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Proteomic Analysis of S-Palmitoylated Proteins in Ocular Lens Reveals Palmitoylation of AQP5 and MP20.
Wang Z, Schey KL
(2018) Invest Ophthalmol Vis Sci 59: 5648-5658
MeSH Terms: Animals, Aquaporin 5, Blotting, Western, Cattle, Chromatography, Liquid, Electrophoresis, Polyacrylamide Gel, Eye Proteins, Immunoblotting, Lens, Crystalline, Lipoylation, Membrane Proteins, Palmitates, Proteomics, Tandem Mass Spectrometry
Show Abstract · Added April 4, 2019
Purpose - The purpose of this study was to characterize the palmitoyl-proteome in lens fiber cells. S-palmitoylation is the most common form of protein S-acylation and the reversible nature of this modification functions as a molecular switch to regulate many biological processes. This modification could play important roles in regulating protein functions and protein-protein interactions in the lens.
Methods - The palmitoyl-proteome of bovine lens fiber cells was investigated by combining acyl-biotin exchange (ABE) chemistry and mass-spectrometry analysis. Due to the possibility of false-positive results from ABE experiment, a method was also developed for direct detection of palmitoylated peptides by mass spectrometry for validating palmitoylation of lens proteins MP20 and AQP5. Palmitoylation levels on AQP5 in different regions of the lens were quantified after iodoacetamide (IAA)-palmitate exchange.
Results - The ABE experiment identified 174 potential palmitoylated proteins. These proteins include 39 well-characterized palmitoylated proteins, 92 previously reported palmitoylated proteins in other tissues, and 43 newly identified potential palmitoylated proteins including two important transmembrane proteins in the lens, AQP5 and MP20. Further analysis by direct detection of palmitoylated peptides confirmed palmitoylation of AQP5 on C6 and palmitoylation of MP20 on C159. Palmitoylation of AQP5 was found to only occur in a narrow region of the inner lens cortex and does not occur in the lens epithelium, in the lens outer cortex, or in the lens nucleus.
Conclusions - AQP5 and MP20 are among 174 palmitoylated proteins found in bovine lens fiber cells. This modification to AQP5 and MP20 may play a role in their translocation from the cytoplasm to cell membranes during fiber cell differentiation.
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14 MeSH Terms
Micro-Data-Independent Acquisition for High-Throughput Proteomics and Sensitive Peptide Mass Spectrum Identification.
Heaven MR, Cobbs AL, Nei YW, Gutierrez DB, Herren AW, Gunawardena HP, Caprioli RM, Norris JL
(2018) Anal Chem 90: 8905-8911
MeSH Terms: Algorithms, Chromatography, Liquid, Databases, Protein, Escherichia coli, Escherichia coli Proteins, HeLa Cells, High-Throughput Screening Assays, Humans, Peptides, Proteome, Proteomics, Software, Tandem Mass Spectrometry, Workflow
Show Abstract · Added August 27, 2018
State-of-the-art strategies for proteomics are not able to rapidly interrogate complex peptide mixtures in an untargeted manner with sensitive peptide and protein identification rates. We describe a data-independent acquisition (DIA) approach, microDIA (μDIA), that applies a novel tandem mass spectrometry (MS/MS) mass spectral deconvolution method to increase the specificity of tandem mass spectra acquired during proteomics experiments. Using the μDIA approach with a 10 min liquid chromatography gradient allowed detection of 3.1-fold more HeLa proteins than the results obtained from data-dependent acquisition (DDA) of the same samples. Additionally, we found the μDIA MS/MS deconvolution procedure is critical for resolving modified peptides with relatively small precursor mass shifts that cause the same peptide sequence in modified and unmodified forms to theoretically cofragment in the same raw MS/MS spectra. The μDIA workflow is implemented in the PROTALIZER software tool which fully automates tandem mass spectral deconvolution, queries every peptide with a library-free search algorithm against a user-defined protein database, and confidently identifies multiple peptides in a single tandem mass spectrum. We also benchmarked μDIA against DDA using a 90 min gradient analysis of HeLa and Escherichia coli peptides that were mixed in predefined quantitative ratios, and our results showed μDIA provided 24% more true positives at the same false positive rate.
