94 results found
Rains JGD, ODonnelly K, Oliver T, et al., 2019, Bicarbonate inhibition of carbonic anhydrase mimics hinders catalytic efficiency: Elucidating the mechanism and gaining insight toward improving speed and efficiency, ACS Catalysis, Vol: 9, Pages: 1353-1365, ISSN: 2155-5435
Carbonic anhydrase (CA) mimics are often studied with a focus on the hydration of CO2 for atmospheric carbon capture. Consequently, the reverse reaction (dehydration of HCO3–) has received minimal attention, so much so that the rate-limiting step of the dehydration reaction in CA mimics is currently unknown. The rate-limiting step of the hydration reaction is reported to be the bicarbonate-bound intermediate step, and thus is susceptible to product inhibition. It is not, however, clear if this inhibition is a consequence of an increase in the rate of the competing dehydration reaction or resulting from the strong affinity of bicarbonate to the mimic. To address this, insight into the dehydration reaction kinetics is needed. We therefore report the most comprehensive study of a CA mimic to date. The dehydration profile of the fastest small-molecule CA mimic, ZnL1S, was characterized, and consequently evidence for the rate-limiting step for the dehydration reaction was seen to be the bicarbonate-bound intermediate step, much like the hydration reaction. This experimental validation of the rate-limiting step was achieved through a variety of methods including NMR experiments and the effect of inhibitors, substrate concentration, and metal center on activity. With this understanding, an improvement in the favorability of the rate-limiting step was achieved, resulting in decreased bicarbonate inhibition. Thus, an increase in the mimic’s kcat for both reactions was observed, resulting in the largest rate constants of any small-molecule CA mimic reported to date (28 093 and 579 M–1 s–1 for hydration and dehydration, respectively). Enzyme-like kcat/km values were obtained for ZnL1S (5.9 × 105 M–1 s–1 for CO2 hydration), and notably there is only a difference of 2.5 orders of magnitude from the enzyme, the closest of any CA mimic reported in the literature. The results from this work can be applied to the development and improvement
Woscholski R, Larijani B, 2017, Last issue of journal of chemical biology., J Chem Biol, Vol: 10, ISSN: 1864-6158
Cilibrizzi A, Fedorova M, Collins J, et al., 2017, A tri-functional vanadium(IV) complex to detect cysteine oxidation, DALTON TRANSACTIONS, Vol: 46, Pages: 6994-7004, ISSN: 1477-9226
The development of effective molecular probes to detect and image the levels of oxidative stress in cells remains a challenge. Herein we report the design, synthesis and preliminary biological evaluation of a novel optical probe to monitor oxidation of thiol groups in cysteine-based phosphatases (CBPs). Following orthogonal protecting approaches we synthesised a new vanadyl complex designed to bind to CBPs. This complex is functionalised with a well-known dimedone derivative (to covalently trap sulfenic acids, SOHs) and a coumarin-based fluorophore for optical visualization. We show that this new probe efficiently binds to a range of phosphatases in vitro with nanomolar affinity. Moreover, preliminary flow cytometry and microscopy studies in live HCT116 cells show that this probe can successfully image cellular levels of sulfenic acids – one of the species resulting from protein oxidative damage.
Cilibrizzi A, Terenghi M, Fedorova M, et al., 2017, Small-molecule optical probes for cell imaging of protein sulfenylation and their application to monitor cisplatin induced protein oxidation, Sensors and Actuators B: Chemical, Vol: 248, Pages: 437-446, ISSN: 0925-4005
Reactive oxygen species (ROS) are considered versatile second messengers mediating fundamental biological functions. A molecular pathway by which ROS determine functional diversity is the selective oxidation of cysteine residues to form sulfenic acid (SOH) products, known as sulfenylation or S-hydroxylation. This crucial post-translational modification is responsible for the alteration of protein stability, function and signalling. Despite considerable advances on the identification of sulfenic residues on individual proteins, improved methods are needed for direct visualization and accurate quantification of the extent of total protein sulfenylation. Herein we present the synthesis of two new cell-permeable fluorescent probes containing dimedone (a cyclic β-diketone with high specificity for sulfenic acids), and apply them to study oxidation processes in individual cells via microscopy. The low cytotoxicity, cell permeability and optical features of the probes allowed us to visualize and quantify the oxidation of cysteine residues in live cells during H2O2-mediated oxidative burst (i.e. exogenously administered H2O2). We present preliminary cellular imaging studies with these probes to analyse the oxidation process in cells treated with the anticancer drug cisplatin.
