69 results on '"Field SJ"'
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2. Practical Steps to Implementing Health and Safety in General PracticeRACooke, SJMoore. Hereford & WorcesterFHSA1993, 44pp, £22.45 (incl. p&p)
- Author
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Field, SJ and Westrop, FM
- Published
- 1995
- Full Text
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3. A qualitative evaluation of the 'Trailblazers' teaching the teachers programme in mental health.
- Author
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Brown CA, Wakefield SE, Bullock AD, and Field SJ
- Abstract
This article aims to provide an evaluation of the 'Trailblazers' programme, and seeks to identify the factors affecting the impact of the programme. 'Trailblazers' is a learner-centred 'Teaching the Teachers' programme in primary care mental health. In the first programme to be run in the West Midlands, seven pairs of participants attended three residential modules and developed an action plan that was cascaded to local colleagues. A primarily qualitative investigation, exploring the experiences of the first cohort of 14 Trailblazers in the West Midlands, demonstrated that the modules were well received and over 200 other professionals benefited from education sessions delivered by the participants. Most delegates were keen to be involved in future programmes and regional policy-making. The Trailblazers programme demonstrated short-term effectiveness in terms of participant reaction, knowledge and changes in professional practice. [ABSTRACT FROM AUTHOR]
- Published
- 2003
- Full Text
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4. A multienzyme S-nitrosylation cascade regulates cholesterol homeostasis.
- Author
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Stomberski CT, Venetos NM, Zhou HL, Qian Z, Collison BR, Field SJ, Premont RT, and Stamler JS
- Subjects
- Mice, Animals, Proteins metabolism, Oxidoreductases metabolism, Homeostasis, Nitric Oxide metabolism, Membrane Proteins, Proprotein Convertase 9, S-Nitrosothiols metabolism
- Abstract
Accumulating evidence suggests that protein S-nitrosylation is enzymatically regulated and that specificity in S-nitrosylation derives from dedicated S-nitrosylases and denitrosylases that conjugate and remove S-nitrosothiols, respectively. Here, we report that mice deficient in the protein denitrosylase SCoR2 (S-nitroso-Coenzyme A Reductase 2; AKR1A1) exhibit marked reductions in serum cholesterol due to reduced secretion of the cholesterol-regulating protein PCSK9. SCoR2 associates with endoplasmic reticulum (ER) secretory machinery to control an S-nitrosylation cascade involving ER cargo-selection proteins SAR1 and SURF4, which moonlight as S-nitrosylases. SAR1 acts as a SURF4 nitrosylase and SURF4 as a PCSK9 nitrosylase to inhibit PCSK9 secretion, while SCoR2 counteracts nitrosylase activity by promoting PCSK9 denitrosylation. Inhibition of PCSK9 by an NO-based drug requires nitrosylase activity, and small-molecule inhibition of SCoR2 phenocopies the PCSK9-mediated reductions in cholesterol observed in SCoR2-deficient mice. Our results reveal enzymatic machinery controlling cholesterol levels through S-nitrosylation and suggest a distinct treatment paradigm for cardiovascular disease., Competing Interests: Declaration of interests J.S.S. and C.T.S. have patents that relate to discoveries herein. J.S.S. is a co-founder of SNO bio, which develops SNO-based technologies, and serves as a consultant and has an equity stake in NNOXX, a company that is developing NO-related technology., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
- Full Text
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5. Induction of membrane curvature by proteins involved in Golgi trafficking.
- Author
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Makowski SL, Kuna RS, and Field SJ
- Subjects
- Humans, Phosphatidylinositol Phosphates metabolism, Protein Transport physiology, Transport Vesicles, Golgi Apparatus pathology, Lipid Bilayers metabolism, Membrane Proteins metabolism
- Abstract
The Golgi apparatus serves a key role in processing and sorting lipids and proteins for delivery to their final cellular destinations. Vesicle exit from the Golgi initiates with directional deformation of the lipid bilayer to produce a bulge. Several mechanisms have been described by which lipids and proteins can induce directional membrane curvature to promote vesicle budding. Here we review some of the mechanisms implicated in inducing membrane curvature at the Golgi to promote vesicular trafficking to various cellular destinations., Competing Interests: Declaration of Competing interest We have no conflicts to report., (Copyright © 2019 Elsevier Ltd. All rights reserved.)
- Published
- 2020
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6. Efficient Golgi Forward Trafficking Requires GOLPH3-Driven, PI4P-Dependent Membrane Curvature.
- Author
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Rahajeng J, Kuna RS, Makowski SL, Tran TTT, Buschman MD, Li S, Cheng N, Ng MM, and Field SJ
- Subjects
- HEK293 Cells, HeLa Cells, Humans, Intracellular Membranes chemistry, Intracellular Membranes metabolism, Lipid Bilayers chemistry, Lipid Bilayers metabolism, Liposomes chemistry, Membrane Proteins chemistry, Myosins metabolism, Phosphatidylinositols chemistry, Protein Binding, Protein Domains, Golgi Apparatus metabolism, Liposomes metabolism, Membrane Proteins metabolism, Phosphatidylinositols metabolism, Secretory Pathway
- Abstract
Vesicle budding for Golgi-to-plasma membrane trafficking is a key step in secretion. Proteins that induce curvature of the Golgi membrane are predicted to be required, by analogy to vesicle budding from other membranes. Here, we demonstrate that GOLPH3, upon binding to the phosphoinositide PI4P, induces curvature of synthetic membranes in vitro and the Golgi in cells. Moreover, efficient Golgi-to-plasma membrane trafficking critically depends on the ability of GOLPH3 to curve the Golgi membrane. Interestingly, uncoupling of GOLPH3 from its binding partner MYO18A results in extensive curvature of Golgi membranes, producing dramatic tubulation of the Golgi, but does not support forward trafficking. Thus, forward trafficking from the Golgi to the plasma membrane requires the ability of GOLPH3 both to induce Golgi membrane curvature and to recruit MYO18A. These data provide fundamental insight into the mechanism of Golgi trafficking and into the function of the unique Golgi secretory oncoproteins GOLPH3 and MYO18A., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2019
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7. Treating cancer with phosphatidylinositol-3-kinase inhibitors: increasing efficacy and overcoming resistance.
- Author
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Paddock MN, Field SJ, and Cantley LC
- Subjects
- Animals, Humans, Neoplasms metabolism, Neoplasms pathology, Antineoplastic Agents pharmacology, Drug Resistance, Neoplasm drug effects, Neoplasms drug therapy, Phosphatidylinositol 3-Kinases metabolism, Protein Kinase Inhibitors pharmacology
- Abstract
The discovery of the phosphatidylinositol-3-kinase (PI3K) pathway was a major advance in understanding growth factor signaling. The high frequency of PI3K pathway mutations in many cancers has encouraged a new field targeting cancer driver mutations. Although there have been many successes, targeting PI3K itself has proven challenging, in part because of its multiple isoforms with distinct roles. Despite promising preclinical results, development of PI3K inhibitors as pharmacologic anticancer agents has been limited by modest single-agent efficacy and significant adverse effects. If we could overcome these limitations, PI3K inhibitors would be a powerful cancer-fighting tool. Data from phase III clinical trials yields insight into some of the problems with PI3K inhibitors. Recent advances have shed light on the mechanisms of tumor resistance to PI3K inhibitors via feedback pathways that cause elevated insulin levels that then activate the same PI3K pathways that are the targets of inhibition. Improving our understanding of the complex regulatory feedback pathways that activate in response to PI3K inhibition will reveal ways to increase the efficacy of PI3K inhibitors and reduce adverse effects, increasing the usefulness of this class as a treatment option for multiple cancer types., (Copyright © 2019 Paddock et al.)
- Published
- 2019
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8. Golgi Oncoprotein GOLPH3 Gene Expression Is Regulated by Functional E2F and CREB/ATF Promoter Elements.
- Author
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Peñalver-González B, Vallejo-Rodríguez J, Mentxaka G, Fullaondo A, Iglesias-Ara A, Field SJ, and Zubiaga AM
- Subjects
- Animals, Binding Sites, Cell Cycle, Cell Line, Tumor, Gene Expression Regulation, Golgi Apparatus genetics, Golgi Apparatus metabolism, Humans, Membrane Proteins metabolism, Mice, Mutation, NIH 3T3 Cells, Phosphoproteins genetics, Promoter Regions, Genetic, Activating Transcription Factor 2 metabolism, E2F Transcription Factors metabolism, Membrane Proteins chemistry, Membrane Proteins genetics
- Abstract
The Golgi organelle duplicates its protein and lipid content to segregate evenly between two daughter cells after mitosis. However, how Golgi biogenesis is regulated during interphase remains largely unknown. Here we show that messenger RNA (mRNA) expression of GOLPH3 and GOLGA2 , two genes encoding Golgi proteins, is induced specifically in G1 phase, suggesting a link between cell cycle regulation and Golgi growth. We have examined the role of E2F transcription factors, critical regulators of G1 to S progression of the cell cycle, in the expression of Golgi proteins during interphase. We show that promoter activity for GOLPH3 , a Golgi protein that is also oncogenic, is induced by E2F1-3 and repressed by E2F7. Mutation of the E2F motifs present in the GOLPH3 promoter region abrogates E2F1-mediated induction of a GOLPH3 luciferase reporter construct. Furthermore, we identify a critical CREB/ATF element in the GOLPH3 promoter that is required for its steady state and ATF2-induced expression. Interestingly, depletion of GOLPH3 with small interfering RNA (siRNA) delays the G1 to S transition in synchronized U2OS cells. Taken together, our results reveal a link between cell cycle regulation and Golgi function, and suggest that E2F-mediated regulation of Golgi genes is required for the timely progression of the cell cycle.
- Published
- 2019
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9. GOLPH3: a Golgi phosphatidylinositol(4)phosphate effector that directs vesicle trafficking and drives cancer.
- Author
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Kuna RS and Field SJ
- Subjects
- Animals, Biological Transport, Humans, Golgi Apparatus metabolism, Membrane Proteins metabolism, Neoplasms metabolism, Neoplasms pathology, Phosphatidylinositol Phosphates metabolism
- Abstract
GOLPH3 is a peripheral membrane protein localized to the Golgi and its vesicles, but its purpose had been unclear. We found that GOLPH3 binds specifically to the phosphoinositide phosphatidylinositol(4)phosphate [PtdIns(4)P], which functions at the Golgi to promote vesicle exit for trafficking to the plasma membrane. PtdIns(4)P is enriched at the trans -Golgi and so recruits GOLPH3. Here, a GOLPH3 complex is formed when it binds to myosin18A (MYO18A), which binds F-actin. This complex generates a pulling force to extract vesicles from the Golgi; interference with this GOLPH3 complex results in dramatically reduced vesicle trafficking. The GOLPH3 complex has been identified as a driver of cancer in humans, likely through multiple mechanisms that activate secretory trafficking. In this review, we summarize the literature that identifies the nature of the GOLPH3 complex and its role in cancer. We also consider the GOLPH3 complex as a hub with the potential to reveal regulation of the Golgi and suggest the possibility of GOLPH3 complex inhibition as a therapeutic approach in cancer., (Copyright © 2019 Kuna and Field.)
