Publications by authors named "Sew Peak-Chew"

27 Publications

  • Page 1 of 1

Spatial proteomics defines the content of trafficking vesicles captured by golgin tethers.

Nat Commun 2020 11 25;11(1):5987. Epub 2020 Nov 25.

MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK.

Intracellular traffic between compartments of the secretory and endocytic pathways is mediated by vesicle-based carriers. The proteomes of carriers destined for many organelles are ill-defined because the vesicular intermediates are transient, low-abundance and difficult to purify. Here, we combine vesicle relocalisation with organelle proteomics and Bayesian analysis to define the content of different endosome-derived vesicles destined for the trans-Golgi network (TGN). The golgin coiled-coil proteins golgin-97 and GCC88, shown previously to capture endosome-derived vesicles at the TGN, were individually relocalised to mitochondria and the content of the subsequently re-routed vesicles was determined by organelle proteomics. Our findings reveal 45 integral and 51 peripheral membrane proteins re-routed by golgin-97, evidence for a distinct class of vesicles shared by golgin-97 and GCC88, and various cargoes specific to individual golgins. These results illustrate a general strategy for analysing intracellular sub-proteomes by combining acute cellular re-wiring with high-resolution spatial proteomics.
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http://dx.doi.org/10.1038/s41467-020-19840-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7689464PMC
November 2020

Analysis of disulphide bond linkage between CoA and protein cysteine thiols during sporulation and in spores of Bacillus species.

FEMS Microbiol Lett 2020 Dec;367(23)

Department of Structural and Molecular Biology, University College London, Gower St., London WC1E 6BT, UK.

Spores of Bacillus species have novel properties, which allow them to lie dormant for years and then germinate under favourable conditions. In the current work, the role of a key metabolic integrator, coenzyme A (CoA), in redox regulation of growing cells and during spore formation in Bacillus megaterium and Bacillus subtilis is studied. Exposing these growing cells to oxidising agents or carbon deprivation resulted in extensive covalent protein modification by CoA (termed protein CoAlation), through disulphide bond formation between the CoA thiol group and a protein cysteine. Significant protein CoAlation was observed during sporulation of B. megaterium, and increased largely in parallel with loss of metabolism in spores. Mass spectrometric analysis identified four CoAlated proteins in B. subtilis spores as well as one CoAlated protein in growing B. megaterium cells. All five of these proteins have been identified as moderately abundant in spores. Based on these findings and published studies, protein CoAlation might be involved in facilitating establishment of spores' metabolic dormancy, and/or protecting sensitive sulfhydryl groups of spore enzymes.
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http://dx.doi.org/10.1093/femsle/fnaa174DOI Listing
December 2020

Eukaryotic cell biology is temporally coordinated to support the energetic demands of protein homeostasis.

Nat Commun 2020 09 17;11(1):4706. Epub 2020 Sep 17.

Columbia University Medical Center, New York, NY, 10032, USA.

Yeast physiology is temporally regulated, this becomes apparent under nutrient-limited conditions and results in respiratory oscillations (YROs). YROs share features with circadian rhythms and interact with, but are independent of, the cell division cycle. Here, we show that YROs minimise energy expenditure by restricting protein synthesis until sufficient resources are stored, while maintaining osmotic homeostasis and protein quality control. Although nutrient supply is constant, cells sequester and store metabolic resources via increased transport, autophagy and biomolecular condensation. Replete stores trigger increased H export which stimulates TORC1 and liberates proteasomes, ribosomes, chaperones and metabolic enzymes from non-membrane bound compartments. This facilitates translational bursting, liquidation of storage carbohydrates, increased ATP turnover, and the export of osmolytes. We propose that dynamic regulation of ion transport and metabolic plasticity are required to maintain osmotic and protein homeostasis during remodelling of eukaryotic proteomes, and that bioenergetic constraints selected for temporal organisation that promotes oscillatory behaviour.
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http://dx.doi.org/10.1038/s41467-020-18330-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7499178PMC
September 2020

Covalent Aurora A regulation by the metabolic integrator coenzyme A.

Redox Biol 2020 01 5;28:101318. Epub 2019 Sep 5.

