Pterostilbene Changes Epigenetic Marks at Enhancer Regions of Oncogenes in Breast Cancer Cells.

Epigenetic aberrations are linked to sporadic breast cancer. Interestingly, certain dietary polyphenols with anti-cancer effects, such as pterostilbene (PTS), have been shown to regulate gene expression by altering epigenetic patterns. Our group has proposed the involvement of DNA methylation and DNA methyltransferase 3B (DNMT3B) as vital players in PTS-mediated suppression of candidate oncogenes and suggested a role of enhancers as target regions. In the present study, we assess a genome-wide impact of PTS on epigenetic marks at enhancers in highly invasive MCF10CA1a breast cancer cells. Following chromatin immunoprecipitation (ChIP)-sequencing in MCF10CA1a cells treated with 7 µM PTS for 9 days, we discovered that PTS leads to increased binding of DNMT3B at enhancers of 77 genes, and 17 of those genes display an overlapping decrease in the occupancy of trimethylation at lysine 36 of histone 3 (H3K36me3), a mark of active enhancers. We selected two genes, PITPNC1 and LINC00910, and found that their enhancers are hypermethylated in response to PTS. These changes coincided with the downregulation of gene expression. Of importance, we showed that 6 out of 17 target enhancers, including PITPNC1 and LINC00910, are bound by an oncogenic transcription factor OCT1 in MCF10CA1a cells. Indeed, the six enhancers corresponded to genes with established or putative cancer-driving functions. PTS led to a decrease in OCT1 binding at those enhancers, and OCT1 depletion resulted in PITPNC1 and LINC00910 downregulation, further demonstrating a role for OCT1 in transcriptional regulation. Our findings provide novel evidence for the epigenetic regulation of enhancer regions by dietary polyphenols in breast cancer cells.

Pterostilbene leads to DNMT3B-mediated DNA methylation and silencing of OCT1-targeted oncogenes in breast cancer cells.

Transcription factor (TF)-mediated regulation of genes is often disrupted during carcinogenesis. The DNA methylation state of TF-binding sites may dictate transcriptional activity of corresponding genes. Stilbenoid polyphenols, such as pterostilbene (PTS), have been shown to exert anticancer action by remodeling DNA methylation and gene expression. However, the mechanisms behind these effects still remain unclear. Here, the dynamics between oncogenic TF OCT1 binding and de novo DNA methyltransferase DNMT3B binding in PTS-treated MCF10CA1a invasive breast cancer cells has been explored. Using chromatin immunoprecipitation (ChIP) followed by next generation sequencing, we determined 47 gene regulatory regions with decreased OCT1 binding and enriched DNMT3B binding in response to PTS. Most of those genes were found to have oncogenic functions. We selected three candidates, PRKCA, TNNT2, and DANT2, for further mechanistic investigation taking into account PRKCA functional and regulatory connection with numerous cancer-driving processes and pathways, and some of the highest increase in DNMT3B occupancy within TNNT2 and DANT2 enhancers. PTS led to DNMT3B recruitment within PRKCA, TNNT2, and DANT2 at loci that also displayed reduced OCT1 binding. Substantial decrease in OCT1 with increased DNMT3B binding was accompanied by PRKCA promoter and TNNT2 and DANT2 enhancer hypermethylation, and gene silencing. Interestingly, DNA hypermethylation of the genes was not detected in response to PTS in DNMT3B-CRISPR knockout MCF10CA1a breast cancer cells. It indicates DNMT3B-dependent methylation of PRKCA, TNNT2, and DANT2 upon PTS. Our findings provide a better understanding of mechanistic players and their gene targets that possibly contribute to the anticancer action of stilbenoid polyphenols.

The role of disulfide bonds in a Solanum tuberosum saposin-like protein investigated using molecular dynamics.