Vilar Compte R, Wilson N, Mak LH, et al., 2016, A lipophilic copper(II) complex as an optical probe for intracellular detection of NO, Dalton Transactions, Vol: 45, Pages: 18177-18182, ISSN: 1477-9226
A new chemical sensor for cellular imaging of NO is presented. This cell-permeable probe is based on a complex where copper(II) is coordinated to a tridentate ligand substituted with a fluorophore (NBD) and an octyl group. The fluorescent response of this complex towards a range of reactive species (namely NO, NO2-, NO3-, H2O2, ClO-, O2-and ONOO-) has been studied in vitroshowing that the probe is highly selective for NO. The probe is readily taken up by cells and is able to image the cellular concentrations of NO
Furse S, Brooks NJ, Woscholski R, et al., 2016, Pressure-dependent inverse bicontinuous cubic phase formation in a phosphatidylinositol 4-phosphate/phosphatidylcholine system, Chemical Data Collections, Vol: 3-4, Pages: 15-20, ISSN: 2405-8300
In this paper, we report the inositide-driven formation of an inverse bicontinuous cubic phase with space group Ia3d (QIIG, gyroid phase). The system under study consisted of distearoylphosphatidylinositol 4-phosphate (DSPIP) and dioleoylphosphatidylcholine at a molar ratio of 1:49, with a physiological concentration of magnesium ions at pH 7·4. The behaviour of the system was monitored as a function of temperature and pressure. The formation of the phase with Ia3d geometry was recorded repeatably at high pressure, and occurred more readily at higher temperatures. We conclude that the Ia3d phase formed is a thermodynamically stable structure, and that DSPIP is a potent source of membrane curvature that can drive the formation of mesophases with both 2- and 3D geometry.
Vilar Compte R, Collins J, Woscholski R, et al., 2016, Vanadyl complexes with dansyl-labelled di-picolinic acid ligands: synthesis, phosphatase inhibition activity and cellular uptake studies, Dalton Transactions, Vol: 45, Pages: 7104-7113, ISSN: 1477-9226
Vanadium complexes have been previously utilised as potent inhibitors of cysteine based phosphatases (CBPs) . Herein, we present the synthesis and characterisation of two new fluorescently labelled vanadyl complexes (14 and 15 ) with bridged dipicolinic acid ligand. These compounds differ significantly from previous vanadyl complexes with phosphatase inhibition properties in that the metal-chelating part is a single tetradentate unit, which should afford greater stability and scope for synthetic elaboration then the earlier complexes. These new complexes inhibit a selection of cysteine based phosphatases (CBPs) in the nM range with some selectivity. Fluorescence spectroscopic studies (including fluorescence anisotropy) were carried out to demonstrate that the complexes are not simply acting as vanadyl delivery vehicles but they interact with the proteins. Finally, we present preliminary fluorescence microscopy studies to demonstrate that the complexes are cell permeable and localise throughout the cytoplasm of NIH3T3 cells.
Billcliff PG, Noakes CJ, Mehta ZB, et al., 2016, OCRL1 engages with the F-BAR protein pacsin 2 to promote biogenesis of membrane-trafficking intermediates, Molecular Biology of the Cell, Vol: 27, Pages: 90-107, ISSN: 1939-4586
Mutation of the inositol 5-phosphatase OCRL1 causes Lowe syndrome and Dent-2 disease. Loss of OCRL1 function perturbs several cellular processes, including membrane traffic, but the underlying mechanisms remain poorly defined. Here we show that OCRL1 is part of the membrane-trafficking machinery operating at the trans-Golgi network (TGN)/endosome interface. OCRL1 interacts via IPIP27A with the F-BAR protein pacsin 2. OCRL1 and IPIP27A localize to mannose 6-phosphate receptor (MPR)–containing trafficking intermediates, and loss of either protein leads to defective MPR carrier biogenesis at the TGN and endosomes. OCRL1 5-phosphatase activity, which is membrane curvature sensitive, is stimulated by IPIP27A-mediated engagement of OCRL1 with pacsin 2 and promotes scission of MPR-containing carriers. Our data indicate a role for OCRL1, via IPIP27A, in regulating the formation of pacsin 2–dependent trafficking intermediates and reveal a mechanism for coupling PtdIns(4,5)P2 hydrolysis with carrier biogenesis on endomembranes.