- Published
- 2019
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10. Neolymphostin A Is a Covalent Phosphoinositide 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Dual Inhibitor That Employs an Unusual Electrophilic Vinylogous Ester.
- Author
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Castro-Falcón G, Seiler GS, Demir Ö, Rathinaswamy MK, Hamelin D, Hoffmann RM, Makowski SL, Letzel AC, Field SJ, Burke JE, Amaro RE, and Hughes CC
- Subjects
- Enzyme Inhibitors metabolism, Molecular Docking Simulation, Phosphatidylinositol 3-Kinases chemistry, Phosphatidylinositol 3-Kinases metabolism, Protein Conformation, Quinolines metabolism, Enzyme Inhibitors chemistry, Enzyme Inhibitors pharmacology, Esters chemistry, Phosphoinositide-3 Kinase Inhibitors, Quinolines chemistry, Quinolines pharmacology, TOR Serine-Threonine Kinases antagonists & inhibitors
- Abstract
Using a novel chemistry-based assay for identifying electrophilic natural products in unprocessed extracts, we identified the PI3-kinase/mTOR dual inhibitor neolymphostin A from Salinispora arenicola CNY-486. The method further showed that the vinylogous ester substituent on the neolymphostin core was the exact site for enzyme conjugation. Tandem MS/MS experiments on PI3Kα treated with the inhibitor revealed that neolymphostin covalently modified Lys802 with a shift in mass of +306 amu, corresponding to addition of the inhibitor and elimination of methanol. The binding pose of the inhibitor bound to PI3Kα was modeled, and hydrogen-deuterium exchange mass spectrometry experiments supported this model. Against a panel of kinases, neolymphostin showed good selectivity for PI3-kinase and mTOR. In addition, the natural product blocked AKT phosphorylation in live cells with an IC
50 of ∼3 nM. Taken together, neolymphostin is the first reported example of a covalent kinase inhibitor from the bacterial domain of life.- Published
- 2018
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11. MYO18A: An unusual myosin.
- Author
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Buschman MD and Field SJ
- Subjects
- Adaptor Proteins, Signal Transducing genetics, Adaptor Proteins, Signal Transducing metabolism, Animals, Biological Transport, Active, Cell Cycle Proteins genetics, Cell Cycle Proteins metabolism, Cell Membrane genetics, Golgi Apparatus genetics, Humans, Membrane Proteins genetics, Membrane Proteins metabolism, Myosins genetics, Rho Guanine Nucleotide Exchange Factors genetics, Rho Guanine Nucleotide Exchange Factors metabolism, Tumor Suppressor Proteins genetics, Tumor Suppressor Proteins metabolism, p21-Activated Kinases genetics, p21-Activated Kinases metabolism, Cell Membrane metabolism, Golgi Apparatus metabolism, Myosins metabolism
- Abstract
MYO18A is a divergent member of the myosin family characterized by the presence of an amino-terminal PDZ domain. MYO18A has been found in a few different complexes involved in intracellular transport processes. MYO18A is found in a complex with LURAP1 and MRCK that functions in retrograde treadmilling of actin. It also has been found in a complex with PAK2, βPIX, and GIT1, functioning to transport that protein complex from focal adhesions to the leading edge. Finally, a high proportion of MYO18A is found in complex with GOLPH3 at the trans Golgi, where it functions to promote vesicle budding for Golgi-to-plasma membrane trafficking. Interestingly, MYO18A has been implicated as a cancer driver, as have other components of the GOLPH3 pathway. It remains uncertain as to whether or not MYO18A has intrinsic motor activity. While many questions remain, MYO18A is a fascinatingly unique myosin that is essential in higher organisms., (Copyright © 2017 Elsevier Ltd. All rights reserved.)
- Published
- 2018
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12. Spelunking for lipids in caveolae.
- Author
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Field SJ
- Subjects
- Animals, Carrier Proteins chemistry, Carrier Proteins metabolism, Caveolae chemistry, Caveolins chemistry, Caveolins metabolism, Cell Membrane chemistry, Humans, Intracellular Signaling Peptides and Proteins chemistry, Intracellular Signaling Peptides and Proteins metabolism, Muscle Proteins chemistry, Muscle Proteins metabolism, Phosphate-Binding Proteins, Phosphatidylinositol 4,5-Diphosphate chemistry, Phosphatidylserines chemistry, Protein Multimerization, RNA-Binding Proteins chemistry, RNA-Binding Proteins metabolism, Vesicular Transport Proteins, Caveolae metabolism, Cell Membrane metabolism, Phosphatidylinositol 4,5-Diphosphate metabolism, Phosphatidylserines metabolism
- Abstract
Phosphatidylserine (PtdSer) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P
2 ) have been implicated in the maintenance of caveolae, but direct evidence that these lipids are required for normal caveolar structure and dynamics in living cells has been lacking. A new study by Fairn and colleagues uses sophisticated tools to perturb specific lipids in living cells to assess the consequences for caveolae. This study demonstrates disparate roles for these lipids in the stability and mobility of caveolae and points the way for future work to understand how these lipids contribute to the biology of caveolae., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)- Published
- 2017
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13. Emerging themes of regulation at the Golgi.
- Author
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Makowski SL, Tran TT, and Field SJ
- Subjects
- Animals, Cell Proliferation, Cell Transformation, Neoplastic metabolism, Humans, Membrane Proteins metabolism, Protein Processing, Post-Translational, Golgi Apparatus metabolism, Lipid Metabolism, Protein Transport, Signal Transduction
- Abstract
The Golgi is generally recognized for its central role in the secretory pathway to orchestrate protein post-translational modification and trafficking of proteins and lipids to their final destination. Despite the common view of the Golgi as an inert sorting organelle, emerging data demonstrate that important signaling events occur at the Golgi, including those that regulate the trafficking function of the Golgi. The phosphatidylinositol-4-phosphate/GOLPH3/MYO18A/F-actin complex serves as a hub for signals that regulate Golgi trafficking function. Furthermore, the Golgi is increasingly appreciated for its important role in cell growth and in driving oncogenic transformation, as illuminated by the discovery that GOLPH3 and MYO18A are cancer drivers., (Copyright © 2017 Elsevier Ltd. All rights reserved.)
- Published
- 2017
- Full Text
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14. GOLPH3 drives cell migration by promoting Golgi reorientation and directional trafficking to the leading edge.
- Author
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Xing M, Peterman MC, Davis RL, Oegema K, Shiau AK, and Field SJ
- Subjects
- Actins metabolism, Amino Acid Sequence, Cell Culture Techniques, Cell Membrane metabolism, Cell Movement physiology, Cell Polarity, Cytoskeleton, HeLa Cells, Humans, Membrane Proteins physiology, Myosins metabolism, Phosphatidylinositol Phosphates metabolism, Protein Transport physiology, Signal Transduction, Golgi Apparatus metabolism, Membrane Proteins metabolism
- Abstract
The mechanism of directional cell migration remains an important problem, with relevance to cancer invasion and metastasis. GOLPH3 is a common oncogenic driver of human cancers, and is the first oncogene that functions at the Golgi in trafficking to the plasma membrane. Overexpression of GOLPH3 is reported to drive enhanced cell migration. Here we show that the phosphatidylinositol-4-phosphate/GOLPH3/myosin 18A/F-actin pathway that is critical for Golgi-to-plasma membrane trafficking is necessary and limiting for directional cell migration. By linking the Golgi to the actin cytoskeleton, GOLPH3 promotes reorientation of the Golgi toward the leading edge. GOLPH3 also promotes reorientation of lysosomes (but not other organelles) toward the leading edge. However, lysosome function is dispensable for migration and the GOLPH3 dependence of lysosome movement is indirect, via GOLPH3's effect on the Golgi. By driving reorientation of the Golgi to the leading edge and driving forward trafficking, particularly to the leading edge, overexpression of GOLPH3 drives trafficking to the leading edge of the cell, which is functionally important for directional cell migration. Our identification of a novel pathway for Golgi reorientation controlled by GOLPH3 provides new insight into the mechanism of directional cell migration with important implications for understanding GOLPH3's role in cancer., (© 2016 Xing, Peterman, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)
- Published
- 2016
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15. The GOLPH3 pathway regulates Golgi shape and function and is activated by DNA damage.
- Author
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Buschman MD, Xing M, and Field SJ
- Abstract
The Golgi protein GOLPH3 binds to PtdIns(4)P and MYO18A, linking the Golgi to the actin cytoskeleton. The GOLPH3 pathway is essential for vesicular trafficking from the Golgi to the plasma membrane. A side effect of GOLPH3-dependent trafficking is to generate the extended ribbon shape of the Golgi. Perturbation of the pathway results in changes to both Golgi morphology and secretion, with functional consequences for the cell. The cellular response to DNA damage provides an example of GOLPH3-mediated regulation of the Golgi. Upon DNA damage, DNA-PK phosphorylation of GOLPH3 increases binding to MYO18A, activating the GOLPH3 pathway, which consequently results in Golgi fragmentation, reduced trafficking, and enhanced cell survival. The PtdIns(4)P/GOLPH3/MYO18A/F-actin pathway provides new insight into the relationship between Golgi morphology and function, and their regulation.
- Published
- 2015
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16. Biallelic mutations in SNX14 cause a syndromic form of cerebellar atrophy and lysosome-autophagosome dysfunction.
- Author
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Akizu N, Cantagrel V, Zaki MS, Al-Gazali L, Wang X, Rosti RO, Dikoglu E, Gelot AB, Rosti B, Vaux KK, Scott EM, Silhavy JL, Schroth J, Copeland B, Schaffer AE, Gordts PL, Esko JD, Buschman MD, Field SJ, Napolitano G, Abdel-Salam GM, Ozgul RK, Sagıroglu MS, Azam M, Ismail S, Aglan M, Selim L, Mahmoud IG, Abdel-Hadi S, Badawy AE, Sadek AA, Mojahedi F, Kayserili H, Masri A, Bastaki L, Temtamy S, Müller U, Desguerre I, Casanova JL, Dursun A, Gunel M, Gabriel SB, de Lonlay P, and Gleeson JG
- Subjects
- Animals, Atrophy genetics, Autophagy, Child, Preschool, Female, Gene Frequency, Humans, Infant, Lod Score, Lysosomal Storage Diseases genetics, Male, Mutation, Syndrome, Zebrafish, Cerebellar Diseases genetics, Cerebellum pathology, Lysosomes metabolism, Phagosomes metabolism, Sorting Nexins genetics, Spinocerebellar Ataxias genetics
- Abstract
Pediatric-onset ataxias often present clinically as developmental delay and intellectual disability, with prominent cerebellar atrophy as a key neuroradiographic finding. Here we describe a new clinically distinguishable recessive syndrome in 12 families with cerebellar atrophy together with ataxia, coarsened facial features and intellectual disability, due to truncating mutations in the sorting nexin gene SNX14, encoding a ubiquitously expressed modular PX domain-containing sorting factor. We found SNX14 localized to lysosomes and associated with phosphatidylinositol (3,5)-bisphosphate, a key component of late endosomes/lysosomes. Patient-derived cells showed engorged lysosomes and a slower autophagosome clearance rate upon autophagy induction by starvation. Zebrafish morphants for snx14 showed dramatic loss of cerebellar parenchyma, accumulation of autophagosomes and activation of apoptosis. Our results characterize a unique ataxia syndrome due to biallelic SNX14 mutations leading to lysosome-autophagosome dysfunction.
