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Durham University

Biophysical Sciences Institute

Publications

Recent Publications


Published in Molecular and Cellular Proteomics, 1st June 2019 (18 (6) 1123-1137)

Reductive Stress Selectively Disrupts Collagen Homeostasis and Modifies Growth Factor-independent Signaling Through the MAPK/Akt Pathway in Human Dermal Fibroblasts

Naomi A. Carne, Steven Bell, Adrian P. Brown, Arto Määttä, Michael J. Flagler, Adam M. Benham

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Abstract:

Redox stress is a well-known contributor to aging and diseases in skin. Reductants such as dithiothreitol (DTT) can trigger a stress response by disrupting disulfide bonds. However, the quantitative response of the cellular proteome to reductants has not been explored, particularly in cells such as fibroblasts that produce extracellular matrix proteins. Here, we have used a robust, unbiased, label-free SWATH-MS proteomic approach to quantitate the response of skin fibroblast cells to DTT in the presence or absence of the growth factor PDGF. Of the 4487 proteins identified, only 42 proteins showed a statistically significant change of 2-fold or more with reductive stress. Our proteomics data show that reductive stress results in the loss of a small subset of reductant-sensitive proteins (including the collagens COL1A1/2 and COL3A1, and the myopathy-associated collagens COL6A1/2/3), and the down-regulation of targets downstream of the MAPK pathway. We show that a reducing environment alters signaling through the PDGF-associated MAPK/Akt pathways, inducing chronic dephosphorylation of ERK1/2 at Thr202/Tyr204 and phosphorylation of Akt at Ser473 in a growth factor-independent manner. Our data highlights collagens as sentinel molecules for redox stress downstream of MAPK/Akt, and identifies intervention points to modulate the redox environment to target skin diseases and conditions associated with erroneous matrix deposition.


The paradoxical cage made out of 24 nearly regular hendecagonal faces. The face edge lengths are identical within 0.5% and the angles within 0.27%

Published in Nature, 8th May 2019. (vol 569, pg 438–442)

An ultra-stable gold-coordinated protein cage displaying reversible assembly

Ali D. Malay, Naoyuki Miyazaki, Artur Biela, Soumyananda Chakraborti, Karolina Majsterkiewicz, Izabela Stupka, Craig S. Kaplan, Agnieszka Kowalczyk, Bernard M.A.G. Piette, Georg K.A. Hochberg, Di Wu, Tomasz P. Wrobel, Adam Fineberg, Manish S. Kushwah, Mitja Kelemen, Primož Vavpetič, Primož Pelicon, Philipp Kukura, Justin L.P. Benesch, Kenji Iwasaki, Jonathan G. Heddle

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Abstract:

Symmetrical protein cages have evolved to fulfil diverse roles in nature, including compartmentalization and cargo delivery, and have inspired synthetic biologists to create novel protein assemblies via the precise manipulation of protein–protein interfaces. Despite the impressive array of protein cages produced in the laboratory, the design of inducible assemblies remains challenging. Here we demonstrate an ultra-stable artificial protein cage, the assembly and disassembly of which can be controlled by metal coordination at the protein–protein interfaces. The addition of a gold (I)- triphenylphosphine compound to a cysteine-substituted, 11-mer protein ring triggers supramolecular self-assembly, which generates monodisperse cage structures with masses greater than 2 MDa. The geometry of these structures is based on the Archimedean snub cube and is, to our knowledge, unprecedented.
Cryo-electron microscopy confirms that the assemblies are held together by 120 S–Au–S staples between the protein oligomers, and exist in two chiral forms. The cage shows extreme chemical and thermal stability, yet it readily disassembles upon exposure to reducing agents. As well as gold, mercury is also found to enable formation of the protein cage. This work establishes an approach for linking protein components into robust, higher-order structures, and expands the design space available for pramolecular assemblies to include previously unexplored geometries.

