutant in cells inhibited the increase in phosphorylation at Ser-56 and ratios of soluble vimentin to insoluble vimentin (an index of vimentin disassembly), and the dissociation of CAS from cytoskeletal vimentin in response to 5-HT activation as compared to cells expressing wild type vimentin. Because CAS may be involved in PAK activation, PAK phosphorylation was evaluated in cells expressing S56A vimentin mutant. Expression of vimentin S56A mutant depressed PAK phosphorylation at Thr-423 induced by 5-HT. The expression of the vimentin S56A mutant also inhibited the spatial reorientation of vimentin filaments in cells in response to 5-HT stimulation. Our results suggest that vimentin phosphorylation at Ser-56 may inversely regulate PAK activation possibly via the increase in the amount of soluble CAS upon agonist stimulation of smooth muscle cells. Additionally, vimentin phosphorylation at this position is critical for vimentin filament spatial rearrangement elicited by agonists.
- Published
- 2006
38. Yeast-like mRNA Capping Apparatus in Giardia lamblia
- Author
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Matthew Witmer, Melissa A. Altura, Stewart Shuman, Steven M. Singer, Stéphane Hausmann, and Heidi G. Elmendorf
- Subjects
Glycerol ,Guanylyltransferase ,Time Factors ,medicine.disease_cause ,Biochemistry ,RNA triphosphatase ,Luciferases ,Guanosine ,biology ,Hydrogen-Ion Concentration ,Nucleotidyltransferases ,Recombinant Proteins ,Acid Anhydride Hydrolases ,Cyanidioschyzon merolae ,Metals ,Electrophoresis, Polyacrylamide Gel ,Guanosine Triphosphate ,Plasmids ,RNA Caps ,Molecular Sequence Data ,Transfection ,Models, Biological ,Catalysis ,Microbiology ,Evolution, Molecular ,Cations ,parasitic diseases ,Centrifugation, Density Gradient ,medicine ,Animals ,Giardia lamblia ,Amino Acid Sequence ,RNA, Messenger ,Molecular Biology ,DNA Primers ,Messenger RNA ,Dose-Response Relationship, Drug ,Sequence Homology, Amino Acid ,RNA ,Cell Biology ,biology.organism_classification ,Phosphoric Monoester Hydrolases ,Protein Structure, Tertiary ,Protein Biosynthesis ,Rhodophyta ,Protozoa ,Triphosphatase - Abstract
A scheme of eukaryotic phylogeny has been suggested based on the structure and physical linkage of the RNA triphosphatase and RNA guanylyltransferase enzymes that catalyze mRNA cap formation. Here we show that the unicellular pathogen Giardia lamblia encodes an mRNA capping apparatus consisting of separate triphosphatase and guanylyltransferase components, which we characterize biochemically. We also show that native Giardia mRNAs have blocked 5′-ends and that 7-methylguanosine caps promote translation of transfected mRNAs in Giardia in vivo. The Giardia triphosphatase belongs to the tunnel family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi, microsporidia, and protozoa such as Plasmodium and Trypanosoma. The tunnel enzymes adopt a unique active-site fold and are structurally and mechanistically unrelated to the cysteine-phosphatase-type RNA triphosphatases found in metazoans and plants, which comprise part of a bifunctional triphosphataseguanylyltransferase fusion protein. All available evidence now points to the separate tunnel-type triphosphatase and guanylyltransferase as the aboriginal state of the capping apparatus. We identify a putative tunnel-type triphosphatase and a separate guanylyltransferase encoded by the red alga Cyanidioschyzon merolae. These findings place fungi, protozoa, and red algae in a common lineage distinct from that of metazoa and plants.