Department of Structural and Molecular Biology, University College London, London, WC1E 6BT, UK; Department of Cell Signaling, Institute of Molecular Biology and Genetics, Kyiv 143, Ukraine. Electronic address:

Aurora A kinase is a master mitotic regulator whose functions are controlled by several regulatory interactions and post-translational modifications. It is frequently dysregulated in cancer, making Aurora A inhibition a very attractive antitumor target. However, recently uncovered links between Aurora A, cellular metabolism and redox regulation are not well understood. In this study, we report a novel mechanism of Aurora A regulation in the cellular response to oxidative stress through CoAlation. A combination of biochemical, biophysical, crystallographic and cell biology approaches revealed a new and, to our knowledge, unique mode of Aurora A inhibition by CoA, involving selective binding of the ADP moiety of CoA to the ATP binding pocket and covalent modification of Cys290 in the activation loop by the thiol group of the pantetheine tail. We provide evidence that covalent CoA modification (CoAlation) of Aurora A is specific, and that it can be induced by oxidative stress in human cells. Oxidising agents, such as diamide, hydrogen peroxide and menadione were found to induce Thr 288 phosphorylation and DTT-dependent dimerization of Aurora A. Moreover, microinjection of CoA into fertilized mouse embryos disrupts bipolar spindle formation and the alignment of chromosomes, consistent with Aurora A inhibition. Altogether, our data reveal CoA as a new, rather selective, inhibitor of Aurora A, which locks this kinase in an inactive state via a "dual anchor" mechanism of inhibition that might also operate in cellular response to oxidative stress. Finally and most importantly, we believe that these novel findings provide a new rationale for developing effective and irreversible inhibitors of Aurora A, and perhaps other protein kinases containing appropriately conserved Cys residues.
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http://dx.doi.org/10.1016/j.redox.2019.101318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6812009PMC
January 2020

A key metabolic integrator, coenzyme A, modulates the activity of peroxiredoxin 5 via covalent modification.

Mol Cell Biochem 2019 Nov 2;461(1-2):91-102. Epub 2019 Aug 2.

Department of Structural and Molecular Biology, University College London, London, WC1E 6BT, UK.

Peroxiredoxins (Prdxs) are antioxidant enzymes that catalyse the breakdown of peroxides and regulate redox activity in the cell. Peroxiredoxin 5 (Prdx5) is a unique member of Prdxs, which displays a wider subcellular distribution and substrate specificity and exhibits a different catalytic mechanism when compared to other members of the family. Here, the role of a key metabolic integrator coenzyme A (CoA) in modulating the activity of Prdx5 was investigated. We report for the first time a novel mode of Prdx5 regulation mediated via covalent and reversible attachment of CoA (CoAlation) in cellular response to oxidative and metabolic stress. The site of CoAlation in endogenous Prdx5 was mapped by mass spectrometry to peroxidatic cysteine 48. By employing an in vitro CoAlation assay, we showed that Prdx5 peroxidase activity is inhibited by covalent interaction with CoA in a dithiothreitol-sensitive manner. Collectively, these results reveal that human Prdx5 is a substrate for CoAlation in vitro and in vivo, and provide new insight into metabolic control of redox status in mammalian cells.
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http://dx.doi.org/10.1007/s11010-019-03593-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6790197PMC
November 2019

A synthetic genetic polymer with an uncharged backbone chemistry based on alkyl phosphonate nucleic acids.

Nat Chem 2019 06 22;11(6):533-542. Epub 2019 Apr 22.

MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge, UK.

The physicochemical properties of nucleic acids are dominated by their highly charged phosphodiester backbone chemistry. This polyelectrolyte structure decouples information content (base sequence) from bulk properties, such as solubility, and has been proposed as a defining trait of all informational polymers. However, this conjecture has not been tested experimentally. Here, we describe the encoded synthesis of a genetic polymer with an uncharged backbone chemistry: alkyl phosphonate nucleic acids (phNAs) in which the canonical, negatively charged phosphodiester is replaced by an uncharged P-alkyl phosphonodiester backbone. Using synthetic chemistry and polymerase engineering, we describe the enzymatic, DNA-templated synthesis of P-methyl and P-ethyl phNAs, and the directed evolution of specific streptavidin-binding phNA aptamer ligands directly from random-sequence mixed P-methyl/P-ethyl phNA repertoires. Our results establish an example of the DNA-templated enzymatic synthesis and evolution of an uncharged genetic polymer and provide a foundational methodology for their exploration as a source of novel functional molecules.
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http://dx.doi.org/10.1038/s41557-019-0255-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6542681PMC
June 2019

The Atypical MAP Kinase ErkB Transmits Distinct Chemotactic Signals through a Core Signaling Module.

Dev Cell 2019 02 3;48(4):491-505.e9. Epub 2019 Jan 3.

