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

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 H2O2-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

Differentiation state-specific mitochondrial dynamic regulatory networks are revealed by global transcriptional analysis of the developing chicken lens.

The mature eye lens contains a surface layer of epithelial cells called the lens epithelium that requires a functional mitochondrial population to maintain the homeostasis and transparency of the entire lens. The lens epithelium overlies a core of terminally differentiated fiber cells that must degrade their mitochondria to achieve lens transparency. These distinct mitochondrial populations make the lens a useful model system to identify those genes that regulate the balance between mitochondrial homeostasis and elimination. Here we used an RNA sequencing and bioinformatics approach to identify the transcript levels of all genes expressed by distinct regions of the lens epithelium and maturing fiber cells of the embryonic Gallus gallus (chicken) lens. Our analysis detected more than 15,000 unique transcripts expressed by the embryonic chicken lens. Of these, more than 3000 transcripts exhibited significant differences in expression between lens epithelial cells and fiber cells. Multiple transcripts coding for separate mitochondrial homeostatic and degradation mechanisms were identified to exhibit preferred patterns of expression in lens epithelial cells that require mitochondria relative to lens fiber cells that require mitochondrial elimination. These included differences in the expression levels of metabolic (DUT, PDK1, SNPH), autophagy (ATG3, ATG4B, BECN1, FYCO1, WIPI1), and mitophagy (BNIP3L/NIX, BNIP3, PARK2, p62/SQSTM1) transcripts between lens epithelial cells and lens fiber cells. These data provide a comprehensive window into all genes transcribed by the lens and those mitochondrial regulatory and degradation pathways that function to maintain mitochondrial populations in the lens epithelium and to eliminate mitochondria in maturing lens fiber cells

Suppression of PI3K signaling is linked to autophagy activation and the spatiotemporal induction of the lens organelle free zone

The terminal steps of lens cell differentiation require elimination of all organelles to create a central Organelle Free Zone (OFZ) that is required for lens function of focusing images on the retina. Previous studies show that the spatiotemporal elimination of these organelles during development is autophagy-dependent. We now show that the inhibition of PI3K signaling in lens organ culture results in the premature induction of autophagy within 24 h, including a significant increase in LAMP1+ lysosomes, and the removal of lens organelles from the center of the lens. Specific inhibition of just the PI3K/Akt signaling axis was directly linked to the elimination of mitochondria and ER, while pan-PI3K inhibitors that block all PI3K downstream signaling removed all organelles, including nuclei. Therefore, blocking the PI3K/Akt pathway was alone insufficient to remove nuclei. RNAseq analysis revealed increased mRNA levels of the endogenous inhibitor of PI3K activation, PIK3IP1, in differentiating lens fiber cells preceding the induction of OFZ formation. Co-immunoprecipitation confirmed that PIK3IP1 associates with multiple PI3K p110 isoforms just prior to formation of the OFZ, providing a likely endogenous mechanism for blocking all PI3K signaling and activating the autophagy pathway required to form the OFZ during lens development

Autophagy Requirements for Eye Lens Differentiation and Transparency

Recent evidence points to autophagy as an essential cellular requirement for achieving the mature structure, homeostasis, and transparency of the lens. Collective evidence from multiple laboratories using chick, mouse, primate, and human model systems provides evidence that classic autophagy structures, ranging from double-membrane autophagosomes to single-membrane autolysosomes, are found throughout the lens in both undifferentiated lens epithelial cells and maturing lens fiber cells. Recently, key autophagy signaling pathways have been identified to initiate critical steps in the lens differentiation program, including the elimination of organelles to form the core lens organelle-free zone. Other recent studies using ex vivo lens culture demonstrate that the low oxygen environment of the lens drives HIF1a-induced autophagy via upregulation of essential mitophagy components to direct the specific elimination of the mitochondria, endoplasmic reticulum, and Golgi apparatus during lens fiber cell differentiation. Pioneering studies on the structural requirements for the elimination of nuclei during lens differentiation reveal the presence of an entirely novel structure associated with degrading lens nuclei termed the nuclear excisosome. Considerable evidence also indicates that autophagy is a requirement for lens homeostasis, differentiation, and transparency, since the mutation of key autophagy proteins results in human cataract formation

Patterns of Crystallin Gene Expression in Differentiation State Specific Regions of the Embryonic Chicken Lens

