Publications by authors named "Riley Payne"

13 Publications

  • Page 1 of 1

Direct Amidation of Esters by Ball Milling*.

Angew Chem Int Ed Engl 2021 Sep 31;60(40):21868-21874. Epub 2021 Aug 31.

Department of Pharmaceutical and Biological Chemistry, University College London (UCL), School of Pharmacy, 29-39 Brunswick Square, Bloomsbury, London, WC1N 1AX, UK.

The direct mechanochemical amidation of esters by ball milling is described. The operationally simple procedure requires an ester, an amine, and substoichiometric KOtBu and was used to prepare a large and diverse library of 78 amide structures with modest to excellent efficiency. Heteroaromatic and heterocyclic components are specifically shown to be amenable to this mechanochemical protocol. This direct synthesis platform has been applied to the synthesis of active pharmaceutical ingredients (APIs) and agrochemicals as well as the gram-scale synthesis of an active pharmaceutical, all in the absence of a reaction solvent.
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http://dx.doi.org/10.1002/anie.202106412DOI Listing
September 2021

Calcium signaling induces a partial EMT.

EMBO Rep 2021 Sep 29;22(9):e51872. Epub 2021 Jul 29.

Abramson Family Cancer Research Institute and Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Epithelial plasticity, or epithelial-to-mesenchymal transition (EMT), is a well-recognized form of cellular plasticity, which endows tumor cells with invasive properties and alters their sensitivity to various agents, thus representing a major challenge to cancer therapy. It is increasingly accepted that carcinoma cells exist along a continuum of hybrid epithelial-mesenchymal (E-M) states and that cells exhibiting such partial EMT (P-EMT) states have greater metastatic competence than those characterized by either extreme (E or M). We described recently a P-EMT program operating in vivo by which carcinoma cells lose their epithelial state through post-translational programs. Here, we investigate the underlying mechanisms and report that prolonged calcium signaling induces a P-EMT characterized by the internalization of membrane-associated E-cadherin (ECAD) and other epithelial proteins as well as an increase in cellular migration and invasion. Signaling through Gαq-associated G-protein-coupled receptors (GPCRs) recapitulates these effects, which operate through the downstream activation of calmodulin-Camk2b signaling. These results implicate calcium signaling as a trigger for the acquisition of hybrid/partial epithelial-mesenchymal states in carcinoma cells.
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http://dx.doi.org/10.15252/embr.202051872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8419705PMC
September 2021

Coupled transmembrane mechanisms control MCU-mediated mitochondrial Ca uptake.

Proc Natl Acad Sci U S A 2020 09 14;117(35):21731-21739. Epub 2020 Aug 14.

Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104;

Ca uptake by mitochondria regulates bioenergetics, apoptosis, and Ca signaling. The primary pathway for mitochondrial Ca uptake is the mitochondrial calcium uniporter (MCU), a Ca-selective ion channel in the inner mitochondrial membrane. MCU-mediated Ca uptake is driven by the sizable inner-membrane potential generated by the electron-transport chain. Despite the large thermodynamic driving force, mitochondrial Ca uptake is tightly regulated to maintain low matrix [Ca] and prevent opening of the permeability transition pore and cell death, while meeting dynamic cellular energy demands. How this is accomplished is controversial. Here we define a regulatory mechanism of MCU-channel activity in which cytoplasmic Ca regulation of intermembrane space-localized MICU1/2 is controlled by Ca-regulatory mechanisms localized across the membrane in the mitochondrial matrix. Ca that permeates through the channel pore regulates Ca affinities of coupled inhibitory and activating sensors in the matrix. Ca binding to the inhibitory sensor within the MCU amino terminus closes the channel despite Ca binding to MICU1/2. Conversely, disruption of the interaction of MICU1/2 with the MCU complex disables matrix Ca regulation of channel activity. Our results demonstrate how Ca influx into mitochondria is tuned by coupled Ca-regulatory mechanisms on both sides of the inner mitochondrial membrane.
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http://dx.doi.org/10.1073/pnas.2005976117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7474680PMC
September 2020

ER-luminal [Ca] regulation of InsP receptor gating mediated by an ER-luminal peripheral Ca-binding protein.

