Publications
142 results found
Carlsson E, Thwaite JE, Jenner DC, et al., 2016, Bacillus anthracis TIR Domain-Containing Protein Localises to Cellular Microtubule Structures and Induces Autophagy, PLOS One, Vol: 11, ISSN: 1932-6203
Toll-like receptors (TLRs) recognise invading pathogens and mediate downstream immune signalling via Toll/IL-1 receptor (TIR) domains. TIR domain proteins (Tdps) have been identified in multiple pathogenic bacteria and have recently been implicated as negative regulators of host innate immune activation. A Tdp has been identified in Bacillus anthracis, the causative agent of anthrax. Here we present the first study of this protein, designated BaTdp. Recombinantly expressed and purified BaTdp TIR domain interacted with several human TIR domains, including that of the key TLR adaptor MyD88, although BaTdp expression in cultured HEK293 cells had no effect on TLR4- or TLR2- mediated immune activation. During expression in mammalian cells, BaTdp localised to microtubular networks and caused an increase in lipidated cytosolic microtubule-associated protein 1A/1B-light chain 3 (LC3), indicative of autophagosome formation. In vivo intra-nasal infection experiments in mice showed that a BaTdp knockout strain colonised host tissue faster with higher bacterial load within 4 days post-infection compared to the wild type B. anthracis. Taken together, these findings indicate that BaTdp does not play an immune suppressive role, but rather, its absence increases virulence. BaTdp present in wild type B. anthracis plausibly interact with the infected host cell, which undergoes autophagy in self-defence.
Byrne B, Alguel Y, Scull NJ, et al., 2016, Structure of eukaryotic purine/Hþ symporter UapA suggests a role for homodimerization in transport activity, Nature Communications, Vol: 7, Pages: 1-9, ISSN: 2041-1723
The uric acid/xanthine H+ symporter, UapA, is a high-affinity purine transporter from the filamentous fungus Aspergillus nidulans. Here we present the crystal structure of a genetically stabilized version of UapA (UapA-G411VΔ1–11) in complex with xanthine. UapA is formed from two domains, a core domain and a gate domain, similar to the previously solved uracil transporter UraA, which belongs to the same family. The structure shows UapA in an inward-facing conformation with xanthine bound to residues in the core domain. Unlike UraA, which was observed to be a monomer, UapA forms a dimer in the crystals with dimer interactions formed exclusively through the gate domain. Analysis of dominant negative mutants is consistent with dimerization playing a key role in transport. We postulate that UapA uses an elevator transport mechanism likely to be shared with other structurally homologous transporters including anion exchangers and prestin.
Hussain H, Du Y, Scull NJ, et al., 2016, Accessible mannitol-based amphiphiles (MNAs) for membrane protein solubilisation and stabilisation, Chemistry, Vol: 22, ISSN: 0861-9255
Integral membrane proteins are amphipathic molecules crucial for all cellular life. The structural study of these macromolecules starts with protein extraction from the native membranes, followed by purification and crystallisation. Detergents are essential tools for these processes, but detergent-solubilised membrane proteins often denature and aggregate, resulting in loss of both structure and function. In this study, a novel class of agents, designated mannitol-based amphiphiles (MNAs), were prepared and characterised for their ability to solubilise and stabilise membrane proteins. Some of MNAs conferred enhanced stability to four membrane proteins including a G protein-coupled receptor (GPCR), the β2 adrenergic receptor (β2AR), compared to both n-dodecyl-d-maltoside (DDM) and the other MNAs. These agents were also better than DDM for electron microscopy analysis of the β2AR. The ease of preparation together with the enhanced membrane protein stabilisation efficacy demonstrates the value of these agents for future membrane protein research.
Ehsan M, Du Y, Scull NJ, et al., 2016, Highly branched pentasaccharide-bearing amphiphiles for membrane protein studies, Journal of the American Chemical Society, Vol: 138, Pages: 3789-3796, ISSN: 1520-5126
Detergents are essential tools for membrane protein manipulation. Micelles formed by detergent molecules have the ability to encapsulate the hydrophobic domains of membrane proteins. The resulting protein–detergent complexes (PDCs) are compatible with the polar environments of aqueous media, making structural and functional analysis feasible. Although a number of novel agents have been developed to overcome the limitations of conventional detergents, most have traditional head groups such as glucoside or maltoside. In this study, we introduce a class of amphiphiles, the PSA/Es with a novel highly branched pentasaccharide hydrophilic group. The PSA/Es conferred markedly increased stability to a diverse range of membrane proteins compared to conventional detergents, indicating a positive role for the new hydrophilic group in maintaining the native protein integrity. In addition, PDCs formed by PSA/Es were smaller and more suitable for electron microscopic analysis than those formed by DDM, indicating that the new agents have significant potential for the structure–function studies of membrane proteins.
