The substrate quality of CK2 target sites has a determinant role on their function and evolution.

Most biological processes are regulated by signaling modules that bind to short linear motifs. For protein kinases, substrates may have full or only partial matches to the kinase recognition motif, a property known as "substrate quality." However, it is not clear whether differences in substrate quality represent neutral variation or if they have functional consequences. We examine this question for the kinase CK2, which has many fundamental functions. We show that optimal CK2 sites are phosphorylated at maximal stoichiometries and found in many conditions, whereas minimal substrates are more weakly phosphorylated and have regulatory functions. Optimal CK2 sites tend to be more conserved, and substrate quality is often tuned by selection. For intermediate sites, increases or decreases in substrate quality may be deleterious, as we demonstrate for a CK2 substrate at the kinetochore. The results together suggest a strong role for substrate quality in phosphosite function and evolution. A record of this paper's transparent peer review process is included in the supplemental information.

Considerations for studying phosphorylation of the mitotic checkpoint pseudokinase BUBR1.

Budding uninhibited by benzimidazole 1-related protein 1 (BUBR1) is a mitotic checkpoint (better known as the spindle assembly checkpoint) protein that forms part of an inhibitory complex required to delay mitosis when errors occur in the attachment between chromosomes and the mitotic spindle. If these errors remain uncorrected, it could result in unequal distribution of genetic material to each of the nascent daughter cells, leading to potentially disastrous consequences at both the cellular and organismal level. In some higher eukaryotes including vertebrates, BUBR1 has a C-terminal kinase fold that is largely thought to be inactive, whereas in many species this domain has been lost through evolution and the truncated protein is known as mitotic arrest deficient 3 (MAD3). Here we present advice and practical considerations for the design of experiments, their analysis and interpretation to study the functions of the vertebrate BUBR1 during mitosis with emphasis on analysis implicating the pseudokinase domain.

A quantitative and semiautomated method for determining misaligned and lagging chromosomes during mitosis.

Accurate chromosome alignment at metaphase facilitates the equal segregation of sister chromatids to each of the nascent daughter cells. Lack of proper metaphase alignment is an indicator of defective chromosome congression and aberrant kinetochore-microtubule attachments which in turn promotes chromosome missegregation and aneuploidy, hallmarks of cancer. Tools to sensitively, accurately, and quantitatively measure chromosome alignment at metaphase will facilitate understanding of the contribution of chromosome segregation errors to the development of aneuploidy. In this work, we have developed and validated a method based on analytical geometry to measure several indicators of chromosome misalignment. We generated semiautomated and flexible ImageJ2/Fiji pipelines to quantify kinetochore misalignment at metaphase plates as well as lagging chromosomes at anaphase. These tools will ultimately allow sensitive and systematic quantitation of these chromosome segregation defects in cells undergoing mitosis.

BUBR1 Pseudokinase Domain Promotes Kinetochore PP2A-B56 Recruitment, Spindle Checkpoint Silencing, and Chromosome Alignment.

The balance of phospho-signaling at the outer kinetochore is critical for forming accurate attachments between kinetochores and the mitotic spindle and timely exit from mitosis. A major player in determining this balance is the PP2A-B56 phosphatase, which is recruited to the kinase attachment regulatory domain (KARD) of budding uninhibited by benzimidazole 1-related 1 (BUBR1) in a phospho-dependent manner. This unleashes a rapid, switch-like phosphatase relay that reverses mitotic phosphorylation at the kinetochore, extinguishing the checkpoint and promoting anaphase. Here, we demonstrate that the C-terminal pseudokinase domain of human BUBR1 is required to promote KARD phosphorylation. Mutation or removal of the pseudokinase domain results in decreased PP2A-B56 recruitment to the outer kinetochore attenuated checkpoint silencing and errors in chromosome alignment as a result of imbalance in Aurora B activity. Our data, therefore, elucidate a function for the BUBR1 pseudokinase domain in ensuring accurate and timely exit from mitosis.

Mps1 Phosphorylates Its N-Terminal Extension to Relieve Autoinhibition and Activate the Spindle Assembly Checkpoint.

