Multiviral Quartet Nanocages Elicit Broad Anti-Coronavirus Responses for Proactive Vaccinology Rory A. Hills, Tiong Kit Tan, Alexander A. Cohen, Jennifer R. Keeffe, Anthony H. Keeble, Priyanthi N.P. Gnanapragasam, Kaya N. Storm, Michelle L. Hill, Sai Liu, Javier Gilbert-Jaramillo, Madeeha Afzal, Amy Napier, William S. James, Pamela J. Bjorkman, Alain R. Townsend and Mark Howarth. bioRxiv 2023 Feb PDF+SI Abstract There are many pathogens, particularly in bats, with the potential to infect human cells. Here we explore how to produce scalable vaccines with broad protection, so that one can start to prepare vaccines in advance of a related pathogen emerging as a threat. Structural basis for a conserved neutralization epitope on the receptor-binding domain of SARS-CoV-2. Kuan-Ying Huang, Xiaorui Chen, Arpita Mohapatra, Vy H. T. Nguyen, Lisa Schimanski, Tiong Kit Tan, Pramila Rijal, Susan Vester, Rory Hills, Mark Howarth, Jennifer Keeffe, Alexander Cohen, Leesa Kakutani, Yi-min Wu, Md. Shahed-Al-Mahmud, Yu-Chi Chou, Pamela Bjorkman, Alain Townsend, Che Ma. Nature Communications Jan 2023 19;14(1):311 PDF+SI Abstract New variants of SARS-CoV-2 are resistant to antibodies from existing vaccines or therapeutic antibody cocktails. By studying human immune responses, we identify an antibody that “gets under the skin” of the virus, achieving broad recognition by binding to protein features normally buried. CD4-binding site immunogens elicit heterologous anti-HIV-1 neutralizing antibodies in transgenic and wildtype animals Gristick HB, Hartweger H, Loewe M, van Schooten J, Ramos V, Oliveira TY, Nishimura Y, Koranda NS, Wall A, Yao KH, Poston D, Gazumyan A, Wiatr M, Horning M, Keeffe JR, Hoffmann MAG, Yang Z, Abernathy ME, Dam KA, Gao H, Gnanapragasam PNP, Kakutani LM, Pavlovitch-Bedzyk AJ, Seaman MS, Howarth M, McGuire AT, Stamatatos L, Martin MA, West AP Jr, Nussenzweig MC, Bjorkman PJ. Science Immunology. 2023 Feb 17;8(80):eade6364 PDF+SI Abstract It is very difficult to get an HIV vaccine that protects against a wide range of strains. The CD4 binding site on HIV’s envelope protein is a good location for binding of antibodies that neutralize diverse strains. Here the envelope protein was evolved to be better at inducing such antibodies, towards a more protective vaccine. SpySwitch enables pH- or heat-responsive captureand release for plug-and-display nanoassembly Susan K. Vester, Rolle Rahikainen, Irsyad N. A. Khairil Anuar, Rory A. Hills, Tiong Kit Tan and Mark Howarth. Nature Communications 2022 June 13:3714 PDF+SI Abstract SpyTag technology has become established as a powerful tool for vaccine assembly. Here we design/evolve a switchable SpyCatcher to be a valuable approach for purification under mild conditions with high purity. We also find a patient-derived antibody has exceptional breadth of recognition across related bat viruses to SARS-CoV-2. Modular capsid decoration boosts adenovirus vaccine-induced humoral and cellular immunity against SARS-CoV-2 Matthew D. J. Dicks, Louisa M. Rose, Lesley A. H. Bowman, Carl Graham, Katie J. Doores, Michael H. Malim, Simon J. Draper, Mark Howarth, Sumi Biswas Molecular Therapy 2022 Aug PDF+SI Abstract Adenoviruses are important vectors for vaccines and cancer immunotherapy. They are particularly good at inducing cytotoxic T cell responses. This study shows how to covalently decorate the adeno surface to enhance the induction of antibody responses. This display also can shield from antibodies against the adenovirus, which may increase the possibility to use the same vector again and again. Simultaneous identification of viruses and SARS-CoV-2 variants with programmable DNA nanobait Filip Bošković, Jinbo Zhu, Ran Tivony, Alexander Ohmann, Kaikai Chen, Mohammed F. Alawami, Milan Đorđević, Niklas Ermann, Joana Pereira Dias, Michael Fairhead, Mark Howarth, Stephen Baker, Ulrich F. Keyser Nature Nanotechnology 2023 PDF+SI Combining DNA nanotechnology