Mark Howarth lab
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Publications
1. Papers and reviews 2. Patents
Papers and reviews
A peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Bijan Zakeri*, Jacob O. Fierer*, Emrah Celik, Emily C. Chittock, Ulrich Schwarz-Linek, Vincent T. Moy, and Mark Howarth PNAS 2012 in press *joint first author
A peptide filtering relation quantifies MHC class I peptide optimization. Dalchau N, Phillips A, Goldstein LD, Howarth M, Cardelli L, Elliott T, Werner JM. PLoS Computational Biology 2011 Oct;7(10):e1002144. PDF Abstract on Medline Supporting Info. Collaboration with Microsoft and my previous lab in Southampton to model mathematically all the steps in peptide presentation for T cell recognition.
Mechanism of size-dependent segregation at the immune synapse determined with molecular rulers. Alakoskela JM, Koner AL, Rudnicka D, Köhler K, Howarth M, Davis DM. Biophysical Journal 2011 Jun 22;100(12):2865-74. PDF Abstract on Medline Supporting Info. The exact intermembrane spacing when a natural killer cell encounters a target cell is important in triggering killer cell activation. In collaboration with Dan Davis' lab we developed a controlled size-series of fluorescent proteins and nanoparticles for real-time imaging of how molecules above a certain size are excluded from the synapse.
How the biotin-streptavidin interaction was made even stronger: investigation via crystallography and a chimaeric tetramer. Chivers CE, Koner AL, Lowe ED, Howarth M. Biochemical Journal 2011 Apr 1;435(1):55-63. Our first foray into structural biology, to help determine the origin of one of the strongest non-covalent interactions in nature.
The type I IGF receptor translocates to the nucleus of human tumor cells. Aleksic T, Chitnis M, Perestenko O, Gao S, Thomas P, Turner G, Protheroe A, Howarth M, Macaulay V. Cancer Research 2010 Aug 15;70(16):6412-9. PDF Abstract on Medline Supporting Info. The classic model of growth factor receptor signaling is that the activated receptor at the plasma membrane/endosomes, activates down-stream pathways. Components of these pathways then enter the nucleus, leading to changes in gene expression. This paper contributes to a new model where the receptor itself moves to the nucleus and has direct effects on transcription. We show that nuclear IGF1R is associated with a more aggressive cancer, suggesting that approaches to block nuclear translocation could improve survival.
A streptavidin variant with slower biotin dissociation and increased mechanostability. Chivers CE, Crozat E, Chu C, Moy VT, Sherratt DJ, Howarth M. Nature Methods 2010 May;7(5):391-93. PDF Abstract on Medline Supporting Info. Streptavidin binds to the vitamin biotin with one of the strongest non-covalent interactions in nature. Streptavidin is used widely in research and also has given promising results in clinical trials for improving delivery of cancer radiotherapy. We developed a mutant of streptavidin, termed traptavidin, which binds biotin 10-times better. We tested traptavidin, by setting up a molecular car-crash to investigate a motor protein involved in chromosome segregation. (Plasmids for Traptavidin with and without His6-tag available from Addgene repository.)
Separating speed and ability to displace roadblocks during DNA translocation by FtsK. Crozat E, Meglio A, Allemand JF, Chivers CE, Howarth M, Vénien-Bryan C, Grainge I, Sherratt DJ. EMBO Journal 2010 Apr 21;29(8):1423-33. PDF Abstract on Medline Supporting Info. FtsK is a bacterial motor protein that pumps DNA, so that each daughter cell receives one copy of the chromosome. In collaboration with the Sherratt lab we explored through crash-testing how the different active sites in a ring coordinate their firing.
Spontaneous Intermolecular Amide Bond Formation between Side Chains for Irreversible Peptide Targeting. Zakeri B & Howarth M. Journal of the American Chemical Society 2010 Apr 7;132(13):4526-7. PDF Abstract on Medline Supporting Info. Peptide interactions with proteins are usually weak. Here we show how to get a genetically encoded peptide to form an irreversible covalent bond when it binds its protein partner. This may be useful for assembly, for imaging and to enable new protein architectures.
Electrophilic affibodies forming covalent bonds to protein targets. Holm L, Moody P, Howarth M. Journal of Biological Chemistry 2009 Nov;284(47):32906-13 PDF Abstract on Medline Supporting Info. Affibodies are like antibodies but smaller and easier to make. We introduced an artificial reactive group next to the target binding site of an affibody. This enabled the affibody to form a specific covalent bond to its target. By preventing affibody dissociation we could improve detection sensitivity and stability. We are working to make this reaction faster and more general, to enable detection of disease markers present in blood at concentrations too low for current tests.
