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Colorectal cancer (CRC) is a common and highly lethal form of cancer. Although the etiologic role of Fusobacterium nucleatum (F. nucleatum) in the development of CRC has been elucidated, the specific tumor molecules involved in the progression of CRC induced by F. nucleatum have not been identified. This study investigated several miRNAs and genes involved in the progression of F. nucleatum-induced CRC by Affymetrix miRNA microarray technology and GeneChip Human Transcriptome Array 2.0. The results suggest that miR-4474 and miR-4717 are up-regulated in CRC tissues in response to F. nucleatum infection, compared with the control group (paracancerous tissues), while other genes associated with signaling pathways in cancer, including CREB-binding protein (CREBBP), STAT1, PRKACB, CAMK2B, JUN, TP53 and EWSR1, were dysregulated. Bioinformatic analysis identified CREBBP as the primary aberrantly expressed gene in F. nucleatum-induced CRC. Consistent with the microarray analysis results, real-time RT-PCR analysis demonstrated that the expression of miR-4474/4717 was upregulated while that of CREBBP mRNA was downregulated in CRC patients infected with F. nucleatum. Additionally, CREBBP was identified as a novel target of miR-4474/4717. The results of this study suggest that miR-4474 and miR-4717 are involved in the progression of F. nucleatum-induced CRC by posttranscriptionally regulating the target gene CREBBP. [ABSTRACT FROM AUTHOR]

While circulating autoantibodies have been detected in patients with several cardiovascular diseases, such studies have not been performed for chronic thromboembolic pulmonary hypertension (CTEPH) and pulmonary arterial hypertension (PAH). Here we investigated the production of certain auto-antibodies in CTEPH patients. Initial screening was performed in 5 CTEPH patients and 5 healthy donors (HDs) using a ProtoArray Human Protein Microarray v5.1 containing 9,375 human proteins, and we selected 34 antigens recognized by IgG antibodies more strongly in the sera of CTEPH patients than in the sera of HDs. In subsequent second/third analyses, we validated the auto-antibody level using amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) in 96 CTEPH patients and 96 HDs as follows: At the second screening, we used 63 crude peptides derived from those selected 34 antigens and found that the serum levels of autoantibodies for 4 peptides seemed higher in CTEPH patients than in HDs. In third analysis, we used the purified peptides of those selected in second screening and found that serum antibodies against peptides derived from exonuclease 3'-5' domain-containing 2 (EXD2) and phosphorylated adaptor for RNA export (PHAX) were significantly higher in CTEPH patients than in HDs. The serum antibody levels to these antigens were also elevated in PAH patients. The titers against EXD2 peptide decreased after surgical treatment in CTEPH patients. These autoantibodies may be useful as biomarkers of CTEPH and PAH, and further investigations may provide novel insight into the etiology. [ABSTRACT FROM AUTHOR]

Objective: Peptidylarginine deiminase 2 (PAD2) and PAD4 are expressed in the synovium of rheumatoid arthritis (RA) patients and catalyze citrullination of arginine residues in proteins targeted by anti-citrullinated protein antibodies (ACPAs). Little is known about the relative importance of PAD2 and PAD4 in generating citrullinated self-antigens. Here we investigate the ability of PAD2 and PAD4 to generate citrullinated targets for ACPAs in four human proteins. Methods: Synovial fluid (SF) and plasma were collected from 42 RA patients. Human fibrinogen, human alpha-enolase (ENO1), human histone H3, and human serum albumin (HSA) were citrullinated in vitro by PAD2 or PAD4. The total degree of citrullination was determined using the anti-modified citrulline approach. Antibody binding to native and citrullinated proteins was measured by ELISA. Results: ACPAs within pooled SF from multiple RA patients reacted equally well with, and cross-reacted with, PAD2- and PAD4-citrullinated fibrinogen. ACPAs from most individual patient SF and plasma samples bound equally well to PAD2- and PAD4-citrullinated fibrinogen or ENO1. When histone H3 was used as target, PAD4 was generally superior in generating epitopes recognized by ACPAs. No binding to citrullinated HSA was observed. Conclusion: In most patients, PAD2 and PAD4 are equally efficient in generating citrullinated target sites for ACPAs in fibrinogen and ENO1. The binding of autoantibodies to histone H3 was generally higher after citrullination with PAD4 than with PAD2. Citrullinated HSA is not a target for ACPAs. [ABSTRACT FROM AUTHOR]