- Published
- 2015
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17. Identification of furfural resistant strains of Saccharomyces cerevisiae and Saccharomyces paradoxus from a collection of environmental and industrial isolates.
- Author
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Field SJ, Ryden P, Wilson D, James SA, Roberts IN, Richardson DJ, Waldron KW, and Clarke TA
- Abstract
Background: Fermentation of bioethanol using lignocellulosic biomass as a raw material provides a sustainable alternative to current biofuel production methods by utilising waste food streams as raw material. Before lignocellulose can be fermented, it requires physical, chemical and enzymatic treatment in order to release monosaccharides, a process that causes the chemical transformation of glucose and xylose into the cyclic aldehydes furfural and hydroxyfurfural. These furan compounds are potent inhibitors of Saccharomyces fermentation, and consequently furfural tolerant strains of Saccharomyces are required for lignocellulosic fermentation., Results: This study investigated yeast tolerance to furfural and hydroxyfurfural using a collection of 71 environmental and industrial isolates of the baker's yeast Saccharomyces cerevisiae and its closest relative Saccharomyces paradoxus. The Saccharomyces strains were initially screened for growth on media containing 100 mM glucose and 1.5 mg ml(-1) furfural. Five strains were identified that showed a significant tolerance to growth in the presence of furfural, and these were then screened for growth and ethanol production in the presence of increasing amounts (0.1 to 4 mg ml(-1)) of furfural., Conclusions: Of the five furfural tolerant strains, S. cerevisiae National Collection of Yeast Cultures (NCYC) 3451 displayed the greatest furfural resistance and was able to grow in the presence of up to 3.0 mg ml(-1) furfural. Furthermore, ethanol production in this strain did not appear to be inhibited by furfural, with the highest ethanol yield observed at 3.0 mg ml(-1) furfural. Although furfural resistance was not found to be a trait specific to any one particular lineage or population, three of the strains were isolated from environments where they might be continually exposed to low levels of furfural through the ongoing natural degradation of lignocelluloses, and would therefore develop elevated levels of resistance to these furan compounds. Thus, these strains represent good candidates for future studies of genetic variation relevant to understanding and manipulating furfural resistance and in the development of tolerant ethanologenic yeast strains for use in bioethanol production from lignocellulose processing.
- Published
- 2015
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18. GOLPH3 links the Golgi, DNA damage, and cancer.
- Author
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Buschman MD, Rahajeng J, and Field SJ
- Subjects
- Cell Movement genetics, Golgi Apparatus genetics, Golgi Apparatus metabolism, Humans, Neoplasms pathology, Protein Transport genetics, Signal Transduction genetics, DNA Damage genetics, DNA-Activated Protein Kinase genetics, Membrane Proteins genetics, Neoplasms genetics
- Abstract
GOLPH3 is the first example of an oncogene that functions in secretory trafficking at the Golgi. The discovery of GOLPH3's roles in both cancer and Golgi trafficking raises questions about how GOLPH3 and the Golgi contribute to cancer. Our recent investigation of the regulation of GOLPH3 revealed a surprising response by the Golgi upon DNA damage that is mediated by DNA-PK and GOLPH3. These results provide new insight into the DNA damage response with important implications for understanding the cellular response to standard cancer therapeutic agents., (©2015 American Association for Cancer Research.)
- Published
- 2015
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19. DNA damage triggers Golgi dispersal via DNA-PK and GOLPH3.
- Author
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Farber-Katz SE, Dippold HC, Buschman MD, Peterman MC, Xing M, Noakes CJ, Tat J, Ng MM, Rahajeng J, Cowan DM, Fuchs GJ, Zhou H, and Field SJ
- Subjects
- Amino Acid Sequence, Animals, Cell Line, Cell Survival, Cells, Cultured, Humans, Membrane Proteins chemistry, Mice, Molecular Sequence Data, Phosphorylation, Rats, Sequence Alignment, DNA Damage, DNA-Activated Protein Kinase metabolism, Golgi Apparatus metabolism, Membrane Proteins metabolism, Myosins metabolism
- Abstract
The response to DNA damage, which regulates nuclear processes such as DNA repair, transcription, and cell cycle, has been studied thoroughly. However, the cytoplasmic response to DNA damage is poorly understood. Here, we demonstrate that DNA damage triggers dramatic reorganization of the Golgi, resulting in its dispersal throughout the cytoplasm. We further show that DNA-damage-induced Golgi dispersal requires GOLPH3/MYO18A/F-actin and the DNA damage protein kinase, DNA-PK. In response to DNA damage, DNA-PK phosphorylates GOLPH3, resulting in increased interaction with MYO18A, which applies a tensile force to the Golgi. Interference with the Golgi DNA damage response by depletion of DNA-PK, GOLPH3, or MYO18A reduces survival after DNA damage, whereas overexpression of GOLPH3, as is observed frequently in human cancers, confers resistance to killing by DNA-damaging agents. Identification of the DNA-damage-induced Golgi response reveals an unexpected pathway through DNA-PK, GOLPH3, and MYO18A that regulates cell survival following DNA damage., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
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20. GOLPH3L antagonizes GOLPH3 to determine Golgi morphology.
- Author
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Ng MM, Dippold HC, Buschman MD, Noakes CJ, and Field SJ
- Subjects
- 3T3 Cells, Amino Acid Sequence, Animals, Cell Line, Glycosyltransferases metabolism, Golgi Apparatus metabolism, HEK293 Cells, HeLa Cells, Humans, MCF-7 Cells, Membrane Proteins genetics, Mice, Myosins genetics, Protein Transport, RNA Interference, RNA, Small Interfering, Sequence Alignment, Signal Transduction, Golgi Apparatus ultrastructure, Membrane Proteins metabolism, Myosins metabolism, Phosphatidylinositol Phosphates metabolism, Phosphoproteins metabolism
- Abstract
GOLPH3 is a phosphatidylinositol-4-phosphate (PI4P) effector that plays an important role in maintaining Golgi architecture and anterograde trafficking. GOLPH3 does so through its ability to link trans-Golgi membranes to F-actin via its interaction with myosin 18A (MYO18A). GOLPH3 also is known to be an oncogene commonly amplified in human cancers. GOLPH3L is a GOLPH3 paralogue found in all vertebrate genomes, although previously it was largely uncharacterized. Here we demonstrate that although GOLPH3 is ubiquitously expressed in mammalian cells, GOLPH3L is present in only a subset of tissues and cell types, particularly secretory tissues. We show that, like GOLPH3, GOLPH3L binds to PI4P, localizes to the Golgi as a consequence of its PI4P binding, and is required for efficient anterograde trafficking. Surprisingly, however, we find that perturbations of GOLPH3L expression produce effects on Golgi morphology that are opposite to those of GOLPH3 and MYO18A. GOLPH3L differs critically from GOLPH3 in that it is largely unable to bind to MYO18A. Our data demonstrate that despite their similarities, unexpectedly, GOLPH3L antagonizes GOLPH3/MYO18A at the Golgi.
- Published
- 2013
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21. Regulation of Drosophila mesoderm migration by phosphoinositides and the PH domain of the Rho GTP exchange factor Pebble.
- Author
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Murray MJ, Ng MM, Fraval H, Tan J, Liu W, Smallhorn M, Brill JA, Field SJ, and Saint R
- Subjects
- Animals, Binding Sites, Cell Membrane metabolism, Cell Movement, Drosophila genetics, Drosophila metabolism, Guanosine Triphosphate metabolism, Phosphatidylinositol 4,5-Diphosphate metabolism, Phosphatidylinositols metabolism, Phosphorylation, Protein Structure, Tertiary, Signal Transduction, Drosophila Proteins genetics, Drosophila Proteins metabolism, Guanine Nucleotide Exchange Factors genetics, Guanine Nucleotide Exchange Factors metabolism, Mesoderm metabolism
- Abstract
The Drosophila RhoGEF Pebble (Pbl) is required for cytokinesis and migration of mesodermal cells. In a screen for genes that could suppress migration defects in pbl mutants we identified the phosphatidylinositol phosphate (PtdInsP) regulator pi5k59B. Genetic interaction tests with other PtdInsP regulators suggested that PtdIns(4,5)P2 levels are important for mesoderm migration when Pbl is depleted. Consistent with this, the leading front of migrating mesodermal cells was enriched for PtdIns(4,5)P2. Given that Pbl contains a Pleckstrin Homology (PH) domain, a known PtdInsP-binding motif, we examined PtdInsP-binding of Pbl and the importance of the PH domain for Pbl function. In vitro lipid blot assays showed that Pbl binds promiscuously to PtdInsPs, with binding strength associated with the degree of phosphorylation. Pbl was also able to bind lipid vesicles containing PtdIns(4,5)P2 but binding was strongly reduced upon deletion of the PH domain. Similarly, in vivo, loss of the PH domain prevented localisation of Pbl to the cell cortex and severely affected several aspects of early mesoderm development, including flattening of the invaginated tube onto the ectoderm, extension of protrusions, and dorsal migration to form a monolayer. Pbl lacking the PH domain could still localise to the cytokinetic furrow, however, and cytokinesis failure was reduced in pbl(ΔPH) mutants. Taken together, our results support a model in which interaction of the PH-domain of Pbl with PtdIns(4,5)P2 helps localise it to the plasma membrane which is important for mesoderm migration., (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Published
- 2012
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22. Role of phosphatidylinositol 4-phosphate (PI4P) and its binding protein GOLPH3 in hepatitis C virus secretion.