Sadaf A, Mortensen JS, Capaldi S, et al., 2015, A class of rigid linker-bearing glucosides for membrane protein structural study, Chemical Science, Vol: 7, Pages: 1933-1939, ISSN: 2041-6539
Membrane proteins are amphipathic bio-macromolecules incompatible with the polar environments ofaqueous media. Conventional detergents encapsulate the hydrophobic surfaces of membrane proteinsallowing them to exist in aqueous solution. Membrane proteins stabilized by detergent micelles are usedfor structural and functional analysis. Despite the availability of a large number of detergents, only a fewagents are sufficiently effective at maintaining the integrity of membrane proteins to allow successfulcrystallization. In the present study, we describe a novel class of synthetic amphiphiles with a branchedtail group and a triglucoside head group. These head and tail groups were connected via an amide orether linkage by using a tris(hydroxylmethyl)aminomethane (TRIS) or neopentyl glycol (NPG) linker toproduce TRIS-derived triglucosides (TDTs) and NPG-derived triglucosides (NDTs), respectively. Membersof this class conferred enhanced stability on target membrane proteins compared to conventionaldetergents. Because of straightforward synthesis of the novel agents and their favourable effects ona range of membrane proteins, these agents should be of wide applicability to membrane protein science.
Carlsson E, Ding JL, Byrne B, 2015, SARM modulates MyD88-mediated TLR activation through BB-loop dependent TIR-TIR interactions., Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Vol: 1863, Pages: 244-253, ISSN: 0167-4889
Toll-like receptors (TLRs) recognise invading pathogens and initiate an innate immune response by recruiting intracellular adaptor proteins via heterotypic Toll/interleukin-1 receptor (TIR) domain interactions. Of the five TIR domain-containing adaptor proteins identified, Sterile α- and armadillo-motif-containing protein (SARM) is functionally unique; suppressing immune signalling instead of promoting it. Here we demonstrate that the recombinantly expressed and purified SARM TIR domain interacts with both the major human TLR adaptors, MyD88 and TRIF. A single glycine residue located in the BB-loop of the SARM TIR domain, G601, was identified as essential for interaction. A short peptide derived from this domain was also found to interact with MyD88 in vitro. SARM expression in HEK-293 cells was found to significantly suppress lipopolysaccharide (LPS)-mediated upregulation of inflammatory cytokines, IL-8 and TNF-α, an effect lost in the G601A mutant. The same result was observed with cytokine activation initiated by MyD88 expression and stimulation of TLR2 with lipoteichoic acid (LTA), suggesting that SARM is capable of suppressing both TRIF- and MyD88- dependent TLR signalling. Our findings indicate that SARM acts on a broader set of target proteins than previously thought, and that the BB-loop motif is functionally important, giving further insight into the endogenous mechanisms used to suppress inflammation in immune cells.
Byrne B, Kazarian SG, Boulet-Audet M, 2015, Cleaning-in-place for immunoaffinity resin monitored by in situ ATR-FTIR spectroscopy, Analytical and Bioanalytical Chemistry, Vol: 407, Pages: 7111-7122, ISSN: 1618-2650
In the next ten years, the pharmaceutical industry anticipates that revenue from biotherapeutics will overtake those generated from small drug molecules. Despite effectively treating a range of chronic and life-threatening diseases, the high cost of biotherapeutics limits their use. For biotherapeutic monoclonal antibodies (mAbs), an important production cost is the affinity resin used for protein capture. Cleaning-in-place (CIP) protocols aim to optimise the lifespan of the resin by slowing binding capacity decay. Binding assays can determine resin capacity from the mobile phase, but do not reveal the underlying causes of Protein A ligand degradation. The focus needs to be on the stationary phase to examine the effect of CIP on the resin. To directly determine both the local Protein A ligand concentration and conformation on two Protein A resins, we developed a method based on attenuated total reflection (ATR) Fourier Transform Infrared (FTIR) spectroscopy. ATR-FTIR spectroscopic imaging revealed that applying a carefully controlled load to agarose beads produces an even and reproducible contact with the internal reflection element. This allowed detection and quantification of the binding capacity of the stationary phase. ATR-FTIR also showed that Protein A proteolysis does not seem to occur under typical CIP conditions (below 1M NaOH). However, our data revealed that concentrations of NaOH above 0.1 M cause significant changes in Protein A conformation. The addition of >0.4 M trehalose during CIP significantly reduced NaOH-induced ligand unfolding observed for one of the two Protein A resins tested. Such insights could help to optimise CIP protocols in order to extend resin lifetime and reduce mAb production costs.
