Publications by authors named "David Stokes"

101 Publications

The microbiome of deep-sea fish reveals new microbial species and a sparsity of antibiotic resistance genes.

Gut Microbes 2021 Jan-Dec;13(1):1-13

Teagasc Food Research Centre, Fermoy, Ireland.

Adaptation to life in the deep-sea can be dramatic, with fish displaying behaviors and appearances unlike those seen in any other aquatic habitat. However, the extent of which adaptations may have developed at a microbial scale is not as clear. Shotgun metagenomic sequencing of the intestinal microbiome of 32 species of deep-sea fish from across the Atlantic Ocean revealed that many of the associated microbes differ extensively from those previously identified in reference databases. 111 individual metagenome-assembled genomes (MAGs) were constructed representing individual microbial species from the microbiomes of these fish, many of which are potentially novel bacterial taxa and provide a window into the microbial diversity in this underexplored environment. These MAGs also demonstrate how these microbes have adapted to deep-sea life by encoding a greater capacity for several cellular processes such as protein folding and DNA replication that can be inhibited by high pressure. Another intriguing feature was the almost complete lack of genes responsible for acquired resistance to known antibiotics in many of the samples. This highlights that deep-sea fish microbiomes may represent one of few animal-associated microbiomes with little influence from human activity. The ability of the microbes in these samples to bioluminesce is lower than expected given predictions that this trait has an important role in their life cycle at these depths. The study highlights the uniqueness, complexity and adaptation of microbial communities living in one of the largest and harshest environments on Earth.
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http://dx.doi.org/10.1080/19490976.2021.1921924DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8115496PMC
May 2021

An Intracellular Pathway Controlled by the N-terminus of the Pump Subunit Inhibits the Bacterial KdpFABC Ion Pump in High K Conditions.

J Mol Biol 2021 May 2;433(15):167008. Epub 2021 May 2.

PHYLIFE: Physical Life Science, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, Odense 5230 M, Denmark. Electronic address:

The heterotetrameric bacterial KdpFABC transmembrane protein complex is an ion channel-pump hybrid that consumes ATP to import K against its transmembrane chemical potential gradient in low external K environments. The KdpB ion-pump subunit of KdpFABC is a P-type ATPase, and catalyses ATP hydrolysis. Under high external K conditions, K can diffuse into the cells through passive ion channels. KdpFABC must therefore be inhibited in high K conditions to conserve cellular ATP. Inhibition is thought to occur via unusual phosphorylation of residue Ser162 of the TGES motif of the cytoplasmic A domain. It is proposed that phosphorylation most likely traps KdpB in an inactive E1-P like conformation, but the molecular mechanism of phosphorylation-mediated inhibition remains unknown. Here, we employ molecular dynamics (MD) simulations of the dephosphorylated and phosphorylated versions of KdpFABC to demonstrate that phosphorylated KdpB is trapped in a conformation where the ion-binding site is hydrated by an intracellular pathway between transmembrane helices M1 and M2 which opens in response to the rearrangement of cytoplasmic domains resulting from phosphorylation. Cytoplasmic access of water to the ion-binding site is accompanied by a remarkable loss of secondary structure of the KdpB N-terminus and disruption of a key salt bridge between Glu87 in the A domain and Arg212 in the P domain. Our results provide the molecular basis of a unique mechanism of regulation amongst P-type ATPases, and suggest that the N-terminus has a significant role to play in the conformational cycle and regulation of KdpFABC.
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http://dx.doi.org/10.1016/j.jmb.2021.167008DOI Listing
May 2021

Serine phosphorylation regulates the P-type potassium pump KdpFABC.

Elife 2020 09 21;9. Epub 2020 Sep 21.

Skirball Institute, Dept. of Cell Biology, New York University School of Medicine, New York, United States.

KdpFABC is an ATP-dependent K pump that ensures bacterial survival in K-deficient environments. Whereas transcriptional activation of kdpFABC expression is well studied, a mechanism for down-regulation when K levels are restored has not been described. Here, we show that KdpFABC is inhibited when cells return to a K-rich environment. The mechanism of inhibition involves phosphorylation of Ser162 on KdpB, which can be reversed in vitro by treatment with serine phosphatase. Mutating Ser162 to Alanine produces constitutive activity, whereas the phosphomimetic Ser162Asp mutation inactivates the pump. Analyses of the transport cycle show that serine phosphorylation abolishes the K-dependence of ATP hydrolysis and blocks the catalytic cycle after formation of the aspartyl phosphate intermediate (E1~P). This regulatory mechanism is unique amongst P-type pumps and this study furthers our understanding of how bacteria control potassium homeostasis to maintain cell volume and osmotic potential.
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http://dx.doi.org/10.7554/eLife.55480DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7535926PMC
September 2020

The KdpFABC complex - K transport against all odds.

Mol Membr Biol 2019 12;35(1):21-38

c Department of Biology, University of Konstanz , Konstanz , Germany.