Monovalent, reduced-size quantum dots for imaging receptors on living cells. Howarth M, Liu W, Puthenveetil S, Zheng Y, Marshall LF, Schmidt MM, Wittrup KD, Bawendi MG, Ting AY. Nature Methods 2008 May;5(5):397-99. PDF Abstract on Medline Supporting Info. Quantum dots are ultra-bright nanoparticles, which make single molecule imaging routine. A major problem in the field is that one nanoparticle can bind several copies of its target receptor. This stops the receptor moving freely and often activates cell signalling. Here we develop a way to purify quantum dots with precisely defined numbers of proteins attached. We use these non-crosslinking high affinity QDs to image the low density lipoprotein receptor and the tumour marker carcinoembryonic antigen.
Compact Biocompatible Quantum Dots Functionalized for Cellular Imaging. Liu W, Howarth M, Greytak AB, Zheng Y, Nocera DG, Ting AY, Bawendi MG. Journal of the American Chemical Society 2008 Jan 30;130(4):1274-84. PDF Abstract on Medline Supporting Info. It has been challenging to reduce quantum dot size at the same time as maintaining high stability and specificity in their targeting. In this paper we engineer the QD passivating layer to address this challenge and apply these QDs to image the epidermal growth factor receptor, an important regulator of cancer cell division.
Tapasin shapes immunodominance hierarchies according to the kinetic stability of peptide-MHC class I complexes. Thirdborough SM, Roddick JS, Radcliffe JN, Howarth M, Stevenson FK, Elliott T. European Journal of Immunology 2008 Jan 14;38(2):364-369. A development of work from my DPhil (see PNAS 2004 below), showing that tapasin not only changes how well peptides are displayed by MHC class I at the cell surface, but also how well these peptides activate the immune system.
Protein imaging in live mammalian cells by site-specific labeling with biotin ligase and monovalent streptavidin. Howarth M,
Ting AY.
Step-by-step instructions
and trouble-shooting guide, for those applying the approaches we developed for
stable cell labeling with biophysical probes and expression of a streptavidin
with a single high affinity binding site.
(Plasmids for BirA and monovalent streptavidin
expression available from
Addgene
repository.) Note in our current protocol we use 5 min with a stir-bar to
resuspend inclusion bodies and reduce pipetting effort. Giving cells a new sugar-coating Howarth
M, and Ting AY. News and Views article on a paper by Kevin Yarema, a protege of Carolyn Bertozzi, feeding cells a thiol sugar. This enables them to redecorate the cell surface with sialic acids bearing thiols, which changed adhesion of stem cells. We explain how this method works and discuss the importance of manipulating protein glycosylation.
A monovalent
streptavidin with a single femtomolar biotin binding site. Nature Methods 2006, Apr;3(4):267-73 PDF Abstract on Medline News and Views Supp. Info. Journal front cover Streptavidin
is used in almost every biology lab and also by many physicists and
engineering seeking to manipulate molecules on the nanometre scale. Streptavidin
is used so much in biotechnology because of its rapid, selective and stable binding to anything
modified with biotin. However, streptavidin has 4 binding sites. This can
cross-link proteins on the surface of cells and limit one's control in
nanotechnology applications. We made a version where one can control the number
of binding sites from 0-4 but keep the tight binding. PNAS 2005, May;102(21):7583-8
PDF Abstract on Medline Supp. Info. Supporting movie
Quantum dots are inorganic nanoparticles that are so bright that they make it routine to image single proteins moving in living cells. Here we develop the use of biotin ligase to add biotin to neurotransmitter receptors on the neuron surface and then track the receptors with quantum dots.
Site-Specific Labeling of Cell Surface Proteins with Biophysical Probes
using Biotin Ligase. Chen I, Howarth M, Lin W, Ting AY. Nature Methods 2005 Feb;2(2):99-104 PDF Abstract on Medline Supp. Info. It is a great challenge to label proteins with new tags that will probe their function. Biotin ligase recognizes a 15 amino acid tag specifically over other cytosolic and cell surface proteins. We get Biotin ligase to add a ketone analog of biotin to a tagged cell-surface protein. Since there are no ketones on the cell-surface, this enables us to react the ketone with any hydrazide-labeled biophysical probe.