The Solanum tuberosum plant specific insert (StPSI) has a defensive role in potato plants, with the requirements of acidic pH and anionic lipids. The StPSI contains a set of three highly conserved disulfide bonds that bridge the protein's helical domains. Removal of these bonds leads to enhanced membrane interactions. This work examined the effects of their sequential removal, both individually and in combination, using all-atom molecular dynamics to elucidate the role of disulfide linkages in maintaining overall protein tertiary structure. The tertiary structure was found to remain stable at both acidic (active) and neutral (inactive) pH despite the removal of disulfide linkages. The findings include how the dimer structure is stabilized and the impact on secondary structure on a residue-basis as a function of disulfide bond removal. The StPSI possesses an extensive network of inter-monomer hydrophobic interactions and intra-monomer hydrogen bonds, which is likely the key to the stability of the StPSI by stabilizing local secondary structure and the tertiary saposin-fold, leading to a robust association between monomers, regardless of the disulfide bond state. Removal of disulfide bonds did not significantly impact secondary structure, nor lead to quaternary structural changes. Instead, disulfide bond removal induces regions of amino acids with relatively higher or lower variation in secondary structure, relative to when all the disulfide bonds are intact. Although disulfide bonds are not required to preserve overall secondary structure, they may have an important role in maintaining a less plastic structure within plant cells in order to regulate membrane affinity or targeting.

Protein Structure Insights into the Bilayer Interactions of the Saposin-Like Domain of Solanum tuberosum Aspartic Protease.

Many plant aspartic proteases contain a saposin-like domain whose principal functions are intracellular sorting and host defence. Its structure is characterised by helical segments cross-linked by three highly conserved cystines. The present study on the saposin-like domain of Solanum tuberosum aspartic protease revealed that acidification from inactive to active conditions causes dimerisation and a strand-to-helix secondary structure transition independent of bilayer interaction. Bilayer fusion was shown to occur under reducing conditions yielding a faster shift to larger vesicle sizes relative to native conditions, implying that a lower level structural motif might be bilayer-active. Characterisation of peptide sequences based on the domain's secondary structural regions showed helix-3 to be active (~4% of the full domain's activity), and mutation of its sole positively charged residue resulted in loss of activity and disordering of structure. Also, the peptides' respective circular dichroism spectra suggested that native folding within the full domain is dependent on surrounding structure. Overall, the present study reveals that the aspartic protease saposin-like domain active structure is an open saposin fold dimer whose formation is pH-dependent, and that a bilayer-active motif shared among non-saposin membrane-active proteins including certain plant defence proteins is nested within an overall structure essential for native functionality.

Randomized controlled trial assessing the efficacy of a reusable fish-shaped iron ingot to increase hemoglobin concentration in anemic, rural Cambodian women.

Background: Anemia affects 45% of women of childbearing age in Cambodia. Iron supplementation is recommended in populations in which anemia prevalence is high. However, there are issues of cost, distribution, and adherence. A potential alternative is a reusable fish-shaped iron ingot, which, when added to the cooking pot, leaches iron into the fluid in which it is prepared.Objective: We sought to determine whether there was a difference in hemoglobin concentrations in rural Cambodian anemic women (aged 18-49 y) who cooked with the iron ingot or consumed a daily iron supplement compared with a control after 1 y.Design: In Preah Vihear, 340 women with mild or moderate anemia were randomly assigned to 1) an iron-ingot group, 2) an iron-supplement (18 mg/d) group, or 3) a nonplacebo control group. A venous blood sample was taken at baseline and at 6 and 12 mo. Blood was analyzed for hemoglobin, serum ferritin, and serum transferrin receptor. Hemoglobin electrophoresis was used to detect structural hemoglobin variants.Results: Anemia prevalence was 44% with the use of a portable hemoglobinometer during screening. At baseline, prevalence of iron deficiency was 9% on the basis of a low serum ferritin concentration. There was no significant difference in mean hemoglobin concentrations between the iron-ingot group (115 g/L; 95% CI: 113, 118 g/L; P = 0.850) or iron-supplement group (115 g/L; 95% CI: 113, 117 g/L; P = 0.998) compared with the control group (115 g/L; 95% CI: 113, 117 g/L) at 12 mo. Serum ferritin was significantly higher in the iron-supplement group (73 µg/L; 95% CI: 64, 82 µg/L; P = 0.002) than in the control group at 6 mo; however, this significance was not maintained at 12 mo (73 µg/L; 95% CI: 58, 91 µg/L; P = 0.176).Conclusions: Neither the iron ingot nor iron supplements increased hemoglobin concentrations in this population at 6 or 12 mo. We do not recommend the use of the fish-shaped iron ingot in Cambodia or in countries where the prevalence of iron deficiency is low and genetic hemoglobin disorders are high. This trial was registered at clinicaltrials.gov as NCT02341586.