In bacteria, K is used to maintain cell volume and osmotic potential. Homeostasis normally involves a network of constitutively expressed transport systems, but in K deficient environments, the KdpFABC complex uses ATP to pump K into the cell. This complex appears to be a hybrid of two types of transporters, with KdpA descending from the superfamily of K transporters and KdpB belonging to the superfamily of P-type ATPases. Studies of enzymatic activity documented a catalytic cycle with hallmarks of classical P-type ATPases and studies of ion transport indicated that K import into the cytosol occurred in the second half of this cycle in conjunction with hydrolysis of an aspartyl phosphate intermediate. Atomic structures of the KdpFABC complex from X-ray crystallography and cryo-EM have recently revealed conformations before and after formation of this aspartyl phosphate that appear to contradict the functional studies. Specifically, structural comparisons with the archetypal P-type ATPase, SERCA, suggest that K transport occurs in the first half of the cycle, accompanying formation of the aspartyl phosphate. Further controversy has arisen regarding the path by which K crosses the membrane. The X-ray structure supports the conventional view that KdpA provides the conduit, whereas cryo-EM structures suggest that K moves from KdpA through a long, intramembrane tunnel to reach canonical ion binding sites in KdpB from which they are released to the cytosol. This review discusses evidence supporting these contradictory models and identifies key experiments needed to resolve discrepancies and produce a unified model for this fascinating mechanistic hybrid.
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http://dx.doi.org/10.1080/09687688.2019.1638977DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6681667PMC
December 2019

Dendritic cell-expressed common gamma-chain recruits IL-15 for trans-presentation at the murine immunological synapse.

Wellcome Open Res 2018 17;3:84. Epub 2018 Oct 17.

Great Ormond Street Hospital for Children NHS Foundation Trust, London, WC1N 3JH, UK.

Mutations of the common cytokine receptor gamma chain (γc) cause Severe Combined Immunodeficiency characterized by absent T and NK cell development. Although stem cell therapy restores these lineages, residual immune defects are observed that may result from selective persistence of γc-deficiency in myeloid lineages. However, little is known about the contribution of myeloid-expressed γc to protective immune responses.  Here we examine the importance of γc for myeloid dendritic cell (DC) function. We utilize a combination of DC/T-cell co-culture assays and a novel lipid bilayer system mimicking the T cell surface to delineate the role of DC-expressed γc during DC/T-cell interaction. We observed that γc in DC was recruited to the contact interface following MHCII ligation, and promoted IL-15Rα colocalization with engaged MHCII. Unexpectedly, trans-presentation of IL-15 was required for optimal CD4+T cell activation by DC and depended on DC γc expression. Neither recruitment of IL-15Rα nor IL-15 trans-signaling at the DC immune synapse (IS), required γc signaling in DC, suggesting that γc facilitates IL-15 transpresentation through induced intermolecular associations or cytoskeletal reorganization following MHCII ligation. These findings show that DC-expressed γc is required for effective antigen-induced CD4+ T cell activation. We reveal a novel mechanism for recruitment of DC IL-15/IL-15Rα complexes to the IS, leading to CD4+ T cell costimulation through localized IL-15 transpresentation that is coordinated with antigen-recognition.
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http://dx.doi.org/10.12688/wellcomeopenres.14493.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6234741PMC
October 2018

Integrative structure and functional anatomy of a nuclear pore complex.

Nature 2018 03 14;555(7697):475-482. Epub 2018 Mar 14.

Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, New York 10065, USA.

Nuclear pore complexes play central roles as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm. However, their large size and dynamic nature have impeded a full structural and functional elucidation. Here we determined the structure of the entire 552-protein nuclear pore complex of the yeast Saccharomyces cerevisiae at sub-nanometre precision by satisfying a wide range of data relating to the molecular arrangement of its constituents. The nuclear pore complex incorporates sturdy diagonal columns and connector cables attached to these columns, imbuing the structure with strength and flexibility. These cables also tie together all other elements of the nuclear pore complex, including membrane-interacting regions, outer rings and RNA-processing platforms. Inwardly directed anchors create a high density of transport factor-docking Phe-Gly repeats in the central channel, organized into distinct functional units. This integrative structure enables us to rationalize the architecture, transport mechanism and evolutionary origins of the nuclear pore complex.
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http://dx.doi.org/10.1038/nature26003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6022767PMC
March 2018

Structural basis for the alternating access mechanism of the cation diffusion facilitator YiiP.

Proc Natl Acad Sci U S A 2018 03 5;115(12):3042-3047. Epub 2018 Mar 5.

Skirball Institute, Department of Cell Biology, New York University School of Medicine, New York, NY 10016

YiiP is a dimeric antiporter from the cation diffusion facilitator family that uses the proton motive force to transport Zn across bacterial membranes. Previous work defined the atomic structure of an outward-facing conformation, the location of several Zn binding sites, and hydrophobic residues that appear to control access to the transport sites from the cytoplasm. A low-resolution cryo-EM structure revealed changes within the membrane domain that were associated with the alternating access mechanism for transport. In the current work, the resolution of this cryo-EM structure has been extended to 4.1 Å. Comparison with the X-ray structure defines the differences between inward-facing and outward-facing conformations at an atomic level. These differences include rocking and twisting of a four-helix bundle that harbors the Zn transport site and controls its accessibility within each monomer. As previously noted, membrane domains are closely associated in the dimeric structure from cryo-EM but dramatically splayed apart in the X-ray structure. Cysteine crosslinking was used to constrain these membrane domains and to show that this large-scale splaying was not necessary for transport activity. Furthermore, dimer stability was not compromised by mutagenesis of elements in the cytoplasmic domain, suggesting that the extensive interface between membrane domains is a strong determinant of dimerization. As with other secondary transporters, this interface could provide a stable scaffold for movements of the four-helix bundle that confers alternating access of these ions to opposite sides of the membrane.
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http://dx.doi.org/10.1073/pnas.1715051115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5866550PMC
March 2018

Proof of Concept Coded Aperture Miniature Mass Spectrometer Using a Cycloidal Sector Mass Analyzer, a Carbon Nanotube (CNT) Field Emission Electron Ionization Source, and an Array Detector.

J Am Soc Mass Spectrom 2018 02 19;29(2):360-372. Epub 2017 Oct 19.

Department of Electrical and Computer Engineering, Duke University, Durham, NC, 27708, USA.