- Published
- 2005
39. Structural and Biochemical Characterization of CIB1 Delineates a New Family of EF-hand-containing Proteins
- Author
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Joseph D. Ferrara, John Sondek, Alex U. Singer, Laurie Betts, Holly R. Gentry, Cheng Yang, and Leslie V. Parise
- Subjects
Models, Molecular ,Cytoplasm ,Protein Folding ,Protein Conformation ,Molecular Sequence Data ,Electrons ,Platelet Glycoprotein GPIIb-IIIa Complex ,Plasma protein binding ,Biology ,Crystallography, X-Ray ,Ligands ,Biochemistry ,Protein Structure, Secondary ,Protein structure ,Calcium-binding protein ,Escherichia coli ,Humans ,Amino Acid Sequence ,Threonine ,Molecular Biology ,Peptide sequence ,Phylogeny ,Ions ,Neurons ,Sequence Homology, Amino Acid ,EF hand ,Effector ,Calcineurin ,X-Rays ,Calcium-Binding Proteins ,Cell Biology ,Protein Structure, Tertiary ,Multigene Family ,Chromatography, Gel ,Calcium ,Protein folding ,Peptides ,Ultracentrifugation ,Protein Binding ,Signal Transduction - Abstract
CIB1 (CIB) is an EF-hand-containing protein that binds multiple effector proteins, including the platelet alphaIIbbeta3 integrin and several serine/threonine kinases and potentially modulates their function. The crystal structure for Ca(2+)-bound CIB1 has been determined at 2.0 A resolution and reveals a compact alpha-helical protein containing four EF-hands, the last two of which bind calcium ions in the standard fashion seen in many other EF-hand proteins. CIB1 shares high structural similarity with calcineurin B and the neuronal calcium sensor (NCS) family of EF-hand-containing proteins. Most importantly, like calcineurin B and NCS proteins, which possess a large hydrophobic pocket necessary for ligand binding, CIB1 contains a hydrophobic pocket that has been implicated in ligand binding by previous mutational analysis. However, unlike several NCS proteins, Ca(2+)-bound CIB1 is largely monomeric whether bound to a relevant peptide ligand or ligand-free. Differences in structure, oligomeric state, and phylogeny define a new family of CIB1-related proteins that extends from arthropods to humans.
- Published
- 2005
40. N-Acetylglucosamine 6-O-Sulfotransferase-1 Regulates Expression of L-Selectin Ligands and Lymphocyte Homing
- Author
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Steven D. Rosen, Fathy M. El-Fasakhany, Takashi Muramatsu, Mineko Izawa, Kenji Kadomatsu, Kenji Uchimura, Reiji Kannagi, Mark S. Singer, and Naoki Takeda
- Subjects
High endothelial venules ,Oligosaccharides ,Ligands ,Models, Biological ,Biochemistry ,Catalysis ,Epitope ,Epitopes ,Mice ,In vivo ,Cell Adhesion ,Leukocytes ,medicine ,Animals ,Mesenteric lymph nodes ,Lymphocytes ,Cloning, Molecular ,L-Selectin ,Sialyl Lewis X Antigen ,Lymphocyte homing receptor ,Molecular Biology ,biology ,Reverse Transcriptase Polymerase Chain Reaction ,Ligand ,Stem Cells ,Cell Biology ,Embryo, Mammalian ,Immunohistochemistry ,Molecular biology ,Blotting, Southern ,medicine.anatomical_structure ,Gene Expression Regulation ,Microscopy, Fluorescence ,Immunology ,biology.protein ,L-selectin ,Lymph Nodes ,Sulfotransferases ,Peripheral lymph - Abstract
Lymphocyte homing is initiated by the binding of L-selectin on lymphocytes to its ligands on high endothelial venules (HEV). Sialyl 6-sulfo Lewis X is a major capping group of L-selectin ligands. N-Acetylglucosamine (GlcNAc) 6-sulfation is essential for the ligand activity, and is catalyzed by GlcNAc 6-O-sulfotransferases (GlcNAc6STs) of which GlcNAc6ST-1 and GlcNAc6ST-2 are expressed in HEV. Here, we report that mice deficient in GlcNAc6ST-1 were impaired in the elaboration of sialyl 6-sulfo Lewis X in HEV and that an epitope of L-selectin ligands recognized by the MECA-79 anti-body was greatly reduced or abolished in the abluminal aspect of HEV. Lymphocyte homing to peripheral lymph nodes, mesenteric lymph nodes, and Peyer's patches was significantly reduced in GlcNAc6ST-1 null mice. These results demonstrate that GlcNAc6ST-1 is involved in lymphocyte homing in vivo, and indicate that GlcNAc6ST-1 and -2 play complementary roles. The importance of GlcNAc6ST-1 is particularly high-lighted by its involvement in lymphocyte homing to Peyer's patches where GlcNAc6ST-2 expression is undetectable.
