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Journal of Lipid Research Mar 2016Phospholipids (PLs), one of the lipid categories, are not only the primary building blocks of cellular membranes, but also can be split to produce products that function...
Phospholipids (PLs), one of the lipid categories, are not only the primary building blocks of cellular membranes, but also can be split to produce products that function as second messengers in signal transduction and play a pivotal role in numerous cellular processes, including cell growth, survival, and motility. Here, we present an integrated novel method that combines a fast and robust TMS-diazomethane-based phosphate derivatization and isotopic labeling strategy, which enables simultaneous profiling and relative quantification of PLs from biological samples. Our results showed that phosphate methylation allows fast and sensitive identification of the six major PL classes, including their lysophospholipid counterparts, under positive ionization mode. The isotopic labeling of endogenous PLs was achieved by deuterated diazomethane, which was generated through acid-catalyzed hydrogen/deuterium (H/D) exchange and methanolysis of TMS-diazomethane during the process of phosphate derivatization. The measured H/D ratios of unlabeled and labeled PLs, which were mixed in known proportions, indicated that the isotopic labeling strategy is capable of providing relative quantitation with adequate accuracy, reproducibility, and a coefficient of variation of 9.1%, on average. This novel method offers unique advantages over existing approaches and presents a powerful tool for research of PL metabolism and signaling.
Topics: Cell Line, Tumor; Diazomethane; Humans; Isotope Labeling; Lysophospholipids; Metabolomics; Methylation; Phospholipids; Time Factors
PubMed: 26733148
DOI: 10.1194/jlr.M063024 -
Plant Signaling & Behavior May 2016The endophytic fungus Piriformospora indica colonizes Arabidopsis thaliana roots and promotes plant performance, growth and resistance/tolerance against abiotic and...
The endophytic fungus Piriformospora indica colonizes Arabidopsis thaliana roots and promotes plant performance, growth and resistance/tolerance against abiotic and biotic stress. Here we demonstrate that the benefits for the plant increase when the two partners are co-cultivated under stress (limited access to nutrient, exposure to heavy metals and salt, light and osmotic stress, pathogen infection). Moreover, physical contact between P. indica and Arabidopsis roots is necessary for optimal growth promotion, and chemical communication cannot replace the physical contact. Lower nutrient availability down-regulates and higher nutrient availability up-regulates the plant defense system including the expression of pathogenesis-related genes in roots. High light, osmotic and salt stresses support the beneficial interaction between the plant and the fungus. P. indica reduces stomata closure and H2O2 production after Alternaria brassicae infection in leaves and suppresses the defense-related accumulation of the phytohormone jasmonic acid. Thus, shifting the growth conditions toward a stress promotes the mutualistic interaction, while optimal supply with nutrients or low stress diminishes the benefits for the plant in the symbiosis.
Topics: Arabidopsis; Basidiomycota; Cyclopentanes; Diazonium Compounds; Host-Pathogen Interactions; Isoleucine; Light; Metals, Heavy; Nitrates; Osmotic Pressure; Oxylipins; Phosphates; Plant Roots; Plant Shoots; Pyridines; Seedlings; Stress, Physiological; Sulfates
PubMed: 27167761
DOI: 10.1080/15592324.2015.1136763 -
Clinical Cancer Research : An Official... Dec 2021Pyruvate kinase M2 (PKM2) catalyzes the final step in glycolysis, a key process of cancer metabolism. PKM2 is preferentially expressed by glioblastoma (GBM) cells with...
PURPOSE
Pyruvate kinase M2 (PKM2) catalyzes the final step in glycolysis, a key process of cancer metabolism. PKM2 is preferentially expressed by glioblastoma (GBM) cells with minimal expression in healthy brain. We describe the development, validation, and translation of a novel PET tracer to study PKM2 in GBM. We evaluated 1-((2-fluoro-6-[F]fluorophenyl)sulfonyl)-4-((4-methoxyphenyl)sulfonyl)piperazine ([F]DASA-23) in cell culture, mouse models of GBM, healthy human volunteers, and patients with GBM.