Despite many potential applications, miniature mass spectrometers have had limited adoption in the field due to the tradeoff between throughput and resolution that limits their performance relative to laboratory instruments. Recently, a solution to this tradeoff has been demonstrated by using spatially coded apertures in magnetic sector mass spectrometers, enabling throughput and signal-to-background improvements of greater than an order of magnitude with no loss of resolution. This paper describes a proof of concept demonstration of a cycloidal coded aperture miniature mass spectrometer (C-CAMMS) demonstrating use of spatially coded apertures in a cycloidal sector mass analyzer for the first time. C-CAMMS also incorporates a miniature carbon nanotube (CNT) field emission electron ionization source and a capacitive transimpedance amplifier (CTIA) ion array detector. Results confirm the cycloidal mass analyzer's compatibility with aperture coding. A >10× increase in throughput was achieved without loss of resolution compared with a single slit instrument. Several areas where additional improvement can be realized are identified. Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-017-1820-yDOI Listing
February 2018

Image-based model of the spectrin cytoskeleton for red blood cell simulation.

PLoS Comput Biol 2017 Oct 9;13(10):e1005790. Epub 2017 Oct 9.

Courant Institute of Mathematical Sciences, New York University, New York, New York, United States of America.

We simulate deformable red blood cells in the microcirculation using the immersed boundary method with a cytoskeletal model that incorporates structural details revealed by tomographic images. The elasticity of red blood cells is known to be supplied by both their lipid bilayer membranes, which resist bending and local changes in area, and their cytoskeletons, which resist in-plane shear. The cytoskeleton consists of spectrin tetramers that are tethered to the lipid bilayer by ankyrin and by actin-based junctional complexes. We model the cytoskeleton as a random geometric graph, with nodes corresponding to junctional complexes and with edges corresponding to spectrin tetramers such that the edge lengths are given by the end-to-end distances between nodes. The statistical properties of this graph are based on distributions gathered from three-dimensional tomographic images of the cytoskeleton by a segmentation algorithm. We show that the elastic response of our model cytoskeleton, in which the spectrin polymers are treated as entropic springs, is in good agreement with the experimentally measured shear modulus. By simulating red blood cells in flow with the immersed boundary method, we compare this discrete cytoskeletal model to an existing continuum model and predict the extent to which dynamic spectrin network connectivity can protect against failure in the case of a red cell subjected to an applied strain. The methods presented here could form the basis of disease- and patient-specific computational studies of hereditary diseases affecting the red cell cytoskeleton.
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http://dx.doi.org/10.1371/journal.pcbi.1005790DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5654263PMC
October 2017

The transmembrane domain of the p75 neurotrophin receptor stimulates phosphorylation of the TrkB tyrosine kinase receptor.

J Biol Chem 2017 10 17;292(40):16594-16604. Epub 2017 Aug 17.

From the Departments of Cell Biology, Physiology & Neuroscience, and Psychiatry, Skirball Institute of Biomolecular Medicine, New York University Langone Medical Center, New York, New York 10016.

The function of protein products generated from intramembraneous cleavage by the γ-secretase complex is not well defined. The γ-secretase complex is responsible for the cleavage of several transmembrane proteins, most notably the amyloid precursor protein that results in Aβ, a transmembrane (TM) peptide. Another protein that undergoes very similar γ-secretase cleavage is the p75 neurotrophin receptor. However, the fate of the cleaved p75 TM domain is unknown. p75 neurotrophin receptor is highly expressed during early neuronal development and regulates survival and process formation of neurons. Here, we report that the p75 TM can stimulate the phosphorylation of TrkB (tyrosine kinase receptor B). phosphorylation experiments indicated that a peptide representing p75 TM increases TrkB phosphorylation in a dose- and time-dependent manner. Moreover, mutagenesis analyses revealed that a valine residue at position 264 in the rat p75 neurotrophin receptor is necessary for the ability of p75 TM to induce TrkB phosphorylation. Because this residue is just before the γ-secretase cleavage site, we then investigated whether the p75(αγ) peptide, which is a product of both α- and γ-cleavage events, could also induce TrkB phosphorylation. Experiments using TM domains from other receptors, EGFR and FGFR1, failed to stimulate TrkB phosphorylation. Co-immunoprecipitation and biochemical fractionation data suggested that p75 TM stimulates TrkB phosphorylation at the cell membrane. Altogether, our results suggest that TrkB activation by p75(αγ) peptide may be enhanced in situations where the levels of the p75 receptor are increased, such as during brain injury, Alzheimer's disease, and epilepsy.
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http://dx.doi.org/10.1074/jbc.M117.788729DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5633122PMC
October 2017

Crystal structure of the potassium-importing KdpFABC membrane complex.

Nature 2017 06 21;546(7660):681-685. Epub 2017 Jun 21.

Department of Cell Biology, New York University School of Medicine, Skirball Institute, 540 First Avenue, New York, New York 10016, USA.

Cellular potassium import systems play a fundamental role in osmoregulation, pH homeostasis and membrane potential in all domains of life. In bacteria, the kdp operon encodes a four-subunit potassium pump that maintains intracellular homeostasis, cell shape and turgor under conditions in which potassium is limiting. This membrane complex, called KdpFABC, has one channel-like subunit (KdpA) belonging to the superfamily of potassium transporters and another pump-like subunit (KdpB) belonging to the superfamily of P-type ATPases. Although there is considerable structural and functional information about members of both superfamilies, the mechanism by which uphill potassium transport through KdpA is coupled with ATP hydrolysis by KdpB remains poorly understood. Here we report the 2.9 Å X-ray structure of the complete Escherichia coli KdpFABC complex with a potassium ion within the selectivity filter of KdpA and a water molecule at a canonical cation site in the transmembrane domain of KdpB. The structure also reveals two structural elements that appear to mediate the coupling between these two subunits. Specifically, a protein-embedded tunnel runs between these potassium and water sites and a helix controlling the cytoplasmic gate of KdpA is linked to the phosphorylation domain of KdpB. On the basis of these observations, we propose a mechanism that repurposes protein channel architecture for active transport across biomembranes.
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http://dx.doi.org/10.1038/nature22970DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5495170PMC
June 2017

Molecular Architecture of the Major Membrane Ring Component of the Nuclear Pore Complex.

