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Delineating the Molecular Basis of the Calmodulin‒bMunc13-2 Interaction by Cross-Linking/Mass Spectrometry-Evidence for a Novel CaM Binding Motif in bMunc13-2.
Piotrowski C, Moretti R, Ihling CH, Haedicke A, Liepold T, Lipstein N, Meiler J, Jahn O, Sinz A
(2020) Cells 9:
MeSH Terms: Amino Acid Motifs, Amino Acid Sequence, Animals, Binding Sites, Calmodulin, Cattle, Cross-Linking Reagents, Hydrophobic and Hydrophilic Interactions, Mass Spectrometry, Models, Molecular, Nerve Tissue Proteins, Protein Binding, Protein Domains, Rats, Swine
Show Abstract · Added March 21, 2020
Exploring the interactions between the Ca binding protein calmodulin (CaM) and its target proteins remains a challenging task. Members of the Munc13 protein family play an essential role in short-term synaptic plasticity, modulated via the interaction with CaM at the presynaptic compartment. In this study, we focus on the bMunc13-2 isoform expressed in the brain, as strong changes in synaptic transmission were observed upon its mutagenesis or deletion. The CaM‒bMunc13-2 interaction was previously characterized at the molecular level using short bMunc13-2-derived peptides only, revealing a classical 1‒5‒10 CaM binding motif. Using larger protein constructs, we have now identified for the first time a novel and unique CaM binding site in bMunc13-2 that contains an -terminal extension of a classical 1‒5‒10 CaM binding motif. We characterize this motif using a range of biochemical and biophysical methods and highlight its importance for the CaM‒bMunc13-2 interaction.
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Synthesis and Characterization of Site-Specific O -Alkylguanine DNA-Alkyl Transferase-Oligonucleotide Crosslinks.
Ghodke PP, Albertolle ME, Johnson KM, Guengerich FP
(2019) Curr Protoc Nucleic Acid Chem 76: e74
MeSH Terms: Catalysis, Catalytic Domain, Chromatography, Liquid, Copper, Cross-Linking Reagents, Escherichia coli, O(6)-Methylguanine-DNA Methyltransferase, Oligonucleotides, Polymerization, Tandem Mass Spectrometry, Templates, Genetic, Trypsin
Show Abstract · Added March 3, 2020
O -Alkylguanine DNA-alkyltransferase (AGT), a DNA repair protein, can form crosslinks with DNA. The AGT-DNA crosslinks are known to be mutagenic when AGT is heterologously expressed in Escherichia coli, as well as in mammalian cells. To understand the biological consequences, reliable access to AGT-oligonucleotide crosslinks is needed. This article describes the synthesis and characterization of site-specific AGT-oligonucleotide crosslinks at the N2-position of deoxyguanosine and N6-position of deoxyadenosine. We developed a post-oligomerization strategy for the synthesis of propargyl-modified oligonucleotides. Copper-catalyzed azide-alkyne cycloaddition was used as a key step to obtain the iodoacetamide-linked oligonucleotides, which serve as good electrophiles for the crosslinking reaction with cysteine-145 of the active site of AGT. Trypsinization of AGT and hydrolysis of oligonucleotides, combined with analysis by liquid chromatography-tandem mass spectrometry, was utilized to confirm the nucleobase-adducted peptides. This method provides a useful strategy for the synthesis and characterization of site-specific DNA-protein crosslinks, which can be further used to understand proteolytic degradation-coupled DNA repair mechanisms. © 2019 by John Wiley & Sons, Inc.
© 2019 John Wiley & Sons, Inc.
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Peptide probes detect misfolded transthyretin oligomers in plasma of hereditary amyloidosis patients.