with nanopore analysis for sensitive multi- virus detection. Virus-like particles against infectious disease and cancer: guidance for the nanoarchitect Rory A. Hills and Mark Howarth Current Opinion in Biotechnology 2021 Oct 29;73:346-354 Full text Abstract How recent advances in nanoassembly, adjuvants, conjugation have helped to establish principles for designing vaccines and cancer immunotherapies. Gastrobodies are engineered antibody mimetics resilient to pepsin and hydrochloric acid Niels Wicke, Michael R. Bedford, Mark Howarth Communications Biology 2021 Aug 11;4(1):960 PDF+SI Abstract Wouldn’t it be good if protein therapeutics could be eaten? Here we generate a selectable protein scaffold by evolutionary and computational engineering of a plant inhibitor, towards this goal. We show that our binder to a C. difficile toxin is resilient to pH 1 + pepsin, as well as testing stability to other proteases and bile acids. DogCatcher allows loop-friendly protein-protein ligation Anthony H. Keeble, Vikash K. Yadav, Matteo P. Ferla, Claudia C. Bauer, Eulashini Chuntharpursat-Bon, Jin Huang, Robin S. Bon and Mark Howarth Cell Chemical Biology 2022 Feb 17;29(2):339-350. PDF + SI Abstract For molecular assembly (vaccines, enzymes etc.) it is often good to lock on through a loop rather than a terminus. We evolved the DogCatcher protein to be the fastest way to do such a reaction. NeissLock provides an inducible protein anhydride for covalent targeting of endogenous proteins Arne H. A. Scheu*, Sheryl Y. T. Lim*, Felix J. Metzner, Shabaz Mohammed, and Mark Howarth Nature Communications 2021 12:717 * joint first author PDF + SI Abstract Inspired by the surface chemistry of Neisseria meningitidis, we harness a genetically-encoded anhydride to trap protein-protein interactions. This may be useful for decoration of a range of therapeutic modalities. Neutralization of SARS-CoV-2 and zoonotic coronavirus threats by mosaic nanoparticle vaccination Alexander A Cohen, Priyanthi N.P. Gnanapragasam, Yu E. Lee, Susan Ou, Leesa M. Kakutani, Jennifer R Keeffe, Christopher O Barnes, Hung-Jen Wu, Mark Howarth, Anthony P West, Michel C. Nussenzweig, Pamela J Bjorkman. Science 12 Jan 2021 PDF+SI Abstract Various coronaviruses have the potential to cause human outbreaks. Display of RBD mixtures on the same virus-like particle induces antibodies that can neutralize multiple viruses. A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain induces potent neutralising antibody responses Tiong Kit Tan*, Pramila Rijal, Rolle Rahikainen, Anthony Keeble…Mark Howarth*, Alain Townsend*. Nature Communications 2021 12: 542 PDF+SI Abstract *corresponding authors Immunization of different animal models, indicating a strong antibody response to multiple sites on RBD, which should be hard for virus mutants to evade. Overcoming Symmetry Mismatch in Vaccine Nanoassembly via Spontaneous Amidation Rolle Rahikainen, Pramila Rijal, Tiong Kit Tan, Hung-Jen Wu , Anne-Marie C. Andersson, Jordan R. Barrett, Thomas A. Bowden, Simon J. Draper, Alain R. Townsend, Mark Howarth Angewandte Chemie 2020 PDF+SI Abstract Nanoassembly can play a key role in generating effective vaccines for emerging (SARS-CoV-2) and established (Influenza) pandemic threats. Spy-display allowed the nanocage to adapt to the challenge of bacterial or viral antigens with diverse cyclic and dihedral symmetries. Establishing display of both trimeric and tetrameric antigens may lead to an Influenza vaccine with broader protection. Power to the Protein: Enhancing and Combining Activities using the Spy Toolbox Anthony H. Keeble and Mark Howarth Chemical Science 2020 11, 7281 - 7291 PDF Abstract Review bringing together the different possibilities arising from precise unbreakable ligation for imaging, nanotechnology, enzymes etc. Transmembrane protein rotaxanes reveal kinetic traps in the refolding of translocated substrates Jianfei Feng, Pablo