Structure 2017 03 2;25(3):434-445. Epub 2017 Feb 2.

Laboratory of Cellular and Structural Biology, The Rockefeller University, Box 213, 1230 York Avenue, New York, NY 10065, USA. Electronic address:

The membrane ring that equatorially circumscribes the nuclear pore complex (NPC) in the perinuclear lumen of the nuclear envelope is composed largely of Pom152 in yeast and its ortholog Nup210 (or Gp210) in vertebrates. Here, we have used a combination of negative-stain electron microscopy, nuclear magnetic resonance, and small-angle X-ray scattering methods to determine an integrative structure of the ∼120 kDa luminal domain of Pom152. Our structural analysis reveals that the luminal domain is formed by a flexible string-of-pearls arrangement of nine repetitive cadherin-like Ig-like domains, indicating an evolutionary connection between NPCs and the cell adhesion machinery. The 16 copies of Pom152 known to be present in the yeast NPC are long enough to form the observed membrane ring, suggesting how interactions between Pom152 molecules help establish and maintain the NPC architecture.
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http://dx.doi.org/10.1016/j.str.2017.01.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5342941PMC
March 2017

Structure and Function of the Nuclear Pore Complex Cytoplasmic mRNA Export Platform.

Cell 2016 11 10;167(5):1215-1228.e25. Epub 2016 Nov 10.

Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, NY 10065, USA. Electronic address:

The last steps in mRNA export and remodeling are performed by the Nup82 complex, a large conserved assembly at the cytoplasmic face of the nuclear pore complex (NPC). By integrating diverse structural data, we have determined the molecular architecture of the native Nup82 complex at subnanometer precision. The complex consists of two compositionally identical multiprotein subunits that adopt different configurations. The Nup82 complex fits into the NPC through the outer ring Nup84 complex. Our map shows that this entire 14-MDa Nup82-Nup84 complex assembly positions the cytoplasmic mRNA export factor docking sites and messenger ribonucleoprotein (mRNP) remodeling machinery right over the NPC's central channel rather than on distal cytoplasmic filaments, as previously supposed. We suggest that this configuration efficiently captures and remodels exporting mRNP particles immediately upon reaching the cytoplasmic side of the NPC.
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http://dx.doi.org/10.1016/j.cell.2016.10.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5130164PMC
November 2016

Structure of the SLC4 transporter Bor1p in an inward-facing conformation.

Protein Sci 2017 01 21;26(1):130-145. Epub 2016 Oct 21.

Skirball Institute for Biomolecular Medicine, Department of Cell Biology, New York University School of Medicine, New York, New York, 10016.

Bor1p is a secondary transporter in yeast that is responsible for boron transport. Bor1p belongs to the SLC4 family which controls bicarbonate exchange and pH regulation in animals as well as borate uptake in plants. The SLC4 family is more distantly related to members of the Amino acid-Polyamine-organoCation (APC) superfamily, which includes well studied transporters such as LeuT, Mhp1, AdiC, vSGLT, UraA, SLC26Dg. Their mechanism generally involves relative movements of two domains: a core domain that binds substrate and a gate domain that in many cases mediates dimerization. To shed light on conformational changes governing transport by the SLC4 family, we grew helical membrane crystals of Bor1p from Saccharomyces mikatae and determined a structure at ∼6 Å resolution using cryo-electron microscopy. To evaluate the conformation of Bor1p in these crystals, a homology model was built based on the related anion exchanger from red blood cells (AE1). This homology model was fitted to the cryo-EM density map using the Molecular Dynamics (MD) Flexible Fitting method and then relaxed by all-atom MD simulation in explicit solvent and membrane. Mapping of water accessibility indicates that the resulting structure represents an inward-facing conformation. Comparisons of the resulting Bor1p model with the X-ray structure of AE1 in an outward-facing conformation, together with MD simulations of inward-facing and outward-facing Bor1p models, suggest rigid body movements of the core domain relative to the gate domain. These movements are consistent with the rocking-bundle transport mechanism described for other members of the APC superfamily.
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http://dx.doi.org/10.1002/pro.3061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5192975PMC
January 2017

Purification and analysis of endogenous human RNA exosome complexes.

RNA 2016 09 11;22(9):1467-75. Epub 2016 Jul 11.

Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York 10065, USA Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, New York 10016, USA.

As a result of its importance in key RNA metabolic processes, the ribonucleolytic RNA exosome complex has been the focus of intense study for almost two decades. Research on exosome subunit assembly, cofactor and substrate interaction, enzymatic catalysis and structure have largely been conducted using complexes produced in the yeast Saccharomyces cerevisiae or in bacteria. Here, we examine different populations of endogenous exosomes from human embryonic kidney (HEK) 293 cells and test their enzymatic activity and structural integrity. We describe methods to prepare EXOSC10-containing, enzymatically active endogenous human exosomes at suitable yield and purity for in vitro biochemistry and negative stain transmission electron microscopy. This opens the door for assays designed to test the in vitro effects of putative cofactors on human exosome activity and will enable structural studies of preparations from endogenous sources.
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http://dx.doi.org/10.1261/rna.057760.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986900PMC
September 2016

Deducing the symmetry of helical assemblies: Applications to membrane proteins.