Intrinsically disordered regions (IDRs) of proteins play significant biological functional roles despite lacking a well-defined 3D structure. For example, IDRs provide efficient housing for large numbers of post-translational modification (PTM) sites in eukaryotic proteins. Here, we study the distribution of more than 15,000 experimentally determined human methylation, acetylation and ubiquitination sites (collectively termed ‘MAU’ sites) in ordered and disordered regions, and analyse their conservation across 380 eukaryotic species. Conservation signals for the maintenance and novel emergence of MAU sites are examined at 11 evolutionary levels from the whole eukaryotic domain down to the ape superfamily, in both ordered and disordered regions. We discover that MAU PTM is a major driver of conservation for arginines and lysines in both ordered and disordered regions, across the 11 levels, most significantly across the mammalian clade. Conservation of human methylatable arginines is very strongly favoured for ordered regions rather than for disordered, whereas methylatable lysines are conserved in either set of regions, and conservation of acetylatable and ubiquitinatable lysines is favoured in disordered over ordered. Notably, we find evidence for the emergence of new lysine MAU sites in disordered regions of proteins in deuterostomes and mammals, and in ordered regions after the dawn of eutherians. For histones specifically, MAU sites demonstrate an idiosyncratic significant conservation pattern that is evident since the last common ancestor of mammals. Similarly, folding-on-binding (FB) regions are highly enriched for MAU sites relative to either ordered or disordered regions, with ubiquitination sites in FBs being highly conserved at all evolutionary levels back as far as mammals. This investigation clearly demonstrates the complex patterns of PTM evolution across the human proteome and that it is necessary to consider conservation of sequence features at multiple evolutionary levels in order not to get an incomplete or misleading picture. [ABSTRACT FROM AUTHOR]

Protein L-isoaspartyl methyltransferase (PIMT/PCMT1), a product of the pcmt1 gene, catalyzes repair of abnormal L-isoaspartyl linkages in age-damaged proteins. Pcmt1 knockout mice exhibit a profound neuropathology and die 30–60 days postnatal from an epileptic seizure. Here we characterize four new SNP variants of human PIMT with respect to enzymatic activity, thermal stability, and propensity to aggregation. Under standard assay conditions, L191S, A150V, P174H and A65V showed activity losses of 72%, 64%, 61%, and 11% respectively. By differential scanning fluorimetry, melting temperature deviations were -5.2, -4.5, +0.5, and -3.4°C. SDS-PAGE of purified protein reveal significant aggregation of L191S, A150V, and P174H, but not A65V. We also report new data on three unusual PIMT variants among the 13 recently characterized by our laboratory. A7P and I58V were previously found to have 1.8–2.0 times the activity of WT PIMT in the standard assay; however, upon kinetic analysis, we find both variants exhibit reduced catalytic efficiency (Vmax/Km) due to weak isoaspartyl substrate binding. The near complete loss of activity (<1%) seen in R36C was investigated by comparing activity of two artificial variants. R36K shows 4.6X the activity of R36C, while R36A shows no improvement, suggesting the guanidino nitrogens of the R36 play a key role in binding the methyl donor S-adenosyl-L-methionine (AdoMet). The new findings reported here extend the list of human PIMT variants that may contribute to neurological diseases in the young and the decline of CNS function in the aged. [ABSTRACT FROM AUTHOR]

Deciphering a function of a given protein requires investigating various biological aspects. Usually, the protein of interest is expressed with a fusion tag that aids or allows subsequent analyses. Additionally, downregulation or inactivation of the studied gene enables functional studies. Development of the CRISPR/Cas9 methodology opened many possibilities but in many cases it is restricted to non-essential genes. Recombinase-dependent gene integration methods, like the Flp-In system, are very good alternatives. The system is widely used in different research areas, which calls for the existence of compatible vectors and efficient protocols that ensure straightforward DNA cloning and generation of stable cell lines. We have created and validated a robust series of 52 vectors for streamlined generation of stable mammalian cell lines using the FLP recombinase-based methodology. Using the sequence-independent DNA cloning method all constructs for a given coding-sequence can be made with just three universal PCR primers. Our collection allows tetracycline-inducible expression of proteins with various tags suitable for protein localization, FRET, bimolecular fluorescence complementation (BiFC), protein dynamics studies (FRAP), co-immunoprecipitation, the RNA tethering assay and cell sorting. Some of the vectors contain a bidirectional promoter for concomitant expression of miRNA and mRNA, so that a gene can be silenced and its product replaced by a mutated miRNA-insensitive version. Our toolkit and protocols have allowed us to create more than 500 constructs with ease. We demonstrate the efficacy of our vectors by creating stable cell lines with various tagged proteins (numatrin, fibrillarin, coilin, centrin, THOC5, PCNA). We have analysed transgene expression over time to provide a guideline for future experiments and compared the effectiveness of commonly used inducers for tetracycline-responsive promoters. As proof of concept we examined the role of the exoribonuclease XRN2 in transcription termination by RNAseq. [ABSTRACT FROM AUTHOR]