Monopolar spindle 1 (Mps1) is a conserved apical kinase in the spindle assembly checkpoint (SAC) that ensures accurate segregation of chromosomes during mitosis. Mps1 undergoes extensive auto- and transphosphorylation, but the regulatory and functional consequences of these modifications remain unclear. Recent findings highlight the importance of intermolecular interactions between the N-terminal extension (NTE) of Mps1 and the Hec1 subunit of the NDC80 complex, which control Mps1 localization at kinetochores and activation of the SAC. Whether the NTE regulates other mitotic functions of Mps1 remains unknown. Here, we report that phosphorylation within the NTE contributes to Mps1 activation through relief of catalytic autoinhibition that is mediated by the NTE itself. Moreover, we find that this regulatory NTE function is independent of its role in Mps1 kinetochore recruitment. We demonstrate that the NTE autoinhibitory mechanism impinges most strongly on Mps1-dependent SAC functions and propose that Mps1 activation likely occurs sequentially through dimerization of a "prone-to-autophosphorylate" Mps1 conformer followed by autophosphorylation of the NTE prior to maximal kinase activation segment trans-autophosphorylation. Our observations underline the importance of autoregulated Mps1 activity in generation and maintenance of a robust SAC in human cells.

Serum vitamin C levels modulate the lifespan and endoplasmic reticulum stress response pathways in mice synthesizing a nonfunctional mutant WRN protein.

Werner syndrome (WS) is a premature aging disorder caused by mutations in a RecQ-family DNA helicase (WRN). Mice lacking part of the helicase domain of the WRN ortholog exhibit several phenotypic features of WS. In this study, we generated a Wrn mutant line that, like humans, relies entirely on dietary sources of vitamin C (ascorbate) to survive, by crossing them to mice that lack the gulonolactone oxidase enzyme required for ascorbate synthesis. In the presence of 0.01% ascorbate (w/v) in drinking water, double-mutant mice exhibited a severe reduction in lifespan, small size, sterility, osteopenia, and metabolic profiles different from wild-type (WT) mice. Although increasing the dose of ascorbate to 0.4% improved dramatically the phenotypes of double-mutant mice, the metabolic and cytokine profiles were different from age-matched WT mice. Finally, double-mutant mice treated with 0.01% ascorbate revealed a permanent activation of all the 3 branches of the ER stress response pathways due to a severe chronic oxidative stress in the ER compartment. In addition, markers associated with the ubiquitin-proteasome-dependent ER-associated degradation pathway were increased. Augmenting the dose of ascorbate reversed the activation of this pathway to WT levels rendering this pathway a potential therapeutic target in WS.-Aumailley, L., Dubois, M. J., Brennan, T. A., Garand, C., Paquet, E. R., Pignolo, R. J., Marette, A., Lebel, M. Serum vitamin C levels modulate the lifespan and endoplasmic reticulum stress response pathways in mice synthesizing a nonfunctional mutant WRN protein.

Different non-synonymous polymorphisms modulate the interaction of the WRN protein to its protein partners and its enzymatic activities.

Werner syndrome (WS) is characterized by the premature onset of several age-associated pathologies including cancer. The protein defective in WS patients (WRN) is a helicase/exonuclease involved in DNA replication and repair. Here, we present the results of a large-scale proteome analysis that has been undertaken to determine protein partners of different polymorphic WRN proteins found with relatively high prevalence in the human population. We expressed different fluorescently tagged-WRN (eYFP-WRN) variants in human 293 embryonic kidney cells (HEK293) and used a combination of affinity-purification and mass spectrometry to identify different compositions of WRN-associated protein complexes. We found that a WRN variant containing a phenylalanine residue at position 1074 and an arginine at position 1367 (eYFP-WRN(F-R)) possesses more affinity for DNA-PKc, KU86, KU70, and PARP1 than a variant containing a leucine at position 1074 and a cysteine at position 1367 (eYFP-WRN(L-C)). Such results were confirmed in a WRN-deficient background using WS fibroblasts. Interestingly, the exonuclase activity of WRN recovered from immunoprecipitated eYFP-WRN(L-C) variant was lower than the eYFP-WRN(F-R) in WS cells. Finally, HEK293 cells and WS fibroblasts overexpressing the eYFP-WRN(F-R) variant were more resistant to the benzene metabolite hydroquinone than cells expressing the eYFP-WRN(L-C) variant. These results indicate that the protein-protein interaction landscape of WRN is subject to modulation by polymorphic amino acids, a characteristic associated with distinctive cell survival outcome.