Foldase and inhibitor functionalities of the pepsinogen prosegment are encoded within discrete segments of the 44 residue domain.

Pepsin is initially produced as the zymogen pepsinogen, containing a 44 residue prosegment (PS) domain. When folded without the PS, pepsin forms a thermodynamically stable denatured state (refolded pepsin, Rp). To guide native folding, the PS binds to Rp, stabilizes the folding transition state, and binds tightly to native pepsin (Np), thereby driving the folding equilibrium to favor the native state. It is unknown whether these functionalities of the PS are encoded within the entire sequence or within discrete segments. PS residues 1p-29p correspond to a highly conserved region in pepsin-like aspartic proteases and we hypothesized that this segment is critical to PS-catalyzed folding. This notion was tested in the present study by characterizing the ability of various truncated PS peptides to bind Rp, catalyze folding from Rp to Np, and to inhibit Np. Four PS truncations were examined, corresponding to PS residues 1p-16p (PS1-16), 1p-29p (PS1-29), 17p-44p (PS17-44) and 30p-44p (PS30-44). The three PS functionalities could be ascribed primarily to discrete regions within the highly conserved motif: 1p-16p dictated Rp binding, 17p-29p dictated Np binding/inhibition, while the entire 1p-29p dictated transition state binding/catalyzing folding. Conversely, PS30-44 played no obvious role in PS-catalyzed folding; it is hypothesized that this more variable region may serve as a linker between PS1-29 and the mature domain. The high sequence conservation of PS1-29 and its role in catalyzing pepsin folding strongly suggest that there is a conserved PS-catalyzed folding mechanism shared by pepsin-like aspartic proteases with this motif.

Almond protein hydrolysate fraction modulates the expression of proinflammatory cytokines and enzymes in activated macrophages.

Simulated gastrointestinal treatment of almond proteins with pepsin and pancreatic proteases resulting in 16.6% degree of hydrolysis or 1.33 milliequivalent leucine per g protein yielded a hydrolysate that modulated excessive nitric oxide production in lipopolysaccharide-activated RAW264.7 macrophages. After fractionation, a resulting fraction of molecular size > 5 kDa retained the nitric oxide modulatory effect observed initially in the crude hydrolysate. The high molecular size fraction was found to modulate levels of proinflammatory cytokines, interleukin (IL)-6, IL-1ß, and tumour necrosis factor (TNF)-α in the activated cells. Immunoblotting analysis indicated that the hydrolysate fraction decreased the expression levels of inflammatory enzyme indicators, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 in the activated cells. RT-PCR analysis showed that treatment of the activated cells with the hydrolysate fraction resulted in the inhibition of relative gene expressions of proinflammatory IL-6, IL-1ß, TNF-α, iNOS and COX-2. These results indicate a potential application of almond protein hydrolysates against inflammatory conditions, and will contribute to delineating the possible contributions of proteins to health benefits attributed to almond consumption.

The effect of thermal and ultrasonic treatment on amino acid composition, radical scavenging and reducing potential of hydrolysates obtained from simulated gastrointestinal digestion of cowpea proteins.

The effect of thermal and ultrasonic treatment of cowpea proteins (CP) on amino acid composition, radical scavenging and reducing potential of hydrolysates (CPH) obtained from in vitro simulated gastrointestinal digestion of CP was evaluated. Hydrolysis of native and treated CP with gastrointestinal pepsin and pancreatin yielded CPH that displayed antioxidant activities based on oxygen radical scavenging capacity (ORAC), ferric reducing antioxidant power (FRAP) and superoxide radical scavenging activity (SRSA). CPH derived from the treated CP yielded higher ORAC values than CPH from untreated proteins. However, lower significant FRAP and SRSA values were observed for these samples compared to untreated CPH (p < 0.05). Amino acid analysis indicated that CP processing decreased total sulphur-containing amino acids in the hydrolysates, particularly cysteine. The amount of cysteine appeared to be positively related to FRAP and SRSA values of CPH samples, but not ORAC. The results indicated that thermal and ultrasonic processing of CP can reduce the radical scavenging and reducing potential of the enzymatic hydrolysates possibly due to the decreased amounts of cysteine. Since the hydrolysates were generated with gastrointestinal enzymes, it is possible that the resulting compounds are produced to exert some health functions during normal consumption of cowpea.