Autosomal recessive nonsyndromic hearing loss is a genetically heterogeneous disorder. Here, we report a severe-to-profound sensorineural hearing loss locus, DFNB100 on chromosome 5q13.2-q23.2. Exome enrichment followed by massive parallel sequencing revealed a c.2510G>A transition variant in PPIP5K2 that segregated with DFNB100-associated hearing loss in two large apparently unrelated Pakistani families. PPIP5Ks enzymes interconvert 5-IP7 and IP8, two key members of the inositol pyrophosphate (PP-IP) cell-signaling family. Their actions at the interface of cell signaling and bioenergetic homeostasis can impact many biological processes. The c.2510G>A transition variant is predicted to substitute a highly invariant arginine residue with histidine (p.Arg837His) in the phosphatase domain of PPIP5K2. Biochemical studies revealed that the p.Arg837His variant reduces the phosphatase activity of PPIP5K2 and elevates its kinase activity. We found that in mouse inner ear, PPIP5K2 is expressed in the cochlear and vestibular sensory hair cells, supporting cells and spiral ganglion neurons. Mice homozygous for a targeted deletion of the Ppip5k2 phosphatase domain exhibit degeneration of cochlear outer hair cells and elevated hearing thresholds. Our demonstration that PPIP5K2 has a role in hearing in humans indicates that PP-IP signaling is important to hair cell maintenance and function within inner ear. [ABSTRACT FROM AUTHOR]

Skeletal myogenesis is a regulated process in which mononucleated cells, the myoblasts, undergo proliferation and differentiation. Upon differentiation, the cells align with each other, and subsequently fuse to form terminally differentiated multinucleated myotubes. Previous reports have identified the protein osteoglycin (Ogn) as an important component of the skeletal muscle secretome, which is expressed differentially during muscle development. However, the posttranscriptional regulation of Ogn by microRNAs during myogenesis is unknown. Bioinformatic analysis showed that miR-155 potentially targeted the Ogn transcript at the 3´-untranslated region (3´ UTR). In this study, we tested the hypothesis that miR-155 inhibits the expression of the Ogn to regulate skeletal myogenesis. C2C12 myoblast cells were cultured and miR-155 overexpression or Ogn knockdown was induced by transfection with miR-155 mimic, siRNA-Ogn, and negative controls with lipofectamine for 15 hours. Near confluence (80–90%), myoblasts were induced to differentiate myotubes in a differentiation medium. Luciferase assay was used to confirm the interaction between miR-155 and Ogn 3’UTR. RT-qPCR and Western blot analyses were used to confirm that the differential expression of miR-155 correlates with the differential expression of myogenic molecular markers (Myh2, MyoD, and MyoG) and inhibits Ogn protein and gene expression in myoblasts and myotubes. Myoblast migration and proliferation were assessed using Wound Healing and MTT assays. Our results show that miR-155 interacts with the 3’UTR Ogn region and decrease the levels of Ogn in myotubes. The overexpression of miR-155 increased MyoG expression, decreased myoblasts wound closure rate, and decreased Myh2 expression in myotubes. Moreover, Ogn knockdown reduced the expression levels of MyoD, MyoG, and Myh2 in myotubes. These results reveal a novel pathway in which miR-155 inhibits Ogn expression to regulate proliferation and differentiation of C2C12 myoblast cells. [ABSTRACT FROM AUTHOR]

Many proteins work together with others in groups called complexes in order to achieve a specific function. Discovering protein complexes is important for understanding biological processes and predict protein functions in living organisms. Large-scale and throughput techniques have made possible to compile protein-protein interaction networks (PPI networks), which have been used in several computational approaches for detecting protein complexes. Those predictions might guide future biologic experimental research. Some approaches are topology-based, where highly connected proteins are predicted to be complexes; some propose different clustering algorithms using partitioning, overlaps among clusters for networks modeled with unweighted or weighted graphs; and others use density of clusters and information based on protein functionality. However, some schemes still require much processing time or the quality of their results can be improved. Furthermore, most of the results obtained with computational tools are not accompanied by an analysis of false positives. We propose an effective and efficient mining algorithm for discovering highly connected subgraphs, which is our base for defining protein complexes. Our representation is based on transforming the PPI network into a directed acyclic graph that reduces the number of represented edges and the search space for discovering subgraphs. Our approach considers weighted and unweighted PPI networks. We compare our best alternative using PPI networks from Saccharomyces cerevisiae (yeast) and Homo sapiens (human) with state-of-the-art approaches in terms of clustering, biological metrics and execution times, as well as three gold standards for yeast and two for human. Furthermore, we analyze false positive predicted complexes searching the PDBe (Protein Data Bank in Europe) database in order to identify matching protein complexes that have been purified and structurally characterized. Our analysis shows that more than 50 yeast protein complexes and more than 300 human protein complexes found to be false positives according to our prediction method, i.e., not described in the gold standard complex databases, in fact contain protein complexes that have been characterized structurally and documented in PDBe. We also found that some of these protein complexes have recently been classified as part of a Periodic Table of Protein Complexes. The latest version of our software is publicly available at . [ABSTRACT FROM AUTHOR]

