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

Effect of plant-based feed ingredients on osmoregulation in the Atlantic salmon lens

Lenses of adult Atlantic salmon fed with a plant oil and plant protein-based diet (plant diet) were compared to lenses of fish fed a diet based on traditional marine ingredients (marine diet) with respect to biochemical composition and functionality ex vivo. After 12 months of feeding, plant diet-fed fish had smaller lenses with higher water contents and lower concentrations of histidine (His) and N-acetylhistidine (NAH) than fish fed with the marine diet. Cataract development in both dietary groups was minimal and no differences between the groups were observed. Lens fatty acid and lipid class composition differed minimally, although a significant increase in linoleic acid was observed. The lenses were examined for their ability to withstand osmotic disturbances ex vivo. Culture in hypoosmotic and hyperosmotic media led to increase and decrease of lens volume, respectively. Lenses from plant diet-fed fish were less resistant to swelling and shrinking, released less NAH into the culture medium, and accumulated His and NAH at higher rates than lenses from marine diet-fed fish. Culture in hypoosmotic medium resulted in higher cataract scores than in control and hyperosmotic medium. mRNA expression of selected genes, including glutathione peroxidase 4 and SPARC (secreted protein acidic and rich in cysteine), was affected by diet and osmotic treatment. It can be concluded that lenses of farmed Atlantic salmon are affected by the diet composition, both in biochemical composition and physiological functionality in relation to osmoregulation

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

Structural and kinetic analysis of an MsrA–MsrB fusion protein from Streptococcus pneumoniae

Methionine sulphoxide reductases (Msr) catalyse the reduction of oxidized methionine to methionine. These enzymes are divided into two classes, MsrA and MsrB, according to substrate specificity. Although most MsrA and MsrB exist as separate enzymes, in some bacteria these two enzymes are fused to form a single polypeptide (MsrAB). Here, we report the first crystal structure of MsrAB from Streptococcus pneumoniae (SpMsrAB) at 2.4 Å resolution. SpMsrAB consists of an N-terminal MsrA domain, a C-terminal MsrB domain and a linker. The linker is composed of 13 residues and contains one 310-helix and several hydrogen bonds interacting with both MsrA and MsrB domains. Interestingly, our structure includes the MsrB domain complexed with an SHMAEI hexa-peptide that is the N-terminal region of neighbouring MsrA domain. A kinetic analysis showed that the apparent Km of SpMsrAB for the R-form-substrate was 20-fold lower than that for the S-form substrate, indicating that the MsrB domain had a much higher affinity for the substrate than the MsrA domain. Our study reveals the first structure of the MsrAB by providing insights into the formation of a disulphide bridge in the MsrB, the structure of the linker region, and the distinct structural nature of active site of each MsrA and MsrB domain

Identification of MSRA gene on chromosome 8p as a candidate metastasis suppressor for human hepatitis B virus-positive hepatocellular carcinoma

<p>Abstract</p> <p>Background</p> <p>The prognosis of patients with hepatocellular carcinoma (HCC) still remains very dismal, which is mainly due to metastasis. In our previous studies, we found that chromosome 8p deletions might contribute to metastasis of HCC. In this study, we aimed to identify the candidate metastatic suppressor gene on chromosome 8p.</p> <p>Methods</p> <p>Oligo-nucleotide microarrays which included 322 genes on human chromosome 8p were constructed to analyze the difference in gene expression profiles between HCC tissues with and without metastasis. The leading differentially expressed genes were identified and selected for further analysis by real-time PCR and Western blotting. Recombinant expression plasmid vectors for each target gene were constructed and transfected into HCC cells and its <it>in vitro </it>effects on proliferation and invasion of HCC cells were also investigated.</p> <p>Results</p> <p>Sixteen leading differentially expressed genes were identified from the HCC tissues with metastasis compared with those without metastasis (<it>p </it>< 0.01, <it>q </it>< 16 %). Among of the 10 significantly down-regulated genes in HCC with metastasis, methionine sulfoxide reductase A (<it>MSRA</it>) had the lowest <it>p </it>value and false discovery rate (FDR), and was considered as a potential candidate for metastasis suppressor gene. Real-time PCR and Western blotting confirmed that the mRNA and protein expression levels of <it>MSRA </it>were significantly decreased in HCC with metastasis compared with those without metastasis (<it>p </it>< 0.001), and <it>MSRA </it>mRNA level in HCCLM6 cells (with high metastatic potential) was also much lower than that of other HCC cell lines. Transfection of a recombinant expression plasmid vector and overexpression of <it>MSRA </it>gene could obviously inhibit cell colony formation (4.33 ± 2.92 vs. 9.17 ± 3.38, <it>p </it>= 0.008) and invasion (7.40 ± 1.67 vs. 17.20 ± 2.59, <it>p</it>= 0.0001) of HCCLM6 cell line.</p> <p>Conclusion</p> <p><it>MSRA </it>gene on chromosome 8p might possess metastasis suppressor activity in HCC.</p