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Da Zhang, Xiuli Wang, Xiaoyu Tian, Lulu Zhang, Guosheng Yang, Yinghong Tao, Chen Liang, Kun Li, Xiaoqi Yu, Xinjing Tang, Chaoshu Tang, Jing Zhou, Wei Kong, Junbao Du, Yaqian Huang, Hongfang Jin
Endogenous hydrogen sulfide (H2 S) and sulfur dioxide (SO2 ) are regarded as important regulators to control endothelial cell function and protect endothelial cell against various injuries. In our present study, we aimed to investigate the effect of endogenous H2 S on the SO2 generation in the endothelial cells and explore its significance in the endothelial inflammation in vitro and in vivo . The human umbilical vein endothelial cell (HUVEC) line (EA.hy926), primary HUVECs, primary rat pulmonary artery endothelial cells (RPAECs), and purified aspartate aminotransferase (AAT) protein from pig heart were used for in vitro experiments...
2018: Frontiers in Immunology
Cameron D Buchman, Sergio C Chai, Taosheng Chen
Pregnane X receptor (PXR) and the constitutive androstane receptor (CAR) are two members of the nuclear receptor superfamily that play major roles in the expression of various drug metabolism enzymes and are known for their ligand promiscuity. As with other nuclear receptors, PXR and CAR are each composed of a ligand-binding domain (LBD) and a DNA-binding domain (DBD) connected by a hinge region. Areas covered: This review focuses on the information obtained over the last 15+ years from X-ray crystallography studies of the structure of PXR and CAR...
May 14, 2018: Expert Opinion on Drug Metabolism & Toxicology
Patrick S Merkle, Kamil Gotfryd, Michel A Cuendet, Katrine Z Leth-Espensen, Ulrik Gether, Claus J Loland, Kasper D Rand
LeuT, a prokaryotic member of the neurotransmitter:sodium symporter (NSS) family, is an established structural model for mammalian NSS counterparts. We investigate the substrate translocation mechanism of LeuT by measuring the solution-phase structural dynamics of the transporter in distinct functional states by hydrogen/deuterium exchange mass spectrometry (HDX-MS). Our HDX-MS data pinpoint LeuT segments involved in substrate transport and reveal for the first time a comprehensive and detailed view of the dynamics associated with transition of the transporter between outward- and inward-facing configurations in a Na+ - and K+ -dependent manner...
May 2018: Science Advances
Ulrik H Mistarz, Kasper D Rand
Gas-phase hydrogen/deuterium exchange measured by mass spectrometry in a millisecond timeframe after ESI (gas-phase HDX-MS) is a fast and sensitive, yet unharnessed method to analyze the primary- and higher-order structure, intramolecular and intermolecular interactions, surface properties, and charge location of peptides and proteins. During a gas-phase HDX-MS experiment, heteroatom-bound non-amide hydrogens are made to exchange with deuterium during a millisecond timespan after electrospray ionization (ESI) by reaction with the highly basic reagent ND3 , enabling conformational analysis of protein states that are pertinent to the native solution-phase...
May 10, 2018: Methods: a Companion to Methods in Enzymology
Amberley Stephens, Nadezhda Nespovitaya, Maria Zacharopoulou, Clemens F Kaminski, Jonathan James Phillips, Gabriele S Kaminski Schierle
Understanding the mechanisms behind amyloid protein aggregation in diseases such as Parkinson's and Alzheimer's disease is often hampered by the reproducibility of in vitro assays. Yet, understanding the basic mechanisms of protein misfolding is essential for the development of novel therapeutic strategies. We show here, that for the amyloid protein alpha-synuclein (aSyn), a protein involved in Parkinson's disease (PD), chromatographic buffers and storage conditions can significantly interfere with the overall structure of the protein and thus affect protein aggregation kinetics...
May 11, 2018: Analytical Chemistry
Richard Y-C Huang, Steven R O'Neil, Daša Lipovšek, Guodong Chen
Higher-order structure (HOS) characterization of therapeutic protein-drug conjugates for comprehensive assessment of conjugation-induced protein conformational changes is an important consideration in the biopharmaceutical industry to ensure proper behavior of protein therapeutics. In this study, conformational dynamics of a small therapeutic protein, adnectin 1, together with its drug conjugate were characterized by hydrogen/deuterium exchange mass spectrometry (HDX-MS) with different spatial resolutions. Top-down HDX allows detailed assessment of the residue-level deuterium content in the payload conjugation region...
May 7, 2018: Journal of the American Society for Mass Spectrometry
Kyle W Anderson, Elyssia S Gallagher, Jeffrey W Hudgens
Membrane proteins are currently the most common targets for pharmaceuticals. However, characterization of their structural dynamics by hydrogen/deuterium exchange mass spectrometry (HDX-MS) is sparse due to insufficient automated methods to handle full-length membrane proteins in lipid bilayers. Additionally, membrane lipids used to mimic the membrane environment and to solubilize membrane proteins can impair chromatography performance and cause ion suppression in the mass spectrometer. The workflow discussed herein advances HDX-MS capabilities and other MS applications for membrane proteins by providing a fully automated method for HDX-MS analysis based on a phospholipid removal scheme compatible with robotic handling...