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14 MeSH Terms
Quantification of thioether-linked glutathione modifications in human lens proteins.
Wang Z, Schey KL
(2018) Exp Eye Res 175: 83-89
MeSH Terms: Adolescent, Alanine, Aminobutyrates, Cataract, Cellular Senescence, Chromatography, Liquid, Crystallins, Cysteine, Glutathione, Humans, Lens, Crystalline, Middle Aged, Protein Processing, Post-Translational, Serine, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Sulfides, Threonine, Tissue Donors, Young Adult
Show Abstract · Added April 4, 2019
Dehydroalanine (DHA) and dehydrobutyrine (DHB) intermediates, formed through β-elimination, induce protein irreversible glutathionylation and protein-protein crosslinking in human lens fiber cells. In total, irreversible glutathionylation was detected on 52 sites including cysteine, serine and threonine residues in 18 proteins in human lenses. In this study, the levels of GSH modification on three serine residues and four cysteine residues located in seven different lens proteins isolated from different regions and different aged lenses were quantified. The relative levels of modification (modified/nonmodified) were site-specific and age-related, ranging from less than 0.05% to about 500%. The levels of modification on all of the sites quantified in the lens cortex increased with age and GSH modification also increased from cortex to outer nucleus region suggesting an age-related increase of modification. The levels of modification on sites located in stable regions of the proteins such as Cys117 of βA3, Cys80 of βB1 and Cys27 of γS, continued increasing in inner nucleus, but modification on sites located in regions undergoing degradation with age decreased in the inner nucleus suggesting GSH modified proteins were more susceptible to further modification. Irreversible GSH modification in cataract lenses was typically higher than in age-matched normal lenses, but the difference did not reach statistical significance for a majority of sites, with the exception Cys117 of βA3 crystallin in WSF. Except for S59 of αA and αB crystallins, GSH modification did not induce protein insolubility suggesting a possible role for this modification in protection from protein-protein crosslinking.
Copyright © 2018 Elsevier Ltd. All rights reserved.
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Determining Double Bond Position in Lipids Using Online Ozonolysis Coupled to Liquid Chromatography and Ion Mobility-Mass Spectrometry.
Harris RA, May JC, Stinson CA, Xia Y, McLean JA
(2018) Anal Chem 90: 1915-1924
MeSH Terms: Animals, Chickens, Chromatography, Liquid, Eggs, Fatty Acids, Unsaturated, Glycerophospholipids, Isomerism, Lipids, Ozone, Spectrometry, Mass, Electrospray Ionization
Show Abstract · Added December 17, 2018
The increasing focus on lipid metabolism has revealed a need for analytical techniques capable of structurally characterizing lipids with a high degree of specificity. Lipids can exist as any one of a large number of double bond positional isomers, which are indistinguishable by single-stage mass spectrometry alone. Ozonolysis reactions coupled to mass spectrometry have previously been demonstrated as a means for localizing double bonds in unsaturated lipids. Here we describe an online, solution-phase reactor using ozone produced via a low-pressure mercury lamp, which generates aldehyde products diagnostic of cleavage at a particular double bond position. This flow-cell device is utilized in conjunction with structurally selective ion mobility-mass spectrometry. The lamp-mediated reaction was found to be effective for multiple lipid species in both positive and negative ionization modes, and the conversion efficiency from precursor to product ions was tunable across a wide range (20-95%) by varying the flow rate through the ozonolysis device. Ion mobility separation of the ozonolysis products generated additional structural information and revealed the presence of saturated species in a complex mixture. The method presented here is simple, robust, and readily coupled to existing instrument platforms with minimal modifications necessary. For these reasons, application to standard lipidomic workflows is possible and aids in more comprehensive structural characterization of a myriad of lipid species.
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10 MeSH Terms
Cytochrome P450 2A6 and other human P450 enzymes in the oxidation of flavone and flavanone.