Elife 2020 05 18;9. Epub 2020 May 18.

Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, United States.

Modulating cytoplasmic Ca concentration ([Ca]) by endoplasmic reticulum (ER)-localized inositol 1,4,5-trisphosphate receptor (InsPR) Ca-release channels is a universal signaling pathway that regulates numerous cell-physiological processes. Whereas much is known regarding regulation of InsPR activity by cytoplasmic ligands and processes, its regulation by ER-luminal Ca concentration ([Ca]) is poorly understood and controversial. We discovered that the InsPR is regulated by a peripheral membrane-associated ER-luminal protein that strongly inhibits the channel in the presence of high, physiological [Ca]. The widely-expressed Ca-binding protein annexin A1 (ANXA1) is present in the nuclear envelope lumen and, through interaction with a luminal region of the channel, can modify high-[Ca] inhibition of InsPR activity. Genetic knockdown of ANXA1 expression enhanced global and local elementary InsP-mediated Ca signaling events. Thus, [Ca] is a major regulator of InsPR channel activity and InsPR-mediated [Ca] signaling in cells by controlling an interaction of the channel with a peripheral membrane-associated Ca-binding protein, likely ANXA1.
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http://dx.doi.org/10.7554/eLife.53531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7259957PMC
May 2020

Variable Assembly of EMRE and MCU Creates Functional Channels with Distinct Gatekeeping Profiles.

iScience 2020 Apr 10;23(4):101037. Epub 2020 Apr 10.

Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

MCU is a Ca-selective channel that mediates mitochondrial Ca influx. The human channel contains tetrameric pore-forming MCU, regulatory subunits MICU1/2, and EMRE that is required both for channel function and MICU1/2-mediated Ca regulation. A structure of MCU with EMRE revealed a 4:4 stoichiometry, but the stoichiometry in vivo is unknown. Expression of tagged EMRE and MCU at a 1:10 ratio in cells lacking EMRE and MCU restored channel activity but not full channel gatekeeping. Increasing EMRE expression enhanced gatekeeping, raising the cytoplasmic Ca concentration ([Ca]) threshold for channel activation. MCU-EMRE concatemers creating channels with 1EMRE:4MCU restored Ca uptake in cells, whereas cells expressing concatemers that enforced a 4EMRE:4MCU stoichiometry demonstrated enhanced channel gatekeeping. Concatemers enforcing 2EMRE/4MCU recapitulated the activity, gatekeeping, and size of endogenous channels. Thus, MCU does not require four EMRE, with most endogenous channels containing two, but complexes with 1-4 EMRE have activity with full or partial gatekeeping.
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http://dx.doi.org/10.1016/j.isci.2020.101037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7170992PMC
April 2020

Isolation of state-dependent monoclonal antibodies against the 12-transmembrane domain glucose transporter 4 using virus-like particles.

Proc Natl Acad Sci U S A 2018 05 16;115(22):E4990-E4999. Epub 2018 May 16.

Integral Molecular, Philadelphia, PA 19104

The insulin-responsive 12-transmembrane transporter GLUT4 changes conformation between an inward-open state and an outward-open state to actively facilitate cellular glucose uptake. Because of the difficulties of generating conformational mAbs against complex and highly conserved membrane proteins, no reliable tools exist to measure GLUT4 at the cell surface, follow its trafficking, or detect the conformational state of the protein. Here we report the isolation and characterization of conformational mAbs that recognize the extracellular and intracellular domains of GLUT4, including mAbs that are specific for the inward-open and outward-open states of GLUT4. mAbs against GLUT4 were generated using virus-like particles to present this complex membrane protein in its native conformation and using a divergent host species (chicken) for immunization to overcome immune tolerance. As a result, the isolated mAbs recognize conformational epitopes on native GLUT4 in cells, with apparent affinities as high as 1 pM and with specificity for GLUT4 across the human membrane proteome. Epitope mapping using shotgun mutagenesis alanine scanning across the 509 amino acids of GLUT4 identified the binding epitopes for mAbs specific for the states of GLUT4 and allowed the comprehensive identification of the residues that functionally control the GLUT4 inward-open and outward-open states. The mAbs identified here will be valuable molecular tools for monitoring GLUT4 structure, function, and trafficking, for differentiating GLUT4 conformational states, and for the development of novel therapeutics for the treatment of diabetes.
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http://dx.doi.org/10.1073/pnas.1716788115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5984492PMC
May 2018