Vitamin C modulates the metabolic and cytokine profiles, alleviates hepatic endoplasmic reticulum stress, and increases the life span of Gulo-/- mice.

Suboptimal intake of dietary vitamin C (ascorbate) increases the risk of several chronic diseases but the exact metabolic pathways affected are still unknown. In this study, we examined the metabolic profile of mice lacking the enzyme gulonolactone oxidase (Gulo) required for the biosynthesis of ascorbate. Gulo-/- mice were supplemented with 0%, 0.01%, and 0.4% ascorbate (w/v) in drinking water and serum was collected for metabolite measurements by targeted mass spectrometry. We also quantified 42 serum cytokines and examined the levels of different stress markers in liver. The metabolic profiles of Gulo-/- mice treated with ascorbate were different from untreated Gulo-/- and normal wild type mice. The cytokine profiles of Gulo-/-mice, in return, overlapped the profile of wild type animals upon 0.01% or 0.4% vitamin C supplementation. The life span of Gulo-/- mice increased with the amount of ascorbate in drinking water. It also correlated significantly with the ratios of serum arginine/lysine, tyrosine/phenylalanine, and the ratio of specific species of saturated/unsaturated phosphatidylcholines. Finally, levels of hepatic phosphorylated endoplasmic reticulum associated stress markers IRE1α and eIF2α correlated inversely with serum ascorbate and life span suggesting that vitamin C modulates endoplasmic reticulum stress response and longevity in Gulo-/- mice.

Impact of vitamin C on the cardiometabolic and inflammatory profiles of mice lacking a functional Werner syndrome protein helicase.

Werner syndrome (WS) is a premature aging disorder caused by mutations in a DNA helicase/exonuclease. Mice lacking the helicase domain of this protein exhibit metabolic abnormalities that are reversed by vitamin C. In this study, we used a targeted metabolomic approach to identify serum metabolites significantly altered in young mutant mice treated with or without vitamin C. We also measured several serum inflammatory and cardiometabolic factors. We show that young mutant mice exhibit an increase in serum hydroxyproline and plasminogen activator inhibitor-1 (PAI-1), markers of cardiovascular diseases and inflammation, before they exhibit morphological anomalies in different tissues. We also observed an increase in three very long chain lysophosphatidylcholines underlying peroxisome perturbation. Vitamin C reversed the concentrations of these metabolites and PAI-1 to wild type values. Transcriptomic analyses on the liver of mutant mice revealed a decrease in the expression of genes involved in fatty acid degradation compared to wild type animals. Vitamin C treatment increased the expression of genes involved in glutathione metabolism and the synthesis of unsaturated fatty acids in these mice. These results show that changes at the transcriptomic level concord with the alterations of several serum metabolites in these mice. Finally, we found that a mislocalization of the Wrn mutant protein in the liver endoplasmic reticulum fraction increased oxidative stress in that cellular compartment. Vitamin C reversed this oxidative stress. To conclude, this study provides novel potential predictive cardiometabolic biomarkers in WS that will allow the assessment of the impact of vitamin C on patients with WS.

Metabolic and Phenotypic Differences between Mice Producing a Werner Syndrome Helicase Mutant Protein and Wrn Null Mice.