Schonhoft JD, Monteiro C, Plate L, Eisele YS, Kelly JM, Boland D, Parker CG, Cravatt BF, Teruya S, Helmke S, Maurer M, Berk J, Sekijima Y, Novais M, Coelho T, Powers ET, Kelly JW
(2017) Sci Transl Med 9:
MeSH Terms: Amyloidosis, Familial, Benzoxazoles, Case-Control Studies, Cross-Linking Reagents, Diazomethane, Genotype, Humans, Ions, Light, Molecular Probes, Molecular Weight, Peptides, Prealbumin, Protein Folding, Protein Multimerization, Protein Structure, Secondary, Proteolysis, Proteomics, Solubility
Show Abstract · Added March 3, 2020
Increasing evidence supports the hypothesis that soluble misfolded protein assemblies contribute to the degeneration of postmitotic tissue in amyloid diseases. However, there is a dearth of reliable nonantibody-based probes for selectively detecting oligomeric aggregate structures circulating in plasma or deposited in tissues, making it difficult to scrutinize this hypothesis in patients. Hence, understanding the structure-proteotoxicity relationships driving amyloid diseases remains challenging, hampering the development of early diagnostic and novel treatment strategies. We report peptide-based probes that selectively label misfolded transthyretin (TTR) oligomers circulating in the plasma of TTR hereditary amyloidosis patients exhibiting a predominant neuropathic phenotype. These probes revealed that there are much fewer misfolded TTR oligomers in healthy controls, in asymptomatic carriers of mutations linked to amyloid polyneuropathy, and in patients with TTR-associated cardiomyopathies. The absence of misfolded TTR oligomers in the plasma of cardiomyopathy patients suggests that the tissue tropism observed in the TTR amyloidoses is structure-based. Misfolded oligomers decrease in TTR amyloid polyneuropathy patients treated with disease-modifying therapies (tafamidis or liver transplant-mediated gene therapy). In a subset of TTR amyloid polyneuropathy patients, the probes also detected a circulating TTR fragment that disappeared after tafamidis treatment. Proteomic analysis of the isolated TTR oligomers revealed a specific patient-associated signature composed of proteins that likely associate with the circulating TTR oligomers. Quantification of plasma oligomer concentrations using peptide probes could become an early diagnostic strategy, a response-to-therapy biomarker, and a useful tool for understanding structure-proteotoxicity relationships in the TTR amyloidoses.
Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
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The sulfilimine cross-link of collagen IV contributes to kidney tubular basement membrane stiffness.
Bhave G, Colon S, Ferrell N
(2017) Am J Physiol Renal Physiol 313: F596-F602
MeSH Terms: Animals, Basement Membrane, Biomechanical Phenomena, Collagen Type IV, Cross-Linking Reagents, Elastic Modulus, Extracellular Matrix Proteins, Genotype, Imines, Kidney, Mice, Inbred C57BL, Mice, Knockout, Peroxidase, Phenotype, Protein Conformation, Tensile Strength
Show Abstract · Added December 7, 2017
Basement membranes (BMs), a specialized form of extracellular matrix, underlie nearly all cell layers and provide structural support for tissues and interact with cell surface receptors to determine cell behavior. Both macromolecular composition and stiffness of the BM influence cell-BM interactions. Collagen IV is a major constituent of the BM that forms an extensively cross-linked oligomeric network. Its deficiency leads to BM mechanical instability, as observed with glomerular BM in Alport syndrome. These findings have led to the hypothesis that collagen IV and its cross-links determine BM stiffness. A sulfilimine bond (S = N) between a methionine sulfur and a lysine nitrogen cross-links collagen IV and is formed by the matrix enzyme peroxidasin. In peroxidasin knockout mice with reduced collagen IV sulfilimine cross-links, we find a reduction in renal tubular BM stiffness. Thus this work provides the first direct experimental evidence that collagen IV sulfilimine cross-links contribute to BM mechanical properties and provides a foundation for future work on the relationship of BM mechanics to cell function in renal disease.
Copyright © 2017 the American Physiological Society.
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Trapping redox partnerships in oxidant-sensitive proteins with a small, thiol-reactive cross-linker.
