Monitoring in vivo reversible cysteine oxidation in proteins using ICAT and mass spectrometry

Abstract MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)–uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882–ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR–uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

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Development and Pharmacokinetics Study of Antifungal Peptide Nanoliposomes by Liquid Chromatography-tandem Mass Spectrometry

Current Pharmaceutical Analysis ◽

10.2174/1573412914666180307155328 ◽

2019 ◽

Vol 15 (4) ◽

pp. 312-318

Author(s):

Shuoye Yang

Keyword(s):

Mass Spectrometry ◽

Biological Properties ◽

Pharmacokinetic Property ◽

Antifungal Peptide ◽

Simple Liquid ◽

Physico Chemical ◽

Liquid Chromatography Tandem Mass ◽

Pegylated Lipids

Background: The therapeutic ability and application of antifungal peptide (APs) are limited by their physico-chemical and biological properties, the nano-liposomal encapsulation would improve the in vivo circulation and stability. </P><P> Objective: To develop a long-circulating liposomal delivery systems encapsulated APs-CGA-N12 with PEGylated lipids and cholesterol, and investigated through in vivo pharmacokinetics. Methods: The liposomes were prepared and characterized, a rapid and simple liquid chromatographytandem mass spectrometry (LC-MS/MS) assay was developed for the determination of antifungal peptide in vivo, the pharmacokinetic characteristics of APs liposomes were evaluated in rats. Results: Liposomes had a large, unilamellar structure, particle size and Zeta potential ranged from 160 to 185 nm and -0.55 to 1.1 mV, respectively. The results indicated that the plasma concentration of peptides in reference solutions rapidly declined after intravenous administration, whereas the liposomeencapsulated ones showed slower elimination. The AUC(0-∞) was increased by 3.0-fold in liposomes in comparison with standard solution (20 mg·kg-1), the half-life (T1/2) was 1.6- and 1.5-fold higher compared to the reference groups of 20 and 40 mg·kg-1, respectively. Conclusion: Therefore, it could be concluded that liposomal encapsulation effectively improved the bioavailability and pharmacokinetic property of antifungal peptides.

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Urinary metabolites of thromboxane and prostacyclin in diabetes mellitus

Acta Endocrinologica ◽

10.1530/acta.0.1180301 ◽

1988 ◽

Vol 118 (2) ◽

pp. 301-305 ◽

Cited By ~ 6

Author(s):

K. Gréen ◽

O. Vesterqvist ◽

V. Grill

Keyword(s):

Diabetes Mellitus ◽

Mass Spectrometry ◽

Gas Chromatography ◽

Insulin Treatment ◽

Artificial Pancreas ◽

Urinary Metabolites ◽

Gas Chromatography Mass Spectrometry ◽

Diabetic State ◽

In Vivo Synthesis

Abstract. The in vivo synthesis of thromboxane A2 and prostacyclin was estimated in 23 diabetics through measurements of the major urinary metabolites 2,3-dinor-thromboxane B2 and 2,3-dinor-6-keto-PGF1α utilizing gas chromatography-mass spectrometry. Mean excretion was similar to that in non-diabetic subjects. The possible influence of hyperglycemia on the excretion of 2,3-dinor-thromboxane B2 and 2,3-dinor-6-keto-PGF1α was evaluated in three ways: by measuring excretion before and during an acute 9-h normalization of hyperglycemia through an artificial pancreas (Biostator) as well as by comparing excretion before and 7–12 days or 40–180 days after the initiation of insulin treatment. Despite significant reducing effects on hyperglycemia or on levels of hemoglobin A1c, no effects on the excretion of the thromboxane and prostacyclin metabolites could be found. Abnormal formation of thromboxane or prostacyclin is not a generalized feature of the diabetic state.

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N-Terminomics Strategies for Protease Substrates Profiling

Molecules ◽

10.3390/molecules26154699 ◽

2021 ◽

Vol 26 (15) ◽

pp. 4699

Author(s):

Mubashir Mintoo ◽

Amritangshu Chakravarty ◽

Ronak Tilvawala

Keyword(s):

Mass Spectrometry ◽

Protease Activity ◽

Viral Infections ◽

Mass Spectrometry Analysis ◽

Post Translational Modification ◽

Spectrometry Analysis ◽

Physiological Conditions ◽

Inflammatory Conditions ◽

Biological Mixtures

Proteases play a central role in various biochemical pathways catalyzing and regulating key biological events. Proteases catalyze an irreversible post-translational modification called proteolysis by hydrolyzing peptide bonds in proteins. Given the destructive potential of proteolysis, protease activity is tightly regulated. Dysregulation of protease activity has been reported in numerous disease conditions, including cancers, neurodegenerative diseases, inflammatory conditions, cardiovascular diseases, and viral infections. The proteolytic profile of a cell, tissue, or organ is governed by protease activation, activity, and substrate specificity. Thus, identifying protease substrates and proteolytic events under physiological conditions can provide crucial information about how the change in protease regulation can alter the cellular proteolytic landscape. In recent years, mass spectrometry-based techniques called N-terminomics have become instrumental in identifying protease substrates from complex biological mixtures. N-terminomics employs the labeling and enrichment of native and neo-N-termini peptides, generated upon proteolysis followed by mass spectrometry analysis allowing protease substrate profiling directly from biological samples. In this review, we provide a brief overview of N-terminomics techniques, focusing on their strengths, weaknesses, limitations, and providing specific examples where they were successfully employed to identify protease substrates in vivo and under physiological conditions. In addition, we explore the current trends in the protease field and the potential for future developments.

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RNA-Centric Approaches to Profile the RNA–Protein Interaction Landscape on Selected RNAs

Non-Coding RNA ◽

10.3390/ncrna7010011 ◽

2021 ◽

Vol 7 (1) ◽

pp. 11 ◽

Cited By ~ 1

Author(s):

André P. Gerber

Keyword(s):

Mass Spectrometry ◽

Protein Interactions ◽

Regulatory Networks ◽

Rna Binding ◽

Rna Binding Proteins ◽

Protein Complexes ◽

Cell Protein ◽

Transcriptional Regulatory Networks ◽

Technological Advances

RNA–protein interactions frame post-transcriptional regulatory networks and modulate transcription and epigenetics. While the technological advances in RNA sequencing have significantly expanded the repertoire of RNAs, recently developed biochemical approaches combined with sensitive mass-spectrometry have revealed hundreds of previously unrecognized and potentially novel RNA-binding proteins. Nevertheless, a major challenge remains to understand how the thousands of RNA molecules and their interacting proteins assemble and control the fate of each individual RNA in a cell. Here, I review recent methodological advances to approach this problem through systematic identification of proteins that interact with particular RNAs in living cells. Thereby, a specific focus is given to in vivo approaches that involve crosslinking of RNA–protein interactions through ultraviolet irradiation or treatment of cells with chemicals, followed by capture of the RNA under study with antisense-oligonucleotides and identification of bound proteins with mass-spectrometry. Several recent studies defining interactomes of long non-coding RNAs, viral RNAs, as well as mRNAs are highlighted, and short reference is given to recent in-cell protein labeling techniques. These recent experimental improvements could open the door for broader applications and to study the remodeling of RNA–protein complexes upon different environmental cues and in disease.

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