Bae HE, Gotfryd K, Thomas J, et al., 2015, Deoxycholate-Based Glycosides (DCGs) for Membrane Protein Stabilisation, CHEMBIOCHEM, Vol: 16, Pages: 1454-1459, ISSN: 1439-4227
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- Citations: 5
Martzoukou O, Karachaliou M, Yalelis V, et al., 2015, Oligomerization of the UapA purine transporter Is critical for ER-exit, plasma membrane localization and turnover, Journal of Molecular Biology, Vol: 427, Pages: 2679-2696, ISSN: 1089-8638
Central to the process of transmembrane cargo trafficking is the successful folding and exit from the ER (endoplasmic reticulum) through packaging in COPII vesicles. Here, we use the UapA purine transporter of Aspergillus nidulans to investigate the role of cargo oligomerization in membrane trafficking. We show that UapA oligomerizes (at least dimerizes) and that oligomerization persists upon UapA endocytosis and vacuolar sorting. Using a validated bimolecular fluorescence complementation assay, we provide evidence that a UapA oligomerization is associated with ER-exit and turnover, as ER-retained mutants due to either modification of a Tyr-based N-terminal motif or partial misfolding physically associate but do not associate properly. Co-expression of ER-retained mutants with wild-type UapA leads to in trans plasma membrane localization of the former, confirming that oligomerization initiates in the ER. Genetic suppression of an N-terminal mutation in the Tyr motif and mutational analysis suggest that transmembrane α-helix 7 affects the oligomerization interface. Our results reveal that transporter oligomerization is essential for membrane trafficking and turnover and is a common theme in fungi and mammalian cells.
Cho KH, Du Y, Scull NJ, et al., 2015, Novel Xylene-linked Maltoside Amphiphiles (XMAs) for membrane protein stabilisation, Chemistry - A European Journal, Vol: 21, Pages: 10008-10013, ISSN: 0947-6539
Membrane proteins are key functional players in biological systems. These biomacromolecules contain both hydrophilic and hydrophobic regions and thus amphipathic molecules are necessary to extract membrane proteins from their native lipid environments and stabilise them in aqueous solutions. Conventional detergents are commonly used for membrane protein manipulation, but membrane proteins surrounded by these agents often undergo denaturation and aggregation. In this study, a novel class of maltoside-bearing amphiphiles, with a xylene linker in the central region, designated xylene-linked maltoside amphiphiles (XMAs) was developed. When these novel agents were evaluated with a number of membrane proteins, it was found that XMA-4 and XMA-5 have particularly favourable efficacy with respect to membrane protein stabilisation, indicating that these agents hold significant potential for membrane protein structural study.
Carrara G, Saraiva N, Parsons M, et al., 2015, Golgi anti-apoptotic proteins are highly conserved ion channels that affect apoptosis and cell migration, Journal of Biological Chemistry, Vol: 290, Pages: 11785-11801, ISSN: 0021-9258
Golgi anti-apoptotic proteins (GAAPs) are multitransmembrane proteins that are expressed in the Golgi apparatus and are able to homo-oligomerize. They are highly conserved throughout eukaryotes and are present in some prokaryotes and orthopoxviruses. Within eukaryotes, GAAPs regulate the Ca2+ content of intracellular stores, inhibit apoptosis, and promote cell adhesion and migration. Data presented here demonstrate that purified viral GAAPs (vGAAPs) and human Bax inhibitor 1 form ion channels and that vGAAP from camelpox virus is selective for cations. Mutagenesis of vGAAP, including some residues conserved in the recently solved structure of a related bacterial protein, BsYetJ, altered the conductance (E207Q and D219N) and ion selectivity (E207Q) of the channel. Mutation of residue Glu-207 or -178 reduced the effects of GAAP on cell migration and adhesion without affecting protection from apoptosis. In contrast, mutation of Asp-219 abrogated the anti-apoptotic activity of GAAP but not its effects on cell migration and adhesion. These results demonstrate that GAAPs are ion channels and define residues that contribute to the ion-conducting pore and affect apoptosis, cell adhesion, and migration independently.Background: GAAPs regulate intracellular Ca2+ fluxes, cell migration, and apoptosis.Results: GAAP forms a cation-selective channel, and residues involved in its ion-conducting properties were identified.Conclusion: Mutations within the pore demonstrate that GAAP effects on apoptosis and migration are separable.Significance: Characterization of the pore region of GAAP provides insight into the mechanism of action of this novel and highly conserved ion channel.