J Struct Biol 2016 08 30;195(2):167-178. Epub 2016 May 30.

Skirball Institute for Biomolecular Medicine, Department of Cell Biology, New York University School of Medicine, New York, NY 10016, United States.

Helical reconstruction represents a convenient and powerful approach for structure determination of macromolecules that assemble into helical arrays. In the case of membrane proteins, formation of tubular crystals with helical symmetry represents an attractive alternative, especially when their small size precludes the use of single-particle analysis. An essential first step for helical reconstruction is to characterize the helical symmetry. This process is often daunting, due to the complexity of helical diffraction and to the low signal-to-noise ratio in images of individual assemblies. Furthermore, the large diameters of the tubular crystals produced by membrane proteins exacerbates the innate ambiguities that, if not resolved, will produce incorrect structures. In this report, we describe a set of tools that can be used to eliminate ambiguities and to validate the choice of symmetry. The first approach increases the signal-to-noise ratio along layer lines by incoherently summing data from multiple helical assemblies, thus producing several candidate indexing schemes. The second approach compares the layer lines from images with those from synthetic models built with the various candidate schemes. The third approach uses unit cell dimensions measured from collapsed tubes to distinguish between these candidate schemes. These approaches are illustrated with tubular crystals from a boron transporter from yeast, Bor1p, and a β-barrel channel from the outer membrane of E. coli, OmpF.
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http://dx.doi.org/10.1016/j.jsb.2016.05.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4944209PMC
August 2016

Filter paper-based insulin-like growth factor assay.

J AAPOS 2015 Aug 30;19(4):363-5. Epub 2015 Jul 30.

Division of Ophthalmology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania; Scheie Eye Institute, Department of Ophthalmology, University of Pennsylvania Perelman School of Medicine, Philadelphia. Electronic address:

This study assessed validity, reliability, and feasibility of a filter paper blood spot insulin-like growth factor 1 (IGF-1) assay for retinopathy of prematurity (ROP) research. Blood samples were collected from 45 healthy children. Half of each sample was spun to obtain serum; half was applied to filter cards and stored for varying times and at different temperatures. IGF-1 assays were performed using a commercially available kit. Intraclass correlation between blood spot and serum IGF-1 values was high (0.97) for validity, and the mean differences were low for test-retest reliability. Time (up to 25 days) and temperature (4° C to 37° C) had no significant effect on sample stability. Feasibility was further assessed in a second cohort study of 74 premature infants being screened for ROP. A total of 817 filter card samples were successfully collected and transported to a central lab, where IGF-1 assays were successfully performed.
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http://dx.doi.org/10.1016/j.jaapos.2015.03.022DOI Listing
August 2015

Utility service: Building a high-performance central energy plant.

Authors:
David Stokes

Health Facil Manage 2014 Apr;27(4):23-7

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April 2014

Cyclooxygenase-2 catalysis and inhibition in lipid bilayer nanodiscs.

Arch Biochem Biophys 2014 Mar 3;546:33-40. Epub 2014 Feb 3.

Department of Structural Biology, The State University of New York at Buffalo, Buffalo, NY 14203, USA; Hauptman-Woodward Medical Research Institute, Buffalo, NY 14203, USA. Electronic address:

Cyclooxygenases (COX-1 and COX-2) oxygenate arachidonic acid (AA) to generate prostaglandins. The enzymes associate with one leaflet of the membrane bilayer. We utilized nanodisc technology to investigate the function of human (hu) COX-2 and murine (mu) COX-2 in a lipid bilayer environment. huCOX-2 and muCOX-2 were incorporated into nanodiscs composed of POPC, POPS, DOPC, or DOPS phospholipids. Size-exclusion chromatography and negative stain electron microscopy confirm that a single COX-2 homodimer is incorporated into the nanodisc scaffold. Nanodisc-reconstituted COX-2 exhibited similar kinetic profiles for the oxygenation of AA, eicosapentaenoic acid, and 1-arachidonoyl glycerol compared to those derived using detergent solubilized enzyme. Moreover, changing the phospholipid composition of the nanodisc did not alter the ability of COX-2 to oxygenate AA or to be inhibited by various nonselective NSAIDs or celecoxib. The cyclooxygenase activity of nanodisc-reconstituted COX-2 was reduced by aspirin acetylation and potentiated by the nonsubstrate fatty acid palmitic acid to the same extent as detergent solubilized enzyme, independent of phospholipid composition. The stabilization and maintenance of activity afforded by the incorporation of the enzyme into nanodiscs generates a native-like lipid bilayer environment to pursue studies of COX utilizing solution-based techniques that are otherwise not tractable in the presence of detergents.
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http://dx.doi.org/10.1016/j.abb.2014.01.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3967765PMC
March 2014

Polarized release of T-cell-receptor-enriched microvesicles at the immunological synapse.

Nature 2014 Mar 2;507(7490):118-23. Epub 2014 Feb 2.

1] Department of Pathology, New York University School of Medicine, 540 First Avenue, New York, New York 10016, USA [2] Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, The University of Oxford, Roosevelt Drive, Headington, Oxford OX3 7FY, UK.

