DNA binding of the p21 repressor ZBTB2 is inhibited by cytosine hydroxymethylation

Recent studies have demonstrated that the modified base 5-hydroxymethylcytosine (5-hmC) is detectable at various rates in DNA extracted from human tissues. This oxidative product of 5-methylcytosine (5-mC) constitutes a new and important actor of epigenetic mechanisms. We designed a DNA pull down assay to trap and identify nuclear proteins bound to 5-hmC and/or 5-mC. We applied this strategy to three cancerous cell lines (HeLa, SH-SY5Y and UT7-MPL) in which we also measured 5-mC and 5-hmC levels by HPLC-MS/MS. We found that the putative oncoprotein Zinc finger and BTB domain-containing protein 2 (ZBTB2) is associated with methylated DNA sequences and that this interaction is inhibited by the presence of 5-hmC replacing 5-mC. As published data mention ZBTB2 recognition of p21 regulating sequences, we verified that this sequence specific binding was also alleviated by 5-hmC. ZBTB2 being considered as a multifunctional cell proliferation activator, notably through p21 repression, this work points out new epigenetic processes potentially involved in carcinogenesis.     

Total ApoE and ApoE4 Isoform Assays in an Alzheimer's Disease Case-control Study by Targeted Mass Spectrometry (n = 669): A Pilot Assay for Methionine-containing Proteotypic Peptides