- Published
- 2004
41. Purification and Characterization of NafY (Apodinitrogenase γ Subunit) from Azotobacter vinelandii
- Author
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Steven W. Singer, Luis M. Rubio, and Paul W. Ludden
- Subjects
Iron-Sulfur Proteins ,Molybdoferredoxin ,Immunoblotting ,Molecular Sequence Data ,Normal Distribution ,Plasma protein binding ,Biochemistry ,Protein Structure, Secondary ,Article ,Cofactor ,Residue (chemistry) ,chemistry.chemical_compound ,Bacterial Proteins ,Nitrogenase ,Amino Acid Sequence ,Molecular Biology ,Peptide sequence ,Glutathione Transferase ,Azotobacter vinelandii ,Chromatography ,Cofactor binding ,Dose-Response Relationship, Drug ,Sequence Homology, Amino Acid ,biology ,Mutagenesis ,Electron Spin Resonance Spectroscopy ,Cell Biology ,biology.organism_classification ,Protein Structure, Tertiary ,Kinetics ,Monomer ,chemistry ,Chromatography, Gel ,Mutagenesis, Site-Directed ,biology.protein ,Electrophoresis, Polyacrylamide Gel ,Protein Binding - Abstract
The formation of an active dinitrogenase requires the synthesis and the insertion of the iron-molybdenum cofactor (FeMo-co) into a presynthesized apodinitrogenase. In Azotobacter vinelandii, NafY (also known as gamma protein) has been proposed to be a FeMo-co insertase because of its ability to bind FeMo-co and apodinitrogenase. Here we report the purification and biochemical characterization of NafY and reach the following conclusions. First, NafY is a 26-kDa monomeric protein that binds one molecule of FeMo-co with very high affinity (K(d) approximately equal to 60 nm); second, the NafY-FeMo-co complex exhibits a S = 3/2 EPR signal with features similar to the signals for extracted FeMo-co and the M center of dinitrogenase; third, site-directed mutagenesis of nafY indicates that the His(121) residue of NafY is involved in cofactor binding; and fourth, NafY binding to apodinitrogenase or to FeMo-co does not require the presence of any additional protein. In addition, we have obtained evidence that suggests the ability of NafY to bind NifB-co, an FeS cluster of unknown structure that is a biosynthetic precursor to FeMo-co.
- Published
- 2004
42. Roles of the Proline-rich Domain in SLP-76 Subcellular Localization and T Cell Function
- Author
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Lawrence E. Samelson, Peggy Myung, Amrom E. Obstfeld, Martha S. Jordan, Jennifer N. Wu, Andrew L. Singer, Stephen C. Bunnell, and Gary A. Koretzky
- Subjects
Antigens, Differentiation, T-Lymphocyte ,Time Factors ,genetic structures ,T-Lymphocytes ,Lymphocyte Activation ,Biochemistry ,Jurkat cells ,Jurkat Cells ,Luciferases ,Genes, Dominant ,Alanine ,biology ,Signal transducing adaptor protein ,Flow Cytometry ,Cell biology ,GRB2 ,Signal transduction ,Plasmids ,Signal Transduction ,Subcellular Fractions ,Proto-oncogene tyrosine-protein kinase Src ,Binding domain ,Proline ,Blotting, Western ,Arginine ,Transfection ,Models, Biological ,Cell Line ,src Homology Domains ,Membrane Microdomains ,Antigens, CD ,Humans ,Cell Lineage ,Lectins, C-Type ,Molecular Biology ,Adaptor Proteins, Signal Transducing ,Lysine ,Cell Biology ,Hematopoietic Stem Cells ,Phosphoproteins ,equipment and supplies ,Subcellular localization ,Precipitin Tests ,eye diseases ,Protein Structure, Tertiary ,Phosphoprotein ,Mutation ,biology.protein ,Calcium ,sense organs ,Gene Deletion - Abstract
The adaptor protein Src homology (SH)2 domain-containing and leukocyte-specific phosphoprotein of 76 kDa (SLP-76) is critical for signal transduction in multiple hematopoietic lineages. It links proximal and distal T cell receptor signaling events through its function as a molecular scaffold in the assembly of multimolecular signaling complexes. Here we studied the functional roles of sub-domains within the SLP-76 proline-rich region, specifically the Gads binding domain and the recently defined P1 domain. To gain a further understanding of the functions mediated by this region, we used three complementary approaches as follows: reconstitution of SLP-76-deficient cells with functional domain deletion mutants, blocking molecular associations through the expression of a dominant negative protein fragment, and directed localization of SLP-76 to assess the role of the domains in SLP-76 recruitment. We find the Gads binding domain and the P1 domain are both necessary for optimal SLP-76 function, and in the absence of these two regions, SLP-76 is functionally inert. Furthermore, we provide direct evidence that SLP-76 localization and, in turn, function are dependent upon association with Gads.
