Identification and characterization of a novel ubiquitous nucleolar protein ‘NARR’ encoded by a gene overlapping the rab34 oncogene

There are only few reports on protein products originating from overlapping mammalian genes even though computational predictions suggest that an appreciable fraction of mammalian genes could potentially overlap. Mass spectrometry-based proteomics has now acquired the tools to probe proteins in an unbiased manner, providing direct evidence of the output of the genomic and gene expression machinery. In particular, proteomics can refine gene predictions and discover novel gene-processing events and gene arrangements. Here, we report the mass spectrometric discovery and biochemical validation of the novel protein encoded by a gene overlapping rab34 oncogene. The novel protein is highly conserved in mammals. In humans, it contains 13 distinct Nine-Amino acid Residue-Repeats (NARR) with the consensus sequence PRVIV(S/T)PR in which the serine or threonine residues are phosphorylated during M-phase. NARR is ubiquitously expressed and resides in nucleoli where it colocalizes with ribosomal DNA (rDNA) gene clusters. Its distribution only partially overlaps with upstream binding factor, one of the main regulators of RNA Polymerase I activity, and is entirely uncoupled from it in mitotic cells and upon inhibition of transcription. NARR only partially colocalizes with fibrillarin, the pre-ribosomal RNA-processing protein, positioning NARR in a separate niche within the rDNA cluster

Classification of Inhibitors of Hepatic Organic Anion Transporting Polypeptides (OATPs): Influence of Protein Expression on Drug–Drug Interactions

ABSTRACT: The hepatic organic anion transporting poly-peptides (OATPs) influence the pharmacokinetics of several drug classes and are involved in many clinical drug−drug interactions. Predicting potential interactions with OATPs is, therefore, of value. Here, we developed in vitro and in silico models for identification and prediction of specific and general inhibitors of OATP1B1, OATP1B3, and OATP2B1. The maximal transport activity (MTA) of each OATP in human liver was predicted from transport kinetics and protein quantification. We then used MTA to predict the effects of a subset of inhibitors on atorvastatin uptake in vivo. Using a data set of 225 drug-like compounds, 91 OATP inhibitors were identified. In silico models indicated that lipophilicity and polar surface area are key molecular features of OATP inhibition. MTA predictions identified OATP1B1 and OATP1B3 as major determinants of atorvastatin uptake in vivo. The relative contributions to overall hepatic uptake varied with isoform specificities of the inhibitors

Examination of Late Palaeolithic archaeological sites in northern Europe for the preservation of cryptotephra layers

We report the first major study of cryptotephra (non-visible volcanic ash layers) on Late Palaeolithic archaeological sites in northern Europe. Examination of 34 sites dating from the Last Termination reveals seven with identifiable cryptotephra layers. Preservation is observed in minerogenic and organic deposits, although tephra is more common in organic sediments. Cryptotephra layers normally occur stratigraphically above or below the archaeology. Nearby off-site palaeoclimate archives (peat bogs and lakes <0.3 km distant) were better locations for detecting tephra. However in most cases the archaeology can only be correlated indirectly with such cryptotephras. Patterns affecting the presence/absence of cryptotephra include geographic position of sites relative to the emitting volcanic centre; the influence of past atmospherics on the quantity, direction and patterns of cryptotephra transport; the nature and timing of local site sedimentation; sampling considerations and subsequent taphonomic processes. Overall, while tephrostratigraphy has the potential to improve significantly the chronology of such sites many limiting factors currently impacts the successful application

Multiple-Enzyme-Digestion Strategy Improves Accuracy and Sensitivity of Label- and Standard-Free Absolute Quantification to a Level That Is Achievable by Analysis with Stable Isotope-Labeled Standard Spiking

Quantification of individual proteins is an essential task in understanding biological processes. For example, determination of concentrations of proteins transporting and metabolizing xenobiotics is a prerequisite for drug disposition predictions in humans based on in vitro data. So far, this task has frequently been accomplished by targeted proteomics. This type of analyses requires preparation of stable isotope labeled standards for each protein of interest. The selection of appropriate standard peptides is usually tedious and the number of proteins that can be studied in a single experiment by these approaches is limited. In addition, incomplete digestion of proteins often affects the accuracy of the quantification. To circumvent these constrains in proteomic protein quantification, label- and standard-free approaches, such as "total protein approach" (TPA) have been proposed. Here we directly compare an approach using stable isotope labeled (SIL) standards and TPA for quantification of transporters and enzymes in human liver samples within the same LC-MS/MS runs. We show that TPA is a convenient alternative to SIL-based methods. Optimization of the sample preparation beyond commonly used single tryptic digestion, by adding consecutive cleavage steps, improves accuracy and reproducibility of the TPA method to a level, which is achievable by analysis using stable isotope-labeled standard spiking