Allelic polymorphism of the apolipoprotein E (ApoE) gene (ApoE ε2, ApoE ε3 and ApoE ε4 alleles) gives rise to three protein isoforms (ApoE2, ApoE3 and ApoE4) that differ by 1 or 2 amino acids. Inheritance of the ApoE ε4 allele is a risk factor for developing Alzheimer''s disease (AD). The potential diagnostic value of ApoE protein levels in biological fluids (i.e. cerebrospinal fluid, plasma and serum) for distinguishing between AD patients and healthy elderly subjects is subject to great controversy. Although a recent study reported subnormal total ApoE and ApoE4 levels in the plasma of AD patients, other studies have found normal or even elevated protein levels (versus controls). Because all previously reported assays were based on immunoenzymatic techniques, we decided to develop an orthogonal assay based on targeted mass spectrometry by tracking (i) a proteotypic peptide common to all ApoE isoforms and (ii) a peptide that is specific for the ε4 allele. After trypsin digestion, the ApoE4-specific peptide contains an oxidation-prone methionine residue. The endogenous methionine oxidation level was evaluated in a small cohort (n = 68) of heterozygous ε3ε4 carriers containing both healthy controls and AD patients. As expected, the proportion of oxidized residues varied from 0 to 10%, with an average of 5%. We therefore developed a standardized strategy for the unbiased, absolute quantification of ApoE4, based on performic acid oxidization of methionine. Once the sample workflow had been thoroughly validated, it was applied to the concomitant quantification of total ApoE and ApoE4 isoform in a large case-control study (n = 669). The final measurements were consistent with most previously reported ApoE concentration values and confirm the influence of the different alleles on the protein expression level. Our results illustrate (i) the reliability of selected reaction monitoring-based assays and (ii) the value of the oxidization step for unbiased monitoring of methionine-containing proteotypic peptides. Furthermore, a statistical analysis indicated that neither total ApoE and ApoE4 levels nor the ApoE/ApoE4 ratio correlated with the diagnosis of AD. These findings reinforce the conclusions of previous studies in which plasma ApoE levels had no obvious clinical significance.Apolipoprotein E (ApoE) is a 299-amino acid protein associated with lipoproteins in the plasma and the cerebrospinal fluid (1). The three major genetic variants of ApoE in the general population isoforms (E2, E3, and E4, encoded by the ε2, ε3 and ε4 alleles, respectively) differ by a single amino acid: E2 (cys112, cys158), E3 (cys112, arg158), and E4 (arg112, arg158). Among the different susceptibility genes associated with the risk of late-onset Alzheimer''s disease (AD)¹ and other neurological conditions, ApoE has been identified as a strong genetic determinant (1). Moreover, the risk of AD is strongly correlated with ApoE4 allele because the presence of one or two copies of the allele increases risk of late-onset AD by about three or 12 times, respectively. Furthermore, the presence of one or two copies of ApoE4 allele correlates with an earlier age of onset by about 10–20 years with regard to noncarriers in patients with late-onset disease (2–4).The diagnostic value of an ApoE assay in AD is however subject to debate. Indeed, supranormal (5), subnormal (6), and nondiscriminating (7–10) plasma concentrations of ApoE in AD cohorts have been reported in many studies based on immunoassay techniques. We wondered whether this ambiguity might be resolved by applying quantitative mass spectrometry (MS). Indeed, the combination of standardized, stable isotope dilution with targeted MS in selected reaction monitoring (SRM) mode is now widely accepted as a valuable alternative to immunoassays for accurate protein quantification (11–17) and may constitute the eagerly awaited bridge between the discovery and verification phases for candidate biomarker panels (18–22). A range of pilot studies have evaluated the sensitivity that can be achieved by SRM monitoring (23, 24); when tracking protein in a whole plasma hydrolysate, limits of quantification (LOQs) in the low μg/ml range are frequently reported (12). However, LOQs in the low nanogram/ml range or the detection of proteins expressed with a low copy number per cell require either immuno-enrichment of the target protein or fractionation of proteotypic peptides by cation exchange (24, 25) or off-gel electrophoresis (26). A generic strategy based on stable isotope standards and capture by antipeptide antibodies (referred to as SISCAPA) is a promising alternative to more traditional enrichment strategies. Numerous reports have already demonstrated the ability of single or multiplexed immuno-SRM to achieve limits of quantification of 1 nanogram/ml or below when starting from less than 100 μl of biofluid (27). Precision, reproducibility and robustness are also key parameters in the development of SRM-based assay platforms dedicated to the clinical verification phase. Indeed, accuracy may even be a secondary consideration at this point, because the main objective is to discriminate between specific and nondiscriminating biomarker candidates within the clinical panel. Various pilot studies of the intra- and interclass performances of SRM monitoring have demonstrated acceptable coefficients of variation and levels of imprecision (below 20%) and, importantly, have pinpointed the main pitfalls that can distort assay results (28–33). Indeed, performance levels and reliability of an assay can be drastically compromised by intraclass variance within the proteotypic peptide fraction, which is caused by chemical modifications of certain amino acid side chains. In particular, peptides containing methionine residues are usually not recommended for SRM-based assays, because the sulfur atom is prone to oxidation (34–36) both in vivo (37, 38) and during sample handling or storage (giving rise to methionine sulfoxide). Unfortunately, peptides containing one or more methionine residues are sometimes the only ones available. By way of an example, 17% of the 625 proteins identified by only one peptide in Human Plasma PeptideAtlas database contain at least one methionine. Hence, quantification of this type of proteotypic peptide is challenging because the ratio between nonoxidized and mono-oxidized sulfoxide forms of heavy-labeled standards also changes during storage. To circumvent this limitation, complete oxidization of the methionine-containing peptide population with chemical oxidant has been attempted but was not sufficiently robust or reproducible over several weeks in the context of large clinical study (39, 40).Here, we report on the absolute quantification of total ApoE and ApoE4 isoform peptides by liquid chromatography (LC)-SRM targeted MS in a case-control study (n = 669). Because ApoE4-specific trypsin peptide possesses a single methionine residue (LGADMEDVR), oxidation was performed immediately after enzymatic digestion. The completeness of was carefully checked. We continuously assessed the overall performance of the assay by introducing quality control samples throughout the clinical cohort study, in order to ensure the validity of statistical comparisons between healthy controls and groups of AD patients.     

A genome-wide collection of Mos1 transposon insertion mutants for the C. elegans research community

Methods that use homologous recombination to engineer the genome of C. elegans commonly use strains carrying specific insertions of the heterologous transposon Mos1. A large collection of known Mos1 insertion alleles would therefore be of general interest to the C. elegans research community. We describe here the optimization of a semi-automated methodology for the construction of a substantial collection of Mos1 insertion mutant strains. At peak production, more than 5,000 strains were generated per month. These strains were then subject to molecular analysis, and more than 13,300 Mos1 insertions characterized. In addition to targeting directly more than 4,700 genes, these alleles represent the potential starting point for the engineered deletion of essentially all C. elegans genes and the modification of more than 40% of them. This collection of mutants, generated under the auspices of the European NEMAGENETAG consortium, is publicly available and represents an important research resource.     