Cell Biology Division, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK. Electronic address:

Signaling from chemoattractant receptors activates the cytoskeleton of crawling cells for chemotaxis. We show using phosphoproteomics that different chemoattractants cause phosphorylation of the same core set of around 80 proteins in Dictyostelium cells. Strikingly, the majority of these are phosphorylated at an [S/T]PR motif by the atypical MAP kinase ErkB. Unlike most chemotactic responses, ErkB phosphorylations are persistent and do not adapt to sustained stimulation with chemoattractant. ErkB integrates dynamic autophosphorylation with chemotactic signaling through G-protein-coupled receptors. Downstream, our phosphoproteomics data define a broad panel of regulators of chemotaxis. Surprisingly, targets are almost exclusively other signaling proteins, rather than cytoskeletal components, revealing ErkB as a regulator of regulators rather than acting directly on the motility machinery. ErkB null cells migrate slowly and orientate poorly over broad dynamic ranges of chemoattractant. Our data indicate a central role for ErkB and its substrates in directing chemotaxis.
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http://dx.doi.org/10.1016/j.devcel.2018.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397043PMC
February 2019

Protein CoAlation and antioxidant function of coenzyme A in prokaryotic cells.

Biochem J 2018 06 6;475(11):1909-1937. Epub 2018 Jun 6.

Department of Structural and Molecular Biology, University College London, London WC1E 6BT, U.K.

In all living organisms, coenzyme A (CoA) is an essential cofactor with a unique design allowing it to function as an acyl group carrier and a carbonyl-activating group in diverse biochemical reactions. It is synthesized in a highly conserved process in prokaryotes and eukaryotes that requires pantothenic acid (vitamin B5), cysteine and ATP. CoA and its thioester derivatives are involved in major metabolic pathways, allosteric interactions and the regulation of gene expression. A novel unconventional function of CoA in redox regulation has been recently discovered in mammalian cells and termed protein CoAlation. Here, we report for the first time that protein CoAlation occurs at a background level in exponentially growing bacteria and is strongly induced in response to oxidizing agents and metabolic stress. Over 12% of gene products were shown to be CoAlated in response to diamide-induced stress CoAlation of glyceraldehyde-3-phosphate dehydrogenase was found to inhibit its enzymatic activity and to protect the catalytic cysteine 151 from overoxidation by hydrogen peroxide. These findings suggest that in exponentially growing bacteria, CoA functions to generate metabolically active thioesters, while it also has the potential to act as a low-molecular-weight antioxidant in response to oxidative and metabolic stress.
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http://dx.doi.org/10.1042/BCJ20180043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5989533PMC
June 2018

NADH Shuttling Couples Cytosolic Reductive Carboxylation of Glutamine with Glycolysis in Cells with Mitochondrial Dysfunction.

Mol Cell 2018 02;69(4):581-593.e7

Medical Research Council Cancer Unit, University of Cambridge, Hutchison/MRC Research Centre, Box 197, Cambridge Biomedical Campus, Cambridge CB2 0XZ, UK. Electronic address:

The bioenergetics and molecular determinants of the metabolic response to mitochondrial dysfunction are incompletely understood, in part due to a lack of appropriate isogenic cellular models of primary mitochondrial defects. Here, we capitalize on a recently developed cell model with defined levels of m.8993T>G mutation heteroplasmy, mTUNE, to investigate the metabolic underpinnings of mitochondrial dysfunction. We found that impaired utilization of reduced nicotinamide adenine dinucleotide (NADH) by the mitochondrial respiratory chain leads to cytosolic reductive carboxylation of glutamine as a new mechanism for cytosol-confined NADH recycling supported by malate dehydrogenase 1 (MDH1). We also observed that increased glycolysis in cells with mitochondrial dysfunction is associated with increased cell migration in an MDH1-dependent fashion. Our results describe a novel link between glycolysis and mitochondrial dysfunction mediated by reductive carboxylation of glutamine.
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http://dx.doi.org/10.1016/j.molcel.2018.01.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5823973PMC
February 2018

Protein CoAlation: a redox-regulated protein modification by coenzyme A in mammalian cells.

Biochem J 2017 07 11;474(14):2489-2508. Epub 2017 Jul 11.

Department of Structural and Molecular Biology, University College London, London WC1E 6BT, U.K.

Coenzyme A (CoA) is an obligatory cofactor in all branches of life. CoA and its derivatives are involved in major metabolic pathways, allosteric interactions and the regulation of gene expression. Abnormal biosynthesis and homeostasis of CoA and its derivatives have been associated with various human pathologies, including cancer, diabetes and neurodegeneration. Using an anti-CoA monoclonal antibody and mass spectrometry, we identified a wide range of cellular proteins which are modified by covalent attachment of CoA to cysteine thiols (CoAlation). We show that protein CoAlation is a reversible post-translational modification that is induced in mammalian cells and tissues by oxidising agents and metabolic stress. Many key cellular enzymes were found to be CoAlated and in ways that modified their activities. Our study reveals that protein CoAlation is a widespread post-translational modification which may play an important role in redox regulation under physiological and pathophysiological conditions.
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http://dx.doi.org/10.1042/BCJ20170129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5509381PMC
July 2017

Catalysts from synthetic genetic polymers.