May 9, 2018: Analytical Chemistry
Luke Berry, Angela Patterson, Natasha Pence, John W Peters, Brian Bothner
The protocol detailed here describes a way to perform hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) on oxygen sensitive proteins. HDX-MS is a powerful tool for studying the protein structure-function relationship. Applying this technique to anaerobic proteins provides insight into the mechanism of proteins that perform oxygen sensitive chemistry. A problem when using HDX-MS to study anaerobic proteins is that there are many parts that require constant movement into and out of an anaerobic chamber...
March 20, 2018: Bio-protocol
Irina Oganesyan, Cristina Lento, Derek J Wilson
Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) emerged as a tool for biochemistry and structural biology around 25 years ago. It has since become a key approach for studying protein dynamics, protein-ligand interactions, membrane proteins and intrinsically disordered proteins (IDPs). In HDX labeling, proteins are exposed to deuterated solvent (usually D2 O) for a variable 'labeling time', resulting in isotope exchange of unprotected labile protons on the amide backbone and amino acid side chains. By comparing the levels of deuterium uptake in different regions of a protein, information on conformational and dynamic changes in the system can be acquired...
April 25, 2018: Methods: a Companion to Methods in Enzymology
M Rafalik, M Spodzieja, A S Kołodziejczyk, S Rodziewicz-Motowidło, A Szymańska, A Grubb, P Czaplewska
Human cystatin C (hCC) is a cysteine proteinase inhibitor involved in pathophysiological processes of dimerization and amyloid formation. These processes are directly associated with a number of neurodegenerative disorders such as Alzheimer disease or hereditary cystatin C amyloid angiopathy (HCCAA). One of the ideas on how to prevent amyloid formation is to use immunotherapy. HCC3 is one of a group of antibodies binding to hCC and reducing the in vitro formation of cystatin C dimers. Therefore, identification of the binding sites in the hCC-HCC3 complex may facilitate a search of effective drugs against HCCAA as well as understanding the mechanisms of neurodegenerative disorders...
April 20, 2018: Journal of Proteomics
Danilo Donnarumma, Claudio Maestri, Pietro Ivan Giammarinaro, Luigi Capriotti, Erika Bartolini, Daniele Veggi, Roberto Petracca, Maria Scarselli, Nathalie Norais
Hydrogen Deuterium exchange (HDx) associated with Mass Spectrometry (MS) is emerging as a powerful tool to provide conformational information on membrane proteins. Unfortunately, as for X-ray diffraction and NMR, HDx performed on reconstituted in vitro systems might not always reflect the in vivo environment. Outer Membrane Vesicles naturally released by E. coli were used to carry out analysis of native OmpF through HDx-MS. A new protocol compatible with HDx analysis and avoiding hindrance from the lipid contents was set-up...
April 5, 2018: Journal of Proteome Research
Brent A Kochert, Roxana E Iacob, Thomas E Wales, Alexandros Makriyannis, John R Engen
Hydrogen-deuterium exchange (HDX) mass spectrometry (MS) can provide valuable information about binding, allostery, and other conformational effects of interaction in protein complexes. For protein-ligand complexes, where the ligand may be a small molecule, peptide, nucleotide, or another protein(s), a typical experiment measures HDX in the protein alone and then compares that with HDX for the protein when part of the complex. Multiple factors are critical in the design and implementation of such experiments, including thoughtful consideration of the percent protein bound, the effects of the labeling protocol on the protein complex, and the dynamic range of the analysis method...
2018: Methods in Molecular Biology
Eva Illes-Toth, Don L Rempel, Michael L Gross
Alpha-synuclein (aS) forms toxic intermediates ranging from small oligomers and protofibrils to large amyloid fibrils. Understanding the time course of aS fibril formation and the role played by its regions is critical for therapeutic intervention. Here, we used pulsed hydrogen-deuterium-exchange and mass spectrometry (HDX-MS) for the first time to probe kinetic intermediates of the full aS aggregation in vitro, achieving kinetic snapshots containing spatially resolved protein information about critical stages...
March 30, 2018: ACS Chemical Neuroscience
Zhening Zhang, Wenguang G Liang, Lucas J Bailey, Yong Zi Tan, Hui Wei, Andrew Wang, Mara Farcasanu, Virgil A Woods, Lauren A McCord, David Lee, Weifeng Shang, Rebecca Deprez-Poulain, Benoit Deprez, David R Liu, Akiko Koide, Shohei Koide, Anthony A Kossiakoff, Sheng Li, Bridget Carragher, Clinton S Potter, Wei-Jen Tang
Insulin degrading enzyme (IDE) plays key roles in degrading peptides vital in type 2 diabetes, Alzheimer's, inflammation, and other human diseases. However, the process through which IDE recognizes peptides that tend to form amyloid fibrils remained unsolved. We used cryoEM to understand both the apo- and insulin-bound dimeric IDE states, revealing that IDE displays a large opening between the homologous ~55 kDa N- and C-terminal halves to allow selective substrate capture based on size and charge complementarity...