Improved characterization of the insulin secretory granule proteomes

Insulin secretory granules (ISGs) are pivotal organelles of pancreatic ß-cells and represent a key participant to glucose homeostasis. Indeed, insulin is packed and processed within these vesicles before its release by exocytosis. It is therefore crucial to acquire qualitative and quantitative data on the ISG proteome, in order to increase our knowledge on ISG biogenesis, maturation and exocytosis. Despites efforts made in the past years, the coverage of the ISG proteome is still incomplete and comprises many potential protein contaminants most likely coming from suboptimal sample preparations. We developed here a 3-step gradient purification procedure combined to Stable Isotope Labeling with Amino acids in Cell culture (SILAC) to further characterize the ISG protein content. Our results allowed to build three complementary proteomes containing 1/ proteins which are enriched in mature ISGs, 2/ proteins sharing multiple localizations including ISGs, and finally 3/ proteins sorted out from immature ISGs and/or co-purifying contaminants. As a proof of concept, the ProSAAS, a neuronal protein found in ISGs was further characterized and its granular localization proved. ProSAAS might represent a novel potential target allowing to better understand the defaults in insulin processing and secretion observed during type 2 diabetes progression. This article is part of a special issue entitled: Translational Proteomics.     

The hydrophobic segment of Arabidopsis thaliana cluster I diacylglycerol kinases is sufficient to target the proteins to cell membranes

Diacylglycerol kinases (DGKs) catalyze the phosphorylation of diacylglycerol into phosphatidic acid. To fulfill their role in many signalling processes, DGKs must be located at, or in, membranes. Most mammalian DGKs are cytosolic and are recruited to membranes upon stimulation, except for epsilon type DGKs that are permanently membrane-associated through a hydrophobic segment. Nothing is known about the mechanism(s) involved in the membrane localization of plant DGKs. By fusion to fluorescent proteins, we show that two DGKs from cluster I in Arabidopsis thaliana possess amino-terminal hydrophobic segments that are sufficient to address them to endoplasmic reticulum membranes.     

The Metalloprotease Meprinβ Processes E-Cadherin and Weakens Intercellular Adhesion

Background

Meprin (EC 3.4.24.18), an astacin-like metalloprotease, is expressed in the epithelium of the intestine and kidney tubules and has been related to cancer, but the mechanistic links are unknown.

Methodology/Principal Findings

We used MDCK and Caco-2 cells stably transfected with meprinα and or meprinβ to establish models of renal and intestinal epithelial cells expressing this protease at physiological levels. In both models E-cadherin was cleaved, producing a cell-associated 97-kDa E-cadherin fragment, which was enhanced upon activation of the meprin zymogen and reduced in the presence of a meprin inhibitor. The cleavage site was localized in the extracellular domain adjacent to the plasma membrane. In vitro assays with purified components showed that the 97-kDa fragment was specifically generated by meprinβ, but not by ADAM-10 or MMP-7. Concomitantly with E-cadherin cleavage and degradation of the E-cadherin cytoplasmic tail, the plaque proteins β-catenin and plakoglobin were processed by an intracellular protease, whereas α-catenin, which does not bind directly to E-cadherin, remained intact. Using confocal microscopy, we observed a partial colocalization of meprinβ and E-cadherin at lateral membranes of incompletely polarized cells at preconfluent or early confluent stages. Meprinβ-expressing cells displayed a reduced strength of cell-cell contacts and a significantly lower tendency to form multicellular aggregates.

Conclusions/Significance

By identifying E-cadherin as a substrate for meprinβ in a cellular context, this study reveals a novel biological role of this protease in epithelial cells. Our results suggest a crucial role for meprinβ in the control of adhesiveness via cleavage of E-cadherin with potential implications in a wide range of biological processes including epithelial barrier function and cancer progression.