The balance of protein synthesis and proteolysis (i.e. proteostasis) is maintained by a complex regulatory network in which mTOR (mechanistic target of rapamycin serine/threonine kinase) pathway and unfolded protein response are prominent positive and negative actors. The interplay between the two systems has been revealed; however the mechanistic details of this crosstalk are largely unknown. The aim of the present study was to investigate the elements of crosstalk during endoplasmic reticulum stress and to verify the key role of GADD34 in the connection with the mTOR pathway. Here, we demonstrate that a transient activation of autophagy is present in endoplasmic reticulum stress provoked by thapsigargin or tunicamycin, which is turned into apoptotic cell death. The transient phase can be characterized by the elevation of the autophagic marker LC3II/I, by mTOR inactivation, AMP-activated protein kinase activation and increased GADD34 level. The switch from autophagy to apoptosis is accompanied with the appearance of apoptotic markers, mTOR reactivation, AMP-activated protein kinase inactivation and a decrease in GADD34. Inhibition of autophagy by 3-methyladenine shortens the transient phase, while inhibition of mTOR by rapamycin or resveratrol prolongs it. Inhibition of GADD34 by guanabenz or transfection of the cells with siGADD34 results in down-regulation of autophagy-dependent survival and a quick activation of mTOR, followed by apoptotic cell death. The negative effect of GADD34 inhibition is diminished when guanabenz or siGADD34 treatment is combined with rapamycin or resveratrol addition. These data confirm that GADD34 constitutes a mechanistic link between endoplasmic reticulum stress and mTOR inactivation, therefore promotes cell survival during endoplasmic reticulum stress. [ABSTRACT FROM AUTHOR]

Most human somatic cells express insufficient levels of telomerase, which can result in telomere shortening and eventually senescence, both of which are hallmarks of ageing. Homology-directed repair (HDR) is important for maintaining proper telomere function in yeast and mammals. In Saccharomyces cerevisiae, Rad52 is required for almost all HDR mechanisms, and telomerase-null cells senesce faster in the absence of Rad52. However, its role in preventing accelerated senescence has been unclear. In this study, we make use of rad52 separation-of-function mutants to find that multiple Rad52-mediated HDR mechanisms are required to delay senescence, including break-induced replication and sister chromatid recombination. In addition, we show that misregulation of histone 3 lysine 56 acetylation, which is known to be defective in sister chromatid recombination, also causes accelerated senescence. We propose a model where Rad52 is needed to repair telomere attrition-induced replication stress. [ABSTRACT FROM AUTHOR]

We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols. [ABSTRACT FROM AUTHOR]

Viral invasion into a host is initially recognized by the innate immune system, mainly through activation of the intracellular cytosolic signaling pathway and coordinated activation of interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-κB) transcription factors that promote type I interferon gene induction. The TANK-binding Kinase 1 (TBK1) phosphorylates and activates IRF3. Here, we show that Optineurin (Optn) dampens the antiviral innate immune response by targeting the deubiquitinating enzyme CYLD to TBK1 in order to inhibit its enzymatic activity. Importantly, we found that this regulatory mechanism is abolished at the G2/M phase as a consequence of the nuclear translocation of CYLD and Optn. As a result, we observed, at this cell division stage, an increased activity and phosphorylation of TBK1 that lead to its relocalization to mitochondria and to enhanced interferon production, suggesting that this process, which relies on Optn function, might be of major importance to mount a preventive antiviral response during mitosis. [ABSTRACT FROM AUTHOR]

The observation that disease associated proteins often interact with each other has fueled the development of network-based approaches to elucidate the molecular mechanisms of human disease. Such approaches build on the assumption that protein interaction networks can be viewed as maps in which diseases can be identified with localized perturbation within a certain neighborhood. The identification of these neighborhoods, or disease modules, is therefore a prerequisite of a detailed investigation of a particular pathophenotype. While numerous heuristic methods exist that successfully pinpoint disease associated modules, the basic underlying connectivity patterns remain largely unexplored. In this work we aim to fill this gap by analyzing the network properties of a comprehensive corpus of 70 complex diseases. We find that disease associated proteins do not reside within locally dense communities and instead identify connectivity significance as the most predictive quantity. This quantity inspires the design of a novel Disease Module Detection (DIAMOnD) algorithm to identify the full disease module around a set of known disease proteins. We study the performance of the algorithm using well-controlled synthetic data and systematically validate the identified neighborhoods for a large corpus of diseases. [ABSTRACT FROM AUTHOR]