March 29, 2018: ELife
Andrea L Wang, Ying Zhou, Michael J Palmieri, Gang G Hao
Recombinant human erythropoietin (EPO) is a therapeutic glycoprotein widely used for treating anemia. EPO glycans carry extensive sialylation and the level of the modification is known to affect receptor binding, protein stability and pharmacokinetics. Nonetheless, a detailed understanding of the effects of sialylation on EPO conformation and dynamics is still lacking. Here we investigate the changes to EPO dynamics following enzymatic trimming of terminal sialic acid by amide hydrogen deuterium exchange mass spectrometry (HDX-MS)...
March 16, 2018: Journal of Pharmaceutical and Biomedical Analysis
Arash Hamidian, Marica Vaapil, Kristoffer von Stedingk, Toshitsugu Fujita, Camilla U Persson, Pontus Eriksson, Srinivas Veerla, Katleen De Preter, Frank Speleman, Hodaka Fujii, Sven Påhlman, Sofie Mohlin
Presence of perivascular neuroblastoma cells with high expression of hypoxia inducible factor (HIF)-2α correlates with distant metastasis and aggressive disease. Regulation of HIFs are traditionally considered to occur post-translationally, but we have recently shown that HIF-2α is unconventionally regulated also at the transcriptional level in neuroblastoma cells. Regulatory factors binding directly to EPAS1 (encoding HIF-2α) to promote transcription are yet to be defined. Here, we employ the novel CRISPR/Cas9-based engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) - mass spectrometry (MS) methodology to, in an unbiased fashion, identify proteins that associate with the EPAS1 promoter under normoxic and hypoxic conditions...
March 22, 2018: Biochemical and Biophysical Research Communications
Martin Lorenz Eisinger, Laiyin Nie, Aline Ricarda Dörrbaum, Julian David Langer, Hartmut Michel
Multidrug resistance (MDR) in bacterial pathogens has become a severe threat to public health. Membrane transporters of the multidrug and toxic compound extrusion (MATE) family contribute critically to MDR, making them promising drug targets. Despite recent advances, structures in different conformations and the mechanistic details of their antiport cycle are still elusive. Here we studied NorM_PS, a representative MATE transporter from Pseudomonas stutzeri, using biochemical assays in combination with hydrogen/deuterium exchange-mass spectrometry (HDX-MS)...
March 16, 2018: Journal of Molecular Biology
Laura J Byrnes, Yingrong Xu, Xiayang Qiu, Justin D Hall, Graham M West
Cystic Fibrosis (CF) is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Mutations associated with CF cause loss-of-function in CFTR leading to salt imbalance in epithelial tissues. Kalydeco (also called VX-770 or ivacaftor) was approved for CF treatment in 2012 but little is known regarding the compound's interactions with CFTR including the site of binding or mechanisms of action. In this study we use hydrogen/deuterium exchange (HDX) coupled with mass spectrometry to assess the conformational dynamics of a thermostabilized form of CFTR in apo and ligand-bound states...
March 16, 2018: Scientific Reports
Kinga Nyíri, Haydyn D T Mertens, Borbála Tihanyi, Gergely N Nagy, Bianka Kőhegyi, Judit Matejka, Matthew J Harris, Judit E Szabó, Veronika Papp-Kádár, Veronika Németh-Pongrácz, Olivér Ozohanics, Károly Vékey, Dmitri I Svergun, Antoni J Borysik, Beáta G Vértessy
Human deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), essential for DNA integrity, acts as a survival factor for tumor cells and is a target for cancer chemotherapy. Here we report that the Staphylococcal repressor protein StlSaPIBov1 (Stl) forms strong complex with human dUTPase. Functional analysis reveals that this interaction results in significant reduction of both dUTPase enzymatic activity and DNA binding capability of Stl. We conducted structural studies to understand the mechanism of this mutual inhibition...
March 12, 2018: Scientific Reports
Yoshitomo Hamuro, Sook Yen E
The technological goal of hydrogen/deuterium exchange-mass spectrometry (HDX-MS) is to determine backbone amide hydrogen exchange rates. The most critical challenge to achieve this goal is obtaining the deuterium incorporation in single-amide resolution, and gas-phase fragmentation may provide a universal solution. The gas-phase fragmentation may generate the daughter ions which differ by a single amino acid and the difference in deuterium incorporations in the two analogous ions can yield the deuterium incorporation at the sub-localized site...
May 2018: Journal of the American Society for Mass Spectrometry
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