Byrne B, 2015, Pichia pastoris as an expression host for membrane protein structural biology, Current Opinion in Structural Biology, Vol: 32, Pages: 9-17, ISSN: 0959-440X
The methylotrophic yeast Pichia pastoris is a widely used recombinant expression host. P. pastoris combines the advantages of ease of use, relatively rapid expression times and low cost with eukaryotic co-translational and post-translational processing systems and lipid composition. The suitability of P. pastoris for high density controlled culture in bioreactors means large amounts of protein can be obtained from small culture volumes. This review details the key features of P. pastoris, which have made it a particularly useful system for the production of membrane proteins, including receptors, channels and transporters, for structural studies. In addition, this review provides an overview of all the constructs and cell strains used to produce membrane proteins, which have yielded high resolution structures.
Cho KH, Husri M, Amin A, et al., 2015, Maltose neopentyl glycol-3 (MNG-3) analogues for membrane protein study, ANALYST, Vol: 140, Pages: 3157-3163, ISSN: 0003-2654
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- Citations: 42
Bertheleme N, Singh S, Dowell S, et al., 2015, Heterologous Expression of G-Protein-Coupled Receptors in Yeast, MEMBRANE PROTEINS - PRODUCTION AND FUNCTIONAL CHARACTERIZATION, Vol: 556, Pages: 141-164, ISSN: 0076-6879
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- Citations: 6
Sadaf A, Cho KH, Byrne B, et al., 2015, Amphipathic Agents for Membrane Protein Study, MEMBRANE PROTEINS - ENGINEERING, PURIFICATION AND CRYSTALLIZATION, Vol: 557, Pages: 57-94, ISSN: 0076-6879
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- Citations: 31
Boulet-Audet M, Byrne B, Kazarian SG, 2014, High-throughput thermal stability analysis of a monoclonal antibody by attenuated total reflection FT-IR spectroscopic imaging, Analytical Chemistry, Vol: 86, Pages: 9786-9793, ISSN: 0003-2700
The use of biotherapeutics, such as monoclonal antibodies, has markedly increased in recent years. It is thus essential that biotherapeutic production pipelines are as efficient as possible. For the production process, one of the major concerns is the propensity of a biotherapeutic antibody to aggregate. In addition to reducing bioactive material recovery, protein aggregation can have major effects on drug potency and cause highly undesirable immunological effects. It is thus essential to identify processing conditions which maximize recovery while avoiding aggregation. Heat resistance is a proxy for long-term aggregation propensity. Thermal stability assays are routinely performed using various spectroscopic and scattering detection methods. Here, we evaluated the potential of macro attenuated total reflection Fourier transform infrared (ATR-FT-IR) spectroscopic imaging as a novel method for the high-throughput thermal stability assay of a monoclonal antibody. This chemically specific visualization method has the distinct advantage of being able to discriminate between monomeric and aggregated protein. Attenuated total reflection is particularly suitable for selectively probing the bottom of vessels, where precipitated aggregates accumulate. With focal plane array detection, we tested 12 different buffer conditions simultaneously to assess the effect of pH and ionic strength on protein thermal stability. Applying the Finke model to our imaging kinetics allowed us to determine the rate constants of nucleation and autocatalytic growth. This analysis demonstrated the greater stability of our immunoglobulin at higher pH and moderate ionic strength, revealing the key role of electrostatic interactions. The high-throughput approach presented here has significant potential for analyzing the stability of biotherapeutics as well as any other biological molecules prone to aggregation.