Published in The Journal of Physical Chemistry, 9th April 2019 (vol 10, 9, pg 2244-2249)

Relative Binding Energies Predict Crystallographic Binding Modes of Ethionamide Booster Lead Compounds

Natalie J. Tatum, Fernanda Duarte , Shina C. L. Kamerlin , and Ehmke Pohl*§

Department of Chemistry, Durham University, South Road, Durham DH1 3LE, U.K.
Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, U.K.
Department of Chemistry - BMC, Uppsala University, BMC Box 576, S-751 23 Uppsala, Sweden
Department of Biosciences, Durham University, Durham DH1 3LE, U.K.
§ Biophysical Sciences Institute, Durham University, Durham DH1 3LE, U.K.
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Abstract:
Transcriptional repressor EthR from Mycobacterium tuberculosis is a valuable target for antibiotic booster drugs. We previously reported a virtual screening campaign to identify EthR inhibitors for development. Two ligand binding orientations were often proposed, though only the top scoring pose was utilized for filtering of the large data set. We obtained biophysically validated hits, some of which yielded complex crystal structures. In some cases, the crystallized binding mode and top scoring mode agree, while for others an alternate ligand binding orientation was found. In this contribution, we combine rigid docking, molecular dynamics simulations, and the linear interaction energy method to calculate binding free energies and derive relative binding energies for a number of EthR inhibitors in both modes. This strategy allowed us to correctly predict the most favorable orientation. Therefore, this widely applicable approach will be suitable to triage multiple binding modes within EthR and other potential drug targets with similar characteristics.


Published in the Journal of the American Chemical Society, 9th February 2019 (vol 141, 8, pg 3430-3434)

Fluorinated Aromatic Monomersas Building Blocks To Control α-Peptoid Conformation and Structure

Diana Gimenez, Guangfeng Zhou, Matthew F.D.Hurley, Juan A.Aguilar, Vincent A.Voelz, Steven Cobb

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Abstract:

Peptoids are peptidomimetics of interest in the fields of drug development and biomaterials. However, obtaining stable secondary structures is challenging, and designing these requires effective control of the peptoid tertiary amide cis/trans equilibrium. Herein, we report new fluorine-containing aromatic monomers that can control peptoid conformation. Specifically, we demonstrate that a fluoro-pyridine group can be used to circumvent the need for monomer chirality to control the cis/trans equilibrium. We also show that incorporation of a trifluoro-methyl group (NCF3Rpe) rather than a methyl group (NRpe) at the α-carbon of a monomer gives rise to a 5-fold increase in cis-isomer preference.


Published in PLOS ONE, 28th November 2018

A statistical framework for radiation dose estimation with uncertainty quantification from the γ-H2AX assay

Jochen Einbeck, Elizabeth A. Ainsbury, Rachel Sales, Stephen Barnard, Felix Kaestle, Manuel Higueras

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Abstract:

Over the last decade, the γ–H2AX focus assay, which exploits the phosphorylation of the H2AX histone following DNA double–strand–breaks, has made considerable progress towards acceptance as a reliable biomarker for exposure to ionizing radiation. While the existing literature has convincingly demonstrated a dose–response effect, and also presented approaches to dose estimation based on appropriately defined calibration curves, a more widespread practical use is still hampered by a certain lack of discussion and agreement on the specific dose–response modelling and uncertainty quantification strategies, as well as by the unavailability of implementations. This manuscript intends to fill these gaps, by stating explicitly the statistical models and techniques required for calibration curve estimation and subsequent dose estimation. Accompanying this article, a web applet has been produced which implements the discussed methods.


Published in ACS Medicinal Chemistry Letters, 9th November 2018 (9 (12) 1297-1300)

Novel Fluorescence Competition Assay for Retinoic Acid Binding Proteins

Charles W.E. Tomlinson, David R. Chisholm, Roy Valentine, Andrew Whiting, Ehmke Pohl

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Abstract:

Vitamin A derived retinoid compounds have multiple, powerful roles in the cellular growth and development cycle and, as a result, have attracted significant attention from both academic and pharmaceutical research in developing and characterizing synthetic retinoid analogues. Simplifying the hit development workflow for retinoid signaling will improve options available for tackling related pathologies, including tumor growth and neurodegeneration. Here, we present a novel assay that employs an intrinsically fluorescent synthetic retinoid, DC271, which allows direct measurement of the binding of nonlabeled compounds to relevant proteins. The method allows for straightforward initial measurement of binding using existing compound libraries and is followed by calculation of binding constants using a dilution series of plausible hits. The ease of use, high throughput format, and measurement of both qualitative and quantitative binding offer a new direction for retinoid-related pharmacological development.


Contact Details

Biophysical Sciences Institute
Durham University.
South Road,
Durham,
DH1 3LE

+44 (0)191 334 2351

admin.bsi@durham.ac.uk