Allan KM, Loberg MA, Chepngeno J, Hurtig JE, Tripathi S, Kang MG, Allotey JK, Widdershins AH, Pilat JM, Sizek HJ, Murphy WJ, Naticchia MR, David JB, Morano KA, West JD
(2016) Free Radic Biol Med 101: 356-366
MeSH Terms: Cross-Linking Reagents, Disulfides, Glutathione Peroxidase, Methionine Sulfoxide Reductases, Oxidants, Oxidation-Reduction, Oxidative Stress, Oxidoreductases Acting on Sulfur Group Donors, Peroxiredoxins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Sulfhydryl Compounds, Sulfones, Thioredoxins, tert-Butylhydroperoxide
Show Abstract · Added April 24, 2017
A broad range of redox-regulated proteins undergo reversible disulfide bond formation on oxidation-prone cysteine residues. Heightened reactivity of the thiol groups in these cysteines also increases susceptibility to modification by organic electrophiles, a property that can be exploited in the study of redox networks. Here, we explored whether divinyl sulfone (DVSF), a thiol-reactive bifunctional electrophile, cross-links oxidant-sensitive proteins to their putative redox partners in cells. To test this idea, previously identified oxidant targets involved in oxidant defense (namely, peroxiredoxins, methionine sulfoxide reductases, sulfiredoxin, and glutathione peroxidases), metabolism, and proteostasis were monitored for cross-link formation following treatment of Saccharomyces cerevisiae with DVSF. Several proteins screened, including multiple oxidant defense proteins, underwent intermolecular and/or intramolecular cross-linking in response to DVSF. Specific redox-active cysteines within a subset of DVSF targets were found to influence cross-linking; in addition, DVSF-mediated cross-linking of its targets was impaired in cells first exposed to oxidants. Since cross-linking appeared to involve redox-active cysteines in these proteins, we examined whether potential redox partners became cross-linked to them upon DVSF treatment. Specifically, we found that several substrates of thioredoxins were cross-linked to the cytosolic thioredoxin Trx2 in cells treated with DVSF. However, other DVSF targets, like the peroxiredoxin Ahp1, principally formed intra-protein cross-links upon DVSF treatment. Moreover, additional protein targets, including several known to undergo S-glutathionylation, were conjugated via DVSF to glutathione. Our results indicate that DVSF is of potential use as a chemical tool for irreversibly trapping and discovering thiol-based redox partnerships within cells.
Copyright © 2016 Elsevier Inc. All rights reserved.
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KCNE1 induces fenestration in the Kv7.1/KCNE1 channel complex that allows for highly specific pharmacological targeting.
Wrobel E, Rothenberg I, Krisp C, Hundt F, Fraenzel B, Eckey K, Linders JT, Gallacher DJ, Towart R, Pott L, Pusch M, Yang T, Roden DM, Kurata HT, Schulze-Bahr E, Strutz-Seebohm N, Wolters D, Seebohm G
(2016) Nat Commun 7: 12795
MeSH Terms: Adamantane, Allosteric Regulation, Animals, Binding Sites, Cross-Linking Reagents, Humans, Ion Channel Gating, KCNQ1 Potassium Channel, Models, Molecular, Mutagenesis, Mutation, Oocytes, Potassium Channel Blockers, Potassium Channels, Voltage-Gated, Tandem Mass Spectrometry, Xenopus laevis
Show Abstract · Added March 24, 2020
Most small-molecule inhibitors of voltage-gated ion channels display poor subtype specificity because they bind to highly conserved residues located in the channel's central cavity. Using a combined approach of scanning mutagenesis, electrophysiology, chemical ligand modification, chemical cross-linking, MS/MS-analyses and molecular modelling, we provide evidence for the binding site for adamantane derivatives and their putative access pathway in Kv7.1/KCNE1 channels. The adamantane compounds, exemplified by JNJ303, are highly potent gating modifiers that bind to fenestrations that become available when KCNE1 accessory subunits are bound to Kv7.1 channels. This mode of regulation by auxiliary subunits may facilitate the future development of potent and highly subtype-specific Kv channel inhibitors.
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Dual drug delivery of tamoxifen and quercetin: Regulated metabolism for anticancer treatment with nanosponges.