The recognition events that mediate adaptive cellular immunity and regulate antibody responses depend on intercellular contacts between T cells and antigen-presenting cells (APCs). T-cell signalling is initiated at these contacts when surface-expressed T-cell receptors (TCRs) recognize peptide fragments (antigens) of pathogens bound to major histocompatibility complex molecules (pMHC) on APCs. This, along with engagement of adhesion receptors, leads to the formation of a specialized junction between T cells and APCs, known as the immunological synapse, which mediates efficient delivery of effector molecules and intercellular signals across the synaptic cleft. T-cell recognition of pMHC and the adhesion ligand intercellular adhesion molecule-1 (ICAM-1) on supported planar bilayers recapitulates the domain organization of the immunological synapse, which is characterized by central accumulation of TCRs, adjacent to a secretory domain, both surrounded by an adhesive ring. Although accumulation of TCRs at the immunological synapse centre correlates with T-cell function, this domain is itself largely devoid of TCR signalling activity, and is characterized by an unexplained immobilization of TCR-pMHC complexes relative to the highly dynamic immunological synapse periphery. Here we show that centrally accumulated TCRs are located on the surface of extracellular microvesicles that bud at the immunological synapse centre. Tumour susceptibility gene 101 (TSG101) sorts TCRs for inclusion in microvesicles, whereas vacuolar protein sorting 4 (VPS4) mediates scission of microvesicles from the T-cell plasma membrane. The human immunodeficiency virus polyprotein Gag co-opts this process for budding of virus-like particles. B cells bearing cognate pMHC receive TCRs from T cells and initiate intracellular signals in response to isolated synaptic microvesicles. We conclude that the immunological synapse orchestrates TCR sorting and release in extracellular microvesicles. These microvesicles deliver transcellular signals across antigen-dependent synapses by engaging cognate pMHC on APCs.
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http://dx.doi.org/10.1038/nature12951DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3949170PMC
March 2014

Three-dimensional reconstruction of intact human integrin αIIbβ3: new implications for activation-dependent ligand binding.

Blood 2013 Dec 17;122(26):4165-71. Epub 2013 Oct 17.

Laboratory of Blood and Vascular Biology, Rockefeller University, New York, NY;

Integrin αIIbβ3 plays a central role in hemostasis and thrombosis. We provide the first 3-dimensional reconstruction of intact purified αIIbβ3 in a nanodisc lipid bilayer. Unlike previous models, it shows that the ligand-binding head domain is on top, pointing away from the membrane. Moreover, unlike the crystal structure of the recombinant ectodomain, the lower legs are not parallel, straight, and adjacent. Rather, the αIIb lower leg is bent between the calf-1 and calf-2 domains and the β3 Integrin-Epidermal Growth Factor (I-EGF) 2 to 4 domains are freely coiled rather than in a cleft between the β3 headpiece and the αIIb lower leg. Our data indicate an important role for the region that links the distal calf-2 and β-tail domains to their respective transmembrane (TM) domains in transmitting the conformational changes in the TM domains associated with inside-out activation.
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http://dx.doi.org/10.1182/blood-2013-04-499194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3868924PMC
December 2013

Dominant form of congenital hyperinsulinism maps to HK1 region on 10q.

Horm Res Paediatr 2013 13;80(1):18-27. Epub 2013 Jul 13.

Division of Endocrinology and Diabetes, The Children's Hospital of Philadelphia, Philadelphia, Pa. 19104, USA.

Background/aims: In a family with congenital hyperinsulinism (HI), first described in the 1950s by McQuarrie, we examined the genetic locus and clinical phenotype of a novel form of dominant HI.

Methods: We surveyed 25 affected individuals, 7 of whom participated in tests of insulin dysregulation (24-hour fasting, oral glucose and protein tolerance tests). To identify the disease locus and potential disease-associated mutations we performed linkage analysis, whole transcriptome sequencing, whole genome sequencing, gene capture, and next generation sequencing.

Results: Most affecteds were diagnosed with HI before age one and 40% presented with a seizure. All affecteds responded well to diazoxide. Affecteds failed to adequately suppress insulin secretion following oral glucose tolerance test or prolonged fasting; none had protein-sensitive hypoglycemia. Linkage analysis mapped the HI locus to Chr10q21-22, a region containing 48 genes. Three novel noncoding variants were found in hexokinase 1 (HK1) and one missense variant in the coding region of DNA2.

Conclusion: Dominant, diazoxide-responsive HI in this family maps to a novel locus on Chr10q21-22. HK1 is the more attractive disease gene candidate since a mutation interfering with the normal suppression of HK1 expression in beta-cells could readily explain the hypoglycemia phenotype of this pedigree.
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http://dx.doi.org/10.1159/000351943DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3876732PMC
March 2014

Strength training increases endurance time to exhaustion during high-intensity exercise despite no change in critical power.

J Strength Cond Res 2014 Mar;28(3):601-9

1School of Nutrition and Health Promotion, Arizona State University, Phoenix, Arizona; 2Department of Human Services, University of Virginia, Charlottesville, Virginia; 3Department of Kinesiology, James Madison University, Harrisonburg, Virginia; and 4School of Sport and Exercise, Massey University, Palmerston North, New Zealand.