Martin-Baniandres, Michael J. Booth, Gianluca Veggiani, Mark Howarth, Hagan Bayley and David Rodriguez- Larrea Communications Biology, 2020 Apr 3;3(1):159. PDF+SI Abstract on Medline Nanopores have produced a revolution in the sequencing of DNA. This paper develops their ability to be used for protein analysis. SnoopLigase-Mediated Peptide-Peptide Conjugation and Purification. Buldun CM, Khairil Anuar INA, Howarth M. Methods Mol Biol. 2021;2208:13-31. Abstract on Medline Approaching infinite affinity through engineering of peptide–protein interaction Anthony H. Keeble, Paula Turkki, Samuel Stokes, Irsyad N. A. Khairil Anuar, Rolle Rahikainen, Vesa P. Hytönen and Mark Howarth PNAS 2019 December 26, 116 (52) 26523-26533. PDF+SI Abstract on Medline Design through computation and evolution of an interaction forming an amide bond with kinetics near the diffusion limit. We explore the system by HDX mass spectrometry, stop-flow FRET, flow cytometry and reconstitution of cellular mechanosensing. (See note on codon usage at Reagents page). Spy&Go purification of SpyTag-proteins using pseudo-SpyCatcher to access an oligomerization toolbox Irsyad N. A. Khairil Anuar, Anusuya Banerjee, Anthony H. Keeble, Alberto Carella, Georgi I. Nikov & Mark Howarth Nature Communications 2019 volume 10, Article number: 1734 PDF+SI Abstract on Medline A new route for affinity purification + platforms to plug in dimer to heptamer assembly, applied to activating cancer cell apoptosis. See our SpySwitch paper above for further optimized purification. SnoopLigase peptide-peptide conjugation enables modular vaccine assembly Anne-Marie C. Andersson, Can M. Buldun, David J. Pattinson, Simon J. Draper & Mark Howarth Sci Rep 2019 March, 9:4625 PDF+SI Abstract on Medline Covalent assembly for immunization, applied to enhance immune responses, particularly for malaria antigens or cancer neoepitopes. Insider information on successful covalent protein coupling with help from SpyBank Keeble AH, Howarth M. Methods in Enzymology 2019;617:443-461. PDF Abstract on Medline SpyInfo page Advice and survey on using SpyTag in various applications. (See note on codon usage at Reagents page). Assembling and decorating hyaluronan hydrogels with twin protein superglues to mimic cell-cell interactions Wieduwild R, Howarth M. Biomaterials. 2018 Jul 17;180:253-264. PDF Abstract on Medline Modular decoration of extracellular matrix in 3D cell culture to explore how adhesion signals program cancer behaviour. Engineering a Rugged Nanoscaffold To Enhance Plug-and-Display Vaccination. Bruun TUJ*, Andersson AC*, Draper SJ, Howarth M. ACS Nano. 2018 Jul 26. doi: 10.1021/acsnano.8b02805. PDF+SI Abstract on Medline *joint first author Rational enhancement of computationally- designed nanoparticle so easy to increase antigen immunogenicity, for malaria and other vaccine challenges. Nanobiotechnology: Baby steps and giant strides towards molecular mastery. Howarth M, Jaramillo A. Current Opinion in Biotechnology. 2018 Jun;51:iv-vi. doi: 10.1016/j.copbio.2018.05.001. New routes and opportunities for modular construction of particulate vaccines: stick, click and glue. Brune KD, Howarth M. Frontiers in Immunology 2018 Jun 26;9:1432. PDF Abstract on Medline Including personalized cancer vaccines, rapid response to pandemic threats and in-field vaccine assembly. SnoopLigase catalyzes peptide-peptide locking and enables solid-phase conjugate isolation. Buldun CM*, Jean JX*, Bedford MR, Howarth M. Journal of the American Chemical Society. 