Lockhart JN, Stevens DM, Beezer DB, Kravitz A, Harth E
(2015) J Control Release 220: 751-7
MeSH Terms: Animals, Antineoplastic Combined Chemotherapy Protocols, Biological Availability, Biotransformation, Breast Neoplasms, Cell Line, Tumor, Cell Survival, Chemistry, Pharmaceutical, Cross-Linking Reagents, Cytochrome P-450 CYP3A, Delayed-Action Preparations, Dose-Response Relationship, Drug, Drug Carriers, Drug Stability, Female, Gastric Juice, Glucuronosyltransferase, Intestinal Secretions, Kinetics, Mice, Nanomedicine, Nanoparticles, Particle Size, Polyesters, Quercetin, Solubility, Tamoxifen
Show Abstract · Added February 15, 2016
We report the synthesis and encapsulation of polyester nanosponge particles (NPs) co-loaded with tamoxifen (TAM) and quercetin (QT) to investigate the loading, release and in vitro metabolism of a dual drug formulation. The NPs are made in two variations, 4% and 8% crosslinking densities, to evaluate the effects on metabolism and release kinetics. The NP-4% formulation with a particle size of 89.3 ± 14.8 nm was found to have loading percentages of 6.91 ± 0.13% TAM and 7.72 ± 0.15% QT after targeting 10% (w/w) each. The NP-8% formulation with a particle size of 91.5 ± 9.8 nm was found to have loading percentages of 7.26 ± 0.10% TAM and 7.80 ± 0.12% QT. The stability of the formulation was established in simulated gastrointestinal fluids, and the metabolism of TAM was shown to be reduced 2-fold and 3-fold for NP-4%s and NP-8%s, respectively, while QT metabolism was reduced 3 and 4-fold. The implications for improved bioavailability of the NP formulations were supported by cytotoxicity results that showed a similar efficacy to free dual drug formulations and even enhanced anti-cancer effects in the recovery condition. This work demonstrates the suitability of the nanosponges not only as a dual release drug delivery system but also enabling a regulated metabolism through the capacity of a nanonetwork. The variation in crosslinking enables a dual release with tailored release kinetics and suggests improved bioavailability aided by a reduced metabolism.
Copyright © 2015 Elsevier B.V. All rights reserved.
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27 MeSH Terms
The Ancient Immunoglobulin Domains of Peroxidasin Are Required to Form Sulfilimine Cross-links in Collagen IV.
Ero-Tolliver IA, Hudson BG, Bhave G
(2015) J Biol Chem 290: 21741-8
MeSH Terms: Collagen Type IV, Cross-Linking Reagents, Evolution, Molecular, Extracellular Matrix, Extracellular Matrix Proteins, HEK293 Cells, Heme, Humans, Imines, Immunoglobulins, Models, Biological, Peroxidase, Peroxidases, Protein Binding, Protein Structure, Tertiary
Show Abstract · Added August 12, 2015
The collagen IV sulfilimine cross-link and its catalyzing enzyme, peroxidasin, represent a dyad critical for tissue development, which is conserved throughout the animal kingdom. Peroxidasin forms novel sulfilimine bonds between opposing methionine and hydroxylysine residues to structurally reinforce the collagen IV scaffold, a function critical for basement membrane and tissue integrity. However, the molecular mechanism underlying cross-link formation remains unclear. In this work, we demonstrate that the catalytic domain of peroxidasin and its immunoglobulin (Ig) domains are required for efficient sulfilimine bond formation. Thus, these molecular features underlie the evolutionarily conserved function of peroxidasin in tissue development and integrity and distinguish peroxidasin from other peroxidases, such as myeloperoxidase (MPO) and eosinophil peroxidase (EPO).
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
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Pendant allyl crosslinking as a tunable shape memory actuator for vascular applications.