DNA Transfection Screening from Single Beads. Yingyongnarongkul BE, Howarth M, Elliott T, Bradley M. Journal of Combinatorial Chemistry 2004 Sep-Oct;6(5):753-60. PDF Supporting info. Abstract on Medline
Solid-phase synthesis of 89 polyamine-based cationic lipids for DNA delivery to mammalian cells. Yingyongnarongkul BE, Howarth M, Elliott T, Bradley M. Chemistry 2004 Jan 23;10(2):463-73. One of the greatest challenges in medicine is to get DNA expressed long term in a wide number of cells, for gene therapy. Even in the laboratory it is still a challenge to get DNA into many cell-types. However, it is hard to predict which compound will be best at introducing DNA into cells, known as DNA transfection. Mark Bradley's group are experts in combinatorial synthesis, where new compounds are made in a highly parallel fashion, rather than one at a time. They turned their attention to combinatorial synthesis of transfection reagents and we helped them in the testing and design of these reagents. We look at an uncommon class of transfection agent with only one lipid tail and show that the amount of compound made on a single polymer bead is sufficient to test transfection activity.
Tapasin enhances MHC class I antigen presentation according to peptide half-life. Howarth M, Williams A, Tolstrup AB, Elliott T. PNAS
2004 Aug;101(32):11737-11742 PDF Abstract
on Medline Elliott
lab MHC class I presents peptides that are seen by killer T cells and controls their activation and killing. Tapasin is an ER chaperone that associates with newly synthesized MHC class I. Here we show that the interaction with tapasin changes the repertoire of peptides that MHC class I presents, to favour stable peptides, with possible consequences for autoimmune disease and killing of bacteria and viruses.
The processing of antigens delivered as DNA vaccines. Howarth M, Elliott T. Immunological Reviews 2004 199:27-39 DNA vaccination involves stimulating an immune response with DNA encoding pathogenic genes or gene fragments, rather than immunizing with protein or attenuated virus, as is more conventional. Trials of DNA vaccination are taking place for many diseases, including skin cancer and HIV. This review discusses how our basic knowledge of MHC class I and II antigen presentation may explain DNA vaccination results and guide future strategies.
Assembly and antigen-presenting function of MHC class I molecules in cells lacking the ER chaperone calreticulin. Gao B, Adhikari R, Howarth M, Nakamura K, Gold MC, Hill AB, Knee R, Michalak M, Elliott T. Immunity 2002 Jan;16(1):99-109 Calreticulin is a multi-talented
protein, involved in calcium homeostasis, gene regulation
Processing and presentation of glycoproteins in the MHC class I and II antigen presentation pathways. Golgher DB, Elliott T, Howarth M. Chapter in "Immunobiology of Carbohydrates", Landes Biosciences, 2002. Link on Google books (can click through the pages) MHC class I presents peptides to killer T cells and MHC class II presents peptides to helper T cells. Many cellular proteins are glycosylated, yet how this affects what peptides are presented by MHC is little understood and challenging to study. It may have relevance to immune responses in conditions such as allergy and cancer. We also point out many of the key questions still unanswered.
The quantity of naturally processed peptides stably bound by HLA-A*0201 is significantly reduced in the absence of tapasin. Barber LD, Howarth M, Bowness P, Elliott T. Tissue Antigens 2001 Dec;58(6):363-8 MHC class I presents a sample of peptides derived from cellular proteins, allowing killer T cells to monitor what is going on inside the cell. It is possible to profile these peptides by eluting them with acid and then HPLC analysis. Here we see the effect of the ER chaperone tapasin on the abundance and nature of peptides eluted from the most common HLA allele in Western populations.
Patents filed
Peptide tag systems that spontaneously form an irreversible link to protein partners via isopeptide bonds. Howarth M, Zakeri B. 2010, United Kingdom Patent Application (described by Isis Innovation)
High Stability Streptavidin Mutant Proteins. Howarth M, Chivers C. 2009, United Kingdom Patent Application (regarding a lower off-rate variant; described in issue of Isis Innovation news, page 22-23)
Controlled modification of semiconductor nanocrystals. Ting AY, Bawendi MG, Howarth M, Liu W. 2007, US Patent and Trademark Office.
Monovalent Streptavidin compositions. Ting AY, Howarth M. 2006, US Patent and Trademark Office.
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Copyright © 2011 Mark Howarth. All rights reserved. |