Werner syndrome (WS) is a premature aging disorder caused by mutations in a RecQ-family DNA helicase, WRN. Mice lacking part of the helicase domain of the WRN orthologue exhibit many phenotypic features of WS, including metabolic abnormalities and a shorter mean life span. In contrast, mice lacking the entire Wrn protein (i.e. Wrn null mice) do not exhibit a premature aging phenotype. In this study, we used a targeted mass spectrometry-based metabolomic approach to identify serum metabolites that are differentially altered in young Wrn helicase mutant and Wrn null mice. An antibody-based quantification of 43 serum cytokines and markers of cardiovascular disease risk complemented this study. We found that Wrn helicase mutants exhibited elevated and decreased levels, respectively, of the anti-inflammatory cytokine IL-10 and the pro-inflammatory cytokine IL-18. Wrn helicase mutants also exhibited an increase in serum hydroxyproline and plasminogen activator inhibitor-1, markers of extracellular matrix remodeling of the vascular system and inflammation in aging. We also observed an abnormal increase in the ratio of very long chain to short chain lysophosphatidylcholines in the Wrn helicase mutants underlying a peroxisome perturbation in these mice. Remarkably, the Wrn mutant helicase protein was mislocalized to the endoplasmic reticulum and the peroxisomal fractions in liver tissues. Additional analyses with mouse embryonic fibroblasts indicated a severe defect of the autophagy flux in cells derived from Wrn helicase mutants compared to wild type and Wrn null animals. These results indicate that the deleterious effects of the helicase-deficient Wrn protein are mediated by the dysfunction of several cellular organelles.

The non-synonymous polymorphism at position 114 of the WRN protein affects cholesterol efflux in vitro and correlates with cholesterol levels in vivo.

Werner syndrome (WS) is a recessive disorder characterized by the premature onset of a number of age-related diseases. The objective of the present study was to examine the degree of associations between non-synonymous coding Single Nucleotide Polymorphisms (SNPs) in the WRN gene and markers of obesity, diabetes, and hypertension using meta-analyses publically available and to test their effect in WS fibroblasts. The P-value, after genomic control correction, for each non-synonymous coding SNP present in the WRN gene was retrieved from the International Consortium for Blood Pressure Genome-Wide Association Study, the Genome Wide Associations Scans for Total Cholesterol, HDL-cholesterol, LDL-cholesterol and triglycerides, and the Meta-Analyses of Glucose and Insulin-related traits Consortium. For SNPs significantly associated with cholesterol traits, we generated expression vectors containing the amino acid changes and measured cholesterol uptake and efflux in transfected WS fibroblasts. One SNP (rs2230009) changing a valine for an isoleucine at position 114 of the WRN protein was nominally associated with cholesterol and LDL-cholesterol measurements (P-values<0.05). Interestingly, a WRN cDNA expression vector bearing a valine at position 114 instead of isoleucine significantly affected cholesterol efflux in WS fibroblasts. These results implicate a functional effect of this WRN polymorphism on cholesterol metabolism.

Down regulation of miR-124 in both Werner syndrome DNA helicase mutant mice and mutant Caenorhabditis elegans wrn-1 reveals the importance of this microRNA in accelerated aging.

Small non-coding microRNAs are believed to be involved in the mechanism of aging but nothing is known on the impact of microRNAs in the progeroid disorder Werner syndrome (WS). WS is a premature aging disorder caused by mutations in a RecQ-like DNA helicase. Mice lacking the helicase domain of the WRN ortholog exhibit many phenotypic features of WS, including a pro-oxidant status and a shorter mean life span.Caenorhabditis elegans (C. elegans) with a nonfunctional wrn-1 DNA helicase also exhibit a shorter life span. Thus, both models are relevant to study the expression of microRNAs involved in WS. In this study, we show that miR-124 expression is lost in the liver of Wrn helicase mutant mice. Interestingly, the expression of this conserved miR-124 in whole wrn-1 mutant worms is also significantly reduced. The loss of mir-124 in C. elegans increases reactive oxygen species formation and accumulation of the aging marker lipofuscin, reduces whole body ATP levels and results in a reduction in life span. Finally, supplementation of vitamin C normalizes the median life span of wrn-1 and mir-124 mutant worms. These results suggest that biological pathways involving WRN and miR-124 are conserved in the aging process across different species.

