Abstract
Introduction: Production of reactive oxygen species through a respiratory burst is critical to the microbicidal activity of the neutrophil. The respiratory burst is initiated by assembly of the components of the NADPH oxidase enzyme system (p47phox, p67phox, p40phox, gp91phox, p22phox and Rac) and expression of the activity of the system to produce superoxide anion (O2 −). We recently identified a neutrophil protein with an approximate MW of 29 kDa which binds to the p67phox, is classified as a peroxiredoxin (Prx) and translocates to the plasma membrane during stimulation of the neutrophil. Additional studies demonstrate that this protein (p29 Prx) increases production of O2 − in a cell-free system of oxidase activity in a specific, stoichiometric manner and that the cysteine residues of p29 Prx, at amino acid positions 47 and 91, are required for this activity. The current studies demonstrate the role of p29 Prx in oxidase activity using the technique of small interfering RNA (siRNA) to degrade specific mRNA and decrease the expression of the protein.
Methods: siRNA probes for p29 Prx were constructed based on standard constraints for unique 19 nucleotide binding sites along with other sequence selection criteria. Six probes were constructed based on the cDNA sequence of p29 Prx; one resulted in significant knockdown of p29 Prx. Inactive siRNA fluorescently labeled was obtained commercially. K562 cells, stably transfected with the p67phox, p47phox, gp91phox and low affinity fMLP receptor, were cultured under standard conditions and expressed p29 Prx message by RT-PCR and protein by Western blot. Transgenic K562 cells were transfected with siRNA or GFP labeled control siRNA with Nucleofector technology. Cell counts and viability were determined by standard techniques. For Western blots, proteins from cell lysates were separated on 10% SDS-PAGE and blotted onto nitrocellulose, and specific proteins were detected with polyclonal antibodies to p29 Prx, actin or p67phox, p47phox, gp91phox, and p22phox by chemiluminescent technique.
Results: After transfection with the active siRNA for p29 Prx, inactive or labeled siRNA, the K562 cells were harvested at 24, 48, and 72 hours. A knockdown by one of the 6 siRNAs resulted in decreased levels of p29 Prx by Western blot. Optimum knockdown was achieved by transfection of 6 μg siRNA and the decrease in p29 Prx observed at 24 hours but was optimum after 48 hours. Under these conditions, a decrease in p29 Prx by 50–60% detected by Western blot was achieved with no differences in levels of actin or any of the phox proteins. The viability of control cells and siRNA transfected cells was not different. Cells transfected with siRNA for p29 Prx which demonstrated a knockdown of this protein exhibited decreased respiratory burst (O2 −) in response to fMLP (measured by chemiluminescence) or PMA (cytochrome c reduction) compared to control porated or transfected cells.
Conclusion: These results correlate with in vitro studies of recombinant p29 Prx in the SDS cell-free system of oxidase activation. Decreased levels of p29 Prx result in decreased oxidase activity on transgenic K562 cells. p29 Prx may be important for the expression of the oxidase enzyme system through its antioxidant or signaling activity.
Cell-free biosynthesis of ω-hydroxy acids boosted by a synergistic combination of alcohol dehydrogenases
