Abstract
Introduction: Prdx6, a member of a new family of peroxidases, is found in neutrophil cytosol associated with p67phox. This protein reduces H2O2 with a conserved cysteine (C47) and also expresses PLA2 activity. In human neutrophils, Prdx6 translocates to plasma membrane after activation and enhances Nox2 activity in a cell-free system of oxidase activity. In transgenic K562 cells and PLB-985 cells, suppression of Prdx6 by siRNA or shRNA decreases Nox2 activity. In these studies, we investigated the association of Prdx6with autophagy in PLB-985 and HEK-293 cells.
Methods: PLB-985 and HEK-293 cells were cultured under standard conditions and were transfected with a plasmid producing stable expression of an shRNA targeting Prdx6. PLB-985 cells were matured with the addition of 1.3% DM50 for 4 days. DNA for wild type (WT) Prdx6 and mutants for the Prdx6 active site (C47S) and PLA2active sites (H26A, S32A, S140A) were cloned into pcDNA3.1 which also had silent mutations making all corresponding mRNAs resistant to shRNA targeting.
Prdx6 knockdown and vector control cells had stable re-expression of the various proteins when cultured with appropriate antibiotics. Superoxide anion (O2-) was determined by SOD inhibitable chemilluminescence with Digenes (National Diagnosistics). Proteins from lysates of various cell lines were separated by 10-12% SDS-PAGE and blotted onto nitrocellulose. Specific proteins were detected standard antibodies and a chemilluminescent technique with quantitation completed using Image J platform. Autophagy in PLB-985 cells with or without suppression of Prdx6 was completed by expression of LC3-II by Western blot after stimulation with serum opsonized zymsoan (5mg/mL). In HEK-293 cells, autophagy was determined by quantification of LC3-II (western blot) under based conditions with the addition of 10µM Chloraquine or starvation conditions with minimal media.
Results: Stable suppression of Prdx6 by shRNA was achieved in both PLB-985 cells (down to 30%) and HEK cells (25%). Re-expression of WT and Prdx6 mutant proteins re-established normal levels. O2-production in response to lµM FMLP was decreased by 44% in KD PLB-985 cells compared to control and re-establishment of oxidase activity depended on PLA2, not Prdx activity.
In PLB-985 cells, LC3-II levels increased (seven fold) over the first 30 minutes after exposure to opsonized zymosan, decreasing to baseline at 60 minutes. Prdx6 suppressed PLB-985 cells showed no change in LC3-II after opsonized zymosan. In HEK-293 cells lacking an active oxidase, suppression of Prdx6 resulted in increased autophagy under basal and starvation conditions. Furthermore, in HEK-293 cells, reintroduction of empty vector or Prdx6 mutated at the Prdx active site (C47S) did not suppress autophagy under basal conditions. However, re-introduction of WT and mutant Prdx6 at the PLA2 active site (D140A) did suppress autophagy demonstrating that this effect was associated with Prdx activity. Similar results were obtained under starvation conditions.
Conclusions: In neutrophil like cells Prdx6 enhanced Nox2 activity through its PLA2 activity and suppression of Prdx6 decreased autophagy as measured by LC3-II accumulation after exposure to a phagocytic stimulus. In non-myeloid HEK-293 cells, suppression of Prdx6 was associated with increased autophagy under basal and starvation conditions, and this effect was dependent on Prdx activity.
Prdx6, then, plays a critical role in neutrophil functions such as Nox2 activity and autophagy. Prdx6 may also be involved in autophagy in non-myeloid cells, and the mechanism for its involvement in autophagy may relate to different activities expressed by this protein.
Disclosures
No relevant conflicts of interest to declare.
A replacement of the active-site aspartic acid residue 293 in mouse cathepsin D affects its intracellular stability, processing and transport in HEK-293 cells