The Werner syndrome gene product (WRN): a repressor of hypoxia-inducible factor-1 activity.

Werner syndrome (WS) is a rare autosomal disease characterized by the premature onset of several age-associated pathologies. The protein defective in WS patients (WRN) is a helicase/exonuclease involved in DNA repair, replication, transcription and telomere maintenance. Hypoxia-inducible factor-1 (HIF-1) is a decisive element for the transcriptional regulation of genes essential for adaptation to low oxygen conditions. HIF-1 is also implicated in the molecular mechanisms of ageing. Here, we show that the cellular depletion of WRN protein (by siRNA targeting) leads to increased HIF-1 complex stabilization and activation. HIF-1 activation in the absence of WRN involves the generation of mitochondrial reactive oxygen species (mtROS) since SkQ1, a mitochondrial-targeted antioxidant, and stigmatellin, an inhibitor of mitochondrial complex III, blocked increased HIF-1 levels. Ascorbate, an essential co-factor involved in HIF-1 stability, was decreased in WRN-depleted cells. Interestingly, expression levels of GLUT1, a known dehydroascorbic acid transporter, were also decreased in WRN-depleted cells. Ascorbate supplementation of WRN-depleted cells led to a dose-dependent inhibition of HIF-1 activation. These results indicate that WRN protein regulates HIF-1 activation by affecting mitochondrial ROS production and intracellular ascorbate levels. This work provides a novel mechanistic link between HIF-1 activity and different age-associated pathologies.

NONO and RALY proteins are required for YB-1 oxaliplatin induced resistance in colon adenocarcinoma cell lines.

BACKGROUND: YB-1 is a multifunctional protein that affects transcription, splicing, and translation. Overexpression of YB-1 in breast cancers causes cisplatin resistance. Recent data have shown that YB-1 is also overexpress in colorectal cancer. In this study, we tested the hypothesis that YB-1 also confers oxaliplatin resistance in colorectal adenocarcinomas. RESULTS: We show for the first time that transfection of YB-1 cDNA confers oxaliplatin resistance in two colorectal cancer cell lines (SW480 and HT29 cell lines). Furthermore, we identified by mass spectrometry analyses important YB-1 interactors required for such oxaliplatin resistance in these colorectal cancer cell lines. A tagged YB-1 construct was used to identify proteins interacting directly to YB-1 in such cells. We then focused on proteins that are potentially involved in colorectal cancer progression based on the Oncomine microarray database. Genes encoding for these YB-1 interactors were also examined in the public NCBI comparative genomic hybridization database to determine whether these genes are localized to regions of chromosomes rearranged in colorectal cancer tissues. From these analyses, we obtained a list of proteins interacting with YB-1 and potentially involved in oxaliplatin resistance. Oxaliplatin dose response curves of SW480 and HT29 colorectal cancer cell lines transfected with several siRNAs corresponding to each of these YB-1 interactors were obtained to identify proteins significantly affecting oxaliplatin sensitivity upon gene silencing. Only the depletion of either NONO or RALY sensitized both colorectal cancer cell lines to oxaliplatin. Furthermore, depletion of NONO or RALY sensitized otherwise oxaliplatin resistant overexpressing YB-1 SW480 or HT29 cells. CONCLUSION: These results suggest knocking down NONO or RALY significant counteracts oxaliplatin resistance in colorectal cancers overexpressing the YB-1 protein.

An integrative approach to identify YB-1-interacting proteins required for cisplatin resistance in MCF7 and MDA-MB-231 breast cancer cells.