Purpose: Transition from lens epithelial cells to lens fiber cell is accompanied by numerous changes in gene expression critical for lens transparency. We identify expression patterns of highly prevalent genes including ubiquitous and enzyme crystallins in the embryonic day 13 chicken lens. Methods: Embryonic day 13 chicken lenses were dissected into central epithelial cell (EC), equatorial epithelial cell (EQ), cortical fiber cell (FP), and nuclear fiber cell (FC) compartments. Total RNA was prepared, subjected to high-throughput unidirectional mRNA sequencing, analyzed, mapped to the chicken genome, and functionally grouped. Results: A total of 77,097 gene-specific transcripts covering 17,450 genes were expressed, of which 10,345 differed between two or more lens subregions. Ubiquitous crystallin gene expression increased from EC to EQ and was similar in FP and FC. Highly expressed crystallin genes fell into three coordinately expressed groups with R2 ≥ 0.93: CRYAA, CRYBB2, CRYAB, and CRYBA2; CRYBB1, CRYBA4, CRYGN, ASL1, and ASL; and CRYBB3 and CRYBA1. The highly expressed transcription factors YBX1, YBX3, PNRC1, and BASP1 were coordinately expressed with the second group of crystallins (r2 \u3e 0.88). Conclusions: Although it is well known that lens crystallin gene expression changes during the epithelial to fiber cell transition, these data identify for the first time three distinct patterns of expression for specific subsets of crystallin genes, each highly correlated with expression of specific transcription factors. The results provide a quantitative basis for designing functional experiments pinpointing the mechanisms governing the landscape of crystallin expression during fiber cell differentiation to attain lens transparency

Autophagy and mitophagy participate in ocular lens organelle degradation

The eye lens consists of a layer of epithelial cells that overlay a series of differentiating fiber cells that upon maturation lose their mitochondria, nuclei and other organelles. Lens transparency relies on the metabolic function of mitochondria contained in the lens epithelial cells and in the immature fiber cells and the programmed degradation of mitochondria and other organelles occurring upon lens fiber cell maturation. Loss of lens mitochondrial function in the epithelium or failure to degrade mitochondria and other organelles in lens fiber cells results in lens cataract formation. To date, the mechanisms that govern the maintenance of mitochondria in the lens and the degradation of mitochondria during programmed lens fiber cell maturation have not been fully elucidated. Here, we demonstrate using electron microscopy and dual-label confocal imaging the presence of autophagic vesicles containing mitochondria in lens epithelial cells, immature lens fiber cells and during early stages of lens fiber cell differentiation. We also show that mitophagy is induced in primary lens epithelial cells upon serum starvation. These data provide evidence that autophagy occurs throughout the lens and that mitophagy functions in the lens to remove damaged mitochondria from the lens epithelium and to degrade mitochondria in the differentiating lens fiber cells for lens development. The results provide a novel mechanism for how mitochondria are maintained to preserve lens metabolic function and how mitochondria are degraded upon lens fiber cell maturation

Multiomics Analysis Reveals Novel Genetic Determinants for Lens Differentiation, Structure, and Transparency

Recent advances in next-generation sequencing and data analysis have provided new gateways for identification of novel genome-wide genetic determinants governing tissue development and disease. These advances have revolutionized our understanding of cellular differentiation, homeostasis, and specialized function in multiple tissues. Bioinformatic and functional analysis of these genetic determinants and the pathways they regulate have provided a novel basis for the design of functional experiments to answer a wide range of long-sought biological questions. A well-characterized model for the application of these emerging technologies is the development and differentiation of the ocular lens and how individual pathways regulate lens morphogenesis, gene expression, transparency, and refraction. Recent applications of next-generation sequencing analysis on well-characterized chicken and mouse lens differentiation models using a variety of omics techniques including RNA-seq, ATAC-seq, whole-genome bisulfite sequencing (WGBS), chip-seq, and CUT&RUN have revealed a wide range of essential biological pathways and chromatin features governing lens structure and function. Multiomics integration of these data has established new gene functions and cellular processes essential for lens formation, homeostasis, and transparency including the identification of novel transcription control pathways, autophagy remodeling pathways, and signal transduction pathways, among others. This review summarizes recent omics technologies applied to the lens, methods for integrating multiomics data, and how these recent technologies have advanced our understanding ocular biology and function. The approach and analysis are relevant to identifying the features and functional requirements of more complex tissues and disease states

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

<p>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.</p

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

<p>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.</p

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

<p>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.</p