Verrastro I, Tveen-Jensen K, Woscholski R, et al., 2016, Reversible oxidation of phosphatase and tensin homolog (PTEN) alters its interactions with signaling and regulatory proteins, Free Radical Biology and Medicine, Vol: 90, Pages: 24-34, ISSN: 0891-5849
Phosphatase and tensin homolog (PTEN) is involved in a number of different cellular processes including metabolism, apoptosis, cell proliferation and survival. It is a redox-sensitive dual-specificity protein phosphatase that acts as a tumor suppressor by negatively regulating the PI3K/Akt pathway. While direct evidence of redox regulation of PTEN downstream signaling has been reported, the effect of PTEN redox status on its protein–protein interactions is poorly understood. PTEN-GST in its reduced and a DTT-reversible H2O2-oxidized form was immobilized on a glutathione-sepharose support and incubated with cell lysate to capture interacting proteins. Captured proteins were analyzed by LC–MSMS and comparatively quantified using label-free methods. 97 Potential protein interactors were identified, including a significant number that are novel. The abundance of fourteen interactors was found to vary significantly with the redox status of PTEN. Altered binding to PTEN was confirmed by affinity pull-down and Western blotting for Prdx1, Trx, and Anxa2, while DDB1 was validated as a novel interactor with unaltered binding. These results suggest that the redox status of PTEN causes a functional variation in the PTEN interactome. The resin capture method developed had distinct advantages in that the redox status of PTEN could be directly controlled and measured.
Murray JI, Woscholski R, Spivey AC, 2015, Organocatalytic Phosphorylation of Alcohols Using Pyridine-N-oxide, Synlett, Vol: 26, Pages: 985-990, ISSN: 1437-2096
Mak LH, Woscholski R, 2015, Targeting PTEN using small molecule inhibitors., Methods, Vol: 77-78C, Pages: 63-68, ISSN: 1046-2023
PTEN (phosphatase and tensin homologue deleted on chromosome 10) is well known as a tumour suppressor. It's PI(3,4,5)P3 lipid phosphatase activity is an important counteracting mechanism in PI 3-kinase (phosphoinositide 3-kinase) signalling. Furthermore, PTEN lies upstream of Akt kinase, a key enzyme in insulin signalling regulating glucose uptake and cell growth. Therefore, PTEN has recently gained attention as a valuable drug target for the treatment of diabetes, stroke, cardiac infarct and fertility. This review summarizes the use of small molecules as PTEN inhibitors. Currently available methodologies and techniques for accessing PTEN inhibition in vitro and in cellulo will be discussed.
Furse S, Mak L, Tate EW, et al., 2015, Synthesis of unsaturated phosphatidylinositol 4-phosphates and the effects of substrate unsaturation on SopB phosphatase activity, ORGANIC & BIOMOLECULAR CHEMISTRY, Vol: 13, Pages: 2001-2011, ISSN: 1477-0520
Murray JI, Woscholski R, Spivey AC, 2014, Highly efficient and selective phosphorylation of amino acid derivatives and polyols catalysed by 2-aryl-4-(dimethylamino)pyridine-N-oxides - towards kinase-like reactivity, CHEMICAL COMMUNICATIONS, Vol: 50, Pages: 13608-13611, ISSN: 1359-7345
Woscholski R, 2014, Chemical intervention tools to probe phosphoinositide-dependent signalling, BIOCHEMICAL SOCIETY TRANSACTIONS, Vol: 42, Pages: 1343-1348, ISSN: 0300-5127
Cilibrizzi A, Collins J, Woscholski R, et al., 2014, New vanadium complexes as optical probes to detect Cys sulfenic modifications in PTEN, 12th European Biological Inorganic Chemistry Conference (EuroBIC), Publisher: Springer Verlag (Germany), Pages: S873-S873, ISSN: 1432-1327
Pulido R, Baker SJ, Barata JT, et al., 2014, A Unified Nomenclature and Amino Acid Numbering for Human PTEN, SCIENCE SIGNALING, Vol: 7, ISSN: 1945-0877
O'Donnelly K, Zhao G, Patel P, et al., 2014, Isolation and kinetic characterisation of hydrophobically distinct populations of form I Rubisco, Planet Methods, Vol: 10, ISSN: 1746-4811