The Y-box binding protein 1 (YB-1) is a multifunctional protein that affects transcription, splicing, and translation. Overexpression of YB-1 in breast cancers causes cisplatin resistance. The exact mechanism by which YB-1 confers cisplatin resistance is unknown. The aim of the present study was to identify, using mass spectrometry, proteins that interact with YB-1 that are important for cisplatin resistance in two breast cancer cell lines, namely MCF7 and MDA-MB-231. A tagged YB-1 construct was used to identify proteins interacting directly with YB-1 in breast cancer cells. We then focused on proteins that are potentially involved in breast cancer progression based on the ONCOMINE public microarray database. Genes encoding for these YB-1-interacting proteins were examined in the public NCBI comparative genomic hybridization database to determine whether they are localized to regions of chromosomes that are rearranged in breast cancer tissues. From these analyses, we generated a list of proteins potentially involved in cisplatin resistance. Cisplatin dose-response curves were constructed in MCF7 and MDA-MB-231 transfected with four siRNA corresponding to each of these YB-1 interactors to identify proteins significantly affecting cisplatin sensitivity upon gene silencing. Depletion of only the X-linked ribosomal protein S4 (RPS4X) resulted in consistent resistance to cisplatin in both cell lines with at least three different siRNA sequences against RPS4X. Further analyses indicated that the knock down of RPS4X decreased DNA synthesis, induced cisplatin resistance, and is equivalent to the overexpression of YB-1 in both MCF7 and MDA-MB-231 cells. These results suggest that the RPS4X/YB-1 complex is a significant potential target to counteract cisplatin resistance in breast cancer.

Proteome-wide identification of WRN-interacting proteins in untreated and nuclease-treated samples.

Werner syndrome (WS) is characterized by the premature onset of several age-associated pathologies. The protein defective in WS patients (WRN) is a helicase/exonuclease involved in DNA repair, replication, telomere maintenance, and transcription. Here, we present the results of a large-scale proteome analysis to determine protein partners of WRN. We expressed fluorescent tagged-WRN (eYFP-WRN) in human 293 embryonic kidney cells and detected interacting proteins by co-immunoprecipitation from cell extract. We identified by mass spectrometry 220 nuclear proteins that complexed with WRN. This number was reduced to 40 when broad-spectrum nucleases were added to the lysate. We consider these 40 proteins as directly interacting with WRN. Some of these proteins have previously been shown to interact with WRN, whereas most are new partners. Among the top 15 hits, we find the new interactors TMPO, HNRNPU, RPS3, RALY, RPS9 DDX21, and HNRNPM. These proteins are likely important components in understanding the function of WRN in preventing premature aging and deserve further investigation. We have confirmed endogenous WRN interaction with endogenous RPS3, a ribosomal protein with endonuclease activities involved in oxidative DNA damage recognition. Our results suggest that the use of nucleases during cell lysis severely restricts interacting protein partners and thus enhances specificity.

Resveratrol improves insulin resistance hyperglycemia and hepatosteatosis but not hypertriglyceridemia, inflammation, and life span in a mouse model for Werner syndrome.

Werner syndrome is a premature aging disorder caused by mutations in a RecQ-like DNA helicase. Mice lacking the helicase domain of the WRN homologue exhibit many features of Werner syndrome, including a pro-oxidant status and a shorter mean life span. Here, we show that resveratrol supplementation improved the hyperglycemia and the insulin resistance phenotype in these Wrn mutant mice. In addition, resveratrol reversed liver steatosis, lipid peroxidaton, and the defenestration phenotypes observed in such mice. Resveratrol, however, did not improve the hypertriglyceridemia, inflammatory stress, nor extend the mean life span of these mutant mice. Microarray and biologic pathway enrichment analyses on liver tissues revealed that resveratrol mainly decreased lipidogenesis and increased genes involved in the insulin signaling pathway and the glutathione metabolism in Wrn mutant mice. Finally, resveratrol-treated mutant mice exhibited an increase in the frequency of lymphoma and of several solid tumors. These results indicate that resveratrol supplementation might exert at least metabolic benefits for Werner syndrome patients.

Ascorbate improves metabolic abnormalities in Wrn mutant mice but not the free radical scavenger catechin.