Nature 2015 Feb 1;518(7539):427-30. Epub 2014 Dec 1.

MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK.

The emergence of catalysis in early genetic polymers such as RNA is considered a key transition in the origin of life, pre-dating the appearance of protein enzymes. DNA also demonstrates the capacity to fold into three-dimensional structures and form catalysts in vitro. However, to what degree these natural biopolymers comprise functionally privileged chemical scaffolds for folding or the evolution of catalysis is not known. The ability of synthetic genetic polymers (XNAs) with alternative backbone chemistries not found in nature to fold into defined structures and bind ligands raises the possibility that these too might be capable of forming catalysts (XNAzymes). Here we report the discovery of such XNAzymes, elaborated in four different chemistries (arabino nucleic acids, ANA; 2'-fluoroarabino nucleic acids, FANA; hexitol nucleic acids, HNA; and cyclohexene nucleic acids, CeNA) directly from random XNA oligomer pools, exhibiting in trans RNA endonuclease and ligase activities. We also describe an XNA-XNA ligase metalloenzyme in the FANA framework, establishing catalysis in an entirely synthetic system and enabling the synthesis of FANA oligomers and an active RNA endonuclease FANAzyme from its constituent parts. These results extend catalysis beyond biopolymers and establish technologies for the discovery of catalysts in a wide range of polymer scaffolds not found in nature. Evolution of catalysis independent of any natural polymer has implications for the definition of chemical boundary conditions for the emergence of life on Earth and elsewhere in the Universe.
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http://dx.doi.org/10.1038/nature13982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4336857PMC
February 2015

The N-terminal acetylation of Sir3 stabilizes its binding to the nucleosome core particle.

Nat Struct Mol Biol 2013 Sep 11;20(9):1119-21. Epub 2013 Aug 11.

Structural Studies Division, Medical Research Council-Laboratory of Molecular Biology, Cambridge, UK.

The N-terminal acetylation of Sir3 is essential for heterochromatin establishment and maintenance in yeast, but its mechanism of action is unknown. The crystal structure of the N-terminally acetylated BAH domain of Saccharomyces cerevisiae Sir3 bound to the nucleosome core particle reveals that the N-terminal acetylation stabilizes the interaction of Sir3 with the nucleosome. Additionally, we present a new method for the production of protein-nucleosome complexes for structural analysis.
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http://dx.doi.org/10.1038/nsmb.2641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3818696PMC
September 2013

Identification of a eukaryotic reductive dechlorinase and characterization of its mechanism of action on its natural substrate.

Chem Biol 2011 Oct;18(10):1252-60

Laboratory of Molecular Biology, Medical Research Council, Cambridge CB2 0QH, UK.

Chlorinated compounds are important environmental pollutants whose biodegradation may be limited by inefficient dechlorinating enzymes. Dictyostelium amoebae produce a chlorinated alkyl phenone called DIF which induces stalk cell differentiation during their multicellular development. Here we describe the identification of DIF dechlorinase. DIF dechlorinase is active when expressed in bacteria, and activity is lost from Dictyostelium cells when its gene, drcA, is knocked out. It has a K(m) for DIF of 88 nM and K(cat) of 6.7 s(-1). DrcA is related to glutathione S-transferases, but with a key asparagine-to-cysteine substitution in the catalytic pocket. When this change is reversed, the enzyme reverts to a glutathione S-transferase, thus suggesting a catalytic mechanism. DrcA offers new possibilities for the rational design of bioremediation strategies.
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http://dx.doi.org/10.1016/j.chembiol.2011.08.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3205185PMC
October 2011

The acetyltransferase activity of the bacterial toxin YopJ of Yersinia is activated by eukaryotic host cell inositol hexakisphosphate.

J Biol Chem 2010 Jun 29;285(26):19927-34. Epub 2010 Apr 29.

Medical Research Council (MRC), Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.