BackgroundRubisco (Ribulose-1,5-bisphosphate carboxylase/oxygenase) is a Calvin Cycle enzyme involved in CO2 assimilation. It is thought to be a major cause of photosynthetic inefficiency, suffering from both a slow catalytic rate and lack of specificity due to a competing reaction with oxygen. Revealing and understanding the engineering rules that dictate Rubisco’s activity could have a significant impact on photosynthetic efficiency and crop yield.ResultsThis paper describes the purification and characterisation of a number of hydrophobically distinct populations of Rubisco from both Spinacia oleracea and Brassica oleracea extracts. The populations were obtained using a novel and rapid purification protocol that employs hydrophobic interaction chromatography (HIC) as a form I Rubisco enrichment procedure, resulting in distinct Rubisco populations of expected enzymatic activities, high purities and integrity.ConclusionsWe demonstrate here that HIC can be employed to isolate form I Rubisco with purities and activities comparable to those obtained via ion exchange chromatography (IEC). Interestingly, and in contrast to other published purification methods, HIC resulted in the isolation of a number of hydrophobically distinct Rubisco populations. Our findings reveal a so far unaccounted diversity in the hydrophobic properties within form 1 Rubisco. By employing HIC to isolate and characterise Spinacia oleracea and Brassica oleracea, we show that the presence of these distinct Rubisco populations is not species specific, and we report for the first time the kinetic properties of Rubisco from Brassica oleracea extracts. These observations may aid future studies concerning Rubisco’s structural and functional properties.
Amor B, Yaliraki SN, Woscholski R, et al., 2014, Uncovering allosteric pathways in caspase-1 using Markov transient analysis and multiscale community detection, MOLECULAR BIOSYSTEMS, Vol: 10, Pages: 2247-2258, ISSN: 1742-206X
Miller D, Booth PJ, Seddon JM, et al., 2013, Protocell design through modular compartmentalization, JOURNAL OF THE ROYAL SOCIETY INTERFACE, Vol: 10, ISSN: 1742-5689
Murray JI, Spivey AC, Woscholski R, 2013, Alternative synthetic tools to phospho-specific antibodies for phosphoproteome analysis: progress and prospects., J Chem Biol, Vol: 6, Pages: 175-184, ISSN: 1864-6158
Signal transduction cascades in living systems are often controlled via post-translational phosphorylation and dephosphorylation of proteins. These processes are catalyzed in vivo by kinase and phosphatase enzymes, which consequently play an important role in many disease states, including cancer and immune system disorders. Current techniques for studying the phosphoproteome (isotopic labeling, chromatographic techniques, and phosphospecific antibodies), although undoubtedly very powerful, have yet to provide a generic tool for phosphoproteomic analysis despite the widespread utility such a technique would have. The use of small molecule organic catalysts that can promote selective phosphate esterification could provide a useful alternative to current state-of-the-art techniques for use in, e.g., the labeling and pull-down of phosphorylated proteins. This report reviews current techniques used for phosphoproteomic analysis and the recent use of small molecule peptide-based catalysts in phosphorylation reactions, indicating possible future applications for this type of catalyst as synthetic alternatives to phosphospecific antibodies for phosphoproteome analysis.
Whyte GF, Vilar R, Woscholski R, 2013, Molecular recognition with boronic acids-applications in chemical biology., J Chem Biol, Vol: 6, Pages: 161-174, ISSN: 1864-6158
Small molecules have long been used for the selective recognition of a wide range of analytes. The ability of these chemical receptors to recognise and bind to specific targets mimics certain biological processes (such as protein-substrate interactions) and has therefore attracted recent interest. Due to the abundance of biological molecules possessing polyhydroxy motifs, boronic acids-which form five-membered boronate esters with diols-have become increasingly popular in the synthesis of small chemical receptors. Their targets include biological materials and natural products including phosphatidylinositol bisphosphate, saccharides and polysaccharides, nucleic acids, metal ions and the neurotransmitter dopamine. This review will focus on the many ways in which small chemical receptors based on boronic acids have been used as biochemical tools for various purposes, including sensing and detection of analytes, interference in signalling pathways, enzyme inhibition and cell delivery systems. The most recent developments in each area will be highlighted.