Werner syndrome (WS) is a premature aging disorder caused by mutations in a RecQ-like DNA helicase. Mice lacking the helicase domain of the WRN homologue exhibit many phenotypic features of WS. Importantly, mutant Wrn(Deltahel/Deltahel) mice show abnormal increases in visceral fat deposition and fasting blood triglyceride levels followed by insulin resistance and high blood glucose levels. These mice also exhibit increased heart and liver tissue reactive oxygen species concomitantly with oxidative DNA damage, indicating a pro-oxidant status. We treated mice with either ascorbate or catechin hydrate for 9 months. Vitamin C supplementation reduced oxidative stress in liver and heart tissues and reversed hypertriglyceridemia, hyperglycemia, and insulin resistance and reduced fat weight in mutant Wrn(Deltahel/Deltahel) mice. Although the free scavenger catechin hydrate also reduced oxidative DNA damage in heart and liver tissues, it did not reverse any of the metabolic phenotype aspects in treated mutant mice. Finally, vitamin C and catechin hydrate did not affect the metabolic status of wild-type mice. These results indicate that vitamin C supplementation could be beneficial for WS patients.

Expression profiling of mouse embryonic fibroblasts with a deletion in the helicase domain of the Werner Syndrome gene homologue treated with hydrogen peroxide.

BACKGROUND: Werner Syndrome (WS) is a rare disorder characterized by the premature onset of a number of age-related diseases. The gene responsible for WS encodes a DNA helicase/exonuclease protein believed to affect different aspects of transcription, replication, and/or DNA repair. In addition to genomic instability, human WS cells exhibit oxidative stress. In this report, we have examined the impact of exogenous hydrogen peroxide on the expression profile of mouse embryonic fibroblasts lacking part of the helicase domain of the WRN homologue (here referred to as Wrn Delta hel/Delta hel). RESULTS: Wrn Delta hel/Delta hel mutant mouse embryonic fibroblasts exhibit increased oxidative stress. This was reflected by increased intracellular reactive oxygen species (ROS), increased oxidative damage in genomic DNA, changes in ATP/ADP ratios, and a disruption of the inner mitochondrial transmembrane potential when compared to wild type mouse embryonic fibroblasts. Expression profile analyses of hydrogen peroxide-treated wild type cells have indicated significant decreases in the expression of genes involved in mitosis, glycolysis, fatty acid metabolism, nucleic acid metabolism, and cell cycle control, as well as protein modification and stability. Such decreases in these biological processes were not observed in hydrogen peroxide-treated Wrn Delta hel/Delta hel cells. Importantly, untreated Wrn Delta hel/Delta hel cells already exhibited down regulation of several biological processes decreased in wild type cells that had been treated with hydrogen peroxide. CONCLUSION: Expression profiling of Wrn Delta hel/Delta hel mutant cells revealed a very different response to exogenous addition of hydrogen peroxide in culture compared to wild type cells. This is due in part to the fact that Wrn Delta hel/Delta hel mutant cells already exhibited a modest chronic intracellular oxidative stress.

Vitamin C restores healthy aging in a mouse model for Werner syndrome.

Werner syndrome (WS) is a premature aging disorder caused by mutations in a RecQ-like DNA helicase. Mice lacking the helicase domain of the WRN homologue exhibit many phenotypic features of WS, including a prooxidant status and a shorter mean life span compared to wild-type animals. Here, we show that Wrn mutant mice also develop premature liver sinusoidal endothelial defenestration along with inflammation and metabolic syndrome. Vitamin C supplementation rescued the shorter mean life span of Wrn mutant mice and reversed several age-related abnormalities in adipose tissues and liver endothelial defenestration, genomic integrity, and inflammatory status. At the molecular level, phosphorylation of age-related stress markers like Akt kinase-specific substrates and the transcription factor NF-kappaB, as well as protein kinase Cdelta and Hif-1alpha transcription factor levels, which are increased in the liver of Wrn mutants, were normalized by vitamin C. Vitamin C also increased the transcriptional regulator of lipid metabolism PPARalpha. Finally, microarray and gene set enrichment analyses on liver tissues revealed that vitamin C decreased genes normally up-regulated in human WS fibroblasts and cancers, and it increased genes involved in tissue injury response and adipocyte dedifferentiation in obese mice. Vitamin C did not have such effect on wild-type mice. These results indicate that vitamin C supplementation could be beneficial for patients with WS.