Investigating Thiol-Dependent Processes in the Phagosome of Macrophages
Date
2014-09-23
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Abstract
The work presented here evaluates the control of redox sensitive processes involved in phagosomal protein degradation which include the activities of cysteine cathepsins and gamma-interferon-inducible lysosomal thiol reductase (GILT). The catalytic mechanisms of these enzymes are dependent on reduced active site thiol (SH) groups making them susceptible to oxidative inactivation via reactive oxygen species (ROS). First, we evaluate how phagosomal thiol-dependent processes are influenced by the activation status of the macrophage. We show that interleukin-4 (IL-4) activation of murine bone marrow derived macrophages (BMMØs) results in a dramatic increase in phagosomal protein degradation. This was due to a decrease in oxidative inactivation of cysteine cathepsins and phagosomal disulfide reduction as a result of decreased NADPH oxidase (NOX2) -mediated ROS production. In addition, we found that IL-4 activation resulted in increased expression of cysteine cathepsins S and L which also enhanced phagosomal proteolysis in a NOX2-independent manner. Second, the influence of GILT on phagosomal thiol-dependent protease activity was evaluated in BMMØs to address how cysteine cathepsins are maintained in their reduced active state. We show that during NOX2 inhibition, GILT maintains phagosomal proteolysis in the early phagosome. The effect of GILT on proteolysis was enhanced in IL-4-activated BMMØs (with/without NOX2 inhibition). Furthermore, in a reconstituted system, we observed enhanced cathepsin S activity in the presence of recombinant active GILT. Last, we investigated the involvement of cytosolic redox control systems on phagosomal thiol-dependent processes. A novel phagosome-specific disulfide reduction assay was screened against a small molecule bioactives library. Results of the screen indicated the putative involvement of thioredoxin reductase (TrxR) in supporting phagosomal disulfide reduction. Furthermore, nicotinamide adenine dinucleotide phosphate (NADPH) depletion in BMMØs decreased both phagosomal disulfide reduction and proteolysis, implicating NADPH as a putative source of phagosomal reducing equivalents. Finally, through conditional depletion of the selenocysteine tRNA in BMMØs, we provide evidence for the involvement of selenoproteins, a family of redox enzymes which includes TrxR, in phagosomal disulfide reduction. Overall, the work presented in this dissertation contributes to our understanding of how macrophages regulate redox sensitive protein degradation in the phagosome.
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Immunology
Citation
Balce, D. R. (2014). Investigating Thiol-Dependent Processes in the Phagosome of Macrophages (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca. doi:10.11575/PRISM/26648