- Author
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Bishé B, Syed GH, Field SJ, and Siddiqui A
- Subjects
- Biological Transport, Active genetics, Cell Line, Tumor, Golgi Apparatus genetics, Golgi Apparatus virology, Hepacivirus genetics, Hepatitis C genetics, Humans, Intracellular Membranes metabolism, Intracellular Membranes virology, Membrane Proteins genetics, Myosins genetics, Myosins metabolism, Phosphatidylinositol Phosphates genetics, Virion genetics, Virion metabolism, Golgi Apparatus metabolism, Hepacivirus metabolism, Hepatitis C metabolism, Membrane Proteins metabolism, Phosphatidylinositol Phosphates metabolism, Virus Release physiology
- Abstract
Hepatitis C virus (HCV) RNA replicates within the ribonucleoprotein complex, assembled on the endoplasmic reticulum (ER)-derived membranous structures closely juxtaposed to the lipid droplets that facilitate the post-replicative events of virion assembly and maturation. It is widely believed that the assembled virions piggy-back onto the very low density lipoprotein particles for secretion. Lipid phosphoinositides are important modulators of intracellular trafficking. Golgi-localized phosphatidylinositol 4-phosphate (PI4P) recruits proteins involved in Golgi trafficking to the Golgi membrane and promotes anterograde transport of secretory proteins. Here, we sought to investigate the role of Golgi-localized PI4P in the HCV secretion process. Depletion of the Golgi-specific PI4P pool by Golgi-targeted PI4P phosphatase hSac1 K2A led to significant reduction in HCV secretion without any effect on replication. We then examined the functional role of a newly identified PI4P binding protein GOLPH3 in the viral secretion process. GOLPH3 is shown to maintain a tensile force on the Golgi, required for vesicle budding via its interaction with an unconventional myosin, MYO18A. Silencing GOLPH3 led to a dramatic reduction in HCV virion secretion, as did the silencing of MYO18A. The reduction in virion secretion was accompanied by a concomitant accumulation of intracellular virions, suggesting a stall in virion egress. HCV-infected cells displayed a fragmented and dispersed Golgi pattern, implicating involvement in virion morphogenesis. These studies establish the role of PI4P and its interacting protein GOLPH3 in HCV secretion and strengthen the significance of the Golgi secretory pathway in this process.
- Published
- 2012
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23. Resolving the contributions of the membrane-bound and periplasmic nitrate reductase systems to nitric oxide and nitrous oxide production in Salmonella enterica serovar Typhimurium.
- Author
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Rowley G, Hensen D, Felgate H, Arkenberg A, Appia-Ayme C, Prior K, Harrington C, Field SJ, Butt JN, Baggs E, and Richardson DJ
- Subjects
- Aerobiosis, Anaerobiosis, Cell Hypoxia, Gene Expression Regulation, Bacterial, Nitrates metabolism, Nitrites metabolism, Salmonella typhimurium metabolism, Cell Membrane enzymology, Nitrate Reductases metabolism, Nitrite Reductases metabolism, Nitrous Oxide metabolism, Periplasm enzymology, Salmonella typhimurium enzymology
- Abstract
The production of cytotoxic nitric oxide (NO) and conversion into the neuropharmacological agent and potent greenhouse gas nitrous oxide (N₂O) is linked with anoxic nitrate catabolism by Salmonella enterica serovar Typhimurium. Salmonella can synthesize two types of nitrate reductase: a membrane-bound form (Nar) and a periplasmic form (Nap). Nitrate catabolism was studied under nitrate-rich and nitrate-limited conditions in chemostat cultures following transition from oxic to anoxic conditions. Intracellular NO production was reported qualitatively by assessing transcription of the NO-regulated genes encoding flavohaemoglobin (Hmp), flavorubredoxin (NorV) and hybrid cluster protein (Hcp). A more quantitative analysis of the extent of NO formation was gained by measuring production of N₂O, the end-product of anoxic NO-detoxification. Under nitrate-rich conditions, the nar, nap, hmp, norV and hcp genes were all induced following transition from the oxic to anoxic state, and 20% of nitrate consumed in steady-state was released as N₂O when nitrite had accumulated to millimolar levels. The kinetics of nitrate consumption, nitrite accumulation and N₂O production were similar to those of wild-type in nitrate-sufficient cultures of a nap mutant. In contrast, in a narG mutant, the steady-state rate of N₂O production was ~30-fold lower than that of the wild-type. Under nitrate-limited conditions, nap, but not nar, was up-regulated following transition from oxic to anoxic metabolism and very little N₂O production was observed. Thus a combination of nitrate-sufficiency, nitrite accumulation and an active Nar-type nitrate reductase leads to NO and thence N₂O production, and this can account for up to 20% of the nitrate catabolized.
- Published
- 2012
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24. Receptor tyrosine kinases and TLR/IL1Rs unexpectedly activate myeloid cell PI3kγ, a single convergent point promoting tumor inflammation and progression.
- Author
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Schmid MC, Avraamides CJ, Dippold HC, Franco I, Foubert P, Ellies LG, Acevedo LM, Manglicmot JR, Song X, Wrasidlo W, Blair SL, Ginsberg MH, Cheresh DA, Hirsch E, Field SJ, and Varner JA
- Subjects
- Animals, Cell Adhesion, Cell Movement, Disease Progression, Humans, Integrin alpha4beta1 physiology, Mice, Mice, Inbred C57BL, Neoplasm Metastasis, Neoplasms prevention & control, ras Proteins physiology, Class Ib Phosphatidylinositol 3-Kinase physiology, Inflammation etiology, Neoplasms pathology, Receptor Protein-Tyrosine Kinases physiology, Receptors, Interleukin-1 physiology, Toll-Like Receptors physiology
- Abstract
Tumor inflammation promotes angiogenesis, immunosuppression, and tumor growth, but the mechanisms controlling inflammatory cell recruitment to tumors are not well understood. We found that a range of chemoattractants activating G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and Toll-like/IL-1 receptors (TLR/IL1Rs) unexpectedly initiate tumor inflammation by activating the PI3-kinase isoform p110γ in Gr1+CD11b+ myeloid cells. Whereas GPCRs activate p110γ in a Ras/p101-dependent manner, RTKs and TLR/IL1Rs directly activate p110γ in a Ras/p87-dependent manner. Once activated, p110γ promotes inside-out activation of a single integrin, α4β1, causing myeloid cell invasion into tumors. Pharmacological or genetic blockade of p110γ suppressed inflammation, growth, and metastasis of implanted and spontaneous tumors, revealing an important therapeutic target in oncology., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
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25. Electron transfer to the active site of the bacterial nitric oxide reductase is controlled by ligand binding to heme b₃.
- Author
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Field SJ, Roldan MD, Marritt SJ, Butt JN, Richardson DJ, and Watmough NJ
- Subjects
- Electron Transport, Hydrogen-Ion Concentration, Ligands, Oxidation-Reduction, Catalytic Domain, Heme chemistry, Heme metabolism, Oxidoreductases chemistry, Oxidoreductases metabolism, Paracoccus denitrificans enzymology
- Abstract
The active site of the bacterial nitric oxide reductase from Paracoccus denitrificans contains a dinuclear centre comprising heme b₃ and non heme iron (Fe(B)). These metal centres are shown to be at isopotential with midpoint reduction potentials of E(m) ≈ +80 mV. The midpoint reduction potentials of the other two metal centres in the enzyme, heme c and heme b, are greater than the dinuclear centre suggesting that they act as an electron receiving/storage module. Reduction of the low-spin heme b causes structural changes at the dinuclear centre which allow access to substrate molecules. In the presence of the substrate analogue, CO, the midpoint reduction potential of heme b₃ is raised to a region similar to that of heme c and heme b. This leads us to suggest that reduction of the electron transfer hemes leads to an opening of the active site which allows substrate to bind and in turn raises the reduction potential of the active site such that electrons are only delivered to the active site following substrate binding., (Copyright © 2011 Elsevier B.V. All rights reserved.)
- Published
- 2011
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26. Novel inducers of the envelope stress response BaeSR in Salmonella Typhimurium: BaeR is critically required for tungstate waste disposal.
- Author
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Appia-Ayme C, Patrick E, Sullivan MJ, Alston MJ, Field SJ, AbuOun M, Anjum MF, and Rowley G
- Subjects
- Animals, Bacterial Outer Membrane Proteins, Gene Expression Regulation, Bacterial, Mice, Salmonella typhimurium physiology, Stress, Physiological, Drug Resistance genetics, Multidrug Resistance-Associated Proteins physiology, Protein Kinases physiology, Salmonella typhimurium genetics, Tungsten Compounds metabolism
- Abstract
The RpoE and CpxR regulated envelope stress responses are extremely important for Salmonella Typhimurium to cause infection in a range of hosts. Until now the role for BaeSR in both the Salmonella Typhimurium response to stress and its contribution to infection have not been fully elucidated. Here we demonstrate stationary phase growth, iron and sodium tungstate as novel inducers of the BaeRregulon, with BaeR critically required for Salmonella resistance to sodium tungstate. We show that functional overlap between the resistance nodulation-cell division (RND) multidrug transporters, MdtA, AcrD and AcrB exists for the waste disposal of tungstate from the cell. We also point to a role for enterobactinsiderophores in the protection of enteric organisms from tungstate, akin to the scenario in nitrogen fixing bacteria. Surprisingly, BaeR is the first envelope stress response pathway investigated in S. Typhimurium that is not required for murine typhoid in either ity(S) or ity(R) mouse backgrounds. BaeR is therefore either required for survival in larger mammals such as pigs or calves, an avian host such as chickens, or survival out with the host altogether where Salmonella and related enterics must survive in soil and water.
- Published
- 2011
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27. Accelerated DNA replication in E2F1- and E2F2-deficient macrophages leads to induction of the DNA damage response and p21(CIP1)-dependent senescence.
- Author
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Iglesias-Ara A, Zenarruzabeitia O, Fernandez-Rueda J, Sánchez-Tilló E, Field SJ, Celada A, and Zubiaga AM
- Subjects
- Animals, Bone Marrow Cells cytology, Bone Marrow Cells metabolism, Cell Cycle, Cell Differentiation, Cell Proliferation, Cells, Cultured, Cellular Senescence, Cyclin-Dependent Kinase Inhibitor p21 genetics, E2F1 Transcription Factor genetics, E2F2 Transcription Factor genetics, Flow Cytometry, Immunoblotting, Macrophages cytology, Mice, Mice, Knockout, Time Factors, Cyclin-Dependent Kinase Inhibitor p21 metabolism, DNA Damage, DNA Replication, E2F1 Transcription Factor metabolism, E2F2 Transcription Factor metabolism, Macrophages metabolism
- Abstract
E2F1-3 proteins appear to have distinct roles in progenitor cells and in differentiating cells undergoing cell cycle exit. However, the function of these proteins in paradigms of terminal differentiation that involve continued cell division has not been examined. Using compound E2F1/E2F2-deficient mice, we have examined the effects of E2F1 and E2F2 loss on the differentiation and simultaneous proliferation of bone-marrow-derived cells toward the macrophage lineage. We show that E2F1/E2F2 deficiency results in accelerated DNA replication and cellular division during the initial cell division cycles of bone-marrow-derived cells, arguing that E2F1/E2F2 are required to restrain proliferation of pro-monocyte progenitors during their differentiation into macrophages, without promoting their cell cycle exit. Accelerated proliferation is accompanied by early expression of DNA replication and cell cycle regulators. Remarkably, rapid proliferation of E2F1/E2F2 compound mutant cultures is temporally followed by induction of a DNA damage response and the implementation of a p21(CIP1)-dependent senescence. We further show that differentiating E2F1/E2F2-knockout macrophages do not trigger a DNA damage response pathway in the absence of DNA replication. These findings underscore the relevance of E2F1 and E2F2 as suppressors of hematopoietic progenitor expansion. Our data indicate that their absence in differentiating macrophages initiates a senescence program that results from enforcement of a DNA damage response triggered by DNA hyper-replication.