- Published
- 2004
43. Leon Heppel and the Early Days of RNA Biochemistry
- Author
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Maxine F. Singer
- Subjects
Chemistry ,MEDLINE ,Nucleic Acid Conformation ,RNA ,Historical Article ,Cell Biology ,Computational biology ,History, 20th Century ,RNA Biochemistry ,Molecular Biology ,Biochemistry - Published
- 2003
44. Direct Interaction of Target SNAREs with the Kv2.1 Channel
- Author
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Ilana Lotan, Dodo Chikvashvili, Michal Linial, Herbert Y. Gaisano, Dafna Singer-Lahat, Sharon Tsuk, Youhou Kang, Izhak Michaelevski, and Oded Fili
- Subjects
Voltage-gated ion channel ,Voltage clamp ,Xenopus ,Cell Biology ,Plasma protein binding ,Gating ,Biology ,Syntaxin 1 ,biology.organism_classification ,Biochemistry ,Exocytosis ,Potassium channel ,Cell biology ,nervous system ,Molecular Biology - Abstract
Previously we suggested that interaction between voltage-gated K+ channels and protein components of the exocytotic machinery regulated transmitter release. This study concerns the interaction between the Kv2.1 channel, the prevalent delayed rectifier K+ channel in neuroendocrine and endocrine cells, and syntaxin 1A and SNAP-25. We recently showed in islet beta-cells that the Kv2.1 K+ current is modulated by syntaxin 1A and SNAP-25. Here we demonstrate, using co-immunoprecipitation and immunocytochemistry analyses, the existence of a physical interaction in neuroendocrine cells between Kv2.1 and syntaxin 1A. Furthermore, using concomitant co-immunoprecipitation from plasma membranes and two-electrode voltage clamp analyses in Xenopus oocytes combined with in vitro binding analysis, we characterized the effects of these interactions on the Kv2.1 channel gating pertaining to the assembly/disassembly of the syntaxin 1A/SNAP-25 (target (t)-SNARE) complex. Syntaxin 1A alone binds strongly to Kv2.1 and shifts both activation and inactivation to hyperpolarized potentials. SNAP-25 alone binds weakly to Kv2.1 and probably has no effect by itself. Expression of SNAP-25 together with syntaxin 1A results in the formation of t-SNARE complexes, with consequent elimination of the effects of syntaxin 1A alone on both activation and inactivation. Moreover, inactivation is shifted to the opposite direction, toward depolarized potentials, and its extent and rate are attenuated. Based on these results we suggest that exocytosis in neuroendocrine cells is tuned by the dynamic coupling of the Kv2.1 channel gating to the assembly status of the t-SNARE complex.
- Published
- 2003
45. The Pleckstrin Homology Domain of Phospholipase C-β2 as an Effector Site for Rac
- Author
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Alex U. Singer, Michele R. Wing, Jason T. Snyder, John Sondek, and T. Kendall Harden
- Subjects
rac1 GTP-Binding Protein ,RhoGEF domain ,DNA, Complementary ,Phospholipase C beta ,Rac3 ,RAC1 ,Biosensing Techniques ,GTPase ,Biology ,Biochemistry ,PAK1 ,Animals ,Humans ,Molecular Biology ,Phospholipase C ,Blood Proteins ,Cell Biology ,Phosphoproteins ,Protein Structure, Tertiary ,rac GTP-Binding Proteins ,Cell biology ,Enzyme Activation ,Isoenzymes ,Pleckstrin homology domain ,Type C Phospholipases ,COS Cells ,Guanine nucleotide exchange factor ,Protein Binding - Abstract
Increasing evidence links the activation of Rho family GTPases to the stimulation of lipid hydrolysis catalyzed by phospholipase C (PLC)-beta isozymes. To better define this relationship, members of a library of recombinant Rho GTPases were screened for their capacity to directly engage various purified PLC-beta isozymes. Of the 17 tested members of the Rho family, only the active isoforms of Rac (Rac1, Rac2, and Rac3) both stimulate PLC-beta activity in vivo and bind PLC-beta2 and PLC-beta3, but not PLC-beta1, in vitro. Furthermore, the recognition site for Rac GTPases was localized to the pleckstrin homology (PH) domain of PLC-beta2, and this PH domain is fully sufficient to selectively interact with the active versions of the Rac GTPases, but not with other similar Rho GTPases. Together, these findings present a quantitative evaluation of the direct interactions between Rac GTPases and PLC-beta isozymes and define a novel role for the PH domain of PLC-beta2 as a putative effector site for Rac GTPases.