Bertheleme N, Strege A, Bunting SE, et al., 2014, Arginine 199 and leucine 208 have key roles in the control of adenosine A(2A) receptor signalling function, PLoS One, Vol: 9, Pages: 1-10, ISSN: 1932-6203
One successful approach to obtaining high-resolution crystal structures of G-protein coupled receptors is the introduction of thermostabilising mutations within the receptor. This technique allows the generation of receptor constructs stabilised into different conformations suitable for structural studies. Previously, we functionally characterised a number of mutants of the adenosine A2A receptor, thermostabilised either in an agonist or antagonist conformation, using a yeast cell growth assay and demonstrated that there is a correlation between thermostability and loss of constitutive activity. Here we report the functional characterisation of 30 mutants intermediate between the Rag23 (agonist conformation mutant) and the wild-type receptor using the same yeast signalling assay with the aim of gaining greater insight into the role individual amino acids have in receptor function. The data showed that R199 and L208 have important roles in receptor function; substituting either of these residues for alanine abolishes constitutive activity. In addition, the R199A mutation markedly reduces receptor potency while L208A reduces receptor efficacy. A184L and L272A mutations also reduce constitutive activity and potency although to a lesser extent than the R199A and L208A. In contrast, the F79A mutation increases constitutive activity, potency and efficacy of the receptor. These findings shed new light on the role individual residues have on stability of the receptor and also provide some clues as to the regions of the protein responsible for constitutive activity. Furthermore, the available adenosine A2A receptor structures have allowed us to put our findings into a structural context.
Glassford SE, Byrne B, Kazarian SG, 2013, Recent applications of ATR FTIR spectroscopy and imaging to proteins, BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, Vol: 1834, Pages: 2849-2858, ISSN: 1570-9639
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- Citations: 186
Carlsson E, Zhang M, Ding JL, et al., 2013, Gly601 residue of human SARM is critical for interaction with other TLR adaptor proteins, Annual Congress of the British-Society-for-Immunology, Publisher: WILEY-BLACKWELL, Pages: 180-180, ISSN: 0019-2805
Chae PS, Kruse AC, Gotfryd K, et al., 2013, Novel Tripod Amphiphiles for Membrane Protein Analysis, CHEMISTRY-A EUROPEAN JOURNAL, Vol: 19, Pages: 15645-15651, ISSN: 0947-6539
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- Citations: 42
Bertheleme N, Chae PS, Singh S, et al., 2013, Unlocking the secrets of the gatekeeper: Methods for stabilizing and crystallizing GPCRs, BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES, Vol: 1828, Pages: 2583-2591, ISSN: 0005-2736
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- Citations: 28
Sethurathinam S, Singh LP, Panneerselvam P, et al., 2013, UXT plays dual opposing roles on SARM-induced apoptosis, FEBS LETTERS, Vol: 587, Pages: 3296-3302, ISSN: 0014-5793
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- Citations: 12
Bertheleme N, Singh S, Dowell SJ, et al., 2013, Loss of constitutive activity is correlated with increased thermostability of the human adenosine A2A receptor, BRITISH JOURNAL OF PHARMACOLOGY, Vol: 169, Pages: 988-998, ISSN: 0007-1188
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- Citations: 25
Saraiva N, Prole DL, Carrara G, et al., 2013, Human and Viral Golgi Anti-apoptotic Proteins (GAAPs) Oligomerize via Different Mechanisms and Monomeric GAAP Inhibits Apoptosis and Modulates Calcium, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 288, Pages: 13057-13067
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- Citations: 26
Cho KH, Byrne B, Chae PS, 2013, Hemifluorinated Maltose-Neopentyl Glycol (HF-MNG) Amphiphiles for Membrane Protein Stabilisation, CHEMBIOCHEM, Vol: 14, Pages: 452-455, ISSN: 1439-4227
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- Citations: 31
Bill R, Byrne B, 2013, Foreword for EDICT Special Edition, volume 2, MOLECULAR MEMBRANE BIOLOGY, Vol: 30, Pages: 113-113, ISSN: 0968-7688
Leung J, Cameron AD, Diallinas G, et al., 2013, Stabilizing the heterologously expressed uric acid-xanthine transporter UapA from the lower eukaryote <i>Aspergillus nidulans</i>, MOLECULAR MEMBRANE BIOLOGY, Vol: 30, Pages: 32-42, ISSN: 0968-7688
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- Citations: 13
Rana RR, Zhang M, Spear AM, et al., 2013, Bacterial TIR-containing proteins and host innate immune system evasion, MEDICAL MICROBIOLOGY AND IMMUNOLOGY, Vol: 202, Pages: 1-10, ISSN: 0300-8584
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- Citations: 41
Bill R, Byrne B, 2013, Introduction to EDICT volume 1, MOLECULAR MEMBRANE BIOLOGY, Vol: 30, Pages: 2-2, ISSN: 0968-7688
Chae PS, Rana RR, Gotfryd K, et al., 2013, Glucose-Neopentyl Glycol (GNG) amphiphiles for membrane protein study, CHEMICAL COMMUNICATIONS, Vol: 49, Pages: 2287-2289, ISSN: 1359-7345
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- Citations: 73
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