- Published
- 2010
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28. Regulation of oxysterol-binding protein Golgi localization through protein kinase D-mediated phosphorylation.
- Author
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Nhek S, Ngo M, Yang X, Ng MM, Field SJ, Asara JM, Ridgway ND, and Toker A
- Subjects
- Amino Acid Sequence, Animals, Cell Line, Humans, Molecular Sequence Data, Phosphorylation, Protein Kinase C genetics, Protein Serine-Threonine Kinases genetics, Protein Serine-Threonine Kinases metabolism, Receptors, Steroid genetics, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Sequence Alignment, Serine metabolism, Golgi Apparatus metabolism, Protein Kinase C metabolism, Receptors, Steroid metabolism
- Abstract
Protein kinase D (PKD) plays a critical role at the trans-Golgi network by regulating the fission of transport carriers destined for the plasma membrane. Two known Golgi-localized PKD substrates, PI4-kinase IIIbeta and the ceramide transfer protein CERT, mediate PKD signaling to influence vesicle trafficking to the plasma membrane and sphingomyelin synthesis, respectively. PKD is recruited and activated at the Golgi through interaction with diacylglycerol, a pool of which is generated as a by-product of sphingomyelin synthesis from ceramide. Here we identify a novel substrate of PKD at the Golgi, the oxysterol-binding protein OSBP. Using a substrate-directed phospho-specific antibody that recognizes the optimal PKD consensus motif, we show that PKD phosphorylates OSBP at Ser240 in vitro and in cells. We further show that OSBP phosphorylation occurs at the Golgi. Phosphorylation of OSBP by PKD does not modulate dimerization, sterol binding, or affinity for PI(4)P. Instead, phosphorylation attenuates OSBP Golgi localization in response to 25-hydroxycholesterol and cholesterol depletion, impairs CERT Golgi localization, and promotes Golgi fragmentation.
- Published
- 2010
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29. GOLPH3 bridges phosphatidylinositol-4- phosphate and actomyosin to stretch and shape the Golgi to promote budding.
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Dippold HC, Ng MM, Farber-Katz SE, Lee SK, Kerr ML, Peterman MC, Sim R, Wiharto PA, Galbraith KA, Madhavarapu S, Fuchs GJ, Meerloo T, Farquhar MG, Zhou H, and Field SJ
- Subjects
- Actins metabolism, Animals, Gene Knockdown Techniques, Golgi Apparatus chemistry, HeLa Cells, Humans, Hydrophobic and Hydrophilic Interactions, Membrane Proteins analysis, Membrane Proteins genetics, Myosins metabolism, Phosphatidylinositol Phosphates metabolism, Transport Vesicles metabolism, Golgi Apparatus metabolism, Membrane Proteins metabolism
- Abstract
Golgi membranes, from yeast to humans, are uniquely enriched in phosphatidylinositol-4-phosphate (PtdIns(4)P), although the role of this lipid remains poorly understood. Using a proteomic lipid-binding screen, we identify the Golgi protein GOLPH3 (also called GPP34, GMx33, MIDAS, or yeast Vps74p) as a PtdIns(4)P-binding protein that depends on PtdIns(4)P for its Golgi localization. We further show that GOLPH3 binds the unconventional myosin MYO18A, thus connecting the Golgi to F-actin. We demonstrate that this linkage is necessary for normal Golgi trafficking and morphology. The evidence suggests that GOLPH3 binds to PtdIns(4)P-rich trans-Golgi membranes and MYO18A conveying a tensile force required for efficient tubule and vesicle formation. Consequently, this tensile force stretches the Golgi into the extended ribbon observed by fluorescence microscopy and the familiar flattened form observed by electron microscopy.
- Published
- 2009
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30. Mutations in INPP5E, encoding inositol polyphosphate-5-phosphatase E, link phosphatidyl inositol signaling to the ciliopathies.
- Author
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Bielas SL, Silhavy JL, Brancati F, Kisseleva MV, Al-Gazali L, Sztriha L, Bayoumi RA, Zaki MS, Abdel-Aleem A, Rosti RO, Kayserili H, Swistun D, Scott LC, Bertini E, Boltshauser E, Fazzi E, Travaglini L, Field SJ, Gayral S, Jacoby M, Schurmans S, Dallapiccola B, Majerus PW, Valente EM, and Gleeson JG
- Subjects
- Acetylation, Amino Acid Substitution, Animals, Base Sequence, Brain diagnostic imaging, Case-Control Studies, Catalytic Domain, Cell Line, Chromosomes, Human, Pair 9, Cilia enzymology, Consanguinity, Culture Media, Serum-Free, Fibroblasts metabolism, Fibroblasts ultrastructure, Genetic Linkage, Green Fluorescent Proteins metabolism, Haplotypes, Homozygote, Humans, Hydrolysis, Mice, Mice, Transgenic, Molecular Sequence Data, Mutation, Missense, Phosphatidylinositol 4,5-Diphosphate genetics, Phosphatidylinositol Phosphates genetics, Phosphoric Monoester Hydrolases chemistry, Phosphoric Monoester Hydrolases metabolism, Physical Chromosome Mapping, Pigment Epithelium of Eye cytology, Polymorphism, Single Nucleotide, Protein Structure, Tertiary, Radiography, Serum metabolism, Tubulin metabolism, Cilia pathology, Mutation, Phosphatidylinositols genetics, Phosphoric Monoester Hydrolases genetics, Signal Transduction genetics
- Abstract
Phosphotidylinositol (PtdIns) signaling is tightly regulated both spatially and temporally by subcellularly localized PtdIns kinases and phosphatases that dynamically alter downstream signaling events. Joubert syndrome is characterized by a specific midbrain-hindbrain malformation ('molar tooth sign'), variably associated retinal dystrophy, nephronophthisis, liver fibrosis and polydactyly and is included in the newly emerging group of 'ciliopathies'. In individuals with Joubert disease genetically linked to JBTS1, we identified mutations in the INPP5E gene, encoding inositol polyphosphate-5-phosphatase E, which hydrolyzes the 5-phosphate of PtdIns(3,4,5)P3 and PtdIns(4,5)P2. Mutations clustered in the phosphatase domain and impaired 5-phosphatase activity, resulting in altered cellular PtdIns ratios. INPP5E localized to cilia in major organs affected by Joubert syndrome, and mutations promoted premature destabilization of cilia in response to stimulation. These data link PtdIns signaling to the primary cilium, a cellular structure that is becoming increasingly recognized for its role in mediating cell signals and neuronal function.
- Published
- 2009
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31. The bacterial respiratory nitric oxide reductase.
- Author
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Watmough NJ, Field SJ, Hughes RJ, and Richardson DJ
- Subjects
- Catalytic Domain, Electron Transport, Nitric Oxide metabolism, Oxidation-Reduction, Protons, Oxidoreductases metabolism, Paracoccus denitrificans enzymology
- Abstract
The two-subunit cytochrome bc complex (NorBC) isolated from membranes of the model denitrifying soil bacterium Paracoccus denitrificans is the best-characterized example of the bacterial respiratory nitric oxide reductases. These are members of the super-family of haem-copper oxidases and are characterized by the elemental composition of their active site, which contains non-haem iron rather than copper, at which the reductive coupling of two molecules of nitric oxide to form nitrous oxide is catalysed. The reaction requires the presence of two substrate molecules at the active site along with the controlled input of two electrons and two protons from the same side of the membrane. In the present paper, we consider progress towards understanding the pathways of electron and proton transfer in NOR and how this information can be integrated with evidence for the likely modes of substrate binding at the active site to propose a revised and experimentally testable reaction mechanism.
- Published
- 2009
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32. E2F2 represses cell cycle regulators to maintain quiescence.
- Author
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Infante A, Laresgoiti U, Fernández-Rueda J, Fullaondo A, Galán J, Díaz-Uriarte R, Malumbres M, Field SJ, and Zubiaga AM
- Subjects
- Animals, Cells, Cultured, E2F1 Transcription Factor metabolism, E2F3 Transcription Factor metabolism, G1 Phase, Gene Knockdown Techniques, Mice, Mice, Inbred C57BL, Mice, Knockout, RNA Interference, Resting Phase, Cell Cycle, S Phase, T-Lymphocytes cytology, Cell Cycle Proteins metabolism, E2F2 Transcription Factor metabolism
- Abstract
E2F transcription factors control diverse biological processes through regulation of target gene expression. However, the mechanism by which this regulation is established, and the relative contribution of each E2F member are still poorly defined. We have investigated the role of E2F2 in regulating cellular proliferation. We show that E2F2 is required for the normal G(0)/G(1) phase because targeted disruption of the E2F2 gene causes T cells to enter S phase early and to undergo accelerated cell division. A large set of E2F target genes involved in DNA replication and cell cycle progression (such as Mcm's, cyclins and Cdc2a) that are silent in G(0) and typically transcribed late in G(1) phase are already actively expressed in quiescent T cells and MEFs lacking E2F2. The classic E2F activators, E2F1 and E2F3, are largely dispensable for this process because compound loss of E2F1(-/-) and E2F2(-/-) produces a comparably shortened G(0)/G(1) phase, with early S phase entry. Likewise, shRNA knockdown of E2F3 does not alter significantly the E2F2(-/-) phenotype. Chromatin immunoprecipitation analysis indicates that in wild-type cells the promoters of the aberrantly early-transcribed genes are occupied by E2F2 in G(0), suggesting a direct role for E2F2 in transcriptional repression. We conclude that E2F2 functions to transcriptionally repress cell cycle genes to establish the G(0) state.
- Published
- 2008
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33. Ultrafast ligand binding dynamics in the active site of native bacterial nitric oxide reductase.
- Author
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Kapetanaki SM, Field SJ, Hughes RJ, Watmough NJ, Liebl U, and Vos MH
- Subjects
- Binding Sites, Carbon Monoxide metabolism, Electron Spin Resonance Spectroscopy, Ligands, Nitric Oxide metabolism, Oxidation-Reduction, Protein Binding, Spectrophotometry, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Oxidoreductases chemistry, Oxidoreductases metabolism, Paracoccus denitrificans enzymology
- Abstract
The active site of nitric oxide reductase from Paracoccus denitrificans contains heme and non-heme iron and is evolutionarily related to heme-copper oxidases. The CO and NO dynamics in the active site were investigated using ultrafast transient absorption spectroscopy. We find that, upon photodissociation from the active site heme, 20% of the CO rebinds in 170 ps, suggesting that not all the CO transiently binds to the non-heme iron. The remaining 80% does not rebind within 4 ns and likely migrates out of the active site without transient binding to the non-heme iron. Rebinding of NO to ferrous heme takes place in approximately 13 ps. Our results reveal that heme-ligand recombination in this enzyme is considerably faster than in heme-copper oxidases and are consistent with a more confined configuration of the active site.
