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Redox-sensitive probes for the measurement of redox chemistries within phagosomes of macrophages and dendritic cells.

Balce DR, Yates RM - Redox Biol (2013)

Bottom Line: There is currently much interest in factors that affect redox chemistries within phagosomes of macrophages and dendritic cells.In addition to the antimicrobial role of reactive oxygen species generation within phagosomes, accumulating evidence suggests that phagosomal redox chemistries influence other phagosomal functions such as macromolecular degradation and antigen processing.Hence the systems that control redox homeostasis in these unique environments remain poorly defined.

View Article: PubMed Central - PubMed

Affiliation: Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, Calgary, AB, Canada T2N 4N1 ; Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, AB, Canada T2N 4N1.

ABSTRACT
There is currently much interest in factors that affect redox chemistries within phagosomes of macrophages and dendritic cells. In addition to the antimicrobial role of reactive oxygen species generation within phagosomes, accumulating evidence suggests that phagosomal redox chemistries influence other phagosomal functions such as macromolecular degradation and antigen processing. Whilst the redox chemistries within many sub-cellular compartments are being heavily scrutinized with the increasing use of fluorescent probe technologies, there is a paucity of tools to assess redox conditions within phagosomes. Hence the systems that control redox homeostasis in these unique environments remain poorly defined. This review highlights current redox-sensitive probes that can measure oxidative or reductive activity in phagosomes and discusses their suitability and limitations of use. Probes that are easily targeted to the phagosome by using established approaches are emphasized.

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Related in: MedlinePlus

Structures of the folate-(BODIPY FL)-SS-rhodamine reporter (folate-FRET), BODIPY FL l-Cystine, di-(o-aminobenzoyl) glutathione disulfide (diabz-GSSG) and the TAMRA disulfide dimer (ssTAMRA).
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f0015: Structures of the folate-(BODIPY FL)-SS-rhodamine reporter (folate-FRET), BODIPY FL l-Cystine, di-(o-aminobenzoyl) glutathione disulfide (diabz-GSSG) and the TAMRA disulfide dimer (ssTAMRA).

Mentions: To address this need, several fluorescence-based techniques have been developed to study disulfide reduction in endosomes and lysosomes (ELs) (phagosome-associated vacuolar organelles). In order to measure EL disulfide reduction, Austin et al. conjugated rhodamine red to their disulfide linked antibody-drug compound at a labeling ratio capable of causing the rhodamine red to self-quench [51]. Although they showed efficient dequenching of the conjugate with artificial reductants in vitro, they showed that ELs were not capable of reducing the disulfide linked antibody drug conjugate. This observation was in agreement with others who had previously reported that endosomes were more oxidizing than reducing [44]. In contrast, Yang et al. developed a Förster/Fluorescence Resonance Energy Transfer (FRET) based probe to show that ELs were indeed competent in disulfide reduction [52]. The folate-(BODIPY FL)-SS-rhodamine reporter consisted of folic acid to target folate-receptor-mediated endocytosis and a peptide spacer to which the flurophores were conjugated. The donor fluorophore in the FRET pair was BODIPY FL, and the acceptor fluorophore was tetraethyl rhodamine which was linked to the peptide spacer via a reducible disulfide linker (Fig. 3); thus a loss of FRET would be observed upon reduction. The FRET signal was measured by both flow cytometry and fluorescence confocal microscopy. Although this group specifically measured endosomal reductive events, this probe could potentially be modified for phagosome-specific measurements by changing the receptor ligand or through conjugation to larger particles. The discrepancies between the two studies described above could be due to a number of factors. One possibility is that the redox environment within ELs can vary depending on the route of cargo intake (i.e. IgG vs. folate receptor). Another possibility is that certain probes, although designed around simple disulfide linkers and cleavable by artificial reductants in vitro, may not be recognized by the enzymatic systems in ELs such as GILT due to conformational or steric restraints.