2018 Feb 28;140(8):3008-3018. PDF+SI Abstract on Medline *joint first author Rational and computational design of a new enzyme + its activity in making other enzymes super-resilient. Nanoteamwork: covalent protein assembly beyond duets towards protein ensembles and orchestras. Banerjee A and Howarth M. Current Opinion in Biotechnology 2017 Nov 21;51:16-23 PDF Abstract on Medline Analyzing progress and concepts for building cooperative and harmonious teams in synthetic biology. Amine Landscaping to Maximize Protein-Dye Fluorescence and Ultrastable Protein-Ligand Interaction. Jacobsen MT, Fairhead M, Fogelstrand P, Howarth M. Cell Chemical Biology 2017 Aug 17;24(8):1040-10 PDF Abstract on Medline Optimizing one of the most common protein modifications, NHS on amines, to generate Flavidin, fluorophore-friendly streptavidin for better imaging. Evolving accelerated amidation by SpyTag/SpyCatcher to analyze membrane dynamics. Keeble AH, Banerjee A, Ferla MP, Reddington SC, Khairil Anuar INA, Howarth M. Angewandte Chemie 2017 Dec 22;56(52):16521–16525 PDF Abstract on Medline Other papers/patents using SpyTag Phage display evolution of our bacterial superglue, allowing irreversible fluorescent tracking of an enterohaemorrhagic platform at the bacterial outer membrane. Smart superglue in streptococci? The proof is in the pulling Howarth M. Journal of Biological Chemistry 2017 May 26;292(21):8998-8999 PDF Abstract on Medline News and Views on mechanical proof-reading in bacterial adhesion Dual plug-and-display synthetic assembly using orthogonal reactive proteins for twin antigen immunization Brune KD, Buldun CM, Li Y, Taylor IJ, Brod F, Biswas S, Howarth M. Bioconjugate Chemistry 2017 May 17;28(5):1544-1551 PDF Abstract on Medline Accelerating vaccine development for multi-strain or multi-disease protection. Extracellular self-assembly of functional and tunable protein conjugates from Bacillus subtilis Gilbert C, Howarth M, Harwood CR, Ellis T. ACS Synthetic Biology 2017 Jun 16;6(6):957-967 PDF Abstract on Medline SpyRing enzyme stabilization (for xylanase, relevant to food, paper, biofuel), assembled by one cell or by cell cooperation. Nanoassembly routes stimulate conflicting antibody quantity and quality for transmission blocking malaria vaccines. Leneghan DB, Miura K, Taylor IJ, Li Y, Jin J, Brune KD, Bachmann MF, Howarth M, Long CA, Biswas S. Sci Rep 2017 7:3811 PDF Abstract on Medline Pushing for the best immunogen, how antibody titre and activity differ in stopping mosquitoes getting infected. Controlling multivalent binding through surface chemistry: model study on streptavidin Dubacheva GV, Araya C, Fairhead M, Codée J, Howarth M, Richter RP. Journal of the American Chemical Society 2017 Mar 22;139(11):4157-4167 PDF Full text Abstract on Medline Streptavidin can steal biotinylated lipids from membranes A single molecule assay to probe monovalent and multivalent bonds between hyaluronan and its key leukocyte receptor CD44 under force Bano F, Banerji S, Howarth M, Jackson DG, Richter RP. Sci Rep 2016 Sep 29;6:34176 PDF+SI Abstract on Medline Understanding extracellular matrix interaction for recruiting immune cells and anchoring circulating tumour cells Programmable polyproteams built using twin peptide superglues Veggiani G, Nakamura T, Brenner MD, Gayet RV, Yan J, Robinson CV, Howarth M. PNAS 2016 Feb 2;113(5):1202-7 PDF+SI Abstract on Medline A platform to build molecular teams, here applied to optimize cancer cell killing, combining ligation of a Death Receptor with Growth Factor receptors. Plug-and-Display: decoration of Virus-Like Particles via isopeptide bonds for modular immunization Brune KD, Leneghan DB, Brian IJ, Ishizuka AS, Bachmann MF, Draper SJ, Biswas S, Howarth M. Sci Rep 2016 Jan 19;6:19234 PDF+SI Press release Abstract on Medline A strategy to accelerate vaccine generation, which could have broad application for cancer immunotherapy and infectious diseases. SpyRings Declassified: A Blueprint for Using Isopeptide-Mediated Cyclization to Enhance Enzyme Thermal Resilience C. Schoene, S.P. Bennett, M. Howarth. Methods in Enzymology 2016;580:149-67 PDF Abstract on Medline SpyRing interrogation: analyzing how enzyme resilience can be achieved with phytase and distinct cyclization chemistries Schoene C, Bennett SP, Howarth M. Sci Rep 2016 Feb 10;6:21151 PDF+SI Abstract on Medline