- Published
- 2008
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34. Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance.
- Author
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Yang X, Ongusaha PP, Miles PD, Havstad JC, Zhang F, So WV, Kudlow JE, Michell RH, Olefsky JM, Field SJ, and Evans RM
- Subjects
- Acetylglucosamine metabolism, Acetylglucosamine pharmacology, Animals, COS Cells, Cell Membrane metabolism, Chlorocebus aethiops, Insulin pharmacology, Lipid Metabolism, Liver enzymology, Liver metabolism, Male, Mice, Mice, Inbred C57BL, N-Acetylglucosaminyltransferases chemistry, N-Acetylglucosaminyltransferases genetics, Phosphatidylinositol Phosphates metabolism, Phosphorylation drug effects, Protein Structure, Tertiary, Protein Transport, Insulin Resistance physiology, N-Acetylglucosaminyltransferases metabolism, Phosphatidylinositols metabolism, Second Messenger Systems drug effects
- Abstract
Glucose flux through the hexosamine biosynthetic pathway leads to the post-translational modification of cytoplasmic and nuclear proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc). This tandem system serves as a nutrient sensor to couple systemic metabolic status to cellular regulation of signal transduction, transcription, and protein degradation. Here we show that O-GlcNAc transferase (OGT) harbours a previously unrecognized type of phosphoinositide-binding domain. After induction with insulin, phosphatidylinositol 3,4,5-trisphosphate recruits OGT from the nucleus to the plasma membrane, where the enzyme catalyses dynamic modification of the insulin signalling pathway by O-GlcNAc. This results in the alteration in phosphorylation of key signalling molecules and the attenuation of insulin signal transduction. Hepatic overexpression of OGT impairs the expression of insulin-responsive genes and causes insulin resistance and dyslipidaemia. These findings identify a molecular mechanism by which nutritional cues regulate insulin signalling through O-GlcNAc, and underscore the contribution of this modification to the aetiology of insulin resistance and type 2 diabetes.
- Published
- 2008
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35. The respiratory nitric oxide reductase (NorBC) from Paracoccus denitrificans.
- Author
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Field SJ, Thorndycroft FH, Matorin AD, Richardson DJ, and Watmough NJ
- Subjects
- Azurin metabolism, Biosensing Techniques, Electron Spin Resonance Spectroscopy, Electrons, Nitrate Reductase chemistry, Nitrate Reductase isolation & purification, Nitrate Reductase metabolism, Nitric Oxide metabolism, Nitric Oxide Donors metabolism, Oxidation-Reduction, Recombinant Proteins isolation & purification, Spectrum Analysis methods, Nitrate Reductase physiology, Paracoccus denitrificans enzymology
- Abstract
The two subunit cytochrome bc complex (NorBC) isolated from membranes of the model denitrifying soil bacterium Paracoccus denitrificans is the best characterized example of the bacterial respiratory nitric oxide reductases. These are members of the superfamily of heme-copper oxidases and are characterized by the elemental composition of their active site, which contains nonheme iron rather than copper, at which the reductive coupling of two molecules of nitric oxide to form nitrous oxide is catalyzed. This chapter describes methods for the purification and characterization of both native nitric oxide reductase from P. denitrificans and a recombinant form of the enzyme expressed in Escherichia coli, which enables site-directed mutagenesis of the catalytic subunit NorB. Examples are given of electronic absorption and electron paramagnetic resonance spectra that characterize the enzyme in a number of redox states, along with a method for the routine assay of the complex using its natural electron donor pseudoazurin.
- Published
- 2008
- Full Text
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36. Characterization of Shewanella oneidensis MtrC: a cell-surface decaheme cytochrome involved in respiratory electron transport to extracellular electron acceptors.
- Author
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Hartshorne RS, Jepson BN, Clarke TA, Field SJ, Fredrickson J, Zachara J, Shi L, Butt JN, and Richardson DJ
- Subjects
- Cytochrome c Group isolation & purification, Electron Spin Resonance Spectroscopy, Electron Transport, Potentiometry, Respiration, Bacterial Outer Membrane Proteins chemistry, Cytochrome c Group chemistry, Cytochromes chemistry, Heme chemistry, Shewanella chemistry
- Abstract
MtrC is a decaheme c-type cytochrome associated with the outer cell membrane of Fe(III)-respiring species of the Shewanella genus. It is proposed to play a role in anaerobic respiration by mediating electron transfer to extracellular mineral oxides that can serve as terminal electron acceptors. The present work presents the first spectropotentiometric and voltammetric characterization of MtrC, using protein purified from Shewanella oneidensis MR-1. Potentiometric titrations, monitored by UV-vis absorption and electron paramagnetic resonance (EPR) spectroscopy, reveal that the hemes within MtrC titrate over a broad potential range spanning between approximately +100 and approximately -500 mV (vs. the standard hydrogen electrode). Across this potential window the UV-vis absorption spectra are characteristic of low-spin c-type hemes and the EPR spectra reveal broad, complex features that suggest the presence of magnetically spin-coupled low-spin c-hemes. Non-catalytic protein film voltammetry of MtrC demonstrates reversible electrochemistry over a potential window similar to that disclosed spectroscopically. The voltammetry also allows definition of kinetic properties of MtrC in direct electron exchange with a solid electrode surface and during reduction of a model Fe(III) substrate. Taken together, the data provide quantitative information on the potential domain in which MtrC can operate.
- Published
- 2007
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37. Deletion of the phosphoinositide 3-kinase p110gamma gene attenuates murine atherosclerosis.
- Author
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Chang JD, Sukhova GK, Libby P, Schvartz E, Lichtenstein AH, Field SJ, Kennedy C, Madhavarapu S, Luo J, Wu D, and Cantley LC
- Subjects
- Animals, Apolipoproteins E physiology, Gene Deletion, Lipoproteins, LDL pharmacology, Mice, Mice, Inbred C57BL, Oxidative Stress, Phosphatidylinositol 3-Kinases genetics, Phosphoinositide-3 Kinase Inhibitors, Proto-Oncogene Proteins c-akt physiology, Atherosclerosis prevention & control, Phosphatidylinositol 3-Kinases physiology
- Abstract
Inflammatory cell activation by chemokines requires intracellular signaling through phosphoinositide 3-kinase (PI3-kinase) and the PI3-kinase-dependent protein serine/threonine kinase Akt. Atherosclerosis is a chronic inflammatory process driven by oxidatively modified (atherogenic) lipoproteins, chemokines, and other agonists that activate PI3-kinase. Here we show that macrophage PI3-kinase/Akt is activated by oxidized low-density lipoprotein, inflammatory chemokines, and angiotensin II. This activation is markedly reduced or absent in macrophages lacking p110gamma, the catalytic subunit of class Ib PI3-kinase. We further demonstrate activation of macrophage/foam cell PI3-kinase/Akt in atherosclerotic plaques from apolipoprotein E (apoE)-null mice, which manifest an aggressive form of atherosclerosis, whereas activation of PI3-kinase/Akt was undetectable in lesions from apoE-null mice lacking p110gamma despite the presence of class Ia PI3-kinase. Moreover, plaques were significantly smaller in apoE-/-p110gamma-/- mice than in apoE-/-p110gamma+/+ or apoE-/-p110gamma+/-mice at all ages studied. In marked contrast to the embryonic lethality seen in mice lacking class Ia PI3-kinase, germ-line deletion of p110gamma results in mice that exhibit normal viability, longevity, and fertility, with relatively well tolerated defects in innate immune and inflammatory responses that may play a role in diseases such as atherosclerosis and multiple sclerosis. Our results not only shed mechanistic light on inflammatory signaling during atherogenesis, but further identify p110gamma as a possible target for pharmacological intervention in the primary and secondary prevention of human atherosclerotic cardiovascular disease.
- Published
- 2007
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38. Reductive activation of nitrate reductases.
- Author
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Field SJ, Thornton NP, Anderson LJ, Gates AJ, Reilly A, Jepson BJ, Richardson DJ, George SJ, Cheesman MR, and Butt JN
- Subjects
- Binding Sites, Catalysis, Electron Spin Resonance Spectroscopy, Enzyme Activation, Oxidation-Reduction, Paracoccus pantotrophus enzymology, Spectroscopy, Fourier Transform Infrared, Synechococcus enzymology, Nitrate Reductases chemistry, Nitrate Reductases metabolism
- Abstract
Protein film voltammetry of Paracoccus pantotrophus respiratory nitrate reductase (NarGH) and Synechococcus elongatus assimilatory nitrate reductase (NarB) shows that reductive activation of these enzymes may be required before steady state catalysis is observed. For NarGH complementary spectroscopic studies suggest a structural context for the activation. Catalytic protein film voltammetry at a range of temperatures has allowed quantitation of the activation energies for nitrate reduction. For NarGH with an operating potential of ca. 0.05 V the activation energy of ca. 35 kJ mol-1 is over twice that measured for NarB whose operating potential is ca. -0.35 V.
- Published
- 2005
- Full Text
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39. PtdIns(4,5)P2 functions at the cleavage furrow during cytokinesis.
- Author
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Field SJ, Madson N, Kerr ML, Galbraith KA, Kennedy CE, Tahiliani M, Wilkins A, and Cantley LC
- Subjects
- Actins metabolism, Adaptor Proteins, Signal Transducing, Animals, CHO Cells, Cell Membrane metabolism, Cricetinae, Cricetulus, Genetic Vectors, Green Fluorescent Proteins metabolism, HeLa Cells, Humans, Mice, NIH 3T3 Cells, Phosphatidylinositol 4,5-Diphosphate, Phospholipase C gamma metabolism, Proteins metabolism, Cytokinesis physiology, Phosphatidylinositol Phosphates metabolism
- Abstract
Phosphoinositides play important roles in regulating the cytoskeleton and vesicle trafficking, potentially important processes at the cleavage furrow. However, it remains unclear which, if any, of the phosphoinositides play a role during cytokinesis. A systematic analysis to determine if any of the phosphoinositides might be present or of functional importance at the cleavage furrow has not been published. Several studies hint at a possible role for one or more phosphoinositides at the cleavage furrow. The best of these are genetic data identifying mutations in phosphoinositide-modifying enzymes (a PtdIns(4)P-5-kinase in S. pombe and a PI-4-kinase in D. melanogaster) that interfere with cytokinesis. The genetic nature of these experiments leaves questions as to how direct may be their contribution to cytokinesis. Here we show that a single phosphoinositide, PtdIns(4,5)P2, specifically accumulates at the furrow. Interference with PtdIns(4,5)P2 interferes with adhesion of the plasma membrane to the contractile ring at the furrow. Finally, four distinct interventions to specifically interfere with PtdIns(4,5)P2 each impair cytokinesis. We conclude that PtdIns(4,5)P2 is present at the cleavage furrow and is required for normal cytokinesis at least in part because of a role in adhesion between the contractile ring and the plasma membrane.