Plague, one of the most devastating diseases in human history, is caused by the bacterium Yersinia pestis. The bacteria use a syringe-like macromolecular assembly to secrete various toxins directly into the host cells they infect. One such Yersinia outer protein, YopJ, performs the task of dampening innate immune responses in the host by simultaneously inhibiting the MAPK and NFkappaB signaling pathways. YopJ catalyzes the transfer of acetyl groups to serine, threonine, and lysine residues on target proteins. Acetylation of serine and threonine residues prevents them from being phosphorylated thereby preventing the activation of signaling molecules on which they are located. In this study, we describe the requirement of a host-cell factor for full activation of the acetyltransferase activity of YopJ and identify this activating factor to be inositol hexakisphosphate (IP(6)). We extend the applicability of our results to show that IP(6) also stimulates the acetyltransferase activity of AvrA, the YopJ homologue from Salmonella typhimurium. Furthermore, an IP(6)-induced conformational change in AvrA suggests that IP(6) acts as an allosteric activator of enzyme activity. Our results suggest that YopJ-family enzymes are quiescent in the bacterium where they are synthesized, because bacteria lack IP(6); once injected into mammalian cells by the pathogen these toxins bind host cell IP(6), are activated, and deregulate the MAPK and NFkappaB signaling pathways thereby subverting innate immunity.
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http://dx.doi.org/10.1074/jbc.M110.126581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2888404PMC
June 2010

N-terminal acetylation of the neuronal protein SNAP-25 is revealed by the SMI81 monoclonal antibody.

Biochemistry 2009 Oct;48(40):9582-9

MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.

The monoclonal antibody SMI81 binds SNAP-25, a major player in neurotransmitter release, with high affinity and has previously been used to follow changes in the levels of this protein in neuropsychiatric disorders. We report here that the SMI81 epitope is present at the extreme N-terminus of SNAP-25 and, unusually, cannot be recognized when present as an internal sequence. Although it is known that SNAP-25 can be palmitoylated and phosphorylated in brain, we now reveal the existence of a third modification, acetylation of the N-terminus. This acetylation event greatly increases the efficiency of SMI81 antibody binding. We show that this highly specific antibody can be used for studying brain function in many vertebrate organisms.
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http://dx.doi.org/10.1021/bi9012403DOI Listing
October 2009

Genetically encoding N(epsilon)-acetyllysine in recombinant proteins.

Nat Chem Biol 2008 Apr 17;4(4):232-4. Epub 2008 Feb 17.

Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH England, UK.

N(epsilon)-acetylation of lysine (1) is a reversible post-translational modification with a regulatory role that rivals that of phosphorylation in eukaryotes. No general methods exist to synthesize proteins containing N(epsilon)-acetyllysine (2) at defined sites. Here we demonstrate the site-specific incorporation of N(epsilon)-acetyllysine in recombinant proteins produced in Escherichia coli via the evolution of an orthogonal N(epsilon)-acetyllysyl-tRNA synthetase/tRNA(CUA) pair. This strategy should find wide applications in defining the cellular role of this modification.
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http://dx.doi.org/10.1038/nchembio.73DOI Listing
April 2008

p32 is a novel mammalian Lgl binding protein that enhances the activity of protein kinase Czeta and regulates cell polarity.

J Cell Biol 2007 Aug 6;178(4):575-81. Epub 2007 Aug 6.

Medical Research Council Laboratory for Molecular Cell Biology and Cell Biology Unit, Department of Anatomy and Developmental Biology, University College London, London WC1E 6BT, England, UK.

Lgl (lethal giant larvae) plays an important role in cell polarity. Atypical protein kinase C (aPKC) binds to and phosphorylates Lgl, and the phosphorylation negatively regulates Lgl activity. In this study, we identify p32 as a novel Lgl binding protein that directly binds to a domain on mammalian Lgl2 (mLgl2), which contains the aPKC phosphorylation site. p32 also binds to PKCzeta, and the three proteins form a transient ternary complex. When p32 is bound, PKCzeta is stimulated to phosphorylate mLgl2 more efficiently. p32 overexpression in Madin-Darby canine kidney cells cultured in a 3D matrix induces an expansion of the actin-enriched apical membrane domain and disrupts cell polarity. Addition of PKCzeta inhibitor blocks apical actin accumulation, which is rescued by p32 overexpression. p32 knockdown by short hairpin RNA also induces cell polarity defects. Collectively, our data indicate that p32 is a novel regulator of cell polarity that forms a complex with mLgl2 and aPKC and enhances aPKC activity.
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http://dx.doi.org/10.1083/jcb.200612022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2064465PMC
August 2007

Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion.

Nat Biotechnol 2007 Jul 24;25(7):770-7. Epub 2007 Jun 24.

Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England, UK.