Understanding a generic approach for enhancing enzyme resilience Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher Reddington SC, Howarth M. Current Opinion in Chemical Biology 2015 Oct 27;29:94-99 PDF Abstract on Medline Say it with proteins: an alphabet of crystal structures Howarth M. Nature Structural & Molecular Biology 2015 May;22(5):349 PDF For video, files to download and more information Outreach project. Happy that >1000 views on YouTube, but a while to go before into Justin Bieber territory. Site-Specific Biotinylation of Purified Proteins Using BirA Fairhead M, Howarth M. Methods in Molecular Biology 2015;1266:171-84 PDF Abstract on Medline SpyLigase peptide–peptide ligation polymerizes affibodies to enhance magnetic cancer cell capture Fierer J.O.*, Veggiani G.*, Howarth M. PNAS 2014;111(13):E1176-81 *joint first author PDF+SI Abstract on Medline Developing a new way to irreversibly lock two peptides (via a 3-way split protein) to assemble tentacle-like polymers of affibodies/antibodies for improving sensitivity of isolation of cancerous cells. SpyAvidin Hubs Enable Precise and Ultrastable Orthogonal Nanoassembly Fairhead M, Veggiani G, Lever M, Yan J, Mesner D, Robinson CV, Dushek O, van der Merwe PA, Howarth M. Journal of the American Chemical Society 2014;136(35):12355-63 PDF Supporting Info. Abstract on Medline Robinson Lab van der Merwe lab Combining one of the best interactions in nature with our bacterial superglue. SpyAvidins should help detection of weak interactions, such as for finding anti-cancer T cells. Superglue from Bacteria: Unbreakable Bridges for Protein Nanotechnology Veggiani G., Zakeri B., Howarth M. Trends in Biotechnology 2014 Oct;32(10):506-12 PDF Abstract on Medline Love-Hate ligands for high resolution analysis of strain in ultra-stable protein:small molecule interaction Fairhead M, Shen D, Chan LK, Lowe ED, Donohoe TJ, Howarth M. Bioorganic & Medicinal Chemistry 2014 Oct 1;22(19):5476-86 PDF Supporting Info. Abstract on Medline Donohoe lab PDB entries: SA-LH1, SA-LH3, SA-LH4, A86D-LH4 A novel chemical biology approach to understand how force distorts protein structure. SpyTag/SpyCatcher Cyclization Confers Resilience to Boiling on a Mesophilic Enzyme Schoene C., Fierer J.O., Bennett S.P., Howarth M. Angewandte Chemie 2014 Jun 10;53(24):6101-4. PDF+SI Abstract on Medline BBSRC News story Towards a general way to make enzymes that can tolerate tough conditions (e.g. for diagnostics or for biofuels), we found that locking the tails together with our bacterial superglue gave dramatic increases in resilience. Structural Analysis and Optimization of the Covalent Association between SpyCatcher and a Peptide Tag Li L., Fierer J.O., Rapoport T.A., Howarth M. Journal of Molecular Biology 2014 Jan 23;426(2):309-317 PDF Supporting Info. Abstract on Medline PDB entries: SpyTag/SpyCatcher, SpyTag/SpyCatcherΔN1 Crystal structure of the SpyCatcher/SpyTag complex, a genetically encoded peptide forming a spontaneous isopeptide bond to its protein partner, and minimization of the size of SpyCatcher. Plug-And-Play Pairing Via Defined Divalent Streptavidins Fairhead M, Krndija D, Lowe ED, Howarth M. Journal of Molecular Biology 2014 Jan 9;426(1):199-214 PDF Abstract on Medline PDB entries: cis-divalent, trans-divalent, trans-divalent with biotin-fluorescein Streptavidin is one of the most widely used building blocks in biology and also has given promising results for improving delivery of cancer radiotherapy. We developed charged tags to enable isolation of precise tetramers for robust nanoassembly and validated the structures by crystallography. Cholesterol loading and ultrastable protein interactions determine the level of tumor marker required for optimal isolation of cancer cells. Jain J, Veggiani G, Howarth M. Cancer Research 2013 Apr 1;73(7):2310-21 PDF Abstract on Medline Supporting Info. Detecting the circulating tumor cells in blood