Domart M-C, Hobday TMC, Peddie CJ, et al., 2012, Acute manipulation of diacylglycerol reveals roles in nuclear envelope assembly & endoplasmic reticulum morphology, PLoS ONE, Vol: 7, ISSN: 1932-6203
The functions and morphology of cellular membranes are intimately related and depend not only on their protein content but also on the repertoire of lipids that comprise them. In the absence of in vivo data on lipid asymmetry in endomembranes, it has been argued that motors, scaffolding proteins or integral membrane proteins rather than non-lamellar bilayer lipids such as diacylglycerol (DAG), are responsible for shaping of organelles, local membrane curvature and fusion. The effects of direct alteration of levels of such lipids remain predominantly uninvestigated. Diacylglycerol (DAG) is a well documented second messenger. Here we demonstrate two additional conserved functions of DAG: a structural role in organelle morphology, and a role in localised extreme membrane curvature required for fusion for which proteins alone are insufficient. Acute and inducible DAG depletion results in failure of the nuclear envelope (NE) to reform at mitosis and reorganisation of the ER into multi-lamellar sheets as revealed by correlative light and electron microscopy and 3D reconstructions. Remarkably, depleted cells divide without a complete NE, and unless rescued by 1,2 or 1,3 DAG soon die. Attenuation of DAG levels by enzyme microinjection into echinoderm eggs and embryos also results in alterations of ER morphology and nuclear membrane fusion. Our findings demonstrate that DAG is an in vivo modulator of organelle morphology in mammalian and echinoderm cells, indicating a fundamental role conserved across the deuterostome superphylum.
Mak LH, Knott J, Scott KA, et al., 2012, Arylstibonic acids are potent and isoform-selective inhibitors of Cdc25a and Cdc25b phosphatases, BIOORGANIC & MEDICINAL CHEMISTRY, Vol: 20, Pages: 4371-4376, ISSN: 0968-0896
Wormit A, Butt SM, Chairam I, et al., 2012, Osmosensitive Changes of Carbohydrate Metabolism in Response to Cellulose Biosynthesis Inhibition, PLANT PHYSIOLOGY, Vol: 159, Pages: 105-117, ISSN: 0032-0889
Charalambous K, Booth PJ, Woscholski R, et al., 2012, Engineering de Novo Membrane-Mediated Protein-Protein Communication Networks, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, Vol: 134, Pages: 5746-5749, ISSN: 0002-7863
Furse S, Brooks NJ, Seddon AM, et al., 2012, Lipid membrane curvature induced by distearoyl phosphatidylinositol 4-phosphate, Soft Matter
Mak LH, Georgiades SN, Rosivatz E, et al., 2011, A Small Molecule Mimicking a Phosphatidylinositol (4,5)-Bisphosphate Binding Pleckstrin Homology Domain, ACS Chemical Biology, ISSN: 1554-8929
Georgiades S, Mak L, Angurell I, et al., 2011, Identification of a potent activator of Akt phosphorylation from a novel series of phenolic, picolinic, pyridino, and hydroxamic zinc(II) complexes, Journal of Biological Inorganic Chemistry, Vol: 16, Pages: 195-208, ISSN: 0949-8257
Rosivatz E, Woscholski R, 2011, Removal or masking of phosphatidylinositol(4,5)bisphosphate from the outer mitochondrial membrane causes mitochondrial fragmentation, Cell Signal., Vol: 23, Pages: 478-486
Mitochondria are central players in programmed cell death and autophagy. While phosphoinositides are well established regulators of membrane traffic, cellular signalling and the destiny of certain organelles, their presence and role for mitochondria remain elusive. In this study we show that removal of PtdIns(4,5)P(2) by phosphatases or masking the lipid with PH domains leads to fission of mitochondria and increased autophagy. Induction of general autophagy by amino acid starvation also coincides with the loss of mitochondrial PtdIns(4,5)P(2), suggesting an important role for this lipid in the processes that govern mitophagy. Our findings reveal that PKCalpha can rescue the removal or masking of PtdIns(4,5)P(2), indicating that the inositol lipid is upstream of PKC
Mak L, Vilar R, Woscholski R, 2010, Characterisation of the PTEN inhibitor VO-OHpic, Journal of Chemical Biology, Vol: 3, Pages: 157-163, ISSN: 1864-6158
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