In vivo incorporation of unnatural amino acids by amber codon suppression is limited by release factor-1-mediated peptide chain termination. Orthogonal ribosome-mRNA pairs function in parallel with, but independent of, natural ribosomes and mRNAs. Here we show that an evolved orthogonal ribosome (ribo-X) improves tRNA(CUA)-dependent decoding of amber codons placed in orthogonal mRNA. By combining ribo-X, orthogonal mRNAs and orthogonal aminoacyl-tRNA synthetase/tRNA pairs in Escherichia coli, we increase the efficiency of site-specific unnatural amino acid incorporation from approximately 20% to >60% on a single amber codon and from <1% to >20% on two amber codons. We hypothesize that these increases result from a decreased functional interaction of the orthogonal ribosome with release factor-1. This technology should minimize the functional and phenotypic effects of truncated proteins in experiments that use unnatural amino acid incorporation to probe protein function in vivo.
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http://dx.doi.org/10.1038/nbt1314DOI Listing
July 2007

Phosphorylation of human microtubule-associated protein tau by protein kinases of the AGC subfamily.

FEBS Lett 2007 Jun 11;581(14):2657-62. Epub 2007 May 11.

MRC Laboratory of Molecular Biology, Hills Road, Cambridge, UK.

Intraneuronal inclusions made of hyperphosphorylated microtubule-associated protein tau are a defining neuropathological characteristic of Alzheimer's disease, and of several other neurodegenerative disorders. Many phosphorylation sites in tau are S/TP sites that flank the microtubule-binding repeats. Others are KXGS motifs in the repeats. One site upstream of the repeats lies in a consensus sequence for AGC kinases. This site (S214) is believed to play an important role in the events leading from normal, soluble to filamentous, insoluble tau. Here, we show that all AGC kinases tested phosphorylated S214. RSK1 and p70 S6 kinase also phosphorylated the neighbouring T212, a TP site that conforms weakly to the AGC kinase consensus sequence. MSK1 phosphorylated S214, as well as S262, a KXGS site in the first repeat, and S305 in the second repeat.
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http://dx.doi.org/10.1016/j.febslet.2007.05.009DOI Listing
June 2007

Control of phospholipid synthesis by phosphorylation of the yeast lipin Pah1p/Smp2p Mg2+-dependent phosphatidate phosphatase.

J Biol Chem 2006 Nov 12;281(45):34537-48. Epub 2006 Sep 12.

Cambridge Institute for Medical Research, University of Cambridge, Wellcome Trust/MRC Building, Hills Road, CB2 2XY Cambridge, United Kingdom.

Phosphorylation of the conserved lipin Pah1p/Smp2p in Saccharomyces cerevisiae was previously shown to control transcription of phospholipid biosynthetic genes and nuclear structure by regulating the amount of membrane present at the nuclear envelope (Santos-Rosa, H., Leung, J., Grimsey, N., Peak-Chew, S., and Siniossoglou, S. (2005) EMBO J. 24, 1931-1941). A recent report identified Pah1p as a Mg2+-dependent phosphatidate (PA) phosphatase that regulates de novo lipid synthesis (Han G.-S., Wu, W. I., and Carman, G. M. (2006) J. Biol. Chem. 281, 9210-9218). In this work we use a combination of mass spectrometry and systematic mutagenesis to identify seven Ser/Thr-Pro motifs within Pah1p that are phosphorylated in vivo. We show that phosphorylation on these sites is required for the efficient transcriptional derepression of key enzymes involved in phospholipid biosynthesis. The phosphorylation-deficient Pah1p exhibits higher PA phosphatase-specific activity than the wild-type Pah1p, indicating that phosphorylation of Pah1p controls PA production. Opi1p is a transcriptional repressor of phospholipid biosynthetic genes, responding to PA levels. Genetic analysis suggests that Pah1p regulates transcription of these genes through both Opi1p-dependent and -independent mechanisms. We also provide evidence that derepression of phospholipid biosynthetic genes is not sufficient to induce the nuclear membrane expansion shown in the pah1delta cells.
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http://dx.doi.org/10.1074/jbc.M606654200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1769310PMC
November 2006

The yeast lipin Smp2 couples phospholipid biosynthesis to nuclear membrane growth.

EMBO J 2005 Jun 5;24(11):1931-41. Epub 2005 May 5.

WellcomeTrust/Cancer Research UK Gurdon Institute, Cambridge, UK.