The N-end rule states that half-life of protein is determined by their N-terminal amino acid residue. N-terminal glutamine amidohydrolase (Ntaq) converts N-terminal glutamine to glutamate by eliminating the amine group and plays an essential role in the N-end rule pathway for protein degradation. Here, we report the crystal structure of human Ntaq1 bound with the N-terminus of a symmetry-related Ntaq1 molecule at 1.5 Å resolution. The structure reveals a monomeric globular protein with alpha-beta-alpha three-layer sandwich architecture. The catalytic triad located in the active site, Cys-His-Asp, is highly conserved among Ntaq family and transglutaminases from diverse organisms. The N-terminus of a symmetry-related Ntaq1 molecule bound in the substrate binding cleft and the active site suggest possible substrate binding mode of hNtaq1. Based on our crystal structure of hNtaq1 and docking study with all the tripeptides with N-terminal glutamine, we propose how the peptide backbone recognition patch of hNtaq1 forms nonspecific interactions with N-terminal peptides of substrate proteins. Upon binding of a substrate with N-terminal glutamine, active site catalytic triad mediates the deamination of the N-terminal residue to glutamate by a mechanism analogous to that of cysteine proteases. [ABSTRACT FROM AUTHOR]

Pathogen-host protein-protein interaction (PPI) plays an important role in revealing the underlying pathogenesis of viruses and bacteria. The need of rapidly mapping proteome-wide pathogen-host interactome opens avenues for and imposes burdens on computational modeling. For Salmonella typhimurium, only 62 interactions with human proteins are reported to date, and the computational modeling based on such a small training data is prone to yield model overfitting. In this work, we propose a multi-instance transfer learning method to reconstruct the proteome-wide Salmonella-human PPI networks, wherein the training data is augmented by homolog knowledge transfer in the form of independent homolog instances. We use AdaBoost instance reweighting to counteract the noise from homolog instances, and deliberately design three experimental settings to validate the assumption that the homolog instances are effective to address the problems of data scarcity and data unavailability. The experimental results show that the proposed method outperforms the existing models and some predictions are validated by the findings from recent literature. Lastly, we conduct gene ontology based clustering analysis of the predicted networks to provide insights into the pathogenesis of Salmonella. [ABSTRACT FROM AUTHOR]

Protein disulfide isomerase (PDI) composed of four thioredoxin-like domains a, b, b', and a', is a key enzyme catalyzing oxidative protein folding in the endoplasmic reticulum. Large scale molecular dynamics simulations starting from the crystal structures of human PDI (hPDI) in the oxidized and reduced states were performed. The results indicate that hPDI adopts more compact conformations in solution than in the crystal structures, which are stabilized primarily by inter-domain interactions, including the salt bridges between domains a and b' observed for the first time. A prominent feature of the compact conformations is that the two catalytic domains a and a' can locate close enough for intra-molecular electron transfer, which was confirmed by the characterization of an intermediate with a disulfide between the two domains. Mutations, which disrupt the inter-domain interactions, lead to decreased reductase activity of hPDI. Our molecular dynamics simulations and biochemical experiments reveal the intrinsic conformational dynamics of hPDI and its biological impact. [ABSTRACT FROM AUTHOR]

Human stem cell factor initiates a diverse array of cellular responses, including hematopoiesis, cell proliferation, differentiation, migration and survival. To explore the relationship between its structure and function, we produced recombinant soluble human stem cell factor1–165 (wild type) and human stem cell factor1–141 (C-terminal truncated) in a yeast expression system and compared their biological activities and thermal stabilities. The biological activity of the two proteins was measured as a function of TF-1 cell viability and effects on downstream signaling targets after incubation. We found that these proteins enhanced cell viability and downstream signaling to a similar extent, in a dose-dependent manner. The biological activity of recombinant human stem cell factor1–165 was significantly greater than that of recombinant human stem cell factor1–141 after heating the proteins (100 ng/mL) at 25–110°C for 10 minutes (P<0.05 for all temperatures). In addition, circular dichroism spectral analysis indicated that β-sheet structures were altered in recombinant human stem cell factor1–141 but not recombinant human stem cell factor1–165 after heating at 90°C for 15 or 30 min. Molecular modeling and limited proteolytic digestion were also used to compare the thermo stability between human stem cell factor1–165 and human stem cell factor1–141. Together, these data indicate that stem cell factor1–165 is more thermostable than stem cell factor1–141. [ABSTRACT FROM AUTHOR]