- Published
- 2003
46. On the Binding Preference of Human Groups IIA and X Phospholipases A2 for Membranes with Anionic Phospholipids
- Author
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Sarah L. Keller, Sofiane Bezzine, James G. Bollinger, Michael H. Gelb, Alan G. Singer, and Sarah L. Veatch
- Subjects
Anions ,Vesicle ,Cell Membrane ,Lysine ,Phospholipid ,Cell Biology ,Phosphatidylserine ,Phospholipase ,Biochemistry ,Phospholipases A ,Sphingomyelins ,Substrate Specificity ,Kinetics ,chemistry.chemical_compound ,Cholesterol ,Membrane ,chemistry ,Phosphatidylcholine ,Humans ,Sphingomyelin ,Molecular Biology ,Phospholipids - Abstract
Mammals contain 9-10 secreted phospholipases A(2) (sPLA(2)s) that display widely different affinities for membranes, depending on the phospholipid composition. The much higher enzymatic activity of human group X sPLA(2) (hGX) compared with human group IIA sPLA(2) (hGIIA) on phosphatidylcholine (PC)-rich vesicles is due in large part to the higher affinity of the former enzyme for such vesicles; this result also holds when vesicles contain cholesterol and sphingomyelin. The inclusion of anionic phosphatidylserine in PC vesicles dramatically enhances interfacial binding and catalysis of hGIIA but not of hGX. This is the result of the large number of lysine and arginine residues scattered over the entire surface of hGIIA, which cause the enzyme to form a supramolecular aggregate with multiple vesicles. Thus, high affinity binding of hGIIA to anionic vesicles is a complex process and cannot be attributed to a few basic residues on its interfacial binding surface, as is also evident from mutagenesis studies. The main reason hGIIA binds poorly to PC-rich vesicles is that it lacks a tryptophan residue on its interfacial binding surface, a residue that contributes to the high affinity binding of hGX to PC-rich vesicles. Results show that the lag in the onset of hydrolysis of PC vesicles by hGIIA is due in part to the poor affinity of this enzyme for these vesicles. Binding affinity of hGIIA, hGX, and their mutants to PC-rich vesicles is well correlated to the ability of these enzymes to act on the PC-rich outer plasma membrane of mammalian cells.
- Published
- 2002
47. Crystal Structure of Human Group X Secreted Phospholipase A2
- Author
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Gérard Lambeau, Farideh Ghomashchi, Bao-Zhu Yu, Michael H. Gelb, Mahendra Kumar Jain, Brian J. Bahnson, Alan G. Singer, and Ying H. Pan
- Subjects
biology ,Stereochemistry ,C-terminus ,Allosteric regulation ,Phospholipid ,Active site ,Cell Biology ,Crystal structure ,Biochemistry ,Micelle ,chemistry.chemical_compound ,Membrane ,chemistry ,Hydrolase ,biology.protein ,Molecular Biology - Abstract
The crystal structure of human group X (hGX) secreted phospholipase A2 (sPLA2) has been solved to a resolution of 1.97 A. As expected the protein fold is similar to previously reported sPLA2 structures. The active site architecture, including the positions of the catalytic residues and the first and second shell water around the Ca2+ cofactor, are highly conserved and remarkably similar to the group IB and group IIA enzymes. Differences are seen in the structures following the (1–12)-N-terminal helix and at the C terminus. These regions are proposed to interact with the substrate membrane surface. The opening to the active site slot is considerably larger in hGX than in human group IIA sPLA2. Furthermore, the electrostatic surface potential of the hGX interfacial-binding surface does not resemble that of the human group IIA sPLA2; the former is highly neutral, whereas the latter is highly cationic. The cationic residues on this face of group IB and IIA enzymes have been implicated in membrane binding and ink allostery. In contrast, hGX does not show activation by the anionic charge at the lipid interface when acting on phospholipid vesicles or short-chain phospholipid micelles. Together, the crystal structure and kinetic results of hGX supports the conclusion that it is as active on zwitterionic as on anionic interfaces, and thus it is predicted to target the zwitterionic membrane surfaces of mammalian cells.
