Cyclic peptides arising by evolutionary parallelism via asparaginyl-endopeptidase-mediated biosynthesis.

The cyclic miniprotein Momordica cochinchinensis Trypsin Inhibitor II (MCoTI-II) (34 amino acids) is a potent trypsin inhibitor (TI) and a favored scaffold for drug design. We have cloned the corresponding genes and determined that each precursor protein contains a tandem series of cyclic TIs terminating with the more commonly known, and potentially ancestral, acyclic TI. Expression of the precursor protein in Arabidopsis thaliana showed that production of the cyclic TIs, but not the terminal acyclic TI, depends on asparaginyl endopeptidase (AEP) for maturation. The nature of their repetitive sequences and the almost identical structures of emerging TIs suggest these cyclic peptides evolved by internal gene amplification associated with recruitment of AEP for processing between domain repeats. This is the third example of similar AEP-mediated processing of a class of cyclic peptides from unrelated precursor proteins in phylogenetically distant plant families. This suggests that production of cyclic peptides in angiosperms has evolved in parallel using AEP as a constraining evolutionary channel. We believe this is evolutionary evidence that, in addition to its known roles in proteolysis, AEP is especially suited to performing protein cyclization.

The Arabidopsis B3 domain protein VERNALIZATION1 (VRN1) is involved in processes essential for development, with structural and mutational studies revealing its DNA-binding surface.

The B3 DNA-binding domain is a plant-specific domain found throughout the plant kingdom from the alga Chlamydomonas to grasses and flowering plants. Over 100 B3 domain-containing proteins are found in the model plant Arabidopsis thaliana, and one of these is critical for accelerating flowering in response to prolonged cold treatment, an epigenetic process called vernalization. Despite the specific phenotype of genetic vrn1 mutants, the VERNALIZATION1 (VRN1) protein localizes throughout the nucleus and shows sequence-nonspecific binding in vitro. In this work, we used a dominant repressor tag that overcomes genetic redundancy to show that VRN1 is involved in processes beyond vernalization that are essential for Arabidopsis development. To understand its sequence-nonspecific binding, we crystallized VRN1(208-341) and solved its crystal structure to 1.6 Å resolution using selenium/single-wavelength anomalous diffraction methods. The crystallized construct comprises the second VRN1 B3 domain and a preceding region conserved among VRN1 orthologs but absent in other B3 domains. We established the DNA-binding face using NMR and then mutated positively charged residues on this surface with a series of 16 Ala and Glu substitutions, ensuring that the protein fold was not disturbed using heteronuclear single quantum correlation NMR spectra. The triple mutant R249E/R289E/R296E was almost completely incapable of DNA binding in vitro. Thus, we have revealed that although VRN1 is sequence-nonspecific in DNA binding, it has a defined DNA-binding surface.

Albumins and their processing machinery are hijacked for cyclic peptides in sunflower.

The cyclic peptide sunflower trypsin inhibitor 1 (SFTI-1) blocks trypsin and is a promising drug lead and protein engineering scaffold. We show that SFTI-1 and the newfound SFT-L1 are buried within PawS1 and PawS2, precursors for seed storage protein albumins. Proalbumins are matured by asparaginyl endopeptidase, which we show is required to liberate both ends of SFTI-1 as well as to mature PawS1 albumin. Thus, these peptides emerge from within an albumin precursor by the action of albumin's own processing enzyme.

Integrated analysis of whole blood oxylipin and cytokine responses after bacterial, viral, and T cell stimulation reveals new immune networks.

Oxylipins are major immunomodulating mediators, yet studies of inflammation focus mainly on cytokines. Here, using a standardized whole-blood stimulation system, we characterized the oxylipin-driven inflammatory responses to various stimuli and their relationships with cytokine responses. We performed a pilot study in 25 healthy individuals using 6 different stimuli: 2 bacterial stimuli (LPS and live BCG), 2 viral stimuli (vaccine-grade poly I:C and live H1N1 attenuated influenza), an enterotoxin superantigen and a Null control. All stimuli induced a strong production of oxylipins but most importantly, bacterial, viral, and T cell immune responses show distinct oxylipin signatures. Integration of the oxylipin and cytokine responses for each condition revealed new immune networks improving our understanding of inflammation regulation. Finally, the oxylipin responses and oxylipin-cytokine networks were compared in patients with active tuberculosis or with latent infection. This revealed different responses to BCG but not LPS stimulation highlighting new regulatory pathways for further investigations.