Redox-sensitive probes for the measurement of redox chemistries within phagosomes of macrophages and dendritic cells.

Balce DR, Yates RM - Redox Biol (2013)

Structures of the folate-(BODIPY FL)-SS-rhodamine reporter (folate-FRET), BODIPY FL l-Cystine, di-(o-aminobenzoyl) glutathione disulfide (diabz-GSSG) and the TAMRA disulfide dimer (ssTAMRA).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814946&req=5

f0015: Structures of the folate-(BODIPY FL)-SS-rhodamine reporter (folate-FRET), BODIPY FL l-Cystine, di-(o-aminobenzoyl) glutathione disulfide (diabz-GSSG) and the TAMRA disulfide dimer (ssTAMRA).
Mentions: To address this need, several fluorescence-based techniques have been developed to study disulfide reduction in endosomes and lysosomes (ELs) (phagosome-associated vacuolar organelles). In order to measure EL disulfide reduction, Austin et al. conjugated rhodamine red to their disulfide linked antibody-drug compound at a labeling ratio capable of causing the rhodamine red to self-quench [51]. Although they showed efficient dequenching of the conjugate with artificial reductants in vitro, they showed that ELs were not capable of reducing the disulfide linked antibody drug conjugate. This observation was in agreement with others who had previously reported that endosomes were more oxidizing than reducing [44]. In contrast, Yang et al. developed a Förster/Fluorescence Resonance Energy Transfer (FRET) based probe to show that ELs were indeed competent in disulfide reduction [52]. The folate-(BODIPY FL)-SS-rhodamine reporter consisted of folic acid to target folate-receptor-mediated endocytosis and a peptide spacer to which the flurophores were conjugated. The donor fluorophore in the FRET pair was BODIPY FL, and the acceptor fluorophore was tetraethyl rhodamine which was linked to the peptide spacer via a reducible disulfide linker (Fig. 3); thus a loss of FRET would be observed upon reduction. The FRET signal was measured by both flow cytometry and fluorescence confocal microscopy. Although this group specifically measured endosomal reductive events, this probe could potentially be modified for phagosome-specific measurements by changing the receptor ligand or through conjugation to larger particles. The discrepancies between the two studies described above could be due to a number of factors. One possibility is that the redox environment within ELs can vary depending on the route of cargo intake (i.e. IgG vs. folate receptor). Another possibility is that certain probes, although designed around simple disulfide linkers and cleavable by artificial reductants in vitro, may not be recognized by the enzymatic systems in ELs such as GILT due to conformational or steric restraints.

Bottom Line: There is currently much interest in factors that affect redox chemistries within phagosomes of macrophages and dendritic cells.In addition to the antimicrobial role of reactive oxygen species generation within phagosomes, accumulating evidence suggests that phagosomal redox chemistries influence other phagosomal functions such as macromolecular degradation and antigen processing.Hence the systems that control redox homeostasis in these unique environments remain poorly defined.

View Article: PubMed Central - PubMed

Affiliation: Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, Calgary, AB, Canada T2N 4N1 ; Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, AB, Canada T2N 4N1.

ABSTRACT
There is currently much interest in factors that affect redox chemistries within phagosomes of macrophages and dendritic cells. In addition to the antimicrobial role of reactive oxygen species generation within phagosomes, accumulating evidence suggests that phagosomal redox chemistries influence other phagosomal functions such as macromolecular degradation and antigen processing. Whilst the redox chemistries within many sub-cellular compartments are being heavily scrutinized with the increasing use of fluorescent probe technologies, there is a paucity of tools to assess redox conditions within phagosomes. Hence the systems that control redox homeostasis in these unique environments remain poorly defined. This review highlights current redox-sensitive probes that can measure oxidative or reductive activity in phagosomes and discusses their suitability and limitations of use. Probes that are easily targeted to the phagosome by using established approaches are emphasized.

Show MeSH
Related in: MedlinePlus