- Published
- 2002
48. Groups IV, V, and X Phospholipases A2s in Human Neutrophils
- Author
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Eva Stefanski, Gérard Lambeau, Brian P. Smart, Niels Borregaard, Jay A. Tischfield, Reinhardt Reithmeier, Cornelia Lichtenberger, Norbert Degousee, Thomas F. Lindsay, Farideh Ghomashchi, Jonathan P. Arm, Barry B. Rubin, Alan G. Singer, Michael H. Gelb, and Walter Reinisch
- Subjects
biology ,Leukotriene B4 ,Zymosan ,hemic and immune systems ,Cell Biology ,Phospholipase ,Biochemistry ,chemistry.chemical_compound ,Azurophilic granule ,Phospholipase A2 ,chemistry ,Eicosanoid ,biology.protein ,lipids (amino acids, peptides, and proteins) ,Arachidonic acid ,Molecular Biology ,Eicosanoid Production - Abstract
The bacterial tripeptide formyl-Met-Leu-Phe (fMLP) induces the secretion of enzyme(s) with phospholipase A2 (PLA2) activity from human neutrophils. We show that circulating human neutrophils express groups V and X sPLA2 (GV and GX sPLA2) mRNA and contain GV and GX sPLA2 proteins, whereas GIB, GIIA, GIID, GIIE, GIIF, GIII, and GXII sPLA2s are undetectable. GV sPLA2 is a component of both azurophilic and specific granules, whereas GX sPLA2 is confined to azurophilic granules. Exposure to fMLP or opsonized zymosan results in the release of GV but not GX sPLA2 and most, if not all, of the PLA2 activity in the extracellular fluid of fMLP-stimulated neutrophils is due to GV sPLA2. GV sPLA2 does not contribute to fMLP-stimulated leukotriene B4 production but may support the anti-bacterial properties of the neutrophil, because 10–100 ng per ml concentrations of this enzyme lead to Gram-negative bacterial membrane phospholipid hydrolysis in the presence of human serum. By use of a recently described and specific inhibitor of cytosolic PLA2-α (group IV PLA2α), we show that this enzyme produces virtually all of the arachidonic acid used for the biosynthesis of leukotriene B4 in fMLP- and opsonized zymosan-stimulated neutrophils, the major eicosanoid produced by these pro-inflammatory cells.
- Published
- 2002
49. Identification of a Heregulin Binding Site in HER3 Extracellular Domain
- Author
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David Eisenberg, Elizabeth Singer, Dennis J. Slamon, Tom Horan, and Ralf Landgraf
- Subjects
Neuregulin-1 ,Molecular Sequence Data ,CHO Cells ,Biochemistry ,Receptor tyrosine kinase ,Epidermal growth factor ,Cricetinae ,Animals ,Amino Acid Sequence ,Binding site ,skin and connective tissue diseases ,Molecular Biology ,DNA Primers ,Binding Sites ,Base Sequence ,Sequence Homology, Amino Acid ,biology ,Hydrolysis ,Receptor Protein-Tyrosine Kinases ,Cell Biology ,Ligand (biochemistry) ,Molecular biology ,Transmembrane protein ,Cell biology ,body regions ,A-site ,biology.protein ,Neuregulin ,Oxidation-Reduction ,Binding domain - Abstract
HER3 (also known as c-Erb-b3) is a type I receptor tyrosine kinase similar in sequence to the epidermal growth factor (EGF) receptor. The extracellular segment of this transmembrane receptor contains four domains. Domains I and II are similar in sequence to domains III and IV, respectively, and domains II and IV are cysteine-rich. We show that the EGF-like domain of heregulin (hrg) binds to domains I and II of HER3, in contrast to the EGF receptor, for which prior studies have shown that a construct consisting of domains III and portions of domain IV binds EGF. Next, we identified a putative hrg binding site by limited proteolysis of the recombinant extracellular domains of HER3 (HER3-ECD(I-IV)) in both the presence and absence of hrg. In the absence of hrg, HER3-ECD(I-IV) is cleaved after position Tyr(50), near the beginning of domain I. Binding of hrg to HER3-ECD(I-IV) fully protects position Tyr(50) from proteolysis. To confirm that domain I contains a hrg binding site, we expressed domains I and II (HER3-ECD(I-II)) and find that it binds hrg with 68 nm affinity. These data suggest that domains I and II of HER3-ECD(I-IV) act as a functional unit in folding and binding of hrg. Thus, our biochemical findings reinforce the structural hypothesis of others that HER3-ECD(I-IV) is similar to the insulin-like growth factor-1 receptor (IGF-1R), as follows: 1) The protected cleavage site in HER3-ECD(I-IV) corresponds to a binding footprint in domain I of IGF-1R; 2) HER3-ECD(I-II) binds hrg with a 68 nm dissociation constant, supporting the hypothesis that domain I is involved in ligand binding; and 3) the large accessible surface area (1749 A) of domain L1 of IGF-1R that is buried by domain S1, as well as the presence of conserved contacts in this interface of type 1 RTKs, suggests that domains L1 and S1 of IGF-1R function as a unit as observed for HER3-ECD(I-II). Our results are consistent with the proposal that HER3 has a structure similar to IGF-1R and binds ligand at a site in corresponding domains.