samples is one of the most promising ways to achieve early cancer diagnosis and rapid feedback on cancer treatment. Here we establish the key factors for magnetic cancer cell isolation and use this insight to enhance the isolation of cells expressing low levels of cancer marker. Described for a general audience on the Oxford University Science Blog. Hydroxy-Terminated Conjugated Polymer Nanoparticles Have Near-Unity Bright Fraction and Reveal Cholesterol- Dependence of IGF1R Nanodomains. Koner AL, Krndija D, Hou Q, Sherratt DJ, Howarth M. ACS Nano 2013 Feb 26;7(2):1137-44 PDF Abstract on Medline Supporting Info. Single molecule imaging is a powerful way to gain insight into receptor function. Here we show that Conjugated Polymer Nanoparticles may be the best nanoparticles for single molecule imaging, because, unlike quantum dots, these particles do not blink and there are almost no "ghost" particles which fail to emit. Quantum Dot Targeting with Lipoic Acid Ligase and HaloTag for Single Molecule Imaging on Living Cells. Liu DS, Phipps WS, Loh KH, Howarth M, Ting AY. ACS Nano 2012 Dec 21;6(12):11080-7. PDF Abstract on Medline Supporting Info. Using two re-designed enzymes, a covalent connection can be made between a peptide-tagged cell surface protein and a fluorescent nanoparticle. This enabled two different types of adhesion molecules to be followed at the single molecule level on living cells. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Zakeri B*, Fierer JO*, Celik E, Chittock EC, Schwarz-Linek U, Moy VT, Howarth M. PNAS 2012 Mar 20;109(12):E690-7. PDF Abstract on Medline Supporting Info. *joint first author Graphic design with Myoungshin Kim 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 (SpyTag with SpyCatcher). This may be useful as a cellular padlock, for imaging and to enable new protein architectures. (Plasmids available from Addgene repository or directly from us.) Described for a general audience on the Oxford University Science Blog or by Bijan in a TED talk. Other papers/patents using SpyTag 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. Mechanisms for size-dependent protein segregation at immune synapses assessed 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 (virally infected or cancerous) 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. PDF Abstract on Medline PDB entries: apo-traptavidin biotin-traptavidin 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. 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. (Traptavidin protein and plasmids available.) 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. Sherratt lab Grainge lab 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. Proof of principle for spontaneous covalent bond formation to a peptide, developed from a pilin and based on Lysine reaction with Asparagine. See also our PNAS 2012 paper. 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 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. Bawendi lab 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. PDF Abstract on Medline 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 from DNA vaccination. Protein imaging in live mammalian cells by site-specific labeling with biotin ligase and monovalent streptavidin. Howarth M, Ting AY. Nature Protocols 2008;3(3):534-45. PDF Abstract on Medline 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 protein available.) Note in our current protocol we use 5 min with a stir-bar to resuspend inclusion bodies and reduce pipetting effort, while our J Mol Biol paper 2014 describes an easier chromatographic separation. Giving cells a new sugar-coating. Howarth M, and Ting AY. Nature Chemical Biology 2006 Mar;2(3):127-8 PDF Abstract on Medline News and Views article on a paper by Kevin Yarema 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. Howarth M, Chinnapen DJ-F, Gerrow K., Dorrestein PC, Grandy MR, Kelleher NL, El-Husseini A, Ting AY. Nature Methods 