EXPERIMENTAL DESIGN
[F]DASA-23 was synthesized with a molar activity of 100.47 ± 29.58 GBq/μmol and radiochemical purity >95%. We performed initial testing of [F]DASA-23 in GBM cell culture and human GBM xenografts implanted orthotopically into mice. Next, we produced [F]DASA-23 under FDA oversight, and evaluated it in healthy volunteers and a pilot cohort of patients with glioma.
RESULTS
In mouse imaging studies, [F]DASA-23 clearly delineated the U87 GBM from surrounding healthy brain tissue and had a tumor-to-brain ratio of 3.6 ± 0.5. In human volunteers, [F]DASA-23 crossed the intact blood-brain barrier and was rapidly cleared. In patients with GBM, [F]DASA-23 successfully outlined tumors visible on contrast-enhanced MRI. The uptake of [F]DASA-23 was markedly elevated in GBMs compared with normal brain, and it identified a metabolic nonresponder within 1 week of treatment initiation.
CONCLUSIONS
We developed and translated [F]DASA-23 as a new tracer that demonstrated the visualization of aberrantly expressed PKM2 for the first time in human subjects. These results warrant further clinical evaluation of [F]DASA-23 to assess its utility for imaging therapy-induced normalization of aberrant cancer metabolism.
Topics: Animals; Brain Neoplasms; Diazonium Compounds; Glioblastoma; Glycolysis; Humans; Mice; Positron-Emission Tomography; Pyruvate Kinase; Sulfanilic Acids
PubMed: 34475101
DOI: 10.1158/1078-0432.CCR-21-0544 -
Organic Letters Dec 2020Dialkyldiazirines have emerged as reagents of choice for biological photoaffinity labeling studies. The mechanism of crosslinking has dramatic consequences for...
Dialkyldiazirines have emerged as reagents of choice for biological photoaffinity labeling studies. The mechanism of crosslinking has dramatic consequences for biological applications where instantaneous labeling is desirable, as carbene insertions display different chemoselectivity and are much faster than competing mechanisms involving diazo or ylide intermediates. Here, deuterium labeling and diazo compound trapping experiments are employed to demonstrate that both carbene and diazo mechanisms operate in the reactions of a dialkyldiazirine motif that is commonly utilized for biological applications. For the fraction of intermolecular labeling that does involve a carbene mechanism, direct insertion is not necessarily involved, as products derived from a carbonyl ylide are also observed. We demonstrate that a strained cycloalkyne can intercept diazo compound intermediates and serve as a bioorthogonal probe for studying the contribution of the diazonium mechanism of photoaffinity labeling on a model protein under aqueous conditions.
Topics: Catalysis; Diazomethane; Diazonium Compounds; Indicators and Reagents; Methane; Molecular Structure
PubMed: 33259213
DOI: 10.1021/acs.orglett.0c02714 -
Molecular & Cellular Proteomics : MCP 2021Cross-linking mass spectrometry (XL-MS) is a powerful tool for studying protein-protein interactions and elucidating architectures of protein complexes. While...
Cross-linking mass spectrometry (XL-MS) is a powerful tool for studying protein-protein interactions and elucidating architectures of protein complexes. While residue-specific XL-MS studies have been very successful, accessibility of interaction regions nontargetable by specific chemistries remain difficult. Photochemistry has shown great potential in capturing those regions because of nonspecific reactivity, but low yields and high complexities of photocross-linked products have hindered their identification, limiting current studies predominantly to single proteins. Here, we describe the development of three novel MS-cleavable heterobifunctional cross-linkers, namely SDASO (Succinimidyl diazirine sulfoxide), to enable fast and accurate identification of photocross-linked peptides by MS. The MS-based workflow allowed SDASO XL-MS analysis of the yeast 26S proteasome, demonstrating the feasibility of photocross-linking of large protein complexes for the first time. Comparative analyses have revealed that SDASO cross-linking is robust and captures interactions complementary to residue-specific reagents, providing the foundation for future applications of photocross-linking in complex XL-MS studies.
