Human Rap1 modulates TRF2 attraction to telomeric DNA.

More than two decades of genetic research have identified and assigned main biological functions of shelterin proteins that safeguard telomeres. However, a molecular mechanism of how each protein subunit contributes to the protecting function of the whole shelterin complex remains elusive. Human Repressor activator protein 1 (Rap1) forms a multifunctional complex with Telomeric Repeat binding Factor 2 (TRF2). Rap1-TRF2 complex is a critical part of shelterin as it suppresses homology-directed repair in Ku 70/80 heterodimer absence. To understand how Rap1 affects key functions of TRF2, we investigated full-length Rap1 binding to TRF2 and Rap1-TRF2 complex interactions with double-stranded DNA by quantitative biochemical approaches. We observed that Rap1 reduces the overall DNA duplex binding affinity of TRF2 but increases the selectivity of TRF2 to telomeric DNA. Additionally, we observed that Rap1 induces a partial release of TRF2 from DNA duplex. The improved TRF2 selectivity to telomeric DNA is caused by less pronounced electrostatic attractions between TRF2 and DNA in Rap1 presence. Thus, Rap1 prompts more accurate and selective TRF2 recognition of telomeric DNA and TRF2 localization on single/double-strand DNA junctions. These quantitative functional studies contribute to the understanding of the selective recognition of telomeric DNA by the whole shelterin complex.

Urine Levels of Phthalate Metabolites and Bisphenol A in Relation to Main Metabolic Syndrome Components: Dyslipidemia, Hypertension and Type 2 Diabetes. A Pilot Study.

AIM: Human exposure to organic pollutants (some of them also called endocrine disruptors) can be associated with adverse metabolic health outcomes including type 2 diabetes. The goal of this study was to compare the urine levels of bisphenol A and phthalate metabolites in subgroups of patients with metabolic syndrome composed of patients with and without three important components of metabolic syndrome (hypertension, dyslipidemia and diabetes). METHODS: We have investigated 24 hr urine samples of 168 patients with metabolic syndrome from the Metabolic Outpatient Department of General University Hospital in Prague. Using standard metabolic syndrome criteria, we classified patients as dyslipidemic (n=87), hypertensive (n=96), and type 2 diabetic (n=58). Bisphenol A and 15 metabolites of phthalates were evaluated in relation to creatinine excretion. Samples were analysed with enzymatic cleavage of glucuronide using ultra-high-performance liquid chromatography-electrospray ionization tandem mass spectrometry in one laboratory with external quality control. RESULTS: Four metabolites, mono-n-butyl phthalate, mono-(2-ethyl-5-hydroxyhexyl) phthalate, mono-(2-ethyl-5-oxohexyl) phthalate, and mono-(2-ethyl-5-carboxypentyl) phthalate showed significantly higher levels in diabetic compared to non-diabetic patients (p<0.001, p=0.002, p=0.002, and p=0.005, respectively). The differences remained significant after adjustment to hypertension, dyslipidemia, age, and BMI. No difference was found between either the hypertensive and non-hypertensive or dyslipidemic and non-dyslipidemic patients. There was no significant relation of bisphenol A level to diabetes, hypertension, dyslipidemia, age, and BMI. CONCLUSIONS: Urine levels of four phthalate metabolites were significantly higher in type 2 diabetics independently on specified predictors. Phthalate levels can be in relation to beta cell dysfunction in type 2 diabetic patients but this study is not able to show if the relation is causal.

[Potential sources of phthalates and bisphenol A and their significance in the development of metabolic diseases]. / Potenciální zdroje ftalátu a bisfenolu A a jejich význam u metabolických onemocnení.

Nowadays, there is increasing evidence showing that the development of the metabolic syndrome combining obesity, type 2 diabetes mellitus, arterial hypertension and dyslipidemia involves except of traditional risk factors (overnutrition, lack of physical activity, genetic predisposition) also the effect of environmental organic substances called organic pollutants or endocrine disruptors. These chemicals can be found in plastic covers, paints, flame retardants, exhaust gases, fertilizers as well as diverse daily utensils. Phthalates, used primarily as plasticizers, and bisphenol A, are among the most wide-spread members of this group.The aim of this article is to provide a basic overview of the relationship between phthalates and bisphenol A and the etiopathogenesis of the metabolic syndrome and to highlight their potential sources. According to the analysis of materials used for parenteral nutrition and urinary excretion of phthalate metabolites and bisphenol A in subjects on long-term parenteral nutrition we suppose that currently used medical materials are safe with respect to the exposure to both phthalates and bisphenol A and that home environment, especially cosmetic products, might constitute a more probable source of these substances.

