TXNL6 is a novel oxidative stress-induced reducing system for methionine sulfoxide reductase a repair of α-crystallin and cytochrome C in the eye lens

Abstract

A key feature of many age-related diseases is the oxidative stress-induced accumulation of protein methionine sulfoxide (PMSO) which causes lost protein function and cell death. Proteins whose functions are lost upon PMSO formation can be repaired by the enzyme methionine sulfoxide reductase A (MsrA) which is a key regulator of longevity. One disease intimately associated with PMSO formation and loss of MsrA activity is age-related human cataract. PMSO levels increase in the eye lens upon aging and in age-related human cataract as much as 70% of total lens protein is converted to PMSO. MsrA is required for lens cell maintenance, defense against oxidative stress damage, mitochondrial function and prevention of lens cataract formation. Essential for MsrA action in the lens and other tissues is the availability of a reducing system sufficient to catalytically regenerate active MsrA. To date, the lens reducing system(s) required for MsrA activity has not been defined. Here, we provide evidence that a novel thioredoxin-like protein called thioredoxin-like 6 (TXNL6) can serve as a reducing system for MsrA repair of the essential lens chaperone α-crystallin/sHSP and mitochondrial cytochrome c. We also show that TXNL6 is induced at high levels in human lens epithelial cells exposed to H(2)O(2)-induced oxidative stress. Collectively, these data suggest a critical role for TXNL6 in MsrA repair of essential lens proteins under oxidative stress conditions and that TXNL6 is important for MsrA defense protection against cataract. They also suggest that MsrA uses multiple reducing systems for its repair activity that may augment its function under different cellular conditions.

Figure 1. TXNL6 transcript is detected in the human lens epithelium and lens fiber cells and in other human tissues.

Ethidium bromide stained agarose gels show Panel A - TXNL6, Panel B - Trx 1, Panel C - Trx 2 and Panel D GAPDH (control) transcript from 200 ng of RNA obtained from human tissues - 1. Lens epithelium, 2. Lens Fiber, 3. Retina, 4. Stomach, 5. Kidney, 6.Heart, 7. Colon, and 8. Spleen.

Figure 2. TXNL6 protein is present in human lens epithelium and fiber cells.

SDS-PAGE and immunoblotting of microdissected lens epithelium (Epi) and lens fiber cell total protein extracts (20 µg) with a TXNL6-specific antibody.

Figure 3. TXNL6 is present in protein extracts prepared from the cytoplasm and mitochondria of human lens epithelial cells.

SDS-PAGE and immunoblotting of Panel A - TXNL6, Panel B - MsrA, Panel C - Trx 1, and Panel D -Trx 2 in 5 µg of protein extracts from the cytoplasm and mitochondria of HLE-B3 lens epithelial cells using specific antibodies.

Figure 4. Mitochondrial and cytosolic localization of TXNL6 in human lens epithelial cells.

Co-localization of Panel A - TXNL6 (green), Panel B - Trx 1 (green) and Panel C - Trx 2 (green), mitotracker red – a specific mitochondrial marker and merging of the two images (orange/yellow) in HLE-B3 lens epithelial cells by immunofluoresence microscopy.

Figure 5. TXNL6 mRNA and protein are induced by oxidative stress exposure of human lens epithelial cells.

A. Ethidium bromide stained agarose gels showing TXNL6 and control GAPDH transcript levels in 200 ng RNA isolated from HLE-B3 human lens epithelial cells at 0, 30 min, 6 h and 16 h recovery following a 2 h exposure to 200 µM H₂O₂. B. Western blot showing TXNL6 protein levels in 5 µg of total protein extract isolated from HLE-B3 lens epithelial cells at 0, 6 h, 16 h and 24 h recovery following a 2 h exposure to 200 µM H₂O₂. The coomassie blue stained SDS-PAGE gel is shown as a control for equal protein loading.

Figure 6. TXNL6-mediated MsrA repair of cytochrome c inhibits cytochrome c peroxidase activity.

Representative graph (from 3 independent experiments) of cytochrome c (cyt c) mediated peroxidase activity. The graph shows mean values for an N of 3 ± SD. Cyt c oxidized (cyt c ox) (incubated for 15 min with a 4∶1 molar ratio of HOCl) is 100% peroxidase activity, all other activities are expressed as a percentage of the cyt c ox activity. The untreated cyt c protein has 16% cyt c peroxidase activity compared to cyt c ox. Cyt c ox incubated with MsrA in the absence of a reducing system has 91% activity of the oxidized. Treatment of the cyt c ox (291 µM) with MsrA (1.9 µM for 2 h at 37°C) and TXNL6 (1.39 µM) with thioredoxin reductase (TxrR) and NADPH leads to a statistically significant (p<0.001) 48% decrease in peroxidase activity. Similarly treatment of cyt c ox with MsrA and either Trx 1 or Trx 2 (both 1.39 µM) with TxrR/NADPH also significantly (p<0.001) decreased both peroxidase activities by 52% and 54% respectively. Incubation of cyt c ox with MsrA and TXNL6 in the absence of TxrR/NADPH resulted in just a 14% decrease in cyt c peroxidase activity while incubation of cyt c ox with TXNL6 and TxrR/NADPH in the absence of MsrA resulted in 19% decrease in cyt c peroxidase activity. p values were obtained using Tukey's test following one-way ANOVA.

Figure 7. MsrA repairs oxidized methionines in cytochrome c using TXNL6 as a reducing agent.

Coomassie staining of a Tricine-SDS-PAGE gel following CNBr cleavage of oxidized cyt c (5 µg). Lane 1 untreated cyt c cleaved with CNBr. Lane 2 Oxidized cyt c (0.2 mM oxidized with 0.8 mM HOCl; a 4∶1 Molar ratio) cleaved with CNBr. Lane 3 Oxidized cyt c (291 µM) treated with MsrA (1.9 µM) and DTT (15 mM) for 2 h at 37°C and cleaved with CNBr. Lane 4 Oxidized cyt c (291 µM) treated with MsrA (1.9 µM) and TXNL6 (10 µg; 1.39 µM) for 2 h at 37°C and cleaved with CNBr. RA - reducing agent/system.

Figure 8. MsrA repairs oxidized methionines in α-crystallin using TXNL6 as a reducing agent.

Coomassie staining of a Tricine-SDS-PAGE gel following CNBr cleavage of oxidized α-crystallin (5 µg). Lane 1 untreated α-crystallin cleaved with CNBr. Lane 2 Oxidized α-crystallin (9.09 µM oxidized with 909 µm HOCl; a 100∶1 Molar ratio) cleaved with CNBr. Lane 3 Oxidized α-crystallin (6.4 µM) treated with MsrA (100 nM) and DTT (15 mM) for 2 h at 37°C and cleaved with CNBr. Lane 4 Oxidized α-crystallin (6.4 µM) treated with MsrA (100 nM) and TXNL6 (10 µg; 1.39 µM) for 2 h at 37°C and cleaved with CNBr. Lane 5 Oxidized α-crystallin (6.4 µM) treated with MsrA (100 nM) and TXNL6 (20 µg; 2.78 µM) for 2 h at 37°C and cleaved with CNBr. RA - reducing agent/system.