Rational redesign of porcine pepsinogen containing an antimicrobial peptide.

A novel strategy for the controlled release and localization of bioactive peptides within digestive and immunity-related enzymes was developed. The N-terminus of porcine pepsinogen A was fused to the basic amino acid-rich region of bovine lactoferricin B termed 'tLfcB', a cationic antimicrobial/anticancer peptide. Recombinant tLfcB-porcine pepsinogen A was expressed in soluble form in Escherichia coli as a thioredoxin (Trx) fusion protein. Thioredoxin-tLfcB-porcine pepsinogen A was found to activate autocatalytically under acidic conditions. Recombinant pepsin A derived from the activation of the fusion protein had a catalytic rate and substrate affinity similar to that derived from the recombinant thioredoxin-porcine pepsinogen A control. Pepsin-treated thioredoxin-tLfcB-porcine pepsinogen A yielded increased antimicrobial activity against the Gram-negative bacteria E.coli relative to control suggesting that a second function (antimicrobial activity) was successfully engineered into a functional peptidase. The novel design strategy described herein presents a potential strategy for targeted delivery of antimicrobial or therapeutic peptides in transgenic organisms via re-engineering native proteins critical to plant and animal defense mechanisms.

Multifunctional aspartic peptidase prosegments.

The structure-function relationships of aspartic peptidases (APs) (EC 3.4.23.X) have been extensively investigated, yet much remains to be elucidated regarding the various molecular mechanisms of these enzymes. Over the past years, APs have received considerable interest for food applications (e.g. cheese, fermented foods) and as potential targets for pharmaceutical intervention in human diseases including hypertension, cancer, Alzheimer's disease, AIDS (acquired immune deficiency syndrome), and malaria. A deeper understanding of the structure and function of APs, therefore, will have a direct impact on the design of peptidase inhibitors developed to treat such diseases. Most APs are synthesized as zymogens which contain an N-terminal prosegment (PS) domain that is removed at acidic pH by proteolytic cleavage resulting in the active enzyme. While the nature of the AP PS function is not entirely understood, the PS can be important in processes such as the initiation of correct folding, protein stability, blockage of the active site, pH-dependence of activation, and intracellular sorting of the zymogen. This review summarizes the current knowledge of AP PS function (especially within the A1 family), with particular emphasis on protein folding, cellular sorting, and inhibition.

The acute impact of ingestion of breads of varying composition on blood glucose, insulin and incretins following first and second meals.

Structural characteristics and baking conditions influence the metabolic responses to carbohydrate-containing foods. We hypothesized that consumption of whole wheat or sourdough breads would have a favourable effect on biomarkers of glucose homeostasis after first and second meals, compared with those for white bread. Ten overweight volunteers consumed 50 g available carbohydrate of each of the four breads (white, whole wheat, sourdough, whole wheat barley) followed 3 h later by a standard second meal. Blood was sampled for 3 h following bread ingestion and a further 2 h after the second meal for determination of glucose, insulin, paracetamol (indirect marker of gastric emptying), glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). Glucose and GLP-1 responses to sourdough bread were lower (P < 0.05) than whole wheat and whole wheat barley breads. Glucose area under the curve (AUC) for sourdough bread was lower than those for whole wheat (P < 0.005) and whole wheat barley (P < 0.03) breads for the entire study. GIP AUC after sourdough bread ingestion was lower compared to white (P < 0.004) and whole wheat barley (P < 0.002) breads following the second meal. There were no significant differences in insulin and paracetamol concentrations among the test breads. Ultra-fine grind whole wheat breads did not result in postprandial responses that were lower than those of white bread, but sourdough bread resulted in lower glucose and GLP-1 responses compared to those of these whole wheat breads following both meals.