Case study: Possible differences in phthalates exposure among the Czech, Hungarian, and Slovak populations identified based on the DEMOCOPHES pilot study results.

OBJECTIVE: Phthalates and their metabolites are classified as endocrine modulators. They affect the hormonal balance in both children and adults. The aim of this publication was to compare the urinary levels of phthalate metabolites in selected populations of the Czech Republic (CZ), Slovakia (SK), and Hungary (HU) in relation to the sources of phthalate exposure identified by means of questionnaire (personal care products, floor and wall coverings, plastic toys, and some kinds of foods). METHODS: Data were obtained through the twin projects COPHES (COnsortium to Perform Human biomonitoring on a European Scale) and DEMOCOPHES (DEMOnstration of a study to COordinate and Perform Human biomonitoring on a European Scale) from 2009 to 2012. The target groups were children aged 6-11 years old and their mothers up to 45 years of age. The metabolites of phthalates (monomethyl phthalate (MMP), monoethyl phthalate (MEP), monobenzyl phthalate (MBzP), mono-cyclohexyl phthalate (MCHP), mono-(2-ethylhexyl) phthalate (MEHP), mono-(2-ethyl-5-hydroxyhexyl) phthalate (5OH-MEHP), and mono-(2-ethyl-5-oxohexyl) phthalate (5OXO-MEHP)) were analysed in first morning urine samples. After enzymatic glucuronide cleavage, the urine sample analyses were performed using ultra-high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) in one laboratory that qualified in the External Quality Assessment exercises organised by COPHES. RESULTS: Significant differences in phthalate exposure between countries were revealed for children only but not for mothers. The concentrations of 5-OH-MEHP (P<0.001), 5OXO-MEHP (P<0.001), and their sum (P<0.001) were the highest in SK compared to CZ and HU. The health based guidance values for the sum of DEHP metabolites 5-OH MEHP and 5OXO-MEHP established by the German Commission for biomonitoring of 300 µg/L and 500 µg/L for women adults and children, respectively, were only exceeded in one mother and three boys. A significant difference was also found for MEP (P=0.0149), with the highest concentrations detected in HU. In all countries, the increasing frequency of using personal care products significantly elevated the concentrations of MEP. CONCLUSION: Some differences were observed between countries in the concentrations of individual urinary phthalate metabolites in children. However, the questionnaire results give no direct explanation for the differences between the countries except the variation in using personal care products.

Synergism of the two Myb domains of Tay1 protein results in high affinity binding to telomeres.

Double-stranded regions of the telomeres are recognized by proteins containing Myb-like domains conferring specificity toward telomeric repeats. Although biochemical and structural studies revealed basic molecular principles involved in DNA binding, relatively little is known about evolutionary pathways leading to various types of Myb domain-containing proteins in divergent species of eukaryotes. Recently we identified a novel type of telomere-binding protein YlTay1p from the yeast Yarrowia lipolytica containing two Myb domains (Myb1, Myb2) very similar to the Myb domain of mammalian TRF1 and TRF2. In this study we prepared mutant versions of YlTay1p lacking Myb1, Myb2, or both Myb domains and found that YlTay1p carrying either Myb domain exhibits preferential affinity to both Y. lipolytica (GGGTTAGTCA)(n) and human (TTAGGG)(n) telomeric sequences. Quantitative measurements of the protein binding to telomeric DNA revealed that the presence of both Myb domains is required for a high affinity of YlTay1p to either telomeric repeat. Additionally, we performed detailed thermodynamic analysis of the YlTay1p interaction with its cognate telomeric DNA, which is to our knowledge the first energetic description of a full-length telomeric-protein binding to DNA. Interestingly, when compared with human TRF1 and TRF2 proteins, YlTay1p exhibited higher affinity not only for Y. lipolytica telomeres but also for human telomeric sequences. The duplication of the Myb domain region in YlTay1p thus produces a synergistic effect on its affinity toward the cognate telomeric sequence, alleviating the need for homodimerization observed in TRF-like proteins possessing a single Myb domain.

