Redox-stress response resistance (RRR) mediated by hyperoxidation of peroxiredoxin 2 in senescent cells.

Aging is closely related to redox regulation. In our previous work, we proposed a new concept, "redox-stress response capacity (RRC)," and found that the decline in RRC was a dynamic characteristic of aging. However, the mechanism of RRC decline during aging remains unknown. In this study, using the senescent human fibroblast cell model and Caenorhabditis elegans model, we identified that peroxiredoxin 2 (PRDX2), as a hydrogen peroxide (H2O2) sensor, was involved in mediating RRC. PRDX2 knockdown led to a decline of RRC and accelerated senescence in fibroblasts and prdx-2 mutant C. elegans also showed decreased RRC. The mechanism study showed that the decreased sensor activity of PRDX2 was related to the increase in hyperoxidation of PRDX2 in senescent cells. Moreover, the level of PRDX2 hyperoxidation also increased in old C. elegans. Simultaneous overexpression of both PRDX2 and sulfiredoxin (SRX) rescued the reduced RRC and delayed senescence. The increase in PRDX2 hyperoxidation in senescent cells led to a decrease in its sensor activity, resulting in the decreased cellular response to H2O2, which is similar to the mechanism of insulin resistance due to the lower insulin receptor sensitivity. Treatment of young cells with a high level of H2O2 to induce a higher level of PRDX2-SO3 resulted in mimicking the RRC decline in senescent cells, which is also similar to a model of insulin resistance induced by high levels of insulin. All these results thrillingly indicate that there is an insulin-resistance-like phenomenon in senescent cells, we named it redox-stress response resistance, RRR. RRR in senescent cells is an important new discovery that explains RRC decline during aging and reveals the internal relationship between redox regulation and aging from a new perspective.

Identification and functional analysis of senescence-associated secretory phenotype of premature senescent hepatocytes induced by hexavalent chromium.

Hexavalent chromium [Cr(VI)] is a common heavy metal pollutant that can cause a number of human disease, including inflammation and cancer. Senescent cells can secrete a variety of molecules known as senescence-associated secretory phenotype (SASP). Our previous studies have confirmed that Cr(VI) can induce premature senescence in L02 hepatocytes, but the composition and the function of the related SASP are still unknown. In order to understand the components of SASP secreted by senescent L02 hepatocytes under the action of Cr(VI), we applied LC-MS/MS-based label-free protein quantification. We found that three SASP components including Coactosin-like protein 1 (COTL1), Alpha-enolase (ENO1), and Peroxiredoxin 2 (PRDX2) were up-regulated, which were confirmed by western blotting and qRT-PCR. Evidence suggested that SASP may promote the development of tumor through chronic inflammatory response, therefore we identified and analyzed the potential biological functions and signaling pathways of these three SASP components using GO and KEGG methods. The interaction between SASP components was analyzed by STRING, and verified by Co-IP. We also found that ENO1 and PRDX2, which have direct interaction, can inhibit the growth and proliferation of wildtype hepatocytes and premature senescent hepatocytes, but can promote the proliferation and behavioral changes of liver tumor cells. The present study provides valuable clues for elucidation of the carcinogenic mechanism of Cr(VI), especially for further prevention and targeted treatment of Cr(VI)-related cancer.

Redox-Oligomeric State of Peroxiredoxin-2 and Glyceraldehyde-3-Phosphate Dehydrogenase in Obstructive Sleep Apnea Red Blood Cells under Positive Airway Pressure Therapy.

In this study, we examined the effect of six months of positive airway pressure (PAP) therapy on Obstructive Sleep Apnea (OSA) red blood cell (RBC) proteome by two dimensional difference gel electrophoresis (2D-DIGE) - based proteomics followed by Western blotting (WB) validation. The discovered dysregulated proteins/proteoforms are associated with cell death, H2O2 catabolic/metabolic process, stress response, and protein oligomerization. Validation by nonreducing WB was performed for peroxiredoxin-2 (PRDX2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by using antibodies against the sulfinylated/sulfonylated cysteine of these proteins to better evaluate their redox-oligomeric states under OSA and/or in response to PAP therapy. The results indicated that the redox-oligomeric state of GAPDH and PRDX2 involving overoxidation by sulfinic/sulfonic acids were differentially modulated in OSA RBC, which might be compromising RBC homeostasis. PAP therapy by restoring this modulation induced a higher oligomerization of overoxidized GAPDH and PRDX2 in some patients that could be associated with eryptosis and the chaperone "gain" of function, respectively. This varied response following PAP may result from the complex interplay between OSA and OSA metabolic comorbidity. Hence, information on the redox status of PRDX2 and GAPDH in RBC will help to better recognize OSA subtypes and predict the therapeutic response in these patients. GAPDH monomer combined with body mass index (BMI) and PRDX2 S-S dimer combined with homeostatic model assessment for insulin resistance (HOMA-IR) showed to be very promising biomarkers to predict OSA and OSA severity, respectively.