- Published
- 2001
50. Distinct Arachidonate-releasing Functions of Mammalian Secreted Phospholipase A2s in Human Embryonic Kidney 293 and Rat Mastocytoma RBL-2H3 Cells through Heparan Sulfate Shuttling and External Plasma Membrane Mechanisms
- Author
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Ayako Enomoto, Michael H. Gelb, Ichiro Kudo, Rao S. Koduri, Mimie Seki, Kumiko Yoshihara, Gérard Lambeau, Makoto Murakami, Satoko Shimbara, Farideh Ghomashchi, Alan G. Singer, and Emmanuel Valentin
- Subjects
Time Factors ,Glypican ,Phospholipase ,Biochemistry ,Group V Phospholipases A2 ,Mice ,chemistry.chemical_compound ,Tumor Cells, Cultured ,Mast Cells ,Phosphorylation ,Arachidonic Acid ,Microscopy, Confocal ,biology ,Hydrolysis ,Heparan sulfate ,Hydrogen-Ion Concentration ,Immunohistochemistry ,Leukotriene C4 ,Recombinant Proteins ,Electrophoresis, Polyacrylamide Gel ,Arachidonic acid ,Protein Binding ,Molecular Sequence Data ,Mast-Cell Sarcoma ,Transfection ,Group II Phospholipases A2 ,Models, Biological ,Dinoprostone ,Phospholipases A ,Cell Line ,Phospholipase A2 ,Animals ,Humans ,Amino Acid Sequence ,Platelet Activating Factor ,Molecular Biology ,Sequence Homology, Amino Acid ,Heparin ,Cell Membrane ,HEK 293 cells ,Cell Biology ,Lipid signaling ,Rats ,chemistry ,Mutagenesis, Site-Directed ,biology.protein ,RNA ,Heparan Sulfate Proteoglycans - Abstract
We analyzed the ability of a diverse set of mammalian secreted phospholipase A(2) (sPLA(2)) to release arachidonate for lipid mediator generation in two transfected cell lines. In human embryonic kidney 293 cells, the heparin-binding enzymes sPLA(2)-IIA, -IID, and -V promote stimulus-dependent arachidonic acid release and prostaglandin E(2) production in a manner dependent on the heparan sulfate proteoglycan glypican. In contrast, sPLA(2)-IB, -IIC, and -IIE, which bind weakly or not at all to heparanoids, fail to elicit arachidonate release, and addition of a heparin binding site to sPLA(2)-IIC allows it to release arachidonate. Heparin nonbinding sPLA(2)-X liberates arachidonic acid most likely from the phosphatidylcholine-rich outer plasma membrane in a glypican-independent manner. In rat mastocytoma RBL-2H3 cells that lack glypican, sPLA(2)-V and -X, which are unique among sPLA(2)s in being able to hydrolyze phosphatidylcholine-rich membranes, act most likely on the extracellular face of the plasma membrane to markedly augment IgE-dependent immediate production of leukotriene C(4) and platelet-activating factor. sPLA(2)-IB, -IIA, -IIC, -IID, and -IIE exert minimal effects in RBL-2H3 cells. These results are also supported by studies with sPLA(2) mutants and immunocytostaining and reveal that sPLA(2)-dependent lipid mediator generation occur by distinct (heparanoid-dependent and -independent) mechanisms in HEK293 and RBL-2H3 cells.
- Published
- 2001
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