SuperSILAC mix for quantitative proteomics of human tumor tissue

SILAC-labeled cell lines as internal standard for comprehensive human tumor tissue proteome quantification. Initially, we SILAC-labeled the breast cancer cell line HCC1599 and mixed the lysate with the lysate of mammary carcinoma tissue from an individual with grade II lobular carcinoma. We digested the resulting protein mixture according to the filter-aided sample preparation protocol 10 and separated peptides into six fractions by anion exchange chromatography in a StageTip format 11 . We analyzed each fraction by online reverse-phase chromatography coupled to high-resolution, quantitative mass spectrometric analysis using the LTQ Orbitrap Adding the labeled mixture, rather than the single cell line, to the tumor achieved the same number of quantified proteins in triplicate analysis but drastically improved quantification accuracy We also shortened the analysis time from one day per replicate to four hours per replicate by measuring the digest without prior separation on the new generation LTQ-Orbitrap Velos instrument. Despite the lower We describe a method to accurately quantify human tumor proteomes by combining a mixture of five stable-isotope labeling by amino acids in cell culture (siLac)-labeled cell lines with human carcinoma tissue. this generated hundreds of thousands of isotopically labeled peptides in appropriate amounts to serve as internal standards for mass spectrometry-based analysis. by decoupling the labeling from the measurement, this super-siLac method broadens the scope of siLac-based proteomics. In recent years, mass spectrometry has made great technological progress and is increasingly broadly applied in cell culture-based studies 1,2 . Nevertheless, accurate quantification of human tissue proteomes by high-resolution mass spectrometry methods is still in its infancy 3,4 . In the stable-isotope labeling by amino acids in cell culture (SILAC) method, cell lines are labeled through the incorporation of stable 'heavy' versions of essential amino acids in the cell populations to be compared A few studies have expanded the use of SILAC to tissue analysis. The Neuro2A cell line has been metabolically labeled and compared to total mouse brain 7 . A total of 602 proteins had been quantified, albeit with up to tenfold quantitative ratios between cell line and tissue. Such high ratios between sample and internal standard make accurate quantification difficult because the signal of the lowerabundance peptide in the SILAC pair may be close to the noise level. We have recently quantified proteins from primary mouse hepatocytes using SILAC-labeled Hepa1-6 cells 8 . Furthermore, entire mice can be SILAC-labeled, but this technology is not applicable to human subjects 9 . When applying proteomics to tumor biology, it is imperative to quantify a representative number of proteins, to obtain reproducible results and to study cancer-relevant proteins of low abundance. Therefore, we explored the use of a mix of multipl

Plasma fibrin clot proteomics in healthy subjects : relation to clot permeability and lysis time

Background: Little is known about fibrin clot composition in relation to its structure and lysability. We investigated plasma clots protein composition and its associations with clot properties. Methods: We studied 20 healthy subjects aged 31-49 years in whom plasma fibrin clot permeability (K-s) and clot lysis time (CLT) were determined. A proteomic analysis of plasma fibrin clots was based on quantitative liquid chromatography-mass spectrometry. Results: Among 494 clot-bound proteins identified in all clots, the highest concentrations were for fibrinogen chains (about 64% of the clot mass) and fibronectin (13%). alpha(2)-antiplasmin (2.7%), factor XIIIA (1.2%), complement component C3 (1.2%), and histidine-rich glycoprotein (HRG, 0.61%) were present at relatively high concentrations. Proteins present in concentrations < 0.5% included (pro)thrombin, plasminogen, apolipoproteins, or platelet factor 4 (PF4). Fibrinogen-alpha and -gamma chains were associated with age, while body-mass index with clot-bound apolipoproteins (all p <.05). K-s correlated with fibrinogen-gamma and PF4 amounts within plasma clots. CLT was associated with fibrinogen-alpha and -gamma, PF4, and HRG (all p <.05). Conclusions: This study is the first to show associations of two key measures of clot properties with protein content within plasma clots, suggesting that looser fibrin clots with enhanced lysability contain less fibrinogen-gamma chain, platelet-derived PF4, and HRG. Significance: Our study for the first time suggests that more permeable fibrin clots with enhanced lysability contain less fibrinogen-gamma chain, platelet-derived factor 4, and histidine-rich glycoprotein, which is related to accelerated clot lysis. The current findings might have functional consequences regarding clot structure, stability, and propagation of thrombin generation, and detailed proteomic analysis of clots in various disorders opens new perspective for coagulation and fibrin research

Intracellular Drug Bioavailability : Effect of Neutral Lipids and Phospholipids

Intracellular unbound drug concentrations are the pharmacologically relevant concentrations for targets inside cells. Intracellular drug concentrations are determined by multiple processes, including the extent of drug binding to intracellular structures. The aim of this study was to evaluate the effect of neutral lipid (NL) and phospholipid (PL) levels on intracellular drug disposition. The NL and/or PL content of 3T3-L1 cells were enhanced, resulting in phenotypes (in terms of morphology and proteome) reminiscent of adipocytes (high NL and PL) or mild phospholipidosis (only high PL). Intracellular bioavailability (F-ic) was then determined for 23 drugs in these cellular models and in untreated wild-type cells. A higher PL content led to higher intracellular drug binding and a lower F-ic. The induction of NL did not further increase drug binding but led to altered F-ic due to increased lysosomal pH. Further, there was a good correlation between binding to beads coated with pure PL and intracellular drug binding. In conclusion, our results suggest that PL content is a major determinant of drug binding in cells and that PL beads may constitute a simple alternative to estimating this parameter. Further, the presence of massive amounts of intracellular NLs did not influence drug binding significantly