Topics: Chromatography, Liquid; Cross-Linking Reagents; Diazomethane; Fungal Proteins; Mass Spectrometry; Photochemical Processes; Proteasome Endopeptidase Complex; Saccharomyces cerevisiae; Serum Albumin, Bovine
PubMed: 33915260
DOI: 10.1016/j.mcpro.2021.100084 -
Biosensors & Bioelectronics Jun 2016Fast and accurate detection of microorganisms is of key importance in clinical analysis and in food and water quality monitoring. Salmonella typhimurium is responsible...
Fast and accurate detection of microorganisms is of key importance in clinical analysis and in food and water quality monitoring. Salmonella typhimurium is responsible for about a third of all cases of foodborne diseases and consequently, its fast detection is of great importance for ensuring the safety of foodstuffs. We report the development of a label-free impedimetric aptamer-based biosensor for S. typhimurium detection. The aptamer biosensor was fabricated by grafting a diazonium-supporting layer onto screen-printed carbon electrodes (SPEs), via electrochemical or chemical approaches, followed by chemical immobilisation of aminated-aptamer. FTIR-ATR, contact angle and electrochemical measurements were used to monitor the fabrication process. Results showed that electrochemical immobilisation of the diazonium-grafting layer allowed the formation of a denser aptamer layer, which resulted in higher sensitivity. The developed aptamer-biosensor responded linearly, on a logarithm scale, over the concentration range 1 × 10(1) to 1 × 10(8)CFU mL(-1), with a limit of quantification (LOQ) of 1 × 10(1) CFU mL(-1) and a limit of detection (LOD) of 6 CFU mL(-1). Selectivity studies showed that the aptamer biosensor could discriminate S. typhimurium from 6 other model bacteria strains. Finally, recovery studies demonstrated its suitability for the detection of S. typhimurium in spiked (1 × 10(2), 1 × 10(4) and 1 × 10(6) CFU mL(-1)) apple juice samples.
Topics: Aptamers, Nucleotide; Biosensing Techniques; Diazonium Compounds; Electrochemical Techniques; Food Microbiology; Humans; Limit of Detection; Salmonella typhimurium
PubMed: 26894987
DOI: 10.1016/j.bios.2016.02.024 -
Molecules (Basel, Switzerland) Jan 2023SARS-CoV-2 M is a chymotrypsin-like cysteine protease playing a relevant role during the replication and infectivity of SARS-CoV-2, the coronavirus responsible for...
SARS-CoV-2 M is a chymotrypsin-like cysteine protease playing a relevant role during the replication and infectivity of SARS-CoV-2, the coronavirus responsible for COVID-19. The binding site of M is characterized by the presence of a catalytic Cys145 which carries out the hydrolytic activity of the enzyme. As a consequence, several M inhibitors have been proposed to date in order to fight the COVID-19 pandemic. In our work, we designed, synthesized and biologically evaluated , a novel inhibitor of SARS-CoV-2 M bearing a trifluoromethyl diazirine moiety. displayed in vitro inhibition activity against SARS-CoV-2 M at a low micromolar level (IC = 4.1 μM) in a FRET-based assay. Moreover, an inhibition assay against PL revealed lack of inhibition, assuring the selectivity of the compound for the M. Furthermore, the target compound was docked within the binding site of the enzyme to predict the established intermolecular interactions in silico. was subsequently tested on the HCT-8 cell line to evaluate its effect on human cells' viability, displaying good tolerability, demonstrating the promising biological compatibility and activity of a trifluoromethyl diazirine moiety in the design and development of SARS-CoV-2 M binders.
Topics: Antiviral Agents; Diazomethane; Molecular Docking Simulation; Protease Inhibitors; SARS-CoV-2
PubMed: 36677572
DOI: 10.3390/molecules28020514 -
Molecules (Basel, Switzerland) Dec 2020NAD (nicotinamide adenine dinucleotide)-dependent protein deacylases, namely, the sirtuins, are important cell adaptor proteins that alter cell physiology in response to...