Kakimoto K, Murayama N, Takenaka S, Nagayoshi H, Lim YR, Kim V, Kim D, Yamazaki H, Komori M, Guengerich FP, Shimada T
(2019) Xenobiotica 49: 131-142
MeSH Terms: Chromatography, Liquid, Cytochrome P-450 CYP2A6, Cytochrome P-450 Enzyme System, Flavanones, Flavones, Humans, Kinetics, Mass Spectrometry, Molecular Docking Simulation, Oxidation-Reduction
Show Abstract · Added March 14, 2018
1. We previously reported that flavone and flavanone interact spectrally with cytochrome P450 (P450 or CYP) 2A6 and 2A13 and other human P450s and inhibit catalytic activities of these P450 enzymes. In this study, we studied abilities of CYP1A1, 1A2, 1B1, 2A6, 2A13, 2C9 and 3A4 to oxidize flavone and flavanone. 2. Human P450s oxidized flavone to 6- and 5-hydroxylated flavones, seven uncharacterized mono-hydroxylated flavones, and five di-hydroxylated flavones. CYP2A6 was most active in forming 6-hydroxy- and 5-hydroxyflavones and several mono- and di-hydroxylated products. 3. CYP2A6 was also very active in catalyzing flavanone to form 2'- and 6-hydroxyflavanones, the major products, at turnover rates of 4.8 min and 1.3 min, respectively. Other flavanone metabolites were 4'-, 3'- and 7-hydroxyflavanone, three uncharacterized mono-hydroxylated flavanones and five mono-hydroxylated flavones, including 6-hydroxyflavone. CYP2A6 catalyzed flavanone to produce flavone at a turnover rate of 0.72 min that was ∼3-fold higher than that catalyzed by CYP2A13 (0.29 min). 4. These results indicate that CYP2A6 and other human P450s have important roles in metabolizing flavone and flavanone, two unsubstituted flavonoids, present in dietary foods. Chemical mechanisms of P450-catalyzed desaturation of flavanone to form flavone are discussed.
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Discovery of human cell selective effector molecules using single cell multiplexed activity metabolomics.
Earl DC, Ferrell PB, Leelatian N, Froese JT, Reisman BJ, Irish JM, Bachmann BO
(2018) Nat Commun 9: 39
MeSH Terms: Aged, Bone Marrow, Cell Extracts, Chromatography, Liquid, DNA Damage, Female, Flow Cytometry, Humans, Leukemia, Leukemia, Myeloid, Acute, Lymphocytes, Male, Mass Spectrometry, Metabolome, Metabolomics, Monocytes, Signal Transduction, Streptomyces, Tumor Cells, Cultured, Young Adult
Show Abstract · Added January 4, 2018
Discovering bioactive metabolites within a metabolome is challenging because there is generally little foreknowledge of metabolite molecular and cell-targeting activities. Here, single-cell response profiles and primary human tissue comprise a response platform used to discover novel microbial metabolites with cell-type-selective effector properties in untargeted metabolomic inventories. Metabolites display diverse effector mechanisms, including targeting protein synthesis, cell cycle status, DNA damage repair, necrosis, apoptosis, or phosphoprotein signaling. Arrayed metabolites are tested against acute myeloid leukemia patient bone marrow and molecules that specifically targeted blast cells or nonleukemic immune cell subsets within the same tissue biopsy are revealed. Cell-targeting polyketides are identified in extracts from biosynthetically prolific bacteria, including a previously unreported leukemia blast-targeting anthracycline and a polyene macrolactam that alternates between targeting blasts or nonmalignant cells by way of light-triggered photochemical isomerization. High-resolution cell profiling with mass cytometry confirms response mechanisms and is used to validate initial observations.
3 Communities
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20 MeSH Terms
Dietary Fatty Acids Control the Species of N-Acyl-Phosphatidylethanolamines Synthesized by Therapeutically Modified Bacteria in the Intestinal Tract.