Protein-protein interaction network-based study of viral pathogenesis has been gaining popularity among computational biologists in recent days. In the present study we attempt to investigate the possible pathways of hepatitis-C virus (HCV) infection by integrating the HCV-human interaction network, human protein interactome and human genetic disease association network. We have proposed quasi-biclique and quasi-clique mining algorithms to integrate these three networks to identify infection gateway host proteins and possible pathways of HCV pathogenesis leading to various diseases. Integrated study of three networks, namely HCV-human interaction network, human protein interaction network, and human proteins-disease association network reveals potential pathways of infection by the HCV that lead to various diseases including cancers. The gateway proteins have been found to be biologically coherent and have high degrees in human interactome compared to the other virus-targeted proteins. The analyses done in this study provide possible targets for more effective anti-hepatitis-C therapeutic involvement. [ABSTRACT FROM AUTHOR]

An amyloidogenic region (AR) in a protein sequence plays a significant role in protein aggregation and amyloid formation. We have investigated the sequence complexity of AR that is present in intrinsically disordered human proteins. More than 80% human proteins in the disordered protein databases (DisProt+IDEAL) contained one or more ARs. With decrease of protein disorder, AR content in the protein sequence was decreased. A probability density distribution analysis and discrete analysis of AR sequences showed that ∼8% residue in a protein sequence was in AR and the region was in average 8 residues long. The residues in the AR were high in sequence complexity and it seldom overlapped with low complexity regions (LCR), which was largely abundant in disorder proteins. The sequences in the AR showed mixed conformational adaptability towards α-helix, β-sheet/strand and coil conformations. [ABSTRACT FROM AUTHOR]

Protein phosphorylation is a key mechanism to regulate protein functions. However, the contribution of this protein modification to species divergence is still largely unknown. Here, we studied the evolution of mammalian phosphoregulation by comparing the human and mouse phosphoproteomes. We found that 84% of the positions that are phosphorylated in one species or the other are conserved at the residue level. Twenty percent of these conserved sites are phosphorylated in both species. This proportion is 2.5 times more than expected by chance alone, suggesting that purifying selection is preserving phosphoregulation. However, we show that the majority of the sites that are conserved at the residue level are differentially phosphorylated between species. These sites likely result from false-negative identifications due to incomplete experimental coverage, false-positive identifications and non-functional sites. In addition, our results suggest that at least 5% of them are likely to be true differentially phosphorylated sites and may thus contribute to the divergence in phosphorylation networks between mouse and humans and this, despite residue conservation between orthologous proteins. We also showed that evolutionary turnover of phosphosites at adjacent positions (in a distance range of up to 40 amino acids) in human or mouse leads to an over estimation of the divergence in phosphoregulation between these two species. These sites tend to be phosphorylated by the same kinases, supporting the hypothesis that they are functionally redundant. Our results support the hypothesis that the evolutionary turnover of phosphorylation sites contributes to the divergence in phosphorylation profiles while preserving phosphoregulation. Overall, our study provides advanced analyses of mammalian phosphoproteomes and a framework for the study of their contribution to phenotypic evolution. [ABSTRACT FROM AUTHOR]

Reconstruction of host-pathogen protein interaction networks is of great significance to reveal the underlying microbic pathogenesis. However, the current experimentally-derived networks are generally small and should be augmented by computational methods for less-biased biological inference. From the point of view of computational modelling, data scarcity, data unavailability and negative data sampling are the three major problems for host-pathogen protein interaction networks reconstruction. In this work, we are motivated to address the three concerns and propose a probability weighted ensemble transfer learning model for HIV-human protein interaction prediction (PWEN-TLM), where support vector machine (SVM) is adopted as the individual classifier of the ensemble model. In the model, data scarcity and data unavailability are tackled by homolog knowledge transfer. The importance of homolog knowledge is measured by the ROC-AUC metric of the individual classifiers, whose outputs are probability weighted to yield the final decision. In addition, we further validate the assumption that only the homolog knowledge is sufficient to train a satisfactory model for host-pathogen protein interaction prediction. Thus the model is more robust against data unavailability with less demanding data constraint. As regards with negative data construction, experiments show that exclusiveness of subcellular co-localized proteins is unbiased and more reliable than random sampling. Last, we conduct analysis of overlapped predictions between our model and the existing models, and apply the model to novel host-pathogen PPIs recognition for further biological research. [ABSTRACT FROM AUTHOR]