Folate supplementation differently affects uracil content in DNA in the mouse colon and liver.

High folate intake may increase the risk of cancer, especially in the elderly. The present study examined the effects of ageing and dietary folate on uracil misincorporation into DNA, which has a mutagenic effect, in the mouse colon and liver. Old (18 months; n 42) and young (4 months; n 42) male C57BL/6 mice were pair-fed with four different amino acid-defined diets for 20 weeks: folate deplete (0 mg/kg diet); folate replete (2 mg/kg diet); folate supplemented (8 mg/kg diet); folate deplete (0 mg/kg diet) with thymidine supplementation (1·8 g/kg diet). Thymidylate synthesis from uracil requires folate, but synthesis from thymidine is folate independent. Liver folate concentrations were determined by the Lactobacillus casei assay. Uracil misincorporation into DNA was measured by a GC/MS method. Liver folate concentrations demonstrated a stepwise increase across the spectrum of dietary folate levels in both old (P = 0·003) and young (P < 0·001) mice. Uracil content in colonic DNA was paradoxically increased in parallel with increasing dietary folate among the young mice (P trend = 0·033), but differences were not observed in the old mice. The mean values of uracil in liver DNA, in contrast, decreased with increasing dietary folate among the old mice, but it did not reach a statistically significant level (P < 0·1). Compared with the folate-deplete group, thymidine supplementation reduced uracil misincorporation into the liver DNA of aged mice (P = 0·026). The present study suggests that the effects of folate and thymidine supplementation on uracil misincorporation into DNA differ depending on age and tissue. Further studies are needed to clarify the significance of increased uracil misincorporation into colonic DNA of folate-supplemented young mice.

Ageing, chronic alcohol consumption and folate are determinants of genomic DNA methylation, p16 promoter methylation and the expression of p16 in the mouse colon.

Older age, dietary folate and chronic alcohol consumption are important risk factors for the development of colon cancer. The present study examined the effects of ageing, folate and alcohol on genomic and p16-specific DNA methylation, and p16 expression in the murine colon. Old (aged 18 months; n 70) and young (aged 4 months; n 70) male C57BL/6 mice were pair-fed either a Lieber-DeCarli liquid diet with alcohol (18 % of energy), a Lieber-DeCarli diet with alcohol (18 %) and reduced folate (0.25 mg folate/l) or an isoenergetic control diet (0.5 mg folate/l) for 5 or 10 weeks. Genomic DNA methylation, p16 promoter methylation and p16 gene expression were analysed by liquid chromatography-MS, methylation-specific PCR and real-time RT-PCR, respectively. Genomic DNA methylation was lower in the colon of old mice compared with young mice (P < 0.02) at 10 weeks. Alcohol consumption did not alter genomic DNA methylation in the old mouse colon, whereas it tended to decrease genomic DNA methylation in young mice (P = 0.08). p16 Promoter methylation and expression were higher in the old mouse colon compared with the corresponding young groups. There was a positive correlation between p16 promoter methylation and p16 expression in the old mouse colon (P < 0.02). In young mice the combination of alcohol and reduced dietary folate led to significantly decreased p16 expression compared with the control group (P < 0.02). In conclusion, ageing and chronic alcohol consumption alter genomic DNA methylation, p16 promoter methylation and p16 gene expression in the mouse colon, and dietary folate availability can further modify the relationship with alcohol in the young mouse.

Evaluation of magnesium fluxes in rat erythrocytes using a stable isotope of magnesium.

The mechanisms that maintain intracellular Mg concentration at physiologic levels are not fully understood. In this work, we described for the first time, a new method using 25Mg stable isotopes, that allows simultaneous determination of Mg2+ efflux and Mg2+ influx in non-loaded cells at physiological levels of extracellular Mg. Erythrocytes from rats were suspended as a 10% suspension in NaCl medium or choline medium. The erythrocyte suspension was incubated at 37C, and aliquots of the cell suspension were centrifuged at the beginning of the incubation and after 60 and 120 min. The quantification of 24Mg, 25Mg and 26Mg in supernatants and in erythrocytes were determined by ICP/MS. Simultaneous Mg2+ efflux and Mg2+ influx were calculated from the intra-extracellular distribution of the three isotopes. By this new approach we characterized Mg2+ influx and Mg2+ efflux at 0.4 mM extracellular Mg in both NaCl and choline Cl medium. Mg2+ efflux and Mg2+ influx were largely inhibited by amiloride in NaCl medium and by cinchonine in choline Cl medium. Apparent velocity and LineWeaver-Burk kinetics showed that Mg2+ influx is different from Mg2+ efflux suggesting the involvement of two distinct transport mechanisms. Moreover, modifying extracellular Mg concentrations, to mimic hypo- or hyper-magnesaemia, we showed that Mg2+ efflux and Mg2+ influx increased with extracellular Mg up to 0.8 mM, the physiologic concentration of total extracellular Mg. Our data demonstrate that Mg2+ fluxes are directly related to the levels of extracellular Mg and that in physiological conditions, Na-dependent and Na-independent Mg2+ efflux counterbalance Mg influx to maintain constant intracellular Mg level.