Expression of the Antioxidative Enzyme Peroxiredoxin 2 in Multiple Sclerosis Lesions in Relation to Inflammation.

Multiple sclerosis is a chronic inflammatory disease of the central nervous system, characterized by demyelination and axonal damage as well as neuronal degeneration. Since oxygen-derived free radicals are an important factor leading to tissue damage in inflammatory multiple sclerosis (MS) lesions, research on antioxidative systems is essential to identify endogenous factors which can possibly counteract oxidative damage. As an important scavenging enzyme family, peroxiredoxins (PRDXs) play a crucial role in preventing oxidative damage; however little is known about their expression and function in MS lesions. In the present study we examined the expression of PRDX2 in white matter lesions of MS patients with long-standing, chronic disease. PRDX2 expression was investigated by immunohistochemistry in the context of oxidative stress and inflammation (determined by microglia/macrophage and T cell infiltration) in ten MS autopsy cases as well as seven control autopsy cases. PRDX2 was found to be upregulated in white matter MS lesions mainly in astrocytes, and its expression level was positively correlated with the degree of inflammation and oxidative stress. Our data suggest that PRDX2 expression contributes to the resistance of astrocytes against oxidative damage.

Construction of lentiviral vector of peroxiredoxin 2 gene mediating RNAi and its effects on the proliferation of SW480 cell / 重庆医学

Objective To construct a lentiviral expression vector of peroxiredoxin2(PRDX2) RNA interference (RNAi) and to investigate the effect of siRNA of PRDX2 genes on the proliferation of human colonrectal cancer SW480 cell .Methods RNAi tar‐get sequences were designed and synthesized towards the PRDX2 gene sequences .The lentiviral vector pGC‐EGFP‐shPRDX2 was constructed and identified .The vector was transformed into SW480 cells ,and the transfection efficiency was evaluated by fluores‐cence microscopy .The expression of PRDX2 was detected with Quantitative real‐time PCR (qRT‐PCR) and Western blot in the transfected cells .Cell growth and colony forming ability were detected with MTT and plate cloning technique .Results PRDX2 gene lentiviral vector was successfully established and was proved by gene sequencing .The expression of PRDX2 in mRNA and pro‐tein was significantly reduced(P<0 .05) .The PRDX2 mRNA and protein expression in SW480 transfected with lentiviral were sig‐nificantly reduced (P< 0 .05) ,and the ability of growth and proliferation were significantly reduced(P< 0 .05) .Conclusion PRDX2 gene lentiviral vector could be a stable and reliable tool .The proliferation and growth of SW480 cells transfected by pGC‐EGFP‐shPRDX2 could be effectively suppressed ,which could facilitate further investigation of the roles of PRDX2 gene in the de‐velopment and progression of colorectal cancer .

Construction of Peroxiredoxin 2 gene lentiviral expression vector and effect on colorectal cancer SW480 cells / 中国免疫学杂志

To construct the lentiviral vector containing Peroxiredoxin 2(Prdx2) gene and the colorectal cancer cell line stably transduced with Prdx 2-containing vector , so as to provide a useful tool for studying the role of Prdx 2 in colorectal cancer.Methods: Prdx2 was amplified by PCR and inserted into lentiviral expression vector Ubi-MCS-EGFP-IRES-Puromycin (GV218) to generate Ubi-Prdx2-EGFP-Puromycin(LV-Prdx2) vector.The inserted Prdx2 gene was verified by double enzyme digestion and DNA sequencing.Subsequently ,lentiviruses were produced and transduced into SW 480 cells.EGFP expression was examined under fluorescence microscopy ,the expression of Prdx2 was detected with qRT-PCR and Western blot.Cell growth and colony forming ability were detected with MTT and plate cloning technique.Results: The lentiviral Prdx2 expression vector was successful construc-ted.Overexpression of Prdx2 was verified in SW480 cells with LV-Prdx2 vector.Prdx2 promoted SW480 cell growth and colony forming ability(P<0.05).Conclusion:Ubi-Prdx2-EGFP-Puromycin(LV-Prdx2) vector is successfully constructed,and the SW480/LV-Prdx2 cell line with stable transduction of Prdx2 containing vector is established.Overexpression Prdx2 can significantly promote the proliferation of colorectal cancer SW 480 cells.