Mechanisms of protein misfolding are of increasing interest in the aetiology of neurodegenerative diseases characterized by protein aggregation and tangles including Amyotrophic Lateral Sclerosis (ALS), Alzheimer’s disease (AD), Parkinson’s disease (PD), Lewy Body Dementia (LBD), and Progressive Supranuclear Palsy (PSP). Some forms of neurodegenerative illness are associated with mutations in genes which control assembly of disease related proteins. For example, the mouse sticky mutation sti, which results in undetected mischarging of tRNAAla with serine resulting in the substitution of serine for alanine in proteins causes cerebellar Purkinje cell loss and ataxia in laboratory animals. Replacement of serine 422 with glutamic acid in tau increases the propensity of tau aggregation associated with neurodegeneration. However, the possibility that environmental factors can trigger abnormal folding in proteins remains relatively unexplored. We here report that a non-protein amino acid, β-N-methylamino-L-alanine (BMAA), can be misincorporated in place of l-serine into human proteins. We also report that this misincorporation can be inhibited by l-serine. Misincorporation of BMAA into human neuroproteins may shed light on putative associations between human exposure to BMAA produced by cyanobacteria and an increased incidence of ALS. [ABSTRACT FROM AUTHOR]

To investigate factors contributing to drug side effects, we systematically examine relationships between 4,199 side effects associated with 996 drugs and their 647 human protein targets. We find that it is the number of essential targets, not the number of total targets, that determines the side effects of corresponding drugs. Furthermore, within the context of a three-dimensional interaction network with atomic-resolution interaction interfaces, we find that drugs causing more side effects are also characterized by high degree and betweenness of their targets and highly shared interaction interfaces on these targets. Our findings suggest that both essentiality and centrality of a drug target are key factors contributing to side effects and should be taken into consideration in rational drug design. [ABSTRACT FROM AUTHOR]

Human protein complexes play crucial roles in various biological processes as the functional module. However, the expression features of human protein complexes at the transcriptome cascade are poorly understood. Here, we used the RNA-Seq data from 16 disparate tissues and four types of human cancers to explore the characteristics and dynamics of human protein complexes. We observed that many individual components of human protein complexes can be generated by multiple distinct transcripts. Similar with yeast, the human protein complex constituents are inclined to co-express in diverse tissues. The dominant isoform of the genes involved in protein complexes tend to encode the complex constituents in each tissue. Our results indicate that the protein complex dynamics not only correlate with the presence or absence of complexes, but may also be related to the major isoform switching for complex subunits. Between any two cancers of breast, colon, lung and prostate, we found that only a few of the differentially expressed transcripts associated with complexes were identical, but 5–10 times more protein complexes involved in differentially expressed transcripts were common. Collectively, our study reveals novel properties and dynamics of human protein complexes at the transcriptome cascade in diverse normal tissues and different cancers. [ABSTRACT FROM AUTHOR]

: The study of the function of many human proteins is often hampered by technical limitations, such as cytotoxicity and phenotypes that result from overexpression of the protein of interest together with the endogenous version. Here we present the snoMEN (snoRNA Modulator of gene ExpressioN) vector technology for generating stable cell lines where expression of the endogenous protein can be reduced and replaced by an exogenous protein, such as a fluorescent protein (FP)-tagged version. SnoMEN are snoRNAs engineered to contain complementary sequences that can promote knock-down of targeted RNAs. We have established and characterised two such partial protein replacement human cell lines (snoMEN-PR). Quantitative mass spectrometry was used to analyse the specificity of knock-down and replacement at the protein level and also showed an increased pull-down efficiency of protein complexes containing exogenous, tagged proteins in the protein replacement cell lines, as compared with conventional co-expression strategies. The snoMEN approach facilitates the study of mammalian proteins, particularly those that have so far been difficult to investigate by exogenous expression and has wide applications in basic and applied gene-expression research. [ABSTRACT FROM AUTHOR]

The four divergent serotypes of dengue virus are the causative agents of dengue fever, dengue hemorrhagic fever and dengue shock syndrome. About two-fifths of the world's population live in areas where dengue is prevalent, and thousands of deaths are caused by the viruses every year. Dengue virus is transmitted from one person to another primarily by the yellow fever mosquito, Aedes aegypti. Recent studies have begun to define how the dengue viral proteins interact with host proteins to mediate viral replication and pathogenesis. A combined analysis of these studies, however, suggests that many virus-host protein interactions remain to be identified, especially for the mosquito host. In this study, we used high-throughput yeast two-hybrid screening to identify mosquito and human proteins that physically interact with dengue proteins. We tested each identified host protein against the proteins from all four serotypes of dengue to identify interactions that are conserved across serotypes. We further confirmed many of the interactions using co-affinity purification assays. As in other large-scale screens, we identified some previously detected interactions and many new ones, moving us closer to a complete host - dengue protein interactome. To help summarize and prioritize the data for further study, we combined our interactions with other published data and identified a subset of the host-dengue interactions that are now supported by multiple forms of evidence. These data should be useful for understanding the interplay between dengue and its hosts and may provide candidates for drug targets and vector control strategies. [ABSTRACT FROM AUTHOR]