Age-associated B vitamin deficiency as a determinant of chronic diseases.

The number of elderly individuals is growing rapidly worldwide and degenerative diseases constitute an increasing problem in terms of both public health and cost. Nutrition plays a role in the ageing process and there has been intensive research during the last decade on B vitamin-related risk factors in vascular and neurological diseases and cancers. Data from epidemiological studies indicate that subclinical deficiency in most water-soluble B vitamins may occur gradually during ageing, possibly due to environmental, metabolic, genetic, nutritional and pathological determinants, as well as to lifestyle, gender and drug consumption. Older adults have distinct absorption, cell transport and metabolism characteristics that may alter B vitamin bioavailability. Case-control and longitudinal studies have shown that, concurrent with an insufficient status of certain B vitamins, hyperhomocysteinaemia and impaired methylation reactions may be some of the mechanisms involved before a degenerative pathology becomes evident. The question that arises is whether B vitamin inadequacies contribute to the development of degenerative diseases or result from ageing and disease. The present paper aims to give an overview of these issues at the epidemiological, clinical and molecular levels and to discuss possible strategies to prevent B vitamin deficiency during ageing.

Polymorphisms in uracil-processing genes, but not one-carbon nutrients, are associated with altered DNA uracil concentrations in an urban Puerto Rican population.

BACKGROUND: Five genes--UNG, SMUG1, MBD4, TDG, and DUT--are involved in the repair or prevention of uracil misincorporation into DNA, an anomaly that can cause mutagenic events that lead to cancer. Little is known about the determinants of uracil misincorporation, including the effects of single nucleotide polymorphisms (SNPs) in the abovementioned genes. Because of their metabolic function, folate and other one-carbon micronutrients may be important factors in the control of uracil misincorporation. OBJECTIVES: We sought to identify polymorphisms in uracil-processing genes that are determinants of DNA uracil concentration and to establish whether one-carbon nutrient status can further modify their effects. DESIGN: We examined the relations between 23 selected variants in the 5 uracil-processing genes, uracil concentrations in whole-blood DNA, and one-carbon nutrient (folate, vitamins B-6 and B-12, and riboflavin) status in 431 participants of the Boston Puerto Rican Health Study. RESULTS: Four SNPs in DUT, UNG, and SMUG1 showed a significant association with DNA uracil concentration. The SNPs in SMUG1 (rs2029166 and rs7296239) and UNG (rs34259) were associated with increased uracil concentrations in the variant genotypes (P = 0.011, 0.022, and 0.045, respectively), whereas the DUT SNP (rs4775748) was associated with a decrease (P = 0.023). In this population, one-carbon nutrient status was not associated with DNA uracil concentration, and it did not modify the effect of these 4 identified SNPs. CONCLUSION: Because elevated uracil misincorporation may induce mutagenic lesions, possibly leading to cancer, we propose that the 4 characterized SNPs in DUT, UNG, and SMUG1 may influence cancer risk and therefore deserve further investigation.

Associations between single nucleotide polymorphisms in folate uptake and metabolizing genes with blood folate, homocysteine, and DNA uracil concentrations.