- Published
- 2005
- Full Text
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40. The p85 regulatory subunit of phosphoinositide 3-kinase down-regulates IRS-1 signaling via the formation of a sequestration complex.
- Author
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Luo J, Field SJ, Lee JY, Engelman JA, and Cantley LC
- Subjects
- Animals, CHO Cells, Cricetinae, Cricetulus, Cytosol metabolism, Dimerization, Down-Regulation, Green Fluorescent Proteins genetics, Humans, Insulin Receptor Substrate Proteins, Mice, Phosphatidylinositol 3-Kinases genetics, Phosphatidylinositol 3-Kinases metabolism, Phosphoproteins metabolism, Phosphorylation, Protein Binding, Protein Subunits metabolism, Protein Transport, Receptor, IGF Type 1 agonists, Recombinant Fusion Proteins metabolism, Signal Transduction, Tyrosine metabolism, Phosphatidylinositol 3-Kinases physiology, Phosphoproteins physiology
- Abstract
Phosphoinositide (PI) 3-kinase is required for most insulin and insulin-like growth factor (IGF) 1-dependent cellular responses. The p85 regulatory subunit of PI 3-kinase is required to mediate the insulin-dependent recruitment of PI 3-kinase to the plasma membrane, yet mice with reduced p85 expression have increased insulin sensitivity. To further understand the role of p85, we examined IGF-1-dependent translocation of p85alpha by using a green fluorescence protein (GFP)-tagged p85alpha (EGFP-p85alpha). In response to IGF-1, but not to PDGF signaling, EGFP-p85alpha translocates to discrete foci in the cell. These foci contain the insulin receptor substrate (IRS) 1 adaptor molecule, and their formation requires the binding of p85 to IRS-1. Surprisingly, monomeric p85 is preferentially localized to these foci compared with the p85-p110 dimer, and these foci are not sites of phosphatidylinositol-3,4,5-trisphosphate production. Ultrastructural analysis reveals that p85-IRS-1 foci are cytosolic protein complexes devoid of membrane. These results suggest a mechanism of signal down-regulation of IRS-1 that is mediated by monomeric p85 through the formation of a sequestration complex between p85 and IRS-1.
- Published
- 2005
- Full Text
- View/download PDF
41. Sexual health care training needs of general practitioner trainers: a regional survey.
- Author
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Markham WA, Bullock AD, Matthews P, Firmstone VR, Kelly S, and Field SJ
- Subjects
- Clinical Competence, Female, Humans, Male, Mass Screening statistics & numerical data, Medical History Taking statistics & numerical data, Sexually Transmitted Diseases diagnosis, Sexually Transmitted Diseases prevention & control, Surveys and Questionnaires, United Kingdom, Family Planning Services, Family Practice education, Practice Patterns, Physicians' statistics & numerical data
- Abstract
Objectives: The National Strategy for Sexual Health and HIV aims to facilitate improved patient access to sexual health care, primarily in general practice. This study aimed to identify sexual health care provision in general practitioner (GP) training practices and highlight training and resource implications of the strategy for GPs and prospective GPs., Methods: Data were gathered from interviews with five key representatives (all of whom had a special interest in GP training and/or sexual health care) and a self-completed questionnaire survey of all 374 GP trainers in the West Midlands region. The questionnaire was developed from the interviews and comprised three sections: sample characteristics; current practice; and 30 statements to elicit attitudes, knowledge and training implications. The questionnaire was mailed out in March 2002 with two re-mailings at 2-week intervals., Results: Most GP trainers (79%; n=295) returned completed questionnaires. Most respondents were already offering some 'Level 1 services' or were prepared to including cervical screening (100%; n=295), sexual history taking (95%; n=271), sexually transmitted infection (STI) testing (74%; n=217), HIV testing (68%; n=198) and contraceptive services (71%; n=208). However, most (86%; n=251) needed further information on the Strategy detail and its implications. Training needs in sexual history taking, STI testing and HIV testing were also highlighted. Most GP trainers (62%; n=181) believed GP registrars were relatively unprepared for sexual health care and proposed improved training and assessment. Appropriate nurse training should also be provided., Conclusion: Although 82% (n=242) of respondents would implement the Strategy if properly resourced, considerable training and support needs were identified.
- Published
- 2005
- Full Text
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42. Modification of protein sub-nuclear localization by synthetic phosphoinositides: evidence for nuclear phosphoinositide signaling mechanisms.
- Author
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Gozani O, Field SJ, Ferguson CG, Ewalt M, Mahlke C, Cantley LC, Prestwich GD, and Yuan J
- Subjects
- Animals, Boron Compounds, Cell Line, Cell Nucleus ultrastructure, Mice, Mice, Inbred BALB C, NIH 3T3 Cells, Nuclear Localization Signals physiology, Phosphatidylinositol Phosphates metabolism, Cell Nucleus physiology, Homeodomain Proteins physiology, Phosphatidylinositol Phosphates physiology, Receptors, Cytoplasmic and Nuclear physiology, Signal Transduction physiology, Tumor Suppressor Proteins physiology
- Abstract
PtdInsPs are critical signaling molecules that regulate diverse cellular functions. One method to study PtdInsP biology involves using synthetic PtdInsP analogs to activate endogenous PtdInsP-mediated events in living cells. Such methodology has been successfully employed to explore the role of several PtdInsP-biological outcomes in the cytoplasm. However, this strategy has not previously been used to examine the function of PtdInsPs in the nucleus of live cells, primarily because there has not been a well-defined PtdInsP-binding protein to provide functional nuclear readouts. Here we have shown that synthetic PtdIns(5)P analogs access and function in the nucleus. We have found that these molecules modify the sub-nuclear localization of PHD finger-containing proteins in live cells and in real time. This work demonstrates that synthetic PtdInsPs and PtdInsP derivatives may be powerful tools for probing nuclear PtdInsP functions. Finally, our work supports a model that endogenous PtdInsPs regulate sub-nuclear localization and function of endogenous nuclear PtdInsP-binding proteins.
- Published
- 2005
- Full Text
- View/download PDF
43. Mixed data rate and format transmission (40-Gbit/s non-return-to-zero, 40-Gbit/s duobinary, and 10-Gbit/s non-return-to-zero) by mid-link spectral inversion.
- Author
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Jansen SL, Khoe GD, de Waardt H, Spälter S, Weiske CJ, Schöpflin A, Field SJ, Escobar HE, and Sher MH
- Abstract
A polarization-diverse subsystem based on periodically poled lithium niobate waveguides is used as an optical phase conjugator for compensation for linear and nonlinear distortion. We show successful transmission formats of 13 x 40 Gbit/s non-return-to-zero mixed with 6 x 10 Gbit/s non-return-to-zero and 40-Gbit/s duobinary over 8 x 100 km of standard single-mode fiber. A single phase conjugator is used to conjugate all data formats, including the alternative duobinary format, simultaneously.
- Published
- 2004
- Full Text
- View/download PDF
44. A novel phosphatidylinositol(3,4,5)P3 pathway in fission yeast.
- Author
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Mitra P, Zhang Y, Rameh LE, Ivshina MP, McCollum D, Nunnari JJ, Hendricks GM, Kerr ML, Field SJ, Cantley LC, and Ross AH
- Subjects
- Evolution, Molecular, Mutation, Phosphatidylinositol 3-Kinases genetics, Phosphatidylinositol Phosphates biosynthesis, Phosphoric Monoester Hydrolases metabolism, Phosphoric Monoester Hydrolases physiology, Phosphotransferases (Alcohol Group Acceptor) genetics, Saccharomyces cerevisiae Proteins genetics, Schizosaccharomyces cytology, Schizosaccharomyces enzymology, Schizosaccharomyces ultrastructure, Phosphatidylinositol Phosphates metabolism, Phosphoric Monoester Hydrolases genetics, Schizosaccharomyces metabolism, Schizosaccharomyces pombe Proteins
- Abstract
The mammalian tumor suppressor, phosphatase and tensin homologue deleted on chromosome 10 (PTEN), inhibits cell growth and survival by dephosphorylating phosphatidylinositol-(3,4,5)-trisphosphate (PI[3,4,5]P3). We have found a homologue of PTEN in the fission yeast, Schizosaccharomyces pombe (ptn1). This was an unexpected finding because yeast (S. pombe and Saccharomyces cerevisiae) lack the class I phosphoinositide 3-kinases that generate PI(3,4,5)P3 in higher eukaryotes. Indeed, PI(3,4,5)P3 has not been detected in yeast. Surprisingly, upon deletion of ptn1 in S. pombe, PI(3,4,5)P3 became detectable at levels comparable to those in mammalian cells, indicating that a pathway exists for synthesis of this lipid and that the S. pombe ptn1, like mammalian PTEN, suppresses PI(3,4,5)P3 levels. By examining various mutants, we show that synthesis of PI(3,4,5)P3 in S. pombe requires the class III phosphoinositide 3-kinase, vps34p, and the phosphatidylinositol-4-phosphate 5-kinase, its3p, but does not require the phosphatidylinositol-3-phosphate 5-kinase, fab1p. These studies suggest that a pathway for PI(3,4,5)P3 synthesis downstream of a class III phosphoinositide 3-kinase evolved before the appearance of class I phosphoinositide 3-kinases., (Copyright The Rockerfeller University Press)
- Published
- 2004
- Full Text
- View/download PDF
45. Diabetes and exocrine pancreatic insufficiency in E2F1/E2F2 double-mutant mice.
- Author
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Iglesias A, Murga M, Laresgoiti U, Skoudy A, Bernales I, Fullaondo A, Moreno B, Lloreta J, Field SJ, Real FX, and Zubiaga AM
- Subjects
- Animals, Apoptosis, Cell Differentiation, Cell Division, DNA Replication, DNA-Binding Proteins genetics, Diabetes Mellitus, Type 1 genetics, Diabetes Mellitus, Type 1 pathology, E2F Transcription Factors, E2F1 Transcription Factor, E2F2 Transcription Factor, Exocrine Pancreatic Insufficiency genetics, Exocrine Pancreatic Insufficiency pathology, Islets of Langerhans pathology, Male, Mice, Mice, Knockout, Microscopy, Electron, Pancreas pathology, Trans-Activators genetics, Transcription Factors genetics, Cell Cycle Proteins, DNA-Binding Proteins deficiency, Diabetes Mellitus, Type 1 etiology, Exocrine Pancreatic Insufficiency etiology, Trans-Activators deficiency, Transcription Factors deficiency
- Abstract
E2F transcription factors are thought to be key regulators of cell growth control. Here we use mutant mouse strains to investigate the function of E2F1 and E2F2 in vivo. E2F1/E2F2 compound-mutant mice develop nonautoimmune insulin-deficient diabetes and exocrine pancreatic dysfunction characterized by endocrine and exocrine cell dysplasia, a reduction in the number and size of acini and islets, and their replacement by ductal structures and adipose tissue. Mutant pancreatic cells exhibit increased rates of DNA replication but also of apoptosis, resulting in severe pancreatic atrophy. The expression of genes involved in DNA replication and cell cycle control was upregulated in the E2F1/E2F2 compound-mutant pancreas, suggesting that their expression is repressed by E2F1/E2F2 activities and that the inappropriate cell cycle found in the mutant pancreas is likely the result of the deregulated expression of these genes. Interestingly, the expression of ductal cell and adipocyte differentiation marker genes was also upregulated, whereas expression of pancreatic cell marker genes were downregulated. These results suggest that E2F1/E2F2 activity negatively controls growth of mature pancreatic cells and is necessary for the maintenance of differentiated pancreatic phenotypes in the adult.