Remodelling of the nuclear membrane is essential for the dynamic changes of nuclear architecture at different stages of the cell cycle and during cell differentiation. The molecular mechanism underlying the regulation of nuclear membrane biogenesis is not known. Here we show that Smp2, the yeast homologue of mammalian lipin, is a key regulator of nuclear membrane growth during the cell cycle. Smp2 is phosphorylated by Cdc28/Cdk1 and dephosphorylated by a nuclear/endoplasmic reticulum (ER) membrane-localized CPD phosphatase complex consisting of Nem1 and Spo7. Loss of either SMP2 or its dephosphorylated form causes transcriptional upregulation of key enzymes involved in lipid biosynthesis concurrent with a massive expansion of the nucleus. Conversely, constitutive dephosphorylation of Smp2 inhibits cell division. We show that Smp2 associates with the promoters of phospholipid biosynthetic enzymes in a Nem1-Spo7-dependent manner. Our data suggest that Smp2 is a critical factor in coordinating phospholipid biosynthesis at the nuclear/ER membrane with nuclear growth during the cell cycle.
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http://dx.doi.org/10.1038/sj.emboj.7600672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1142606PMC
June 2005

Sir2 and the acetyltransferase, Pat, regulate the archaeal chromatin protein, Alba.

J Biol Chem 2005 Jun 11;280(22):21122-8. Epub 2005 Apr 11.

Medical Research Council Cancer Cell Unit, Hutchison Medical Research Council Research Centre, Hills Road, Cambridge CB2 2XZ, United Kingdom.

The DNA binding affinity of Alba, a chromatin protein of the archaeon Sulfolobus solfataricus P2, is regulated by acetylation of lysine 16. Here we identify an acetyltransferase that specifically acetylates Alba on this residue. The effect of acetylation is to lower the affinity of Alba for DNA. Remarkably, the acetyltransferase is conserved not only in archaea but also in bacteria where it appears to play a role in metabolic regulation. Therefore, our data suggest that S. solfataricus has co-opted this bacterial regulatory system to generate a rudimentary form of chromatin regulation.
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http://dx.doi.org/10.1074/jbc.M501280200DOI Listing
June 2005

The phosphorylation of subunits of complex I from bovine heart mitochondria.

J Biol Chem 2004 Jun 31;279(25):26036-45. Epub 2004 Mar 31.

Medical Research Council Dunn Human Nutrition Unit, Hills Road, Cambridge CB2 2XY, UK.

In bovine heart mitochondria and in submitochondrial particles, membrane-associated proteins with apparent molecular masses of 18 and 10 kDa become strongly radiolabeled by [(32)P]ATP in a cAMP-dependent manner. The 18-kDa phosphorylated protein is subunit ESSS from complex I and not as previously reported the 18 k subunit (with the N-terminal sequence AQDQ). The phosphorylated residue in subunit ESSS is serine 20. In the 10 kDa band, the complex I subunit MWFE was phosphorylated on serine 55. In the presence of protein kinase A and cAMP, the same subunits of purified complex I were phosphorylated by [(32)P]ATP at the same sites. Subunits ESSS and MWFE both contribute to the membrane arm of complex I. Each has a single hydrophobic region probably folded into a membrane spanning alpha-helix. It is likely that the phosphorylation site of subunit ESSS lies in the mitochondrial matrix and that the site in subunit MWFE is in the intermembrane space. Subunit ESSS has no known role, but subunit MWFE is required for assembly into complex I of seven hydrophobic subunits encoded in the mitochondrial genome. The possible effects of phosphorylation of these subunits on the activity and/or the assembly of complex I remain to be explored.
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http://dx.doi.org/10.1074/jbc.M402710200DOI Listing
June 2004

Interactions between centromere complexes in Saccharomyces cerevisiae.

Mol Biol Cell 2003 Dec 17;14(12):4931-46. Epub 2003 Oct 17.

MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, England.

We have purified two new complexes from Saccharomyces cerevisiae, one containing the centromere component Mtw1p together with Nnf1p, Nsl1p, and Dsn1p, which we call the Mtw1p complex, and the other containing Spc105p and Ydr532p, which we call the Spc105p complex. Further purifications using Dsn1p tagged with protein A show, in addition to the other components of the Mtw1p complex, the two components of the Spc105p complex and the four components of the previously described Ndc80p complex, suggesting that all three complexes are closely associated. Fluorescence microscopy and immunoelectron microscopy show that Nnf1p, Nsl1p, Dsn1p, Spc105p, and Ydr532p all localize to the nuclear side of the spindle pole body and along short spindles. Chromatin immunoprecipitation assays show that all five proteins are associated with centromere DNA. Homologues of Nsl1p and Spc105p in Schizosaccharomyces pombe also localize to the centromere. Temperature-sensitive mutations of Nsl1p, Dsn1p, and Spc105p all cause defects in chromosome segregation. Synthetic-lethal interactions are found between temperature-sensitive mutations in proteins from all three complexes, in agreement with their close physical association. These results show an increasingly complex structure for the S. cerevisiae centromere and a probable conservation of structure between parts of the centromeres of S. cerevisiae and S. pombe.
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http://dx.doi.org/10.1091/mbc.e03-06-0419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC284796PMC
December 2003

An N-acetylglucosaminyltransferase of the Golgi apparatus of the yeast Saccharomyces cerevisiae that can modify N-linked glycans.