2006, Apr;3(4):267-73 PDF Abstract on Medline News and Views Supp. Info. Dorrestein lab 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. Our J Mol Biol paper 2014 describes an easier chromatographic separation of the monovalent and divalent forms. (Monovalent streptavidin protein and plasmids available.) Targeting quantum dots to surface proteins in living cells with biotin ligase. Howarth M, Takao K, Hayashi Y, and Ting AY. PNAS 2005, May;102(21):7583-8 PDF Abstract on Medline Supp. Info./Movie Hayashi Lab 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 Mark Bradley lab 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. PDF Supporting info. Abstract on Medline 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 consequences for autoimmune disease and vaccine design. The processing of antigens delivered as DNA vaccines. Howarth M, Elliott T. Immunological Reviews 2004 199:27-39 PDF Abstract on Medline 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 PDF Abstract on Medline Bin Gao lab Calreticulin is a multi-talented protein, involved in calcium homeostasis, gene regulation, and folding of secretory proteins. Here we tested how the presence of calreticulin changed the loading of MHC class I with peptide and its exit from the endoplasmic reticulum. Processing and presentation of glycoproteins in the MHC class I and II antigen presentation pathways. Golgher DB, Elliott T, Howarth M. Immunobiology of Carbohydrates, chapter, Landes Biosciences, 2002. PDF 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 PDF Abstract on Medline 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. Switchable polypeptide and its use for gentle affinity purification. (SpySwitch) Howarth M, Khairil Anuar I.N.A., Rahikainen R. 2021 UK Intellectual Property Office 2117283.8 Polypeptides that interact with peptide tags at loops or termini and uses thereof. (DogCatcher) Howarth M, Yadav V, Ferla M. 2021 UK Intellectual Property Office 2104999.4 PDF Ligand-binding polypeptides and uses thereof. (Gastrobodies) Howarth M, Wicke N. 2021 UK Intellectual Property Office 2019817.2 PDF System for covalently linking proteins. (NeissLock) Howarth M, Scheu AHA, Lim SYT. 2020 UK Intellectual Property Office 2003683.6 PDF Viruses with modified capsid proteins. Dicks M, Howarth M, Biswas S. 2019 UK Intellectual Property Office 1915905.2 PDF Polypeptide with enhanced rate of spontaneous isopeptide bond formation with its peptide tag partner and uses thereof. (Spy003) Howarth M, Keeble A, Carella A. 2019 UK Intellectual Property Office 1903479.2 PDF Polypeptide and its use in affinity purification. (Spy&Go) Howarth M, Khairil Anuar I.N.A. 2018 UK Intellectual Property Office 1819850.7 PDF Peptide ligase and use thereof. (SnoopLigase) Howarth M, Buldun C. 2017 UK Intellectual Property Office 1705750.6 PDF Proteins and peptide tags with enhanced rate of spontaneous isopeptide bond formation and uses thereof. (Spy002) Howarth M, Keeble A. 2017 UK Intellectual Property Office 1706430.4 PDF Mutant streptavidin with improved fluorescent brightness and uses thereof. Howarth M, Jacobsen MT. 2016 UK Intellectual Property Office 1616051.7 PDF Methods and products for fusion protein synthesis Howarth M, Veggiani G, Gayet R. 2015 UK Intellectual Property Office 1509782.7, United Kingdom Patent application (described by Oxford University Innovation) Peptide tag systems that spontaneously form an irreversible link to protein partners via isopeptide bonds. Howarth M, Zakeri B. 2010, UK Intellectual Property Office 1002362.0 for PDF High Stability Streptavidin Mutant Proteins. (Traptavidin) Howarth M, Chivers C. 2009, UK Intellectual Property Office 0919102.4 PDF Controlled modification of semiconductor nanocrystals. Ting AY, Bawendi MG, Howarth M, Liu W. 2007, US Patent and Trademark Office. for PDF Monovalent Streptavidin compositions. Ting AY, Howarth M. 2006, US Patent and Trademark Office. for PDF