Boire TC, Gupta MK, Zachman AL, Lee SH, Balikov DA, Kim K, Bellan LM, Sung HJ
(2015) Acta Biomater 24: 53-63
MeSH Terms: Animals, Blood Vessel Prosthesis, Blood Vessel Prosthesis Implantation, Cross-Linking Reagents, Disease Models, Animal, Hindlimb, Human Umbilical Vein Endothelial Cells, Humans, Ischemia, Materials Testing, Mice, Polystyrenes
Show Abstract · Added March 18, 2020
UNLABELLED - Thermo-responsive shape memory polymers (SMPs) can be programmed to fit into small-bore incisions and recover their functional shape upon deployment in the body. This property is of significant interest for developing the next generation of minimally-invasive medical devices. To be used in such applications, SMPs should exhibit adequate mechanical strengths that minimize adverse compliance mismatch-induced host responses (e.g. thrombosis, hyperplasia), be biodegradable, and demonstrate switch-like shape recovery near body temperature with favorable biocompatibility. Combinatorial approaches are essential in optimizing SMP material properties for a particular application. In this study, a new class of thermo-responsive SMPs with pendant, photocrosslinkable allyl groups, x%poly(ε-caprolactone)-co-y%(α-allyl carboxylate ε-caprolactone) (x%PCL-y%ACPCL), are created in a robust, facile manner with readily tunable material properties. Thermomechanical and shape memory properties can be drastically altered through subtle changes in allyl composition. Molecular weight and gel content can also be altered in this combinatorial format to fine-tune material properties. Materials exhibit highly elastic, switch-like shape recovery near 37°C. Endothelial compatibility is comparable to tissue culture polystyrene (TCPS) and 100%PCL in vitro and vascular compatibility is demonstrated in vivo in a murine model of hindlimb ischemia, indicating promising suitability for vascular applications.
STATEMENT OF SIGNIFICANCE - With the ongoing thrust to make surgeries minimally-invasive, it is prudent to develop new biomaterials that are highly compatible and effective in this workflow. Thermo-responsive shape memory polymers (SMPs) have great potential for minimally-invasive applications because SMP medical devices (e.g. stents, grafts) can fit into small-bore minimally-invasive surgical devices and recover their functional shape when deployed in the body. To realize their potential, it is imperative to devise combinatorial approaches that enable optimization of mechanical, SM, and cellular responses for a particular application. In this study, a new class of thermo-responsive SMPs is created in a robust, facile manner with readily tunable material properties. Materials exhibit excellent, switch-like shape recovery near body temperature and promising biocompatibility for minimally-invasive vascular applications.
Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
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Protein structure prediction guided by crosslinking restraints--A systematic evaluation of the impact of the crosslinking spacer length.
Hofmann T, Fischer AW, Meiler J, Kalkhof S
(2015) Methods 89: 79-90
MeSH Terms: Animals, Chromatography, Liquid, Cross-Linking Reagents, Forecasting, Horses, Protein Conformation, Protein Folding, Proteins, Tandem Mass Spectrometry
Show Abstract · Added February 5, 2016
Recent development of high-resolution mass spectrometry (MS) instruments enables chemical crosslinking (XL) to become a high-throughput method for obtaining structural information about proteins. Restraints derived from XL-MS experiments have been used successfully for structure refinement and protein-protein docking. However, one formidable question is under which circumstances XL-MS data might be sufficient to determine a protein's tertiary structure de novo? Answering this question will not only include understanding the impact of XL-MS data on sampling and scoring within a de novo protein structure prediction algorithm, it must also determine an optimal crosslinker type and length for protein structure determination. While a longer crosslinker will yield more restraints, the value of each restraint for protein structure prediction decreases as the restraint is consistent with a larger conformational space. In this study, the number of crosslinks and their discriminative power was systematically analyzed in silico on a set of 2055 non-redundant protein folds considering Lys-Lys, Lys-Asp, Lys-Glu, Cys-Cys, and Arg-Arg reactive crosslinkers between 1 and 60Å. Depending on the protein size a heuristic was developed that determines the optimal crosslinker length. Next, simulated restraints of variable length were used to de novo predict the tertiary structure of fifteen proteins using the BCL::Fold algorithm. The results demonstrate that a distinct crosslinker length exists for which information content for de novo protein structure prediction is maximized. The sampling accuracy improves on average by 1.0 Å and up to 2.2 Å in the most prominent example. XL-MS restraints enable consistently an improved selection of native-like models with an average enrichment of 2.1.
Copyright © 2015. Published by Elsevier Inc.
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