Glycobiology 2003 Aug 19;13(8):581-9. Epub 2003 Mar 19.

MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.

The yeast Saccharomyces cerevisiae is widely regarded as being only capable of producing N-linked glycans with high-mannose structures. To investigate the glycan structures made in different mutant strains, we made use of a reporter protein consisting of a version of hen egg lysozyme that contains a single site for N-linked glycosylation. Mass spectrometry analysis of the attached glycans revealed that a large proportion contained an unexpected extra mass corresponding to a single N-acetylhexosamine residue. In addition, the glycosylated lysozyme was recognized by an N-acetylglucosamine specific lectin. The genome of S. cerevisiae contains an uncharacterized open reading frame, YOR320c, that is related to a known N-acetylglucosaminyltransferase. Deletion of this ORF resulted in the disappearance of the extra mass on the N-linked glycans and loss of lectin binding. We show that the protein encoded by YOR320c (which we term Gnt1p) is localized to the Golgi apparatus and has GlcNAc-transferase activity in vitro. The physiological role of Gnt1p is unclear because mutants lacking the protein show no obvious growth or cell wall defects. Nonetheless, these results indicate that heterologous glycoproteins expressed in yeast can receive N-glycans with structures other than high mannose. In addition, they indicate that the lumen of the yeast Golgi contains UDP-GlcNAc, which may facilitate reconstitution of higher eukaryotic N-glycan processing.
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http://dx.doi.org/10.1093/glycob/cwg063DOI Listing
August 2003

RPA is an initiation factor for human chromosomal DNA replication.

Nucleic Acids Res 2003 Mar;31(6):1725-34

Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK.

The initiation of chromosomal DNA replication in human cell nuclei is not well understood because of its complexity. To allow investigation of this process on a molecular level, we have recently established a cell-free system that initiates chromosomal DNA replication in an origin-specific manner under cell cycle control in isolated human cell nuclei. We have now used fractionation and reconstitution experiments to functionally identify cellular factors present in a human cell extract that trigger initiation of chromosomal DNA replication in this system. Initial fractionation of a cytosolic extract indicates the presence of at least two independent and non-redundant initiation factors. We have purified one of these factors to homogeneity and identified it as the single-stranded DNA binding protein RPA. The prokaryotic single-stranded DNA binding protein SSB cannot substitute for RPA in the initiation of human chromosomal DNA replication. Antibodies specific for human RPA inhibit the initiation step of human chromosomal DNA replication in vitro. RPA is recruited to DNA replication foci and becomes phosphorylated concomitant with the initiation step in vitro. These data establish a direct functional role for RPA as an essential factor for the initiation of human chromosomal DNA replication.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC152871PMC
http://dx.doi.org/10.1093/nar/gkg269DOI Listing
March 2003

EpsinR: an ENTH domain-containing protein that interacts with AP-1.

Mol Biol Cell 2003 Feb;14(2):625-41

University of Cambridge, Department of Clinical Biochemistry, Cambridge Institute for Medical Research, United Kingdom.

We have used GST pulldowns from A431 cell cytosol to identify three new binding partners for the gamma-adaptin appendage: Snx9, ARF GAP1, and a novel ENTH domain-containing protein, epsinR. EpsinR is a highly conserved protein that colocalizes with AP-1 and is enriched in purified clathrin-coated vesicles. However, it does not require AP-1 to get onto membranes and remains membrane-associated in AP-1-deficient cells. Moreover, although epsinR binds AP-1 via its COOH-terminal domain, its NH(2)-terminal ENTH domain can be independently recruited onto membranes, both in vivo and in vitro. Brefeldin A causes epsinR to redistribute into the cytosol, and recruitment of the ENTH domain requires GTPgammaS, indicating that membrane association is ARF dependent. In protein-lipid overlay assays, the epsinR ENTH domain binds to PtdIns(4)P, suggesting a possible mechanism for ARF-dependent recruitment onto TGN membranes. When epsinR is depleted from cells by RNAi, cathepsin D is still correctly processed intracellularly to the mature form. This indicates that although epsinR is likely to be an important component of the AP-1 network, it is not necessary for the sorting of lysosomal enzymes.
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http://dx.doi.org/10.1091/mbc.e02-09-0552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC149997PMC
February 2003