Proline modulates the intracellular redox environment and protects mammalian cells against oxidative stress

Abstract

The potential of proline to suppress reactive oxygen species (ROS) and apoptosis in mammalian cells was tested by manipulating intracellular proline levels exogenously and endogenously by overexpression of proline metabolic enzymes. Proline was observed to protect cells against H(2)O(2), tert-butyl hydroperoxide, and a carcinogenic oxidative stress inducer but was not effective against superoxide generators such as menadione. Oxidative stress protection by proline requires the secondary amine of the pyrrolidine ring and involves preservation of the glutathione redox environment. Overexpression of proline dehydrogenase (PRODH), a mitochondrial flavoenzyme that oxidizes proline, resulted in 6-fold lower intracellular proline content and decreased cell survival relative to control cells. Cells overexpressing PRODH were rescued by pipecolate, an analog that mimics the antioxidant properties of proline, and by tetrahydro-2-furoic acid, a specific inhibitor of PRODH. In contrast, overexpression of the proline biosynthetic enzymes Delta(1)-pyrroline-5-carboxylate (P5C) synthetase (P5CS) and P5C reductase (P5CR) resulted in 2-fold higher proline content, significantly lower ROS levels, and increased cell survival relative to control cells. In different mammalian cell lines exposed to physiological H(2)O(2) levels, increased endogenous P5CS and P5CR expression was observed, indicating that upregulation of proline biosynthesis is an oxidative stress response.

Fig. 1

Proline prevents DNA laddering induced by H₂O₂. Agarose (1.5%) gel electrophoresis of DNA extract from HEK 293 cells treated with H₂O₂ (1 mM) for 3 and 6 h in the absence (−) and presence (+) of 5 mM proline. Far left-hand lane shows a 1.0 kB DNA ladder (M) and far right-hand lane shows DNA extract from untreated HEK 293 control cells (C).

Fig. 2

Concentration dependence of proline protection against oxidative stress. HEK 293 cells were incubated with 0.5 mM H₂O₂ (3 h) in the presence of increasing amounts of proline (0–5 mM) supplemented in the medium. The proline concentration in the medium alone is ~ 10 µM.

Fig. 3

Flow cytometry analysis of ROS levels in HEK 293 cells. Fluorescent positives from HEK 293 cells without H₂O₂ stress treatment (−H₂O₂) and HEK 293 cells incubated with H₂O₂ (0.5 mM) for 3 h at 37 °C in the absence (+ H₂O₂) and presence of 5 mM proline (+ H₂O₂ + proline). Cells were treated with DCHF-DA prior to analysis.

Fig. 4

Proline accumulation correlates with increased cell survival. (A) Western analysis of HEK 293 cells transfected with P5CR-pFlag-CMV3, PRODH-pFlag-CMV3, and P5CS-pFlag-CMV3 and vector alone for 24 h at 37 °C. Protein extracts were separated by SDS-PAGE and immunoblotted with an anti-flag FITC conjugate antibody and visualized by fluorescence imaging. Protein standards visualized by the same detection system are shown in the left-hand lane. The anticipated molecular sizes of the pFlag fusion proteins are 34.7 kDa (P5CR), 69.15 kDa (PRODH), and 88.2 kDa (P5CS). (B) Survival rates of transfected HEK 293 cultured cells after 3 h incubation (37 °C) in the absence and presence of H₂O₂ (0.5 mM). HEK 293 cells were transfected with pcDNA3.1 vector alone (control) or with PRODH, P5CS, P5CR, and P5CS/P5CR for 24 h before treatment with H₂O₂. Survival rates were determined by the MTT assay. (C) Proline content in HEK 293 cells after 24 h transfection with pcDNA3.1 vector alone (control), PRODH, P5CS, P5CR, and P5CS/P5CR.

Fig. 5

Flow cytometry analysis of intracellular ROS levels. Fluorescent positives from HEK 293 cells transfected with PRODH, P5CS, P5CR, and P5CS/P5CR and exposed to 0.5 mM H₂O₂ for 3 h at 37 °C. Fluorescent positives from transfected control cells (pcDNA3.1 alone) without H₂O₂ exposure are also shown. The oxidant-sensitive probe DCHF-DA was used to detect intracellular ROS levels.

Fig. 6

Superoxide formation by PRODH. (A) SDS-PAGE analysis of recombinant PRODH expressed in E. coli. Left, protein molecular weight standard; Middle, 3 µg of PRODH preparation; Right, 7 µg of PRODH preparation. The molecular weight of the PRODH band estimated by SDS-PAGE is 67.4 kDa. (B) Recombinant PRODH (20 µg) was incubated in proline:O₂ assay buffer (pH 7.5) with 150 mM proline and cytochrome c (40 µM) for 60 min at 23 °C in the absence and presence of SOD (36 U). Solid curves show the absorbance spectra of the assay mixture at 0 (curve 1) and 60 min (curve 2) without SOD. The dashed curve is the spectrum of the assay mixture supplemented with SOD and incubated for 60 min. The inset shows the rate of cytochrome c reduction in the absence (−THFA) and presence of 5 mM THFA (+ THFA) in the assay mixture.

Fig. 7

Rescue of PRODH toxicity in HEK 293 cells. HEK 293 cells were transfected with PRODH for 24 h in medium alone and medium supplemented with DL-pipecolate (5 mM) or L-THFA (5 mM) prior to treatment with H₂O₂. PRODH transfected HEK 293 cultured cells were then incubated for 3 h in the absence and presence of H₂O₂ (0.5 mM). Survival rates were determined by the MTT assay. HEK 293 cells transfected with pcDNA3.1 vector alone (control) exhibited 96% viability without H₂O₂ treatment.

Fig. 8

Expression profiling of P5CS, P5CR and OAT during H₂O₂ stress treatment. Total RNA was extracted at different time points from mammalian cells incubated at 37 °C from 0–24 h in the absence and presence of H₂O₂ maintained at 20 µM (HEK 293 cells) or 100 µM (HeLa, HepG2, BJAB, and Jurkat cells) in the cell culture medium. RT-PCR products for P5CR, P5CS, OAT and GAPDH transcripts are shown from non-stressed (−H₂O₂) and H₂O₂-stressed (+H₂O₂) cells at 0–24 h of incubation.