CALHM3 Is Essential for Rapid Ion Channel-Mediated Purinergic Neurotransmission of GPCR-Mediated Tastes.

Neuron 2018 05 19;98(3):547-561.e10. Epub 2018 Apr 19.

Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. Electronic address:

Binding of sweet, umami, and bitter tastants to G protein-coupled receptors (GPCRs) in apical membranes of type II taste bud cells (TBCs) triggers action potentials that activate a voltage-gated nonselective ion channel to release ATP to gustatory nerves mediating taste perception. Although calcium homeostasis modulator 1 (CALHM1) is necessary for ATP release, the molecular identification of the channel complex that provides the conductive ATP-release mechanism suitable for action potential-dependent neurotransmission remains to be determined. Here we show that CALHM3 interacts with CALHM1 as a pore-forming subunit in a CALHM1/CALHM3 hexameric channel, endowing it with fast voltage-activated gating identical to that of the ATP-release channel in vivo. Calhm3 is co-expressed with Calhm1 exclusively in type II TBCs, and its genetic deletion abolishes taste-evoked ATP release from taste buds and GPCR-mediated taste perception. Thus, CALHM3, together with CALHM1, is essential to form the fast voltage-gated ATP-release channel in type II TBCs required for GPCR-mediated tastes.
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http://dx.doi.org/10.1016/j.neuron.2018.03.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5934295PMC
May 2018

MICU2 Restricts Spatial Crosstalk between InsPR and MCU Channels by Regulating Threshold and Gain of MICU1-Mediated Inhibition and Activation of MCU.

Cell Rep 2017 Dec;21(11):3141-3154

Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. Electronic address:

Ca entry into mitochondria is mediated by the Ca uniporter-channel complex containing MCU, the Ca-selective pore, and associated regulatory proteins. The roles of MICU proteins are controversial. MICU1 was proposed to be necessary for MCU activity, whereas subsequent studies suggested it inhibits the channel in the low-cytoplasmic Ca ([Ca]) regime, a mechanism referred to as "gatekeeping," that imposes a [Ca] threshold for channel activation at ∼1-3 μM. Here, we measured MCU activity over a wide range of quantitatively controlled and recorded [Ca]. MICU1 alone can mediate gatekeeping as well as highly cooperative activation of MCU activity, whereas the fundamental role of MICU2 is to regulate the threshold and gain of MICU1-mediated inhibition and activation of MCU. Our results provide a unifying model for the roles of the MICU1/2 heterodimer in MCU-channel regulation and suggest an evolutionary role for MICU2 in spatially restricting Ca crosstalk between single inositol 1,4,5-trisphosphate receptor (InsPR) and MCU channels.
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http://dx.doi.org/10.1016/j.celrep.2017.11.064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5734103PMC
December 2017

EMRE Is a Matrix Ca(2+) Sensor that Governs Gatekeeping of the Mitochondrial Ca(2+) Uniporter.

Cell Rep 2016 Jan 7;14(3):403-410. Epub 2016 Jan 7.

Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

The mitochondrial uniporter (MCU) is an ion channel that mediates Ca(2+) uptake into the matrix to regulate metabolism, cell death, and cytoplasmic Ca(2+) signaling. Matrix Ca(2+) concentration is similar to that in cytoplasm, despite an enormous driving force for entry, but the mechanisms that prevent mitochondrial Ca(2+) overload are unclear. Here, we show that MCU channel activity is governed by matrix Ca(2+) concentration through EMRE. Deletion or charge neutralization of its matrix-localized acidic C terminus abolishes matrix Ca(2+) inhibition of MCU Ca(2+) currents, resulting in MCU channel activation, enhanced mitochondrial Ca(2+) uptake, and constitutively elevated matrix Ca(2+) concentration. EMRE-dependent regulation of MCU channel activity requires intermembrane space-localized MICU1, MICU2, and cytoplasmic Ca(2+). Thus, mitochondria are protected from Ca(2+) depletion and Ca(2+) overload by a unique molecular complex that involves Ca(2+) sensors on both sides of the inner mitochondrial membrane, coupled through EMRE.
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http://dx.doi.org/10.1016/j.celrep.2015.12.054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4731249PMC
January 2016

MCUR1, CCDC90A, Is a Regulator of the Mitochondrial Calcium Uniporter.

Cell Metab 2015 Oct;22(4):533-5

Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6085, USA; Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6085, USA. Electronic address:

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http://dx.doi.org/10.1016/j.cmet.2015.09.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384258PMC
October 2015

Thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure-guided surface engineering.

Proteins 2015 Jul 8;83(7):1225-37. Epub 2015 May 8.

Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico.

In this article, we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility of using TGP as a fusion partner in various assays and significantly, in amyloid assays in which the standard fluorescent protein, EGFP, is undesirable because of aberrant oligomerization.
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http://dx.doi.org/10.1002/prot.24699DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4592778PMC
July 2015

Detection of proton movement directly across viral membranes to identify novel influenza virus M2 inhibitors.

J Virol 2013 Oct 24;87(19):10679-86. Epub 2013 Jul 24.

Integral Molecular, Inc., Philadelphia, Pennsylvania, USA.

The influenza virus M2 protein is a well-validated yet underexploited proton-selective ion channel essential for influenza virus infectivity. Because M2 is a toxic viral ion channel, existing M2 inhibitors have been discovered through live virus inhibition or medicinal chemistry rather than M2-targeted high-throughput screening (HTS), and direct measurement of its activity has been limited to live cells or reconstituted lipid bilayers. Here, we describe a cell-free ion channel assay in which M2 ion channels are incorporated into virus-like particles (VLPs) and proton conductance is measured directly across the viral lipid bilayer, detecting changes in membrane potential, ion permeability, and ion channel function. Using this approach in high-throughput screening of over 100,000 compounds, we identified 19 M2-specific inhibitors, including two novel chemical scaffolds that inhibit both M2 function and influenza virus infectivity. Counterscreening for nonspecific disruption of viral bilayer ion permeability also identified a broad-spectrum antiviral compound that acts by disrupting the integrity of the viral membrane. In addition to its application to M2 and potentially other ion channels, this technology enables direct measurement of the electrochemical and biophysical characteristics of viral membranes.
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http://dx.doi.org/10.1128/JVI.01190-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3807417PMC
October 2013

Maturation of the Gag core decreases the stability of retroviral lipid membranes.

Virology 2012 Nov 17;433(2):401-9. Epub 2012 Sep 17.

Integral Molecular, Inc., Philadelphia, PA 19104, United States.

To better understand how detergents disrupt enveloped viruses, we monitored the biophysical stability of murine leukemia virus (MLV) virus-like particles (VLPs) against a panel of commonly used detergents using real-time biosensor measurements. Although exposure to many detergents, such as Triton X-100 and Empigen, results in lysis of VLP membranes, VLPs appeared resistant to complete membrane lysis by a significant number of detergents, including Tween 20, Tween 80, Lubrol, and Saponin. VLPs maintained their structural integrity after exposure to Tween 20 at concentrations up to 500-fold above its CMC. Remarkably, VLPs containing immature cores composed of unprocessed (uncleaved) Gag polyprotein were significantly more resistant to detergent lysis than VLPs with mature cores. Although the maturity of retroviral Gag is known to influence the stability of the protein core structure itself, our studies suggest that the maturity of the Gag core also influences the stability of the lipid bilayer surrounding the core.
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http://dx.doi.org/10.1016/j.virol.2012.08.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3848701PMC
November 2012
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