Functional analysis of the p.(Leu15Pro) and p.(Gly20Arg) sequence changes in the signal sequence of LDL receptor.

The low density lipoprotein receptor (LDLR) is a transmembrane protein that plays a key role in cholesterol metabolism. It contains 860 amino acids including a 21 amino acid long signal sequence, which directs the protein into the endoplasmic reticulum. Mutations in the LDLR gene lead to cholesterol accumulation in the plasma and results in familial hypercholesterolemia (FH). Knowledge of the impact of a mutation on the LDLR protein structure and function is very important for the diagnosis and management of FH. Unfortunately, for a large proportion of mutations this information is still missing. In this study, we focused on the LDLR signal sequence and carried out functional and in silico analyses of two sequence changes, p.(Gly20Arg) and p.(Leu15Pro), localized in this part of the LDLR. Our results revealed that the p.(Gly20Arg) change, previously described as disease causing, has no detrimental effect on protein expression or LDL particle binding. In silico analysis supports this observation, showing that both the wt and p.(Gly20Arg) signal sequences adopt an expected α-helix structure. In contrast, the mutation p.(Leu15Pro) is not associated with functional protein expression and exhibits a structure with disrupted a α-helical arrangement in the signal sequence, which most likely affects protein folding in the endoplasmic reticulum.

Solution for blank and matrix difficulties encountered during phthalate analysis of edible oils by high performance liquid chromatography coupled with tandem mass spectrometry.

Worldwide production of phthalates has led to their undesirable presence in the food chain. Particularly edible oils have become an area of growing concern owing to numerous reported occurrences of phthalates. The analytical methods used in this field face difficulties associated mainly with matrix complexity or phthalate contamination which this study has aimed to describe and resolve. The proposed procedure consisting of liquid-liquid extraction, solid phase extraction and high performance liquid chromatography coupled with tandem mass spectrometry allowed us to analyze simultaneously 6 individual phthalates and 2 phthalate isomeric mixtures. DSC-18 SPE phase was selected for cleanup owing to the most efficient co-extract removal (assessed using high resolution mass spectrometry). Several sources of phthalate contamination were identified, however, the mobile phase was the most serious. The key improvement was achieved by equipping a contamination trap, a 50-mm reverse phase HPLC column, generating a delay between target and mobile phase peaks of the same compounds. RSDs ranging between 2.4 and 16 % confirm good precision and LOQs between 5.5 and 110µgkg(-1) reflect satisfactory blank management. With up to 19 occurrences in 25 analyzed edible oil samples and levels up to 33mgkg(-1), bis(2-ethylhexyl), diisononyl and diisodecyl phthalates were the most important contaminants.

Phthalates exposure indicators determined by urinary phthalate metabolites in healthy non-obese Czech adults: FANTOM study.

It is assumed that human exposure to phthalates may be associated with adverse health effects. The indicators of urinary phthalate metabolite concentrations in healthy adults are limited. In this study, the phthalate metabolites concentrations were detected from 24-h urine collection in non-obese Czech adults (n = 201). Each participant filled in an 80-item questionnaire (FANTOM-SQ 2013) regarding the outdoor and indoor sources of phthalates, the use of personal care products and food intake sources. The concentrations of 15 phthalates metabolites were analysed following enzymatic cleavage of the glucuronide using ultra-high-performance liquid chromatography-electrospray ionisation tandem mass spectrometry (UHPLC-ESI-MS/MS). The indicators of chronic or acute exposure phthalate-containing materials were identified. It is shown that higher fruit consumption was positively and significantly associated with a higher level of total 15 urinary phthalates biomarkers (p < 0.001). Regular meat consumption showed a negative significant association with total 15 phthalates metabolites (p < 0.01). The use of personal care products was significantly and positively correlated with monoethyl phthalate urine concentrations (p < 0.05). The analysis of the dietary behaviour and personal care products use in the Czech non-obese population showed it to be a predictable tool in the level of phthalates exposure when high fruit consumption and personal care products use are linked to higher phthalate metabolite contents in the urine. However, this topic deserves more research.