- Published
- 2004
- Full Text
- View/download PDF
46. Redox-dependent open and closed forms of the active site of the bacterial respiratory nitric-oxide reductase revealed by cyanide binding studies.
- Author
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Grönberg KL, Watmough NJ, Thomson AJ, Richardson DJ, and Field SJ
- Subjects
- Binding Sites, Catalysis, Dose-Response Relationship, Drug, Electrons, Heme chemistry, Iron, Kinetics, Ligands, Nitric Oxide metabolism, Oxygen metabolism, Paracoccus denitrificans enzymology, Potassium Cyanide chemistry, Potassium Cyanide pharmacology, Protein Binding, Time Factors, Ultraviolet Rays, Bacteria enzymology, Cyanides chemistry, Oxidation-Reduction, Oxidoreductases chemistry, Oxygen Consumption
- Abstract
The bacterial respiratory nitric-oxide reductase (NOR) catalyzes the respiratory detoxification of nitric oxide in bacteria and Archaea. It is a member of the well known super-family of heme-copper oxidases but has a [heme Fe-non-heme Fe] active site rather than the [heme Fe-Cu(B)] active site normally associated with oxygen reduction. Paracoccus denitrificans NOR is spectrally characterized by a ligand-to-metal charge transfer absorption band at 595 nm, which arises from the high spin ferric heme iron of a micro-oxo-bridged [heme Fe(III)-O-Fe(III)] active site. On reduction of the nonheme iron, the micro-oxo bridge is broken, and the ferric heme iron is hydroxylated or hydrated, depending on the pH. At present, the catalytic cycle of NOR is a matter of much debate, and it is not known to which redox state(s) of the enzyme nitric oxide can bind. This study has used cyanide to probe the nature of the active site in a number of different redox states. Our observations suggest that the micro-oxo-bridged [heme Fe(III)-O-Fe(III)] active site represents a closed or resting state of NOR that can be opened by reduction of the non-heme iron.
- Published
- 2004
- Full Text
- View/download PDF
47. Identification and characterization of a phosphoinositide phosphate kinase homolog.
- Author
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Chang JD, Field SJ, Rameh LE, Carpenter CL, and Cantley LC
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Blotting, Western, Brain enzymology, Cell Line, Chromosomes, Human, Pair 9, DNA Primers, Humans, Mice, Minor Histocompatibility Antigens, Molecular Sequence Data, Mutagenesis, Site-Directed, Phosphotransferases (Alcohol Group Acceptor) chemistry, Phosphotransferases (Alcohol Group Acceptor) genetics, Protein Biosynthesis, Sequence Homology, Amino Acid, Transcription, Genetic, Phosphotransferases (Alcohol Group Acceptor) metabolism
- Abstract
Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) plays a central role in regulating the actin cytoskeleton as a substrate for phosphoinositide 3-kinase and phospholipase C as well as by binding directly to proteins that control the processes of actin monomer sequestration, filament severing, capping, nucleation, cross-linking, and bundling (Ma, L., Cantley, L. C., Janmey, P. A., and Kirschner, M. W. (1998) J. Cell Biol. 140, 1125-1136; Hinchliffe, K. (2000) Curr. Biol. 10, R104-R1051). Three related phosphatidylinositol 4-phosphate 5-kinases (PI(4)P 5-kinases) have been identified in mammalian cells (types Ialpha, Ibeta, and Igamma) and appear to play distinct roles in actin remodeling. Here we have identified a fourth member of this family by searching the human genome and EST data bases. This new protein, which we have designated phosphatidylinositol phosphate kinase homolog (PIPKH), is expressed at relatively high levels in brain and testis. Immunoprecipitates of PIPKH expressed in mammalian cells contain PI(4)P 5-kinase activity, but this activity is not affected by mutations in residues that inactivate other type I PI(4)P 5-kinases. We show that the PI(4)P 5-kinase activity in PIPKH immunoprecipitates can be explained by the ability of PIPKH to heterodimerize with other type I PI(4)P 5-kinases. Transfection of 293t cells with PIPKH resulted in >8-fold increase in total phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) without a significant net increase in total PI(4,5)P(2). When coexpressed with PIPKH, green fluorescent protein (GFP) fusion construct of the pleckstrin homology domain from Bruton's tyrosine kinase (GFP-BTK-PH) localized in intracellular vesicular structures, suggesting an unusual intracellular site of PI(3,4,5)P(3) production. Finally, expression of PIPKH induced the reorganization of actin from predominantly stress fibers to predominantly foci and comets similar to those observed previously in cells infected with the intracellular pathogen Listeria or transfected with recombinant PIPKIalpha. These results suggest that PIPKH acts as a scaffold to localize and regulate type I PI(4)P 5-kinases and the synthesis of PI(3,4,5)P(3).
- Published
- 2004
- Full Text
- View/download PDF
48. The PHD finger of the chromatin-associated protein ING2 functions as a nuclear phosphoinositide receptor.
- Author
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Gozani O, Karuman P, Jones DR, Ivanov D, Cha J, Lugovskoy AA, Baird CL, Zhu H, Field SJ, Lessnick SL, Villasenor J, Mehrotra B, Chen J, Rao VR, Brugge JS, Ferguson CG, Payrastre B, Myszka DG, Cantley LC, Wagner G, Divecha N, Prestwich GD, and Yuan J
- Subjects
- 1-Phosphatidylinositol 4-Kinase metabolism, Amino Acid Sequence genetics, Base Sequence genetics, Cell Membrane genetics, Cell Membrane metabolism, Cell Nucleus genetics, Genes, Tumor Suppressor, Homeodomain Proteins antagonists & inhibitors, Homeodomain Proteins genetics, Humans, Molecular Sequence Data, Phosphatidylinositol Phosphates metabolism, Protein Binding genetics, Protein Structure, Tertiary genetics, RNA Interference, Receptors, Cytoplasmic and Nuclear genetics, Tumor Cells, Cultured, Tumor Suppressor Protein p53 genetics, Tumor Suppressor Protein p53 metabolism, Apoptosis genetics, Cell Nucleus metabolism, DNA Damage genetics, Eukaryotic Cells metabolism, Homeodomain Proteins metabolism, Receptors, Cytoplasmic and Nuclear metabolism, Signal Transduction genetics, Tumor Suppressor Proteins
- Abstract
Phosphoinositides (PtdInsPs) play critical roles in cytoplasmic signal transduction pathways. However, their functions in the nucleus are unclear, as specific nuclear receptors for PtdInsPs have not been identified. Here, we show that ING2, a candidate tumor suppressor protein, is a nuclear PtdInsP receptor. ING2 contains a plant homeodomain (PHD) finger, a motif common to many chromatin-regulatory proteins. We find that the PHD fingers of ING2 and other diverse nuclear proteins bind in vitro to PtdInsPs, including the rare PtdInsP species, phosphatidylinositol 5-phosphate (PtdIns(5)P). Further, we demonstrate that the ING2 PHD finger interacts with PtdIns(5)P in vivo and provide evidence that this interaction regulates the ability of ING2 to activate p53 and p53-dependent apoptotic pathways. Together, our data identify the PHD finger as a phosphoinositide binding module and a nuclear PtdInsP receptor, and suggest that PHD-phosphoinositide interactions directly regulate nuclear responses to DNA damage.
- Published
- 2003
- Full Text
- View/download PDF
49. Spectral properties of bacterial nitric-oxide reductase: resolution of pH-dependent forms of the active site heme b3.
- Author
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Field SJ, Prior L, Roldan MD, Cheesman MR, Thomson AJ, Spiro S, Butt JN, Watmough NJ, and Richardson DJ
- Subjects
- Binding Sites, Hydrogen-Ion Concentration, Oxidation-Reduction, Oxidoreductases metabolism, Bacteria enzymology, Heme metabolism, Oxidoreductases chemistry
- Abstract
Bacterial nitric-oxide reductase catalyzes the two electron reduction of nitric oxide to nitrous oxide. In the oxidized form the active site non-heme Fe(B) and high spin heme b(3) are mu-oxo bridged. The heme b(3) has a ligand-to-metal charge transfer band centered at 595 nm, which is insensitive to pH over the range of 6.0-8.5. Partial reduction of nitric-oxide reductase yields a three electron-reduced state where only the heme b(3) remains oxidized. This results in a shift of the heme b(3) charge transfer band lambda(max) to longer wavelengths. At pH 6.0 the charge transfer band lambda(max) is 605 nm, whereas at pH 8.5 it is 635 nm. At pH 6.5 and 7.5 the nitric-oxide reductase ferric heme b(3) population is a mixture of both 605- and 635-nm forms. Magnetic circular dichroism spectroscopy suggests that at all pH values examined the proximal ligand to the ferric heme b(3) in the three electron-reduced form is histidine. At pH 8.5 the distal ligand is hydroxide, whereas at pH 6.0, when the enzyme is most active, it is water.
- Published
- 2002
- Full Text
- View/download PDF
50. Lipid research picks up speed on the slopes of Taos.
- Author
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Field SJ, Lamia KA, Rameh LE, and Cantley LC
- Subjects
- Lipid Metabolism, Phosphatidylinositol 3-Kinases physiology, Phosphoprotein Phosphatases physiology, Signal Transduction physiology
- Abstract
The 2002 Keystone Symposium on "Regulation of Cellular Responses by Lipid Mediators" provided a lively and active forum to discuss research in lipid signaling. This meeting review can provide only a glimpse into the diversity of research presented. Here we have chosen to highlight a group of exciting presentations describing novel features of the temporal and spatial regulation of phosphoinositides and their downstream targets.
- Published
- 2002
- Full Text
- View/download PDF
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