Dosoky NS, Guo L, Chen Z, Feigley AV, Davies SS
(2018) ACS Infect Dis 4: 3-13
MeSH Terms: Acyltransferases, Animals, Bacteria, Biomarkers, Biosynthetic Pathways, Chromatography, Liquid, Diet, Fatty Acids, Gastrointestinal Microbiome, Lipid Metabolism, Liver, Male, Mice, Phosphatidylethanolamines, Tandem Mass Spectrometry, Temperature
Show Abstract · Added July 17, 2019
Engineering the gut microbiota to produce specific beneficial metabolites represents an important new potential strategy for treating chronic diseases. Our previous studies with bacteria engineered to produce N-acyl-phosphatidylethanolamines (NAPEs), the immediate precursors of the lipid satiety factors N-acyl-ethanolamides (NAEs), found that colonization of these bacteria inhibited development of obesity in C57BL/6J mice fed a high fat diet. Individual NAE species differ in their bioactivities. Intriguingly, colonization by our engineered bacteria resulted in increased hepatic N-stearoyl-ethanolamide (C18:0NAE) levels despite the apparent inability of these bacteria to biosynthesize its precursor N-stearoyl-phosphatidylethanolamine (C18:0NAPE) in vitro. We therefore sought to identify the factors that allowed C18:0NAPE biosynthesis by the engineered bacteria after colonization of the intestinal tract. We found that the species of NAPE biosynthesized by engineered bacteria depends on the species of dietary fatty acids available in the intestine, suggesting a simple method to fine-tune the therapeutic effects of modified microbiota.
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Prospective study of urinary prostaglandin E2 metabolite and pancreatic cancer risk.
Cui Y, Shu XO, Li HL, Yang G, Wen W, Gao YT, Cai Q, Rothman N, Yin HY, Lan Q, Xiang YB, Zheng W
(2017) Int J Cancer 141: 2423-2429
MeSH Terms: Adult, Aged, Body Mass Index, Case-Control Studies, China, Chromatography, Liquid, Cyclooxygenase 2, Female, Humans, Logistic Models, Male, Middle Aged, Odds Ratio, Pancreatic Neoplasms, Prospective Studies, Prostaglandins, Tandem Mass Spectrometry
Show Abstract · Added April 3, 2018
The cyclooxygenase 2 (COX-2) pathway is upregulated in many pancreatic cancer cells, and it is believed that carcinogenetic effects of COX-2 upregulation are largely through prostaglandin E2 (PGE2) overproduction. We tested this hypothesis by evaluating the association between urinary PGE2 metabolites (PGE-M), a biomarker of in vivo PGE2 overproduction, and pancreatic cancer risk. We conducted a case-control study with 722 subjects (239 cases and 483 controls) nested within two prospective cohort studies, the Shanghai Women's Health Study (SWHS) and Shanghai Men's Health Study (SMHS). Pre-diagnosis urine samples were measured for PGE-M using a liquid chromatography/tandem mass spectrometric method. Conditional logistic regression was used to estimate odds ratio (OR) and 95% confidence intervals (95%CI), with adjustment for potential confounders. Compared to those with the lowest urine level of PGE-M (the first quartile), individuals with higher urine levels of PGE-M had an increased risk of developing pancreatic cancer, with adjusted ORs (95%CI) of 1.63 (0.98-2.73), 1.55 (0.90-2.69) and 1.94 (1.07-3.51), for the second to the fourth quartile groups, respectively (p for trend = 0.054). This dose-response positive association was more evident among those who had BMI <25 kg/m than overweight individuals (p for interaction = 0.058). After excluding cases diagnosed in the first year of follow-up and their matched controls, this positive association persisted (p for trend = 0.037) and the interaction became statistically significant (p for interaction = 0.017). Our study adds additional evidence that the COX-2 pathway is involved in pancreatic carcinogenesis and suggests that urinary PGE-M may serve as a biomarker for predicting pancreatic cancer risk.
© 2017 UICC.
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Kidney function is associated with an altered protein composition of high-density lipoprotein.