The purpose of this study was to determine whether improvements in endurance exercise performance elicited by strength training were accurately reflected by changes in parameters of the power-duration hyperbola for high-intensity exercise. Before and after 8 weeks of strength training (N = 14) or no exercise, control (N = 5), 19 males (age: 20.6 ± 2.0 years; weight: 78.2 ± 15.9 kg) performed a maximal incremental exercise test on a cycle ergometer and also cycled to exhaustion during 4 constant-power exercise bouts. Critical power (CP) and anaerobic work capacity (W') were estimated using nonlinear and linear models. Subjects in the strength training group improved significantly more than controls (p < 0.05) for strength (~30%), power at V[Combining Dot Above]O2peak (7.9%), and time to exhaustion (TTE) for all 4 constant-power tests (~39%). Contrary to our hypothesis, CP did not change significantly after strength training (p > 0.05 for all models). Strength training improved W' (mean range of improvement = +5.8 to +10.0 kJ; p < 0.05) for both linear models. Increases in W' were consistently positively correlated with improvements in TTE, whereas changes in CP were not. Our findings indicate that strength training alters the power-duration hyperbola such that W' is enhanced without any improvement in CP. Consequently, CP may not be robust enough to track changes in endurance capacity elicited by strength training, and we do not recommend it to be used for this purpose. Conversely, W' may be the better indicator of improvement in endurance performance elicited by strength training.
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http://dx.doi.org/10.1519/JSC.0b013e31829e113bDOI Listing
March 2014

Vitamin D bioavailability and catabolism in pediatric chronic kidney disease.

Pediatr Nephrol 2013 Sep 2;28(9):1843-53. Epub 2013 Jun 2.

The Children's Hospital of Philadelphia, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA.

Background: Vitamin D-binding protein (DBP) and catabolism have not been examined in the clinical setting of childhood chronic kidney disease (CKD).

Methods: The concentrations of serum vitamin D {25-hydroxyvitamin D [25(OH)D], 1,25-dihydroxyvitamin D [1,25(OH)(2)D], 24,25-dihydroxyvitamin D [24,25(OH)(2)D]}, DBP, intact parathyroid hormone (iPTH), and fibroblast growth factor-23 (FGF23) were measured in 148 participants with CKD stages 2-5D secondary to congenital anomalies of the kidney/urinary tract (CAKUT), glomerulonephritis (GN), or focal segmental glomerulosclerosis (FSGS). Free and bioavailable 25(OH)D concentrations were calculated using total 25(OH)D, albumin, and DBP concentrations.

Results: The concentrations of all vitamin D metabolites were lower with more advanced CKD (p < 0.001) and glomerular diagnoses (p ≤ 0.002). Among non-dialysis participants, DBP was lower in FSGS versus other diagnoses (FSGS-dialysis interaction p = 0.02). Winter season, older age, FSGS and GN, and higher FGF23 concentrations were independently associated with lower concentrations of free and bioavailable 25(OH)D. Black race was associated with lower total 25(OH)D and DBP, but not free or bioavailable 25(OH)D. 24,25(OH)(2)D was the vitamin D metabolite most strongly associated with iPTH. Lower 25(OH)D and higher iPTH concentrations, black race, and greater CKD severity were independently associated with lower levels of 24,25(OH)(2)D, while higher FGF23 concentrations and GN were associated with higher levels of 24,25(OH)(2)D.

Conclusions: Children with CKD exhibit altered catabolism and concentrations of DBP and free and bioavailable 25(OH)D, and there is an important impact of their underlying disease.
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http://dx.doi.org/10.1007/s00467-013-2493-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4053210PMC
September 2013

Mineral metabolism and cortical volumetric bone mineral density in childhood chronic kidney disease.

J Clin Endocrinol Metab 2013 May 1;98(5):1930-8. Epub 2013 Apr 1.

The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA.

Context: The relationships among cortical volumetric bone mineral density (CortBMD) and comprehensive measures of mineral metabolism have not been addressed in chronic kidney disease (CKD).

Objective: The aim of the study was to identify the determinants of CortBMD in childhood CKD. A secondary objective was to assess whether CortBMD was associated with subsequent fracture.

Design And Participants: This prospective cohort study included 171 children, adolescents, and young adults (aged 5-21 years) with CKD stages 2-5D at enrollment and 89 1 year later.

Outcomes: Serum measures included vitamin D [25-hydroxyvitamin D (25[OH]D), 1,25-dihydroxyvitamin D (1,25(OH)₂D), 24,25-dihydroxyvitamin D], vitamin D-binding protein, intact PTH, fibroblast growth factor 23, calcium, and phosphorus. Tibia quantitative computed tomography measures of CortBMD were expressed as sex-, race-, and age-specific Z-scores based on 675 controls. Multivariable linear regression identified the independent correlates of CortBMD Z-scores and the change in CortBMD Z-scores.

Results: Lower calcium (β = .31/1 mg/dL, P = .01) and 25(OH)D (β = .18/10 ng/mL, P = .04) and higher PTH (β = -.02/10%, P = .002) and 1,25(OH)₂D (β = -.07/10%, P < .001) were independently associated with lower CortBMD Z-scores at baseline. The correlations of total, free, and bioavailable 25(OH)D with CortBMD did not differ. Higher baseline 1,25(OH)₂D (P < .05) and greater increases in PTH (P < .001) were associated with greater declines in CortBMD Z-scores. Greater increases in calcium concentrations were associated with greater increases in CortBMD Z-scores in growing children (interaction P = .009). The hazard ratio for fracture was 1.75 (95% confidence interval 1.15-2.67; P = .009) per SD lower baseline CortBMD.

Conclusions: Greater PTH and 1,25(OH)₂D and lower calcium concentrations were independently associated with baseline and progressive cortical deficits in childhood CKD. Lower CortBMD Z-score was associated with increased fracture risk.
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http://dx.doi.org/10.1210/jc.2012-4188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3644604PMC
May 2013

Structure, dynamics, evolution, and function of a major scaffold component in the nuclear pore complex.

Structure 2013 Apr 14;21(4):560-71. Epub 2013 Mar 14.

Department of Biochemistry, Ullmann Building, Room 409, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.