NAD (nicotinamide adenine dinucleotide)-dependent protein deacylases, namely, the sirtuins, are important cell adaptor proteins that alter cell physiology in response to low calorie conditions. They are thought to mediate the beneficial effects of calorie restriction to extend longevity and improve health profiles. Novel chemical probes are highly desired for a better understanding of sirtuin's roles in various biological processes. We developed a group of remarkably simple activity-based chemical probes for the investigation of active sirtuin content in complex native proteomes. These probes harbor a thioacyllysine warhead, a diazirine photoaffinity tag, as well as a terminal alkyne bioorthogonal functional group. Compared to their benzophenone-containing counterparts, these new probes demonstrated improved labeling efficiency and sensitivity, shortened irradiation time, and reduced background signal. They were applied to the labeling of individual recombinant proteins, protein mixtures, and whole cell lysate. These cell permeable small molecule probes also enabled the cellular imaging of sirtuin activity change. Taken together, our study provides new chemical biology tools and future drug discovery strategies for perturbing the activity of different sirtuin isoforms.
Topics: Chemistry Techniques, Synthetic; Diazomethane; Drug Design; Drug Discovery; Histone Deacetylase Inhibitors; Humans; Isoenzymes; Ligands; Molecular Probes; Molecular Structure; NAD; Sirtuins; Staining and Labeling; Structure-Activity Relationship
PubMed: 33375102
DOI: 10.3390/molecules26010011 -
Molecules (Basel, Switzerland) Mar 2018Tissue adhesives based on polyamidoamine (PAMAM) dendrimer, grafted with UV-sensitive aryldiazirine (PAMAM-g-diazirine) are promising new candidates for light active...
Tissue adhesives based on polyamidoamine (PAMAM) dendrimer, grafted with UV-sensitive aryldiazirine (PAMAM-g-diazirine) are promising new candidates for light active adhesion on soft tissues. Diazirine carbene precursors form interfacial and intermolecular covalent crosslinks with tissues after UV light activation that requires no premixing or inclusion of free radical initiators. However, primary amines on the PAMAM dendrimer surface present a potential risk due to their cytotoxic and immunological effects. PAMAM-g-diazirine formulations with cationic pendant amines converted into neutral amide groups were evaluated. In vitro toxicity is reduced by an order of magnitude upon amine capping while retaining bioadhesive properties. The in vivo immunological response to PAMAM-g-diazirine formulations was found to be optimal in comparison to standard poly(lactic--glycolic acid) (PLGA) thin films.
Topics: Cross-Linking Reagents; Dendrimers; Diazomethane; Membranes, Artificial; Tissue Adhesives; Ultraviolet Rays
PubMed: 29601480
DOI: 10.3390/molecules23040796 -
Expert Review of Proteomics Aug 2006Protein chemistry, such as crosslinking and photoaffinity labeling, in combination with modern mass spectrometric techniques, can provide information regarding... (Review)
Review
Protein chemistry, such as crosslinking and photoaffinity labeling, in combination with modern mass spectrometric techniques, can provide information regarding protein-protein interactions beyond that normally obtained from protein identification and characterization studies. While protein crosslinking can make tertiary and quaternary protein structure information available, photoaffinity labeling can be used to obtain structural data about ligand-protein interaction sites, such as oligonucleotide-protein, drug-protein and protein-protein interaction. In this article, we describe mass spectrometry-based photoaffinity labeling methodologies currently used and discuss their current limitations. We also discuss their potential as a common approach to structural proteomics for providing 3D information regarding the binding region, which ultimately will be used for molecular modeling and structure-based drug design.
Topics: Azides; Benzophenones; Diazomethane; Ligands; Mass Spectrometry; Models, Molecular; Photoaffinity Labels; Protein Interaction Mapping; Proteomics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Ultraviolet Rays
PubMed: 16901199
DOI: 10.1586/14789450.3.4.399