BACKGROUND: Folate is an essential nutrient that supports nucleotide synthesis and biological methylation reactions. Diminished folate status results in chromosome breakage and is associated with several diseases, including colorectal cancer. Folate status is also inversely related to plasma homocysteine concentrations -- a risk factor for cardiovascular disease. OBJECTIVE: We sought to gain further understanding of the genetic determinants of plasma folate and homocysteine concentrations. Because folate is required for the synthesis of thymidine from uracil, the latter accumulating and being misincorporated into DNA during folate depletion, the DNA uracil content was also measured. DESIGN: Thirteen single nucleotide polymorphisms (SNPs) in genes involved in folate uptake and metabolism, including folate hydrolase (FOLH1), folate polyglutamate synthase (FPGS), gamma-glutamyl hydrolase (GGH), methylene tetrahydrofolate reductase (MTHFR), methionine synthase (MTR), proton-coupled folate transporter (PCFT), and reduced folate carrier (RFC1), were studied in a cohort of 991 individuals. RESULTS: The MTHFR 677TT genotype was associated with increased plasma homocysteine and decreased plasma folate. MTHFR 1298A>C and RFC1 intron 5A>G polymorphisms were associated with significantly altered plasma homocysteine concentrations. The FOLH1 1561C>T SNP was associated with altered plasma folate concentrations. The MTHFR 677TT genotype was associated with a approximately 34% lower DNA uracil content (P = 0.045), whereas the G allele of the GGH -124T>G SNP was associated with a stepwise increase in DNA uracil content (P = 0.022). CONCLUSION: Because the accumulation of uracil in DNA induces chromosome breaks, mutagenic lesions, we suggest that, as for MTHFR C677T, the GGH -124 T>G SNP may modulate the risk of carcinogenesis and therefore warrants further attention.

Preferential response of glutathione-related enzymes to folate-dependent changes in the redox state of rat liver.

BACKGROUND: Oxidative stress likely constitutes an important contributing factor in the onset of degenerative diseases associated with folate deficiency. Direct, as well as homocysteine-linked, antioxidant properties of folate could explain its preventive effect on these pathologies. AIM OF THE STUDY: Our study aimed at determining the changes in the redox status of adult rats as a function of folate intake. METHODS: Adult male rats were pair-fed for 4 weeks with a semi-synthetic diet containing 0, 0.5, 1.5, 8 or 20 mg of folic acid/kg. Folate and homocysteine concentrations, redox status markers and antioxidant enzyme activities were measured in the plasma and/or liver of the rats. A principal component analysis of the overall data was performed to draw a general scheme of the changes observed between the conditions. RESULTS: Folate deficiency caused increased homocysteinemia and features of oxidative stress including reduced plasma antioxidant capacity together with increased lipid peroxidation in liver and heart. This was associated with an increase in the specific activity of several enzymes involved in liver glutathione metabolism (glutathione peroxidase, glutathione reductase and glutathione S-transferase), suggesting an adaptive tissue response to the oxidative stress induced by folate deficiency. In contrast, no such variation was observed for hepatic superoxide dismutase and catalase. CONCLUSION: Despite no changes in hepatic levels of total glutathione, our findings indicate that glutathione-dependent antioxidant pathways could be particularly involved in the compensatory mechanism committed by liver to counteract the oxidative stress induced by folate deficiency. They also suggest that folate supplementation may not be associated with a better antioxidant protection of rats.

Proteomic analysis reveals changes in the liver protein pattern of rats exposed to dietary folate deficiency.

Epidemiologic and experimental studies showed that folate deficiency is associated with increased risk of degenerative diseases by enhancing abnormal one-carbon metabolism. We studied the changes in the proteome of liver, the main tissue of folate storage and metabolism, in a rat model of dietary folate depletion. Four-month-old rats were fed for 4 wk an amino acid-defined diet without folate and compared with pair-fed rats given the same diet adequately supplemented with folic acid. Folate deprivation decreased plasma and hepatic folate concentrations dramatically, while increasing homocysteinemia significantly. Using 2-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight MS, we identified 9 spots corresponding to differentially expressed proteins in the liver of folate-deficient rats compared with controls. Among those spots, 4 had a significantly increased volume, whereas the volume of the 5 other spots was decreased. Upregulated proteins included glutathione peroxidase (GPx) 1 and peroxiredoxin 6, 2 enzymes involved in the response to oxidative stress, and MAWD binding protein (MAWDBP), which has been associated with cancer. MAWDBP was simultaneously identified as a second spot with a lower isoelectric point (pI) that vanished almost completely after folate deficiency. Decreased abundance was also observed for cofilin 1, a protein linked to tumorigenesis, and for the GRP 75 precursor and preproalbumin, both of which are responsive to oxidative stress and/or inflammation. Moreover, an enzyme activity assay and/or Western blot analysis of GPx-1 and MAWDBP confirmed the proteomic findings. Our results show that folate deficiency modifies the abundance of several liver proteins consistently with adaptive tissue responses to oxidative and degenerative processes.