The nuclear pore complex, composed of proteins termed nucleoporins (Nups), is responsible for nucleocytoplasmic transport in eukaryotes. Nuclear pore complexes (NPCs) form an annular structure composed of the nuclear ring, cytoplasmic ring, a membrane ring, and two inner rings. Nup192 is a major component of the NPC's inner ring. We report the crystal structure of Saccharomyces cerevisiae Nup192 residues 2-960 [ScNup192(2-960)], which adopts an α-helical fold with three domains (i.e., D1, D2, and D3). Small angle X-ray scattering and electron microscopy (EM) studies reveal that ScNup192(2-960) could undergo long-range transition between "open" and "closed" conformations. We obtained a structural model of full-length ScNup192 based on EM, the structure of ScNup192(2-960), and homology modeling. Evolutionary analyses using the ScNup192(2-960) structure suggest that NPCs and vesicle-coating complexes are descended from a common membrane-coating ancestral complex. We show that suppression of Nup192 expression leads to compromised nuclear transport and hypothesize a role for Nup192 in modulating the permeability of the NPC central channel.
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http://dx.doi.org/10.1016/j.str.2013.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3755625PMC
April 2013

Inward-facing conformation of the zinc transporter YiiP revealed by cryoelectron microscopy.

Proc Natl Acad Sci U S A 2013 Feb 22;110(6):2140-5. Epub 2013 Jan 22.

Laboratory of Cryo-Electron Microscopy, New York Structural Biology Center, New York, NY 10027, USA.

YiiP is a dimeric Zn(2+)/H(+) antiporter from Escherichia coli belonging to the cation diffusion facilitator family. We used cryoelectron microscopy to determine a 13-Å resolution structure of a YiiP homolog from Shewanella oneidensis within a lipid bilayer in the absence of Zn(2+). Starting from the X-ray structure in the presence of Zn(2+), we used molecular dynamics flexible fitting to build a model consistent with our map. Comparison of the structures suggests a conformational change that involves pivoting of a transmembrane, four-helix bundle (M1, M2, M4, and M5) relative to the M3-M6 helix pair. Although accessibility of transport sites in the X-ray model indicates that it represents an outward-facing state, our model is consistent with an inward-facing state, suggesting that the conformational change is relevant to the alternating access mechanism for transport. Molecular dynamics simulation of YiiP in a lipid environment was used to address the feasibility of this conformational change. Association of the C-terminal domains is the same in both states, and we speculate that this association is responsible for stabilizing the dimer that, in turn, may coordinate the rearrangement of the transmembrane helices.
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http://dx.doi.org/10.1073/pnas.1215455110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3568326PMC
February 2013

Regulation of glucagon secretion in normal and diabetic human islets by γ-hydroxybutyrate and glycine.

J Biol Chem 2013 Feb 24;288(6):3938-51. Epub 2012 Dec 24.

Division of Endocrinology, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA.

Paracrine signaling between pancreatic islet β-cells and α-cells has been proposed to play a role in regulating glucagon responses to elevated glucose and hypoglycemia. To examine this possibility in human islets, we used a metabolomic approach to trace the responses of amino acids and other potential neurotransmitters to stimulation with [U-(13)C]glucose in both normal individuals and type 2 diabetics. Islets from type 2 diabetics uniformly showed decreased glucose stimulation of insulin secretion and respiratory rate but demonstrated two different patterns of glucagon responses to glucose: one group responded normally to suppression of glucagon by glucose, but the second group was non-responsive. The non-responsive group showed evidence of suppressed islet GABA levels and of GABA shunt activity. In further studies with normal human islets, we found that γ-hydroxybutyrate (GHB), a potent inhibitory neurotransmitter, is generated in β-cells by an extension of the GABA shunt during glucose stimulation and interacts with α-cell GHB receptors, thus mediating the suppressive effect of glucose on glucagon release. We also identified glycine, acting via α-cell glycine receptors, as the predominant amino acid stimulator of glucagon release. The results suggest that glycine and GHB provide a counterbalancing receptor-based mechanism for controlling α-cell secretory responses to metabolic fuels.
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http://dx.doi.org/10.1074/jbc.M112.385682DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3567647PMC
February 2013

High-throughput methods for electron crystallography.

Methods Mol Biol 2013 ;955:273-96

Department of Cell Biology, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY, USA.

Membrane proteins play a tremendously important role in cell physiology and serve as a target for an increasing number of drugs. Structural information is key to understanding their function and for developing new strategies for combating disease. However, the complex physical chemistry associated with membrane proteins has made them more difficult to study than their soluble cousins. Electron crystallography has historically been a successful method for solving membrane protein structures and has the advantage of providing a native lipid environment for these proteins. Specifically, when membrane proteins form two-dimensional arrays within a lipid bilayer, electron microscopy can be used to collect images and diffraction and the corresponding data can be combined to produce a three-dimensional reconstruction, which under favorable conditions can extend to atomic resolution. Like X-ray crystallography, the quality of the structures are very much dependent on the order and size of the crystals. However, unlike X-ray crystallography, high-throughput methods for screening crystallization trials for electron crystallography are not in general use. In this chapter, we describe two alternative methods for high-throughput screening of membrane protein crystallization within the lipid bilayer. The first method relies on the conventional use of dialysis for removing detergent and thus reconstituting the bilayer; an array of dialysis wells in the standard 96-well format allows the use of a liquid-handling robot and greatly increases throughput. The second method relies on titration of cyclodextrin as a chelating agent for detergent; a specialized pipetting robot has been designed not only to add cyclodextrin in a systematic way, but to use light scattering to monitor the reconstitution process. In addition, the use of liquid-handling robots for making negatively stained grids and methods for automatically imaging samples in the electron microscope are described.
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http://dx.doi.org/10.1007/978-1-62703-176-9_15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3644976PMC
April 2013