Rubinow KB, Henderson CM, Robinson-Cohen C, Himmelfarb J, de Boer IH, Vaisar T, Kestenbaum B, Hoofnagle AN
(2017) Kidney Int 92: 1526-1535
MeSH Terms: Adult, African Americans, Aged, Chromatography, Liquid, Cohort Studies, Dyslipidemias, European Continental Ancestry Group, Female, Glomerular Filtration Rate, Humans, Lipoproteins, HDL, Male, Middle Aged, Proteomics, Renal Insufficiency, Chronic, Risk Factors, Tandem Mass Spectrometry
Show Abstract · Added September 19, 2017
Patients with chronic kidney disease (CKD) exhibit a myriad of metabolic derangements, including dyslipidemia characterized by low plasma concentrations of high-density lipoprotein (HDL)-associated cholesterol. However, the effects of kidney disease on HDL composition have not been comprehensively determined. Here we used a targeted mass spectrometric approach to quantify 38 proteins contained in the HDL particles within a CKD cohort of 509 participants with a broad range of estimated glomerular filtration rates (eGFRs) (CKD stages I-V, and a mean eGFR of 45.5 mL/min/1.73m). After adjusting for multiple testing, demographics, comorbidities, medications, and other characteristics, eGFR was significantly associated with differences in four HDL proteins. Compared to participants with an eGFR of 60 mL/min/1.73m or more, those with an eGFR under 15 mL/min/1.73m exhibited 1.89-fold higher retinol-binding protein 4 (95% confidence interval 1.34-2.67), 1.52-fold higher apolipoprotein C-III (1.25-1.84), 0.70-fold lower apolipoprotein L1 (0.55-0.92), and 0.64-fold lower vitronectin (0.48-0.85). Although the HDL apolipoprotein L1 was slightly lower among African Americans than among Caucasian individuals, the relationship to eGFR did not differ by race. After adjustment, no HDL-associated proteins associated with albuminuria. Thus, modest changes in the HDL proteome provide preliminary evidence for an association between HDL proteins and declining kidney function, but this needs to be replicated. Future analyses will determine if HDL proteomics is indeed a clinical predictor of declining kidney function or cardiovascular outcomes.
Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.
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Identification of Phosphorylation Codes for Arrestin Recruitment by G Protein-Coupled Receptors.
Zhou XE, He Y, de Waal PW, Gao X, Kang Y, Van Eps N, Yin Y, Pal K, Goswami D, White TA, Barty A, Latorraca NR, Chapman HN, Hubbell WL, Dror RO, Stevens RC, Cherezov V, Gurevich VV, Griffin PR, Ernst OP, Melcher K, Xu HE
(2017) Cell 170: 457-469.e13
MeSH Terms: Amino Acid Sequence, Animals, Arrestins, Chromatography, Liquid, Humans, Mice, Models, Molecular, Phosphorylation, Rats, Rhodopsin, Sequence Alignment, Tandem Mass Spectrometry, X-Rays
Show Abstract · Added March 14, 2018
G protein-coupled receptors (GPCRs) mediate diverse signaling in part through interaction with arrestins, whose binding promotes receptor internalization and signaling through G protein-independent pathways. High-affinity arrestin binding requires receptor phosphorylation, often at the receptor's C-terminal tail. Here, we report an X-ray free electron laser (XFEL) crystal structure of the rhodopsin-arrestin complex, in which the phosphorylated C terminus of rhodopsin forms an extended intermolecular β sheet with the N-terminal β strands of arrestin. Phosphorylation was detected at rhodopsin C-terminal tail residues T336 and S338. These two phospho-residues, together with E341, form an extensive network of electrostatic interactions with three positively charged pockets in arrestin in a mode that resembles binding of the phosphorylated vasopressin-2 receptor tail to β-arrestin-1. Based on these observations, we derived and validated a set of phosphorylation codes that serve as a common mechanism for phosphorylation-dependent recruitment of arrestins by GPCRs.
Copyright © 2017 Elsevier Inc. All rights reserved.
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13 MeSH Terms