Cohesin is best known as a multi-subunit protein complex that holds together replicated sister chromatids from S phase until G2. Cohesin also has an important role in the regulation of gene expression. We previously demonstrated that the cohesin complex positively regulates expression of the oncogene MYC. Cell proliferation driven by MYC contributes to many cancers, including breast cancer. The MYC oncogene is estrogen-responsive and a transcriptional target of estrogen receptor alpha (ERa). Estrogen-induced cohesin binding sites coincide with ERa binding at the MYC locus, raising the possibility that cohesin and ERa combine actions to regulate MYC transcription. The objective of this study was to investigate a putative role for cohesin in estrogen induction of MYC expression. We found that siRNA-targeted depletion of a cohesin subunit, RAD21, decreased MYC expression in ER-positive (MCF7 and T47D) and ER-negative (MDA-MB-231) breast cancer cell lines. In addition, RAD21 depletion blocked estradiol-mediated activation of MYC in ER-positive cell lines, and decreased ERa binding to estrogen response elements (EREs) upstream of MYC, without affecting total ERa levels. Treatment of MCF7 cells with estradiol caused enrichment of RAD21 binding at upstream enhancers and at the P2 promoter of MYC. Enriched binding at all sites, except the P2 promoter, was dependent on ERa. Since RAD21 depletion did not affect transcription driven by an exogenous reporter construct containing a naked ERE, chromatin-based mechanisms are likely to be involved in cohesin-dependent MYC transcription. This study demonstrates that ERa activation of MYC can be modulated by cohesin. Together, these results demonstrate a novel role for cohesin in estrogen-mediated regulation of MYC and the first evidence that cohesin plays a role in ERa binding. [ABSTRACT FROM AUTHOR]

Indoleamine 2,3-dioxygenase (IDO) has been identified as an important antimicrobial and immunoregulatory effector molecule essential for the establishment of tolerance by regulating local tryptophan (Trp) concentrations. On the other hand, the immunosuppressive capacity of IDO can have detrimental effects for the host as it can lead to deleterious alterations of the immune response by promoting tolerance to some types of tumors. To suppress this disadvantageous IDO effect, the competitive inhibitor 1-Methyl-Tryptophan (1-MT) is being tested in clinical trials. However, it remains inconclusive which stereoisomer of 1-MT is the more effective inhibitor of IDO-mediated immunosuppression. While IDO enzyme activity is more efficiently inhibited by 1-L-MT in cell-free or in vitro settings, 1-D-MT is superior to 1-L-MT in the enhancement of anti-tumor responses in vivo. Here, we present new data showing that commercially available 1-L-MT lots contain tryptophan in amounts sufficient to compensate for the IDO-mediated tryptophan depletion in vitro. The addition of 1-L-MT abrogated IDO-mediated antimicrobial effects and permitted the growth of the tryptophan-auxotroph microorganisms Staphylococcus aureus and Toxoplasma gondii. Consistent with this, the tryptophan within 1-L-MT lots was sufficient to antagonize IDO-mediated inhibition of T cell responses. Mass spectrometry (MS) analysis revealed not only tryptophan within 1-L-MT, but also the incorporation of this tryptophan in bacterial and human proteins that were generated in the presence of 1-L-MT in otherwise tryptophan-free conditions. In summary, these data reveal that tryptophan within 1-L-MT can affect the results of in vitro studies in an L-stereospecific and IDO-independent way. [ABSTRACT FROM AUTHOR]

Background: MDM4, also called MDMX or HDMX in humans, is an important negative regulator of the p53 tumor suppressor. MDM4 is overexpressed in about 17% of all cancers and more frequently in some types, such as colon cancer or retinoblastoma. MDM4 is known to be post-translationally regulated by MDM2- mediated ubiquitination to decrease its protein levels in response to genotoxic stress, resulting in accumulation and activation of p53. At the transcriptional level, MDM4 gene regulation has been less clearly understood. We have reported that DNA damage triggers loss of MDM4 mRNA and a concurrent increase in p53 activity. These experiments attempt to determine a mechanism for down-regulation of MDM4 mRNA. Methodology/Principal Findings: Here we report that MDM4 mRNA is a target of hsa-mir-34a (miR-34a). MDM4 mRNA contains a lengthy 3' untranslated region; however, we find that it is a miR-34a site within the open reading frame (ORF) of exon 11 that is responsible for the repression. Overexpression of miR- 34a, but not a mutant miR-34a, is sufficient to decrease MDM4 mRNA levels to an extent identical to those of known miR-34a target genes. Likewise, MDM4 protein levels are decreased by miR-34a overexpression. Inhibition of endogenous miR-34a increased expression of miR-34a target genes and MDM4. A portion of MDM4 exon 11 containing this 8mer-A1 miR-34a site fused to a luciferase reporter gene is sufficient to confer responsiveness, being inhibited by additional expression of exogenous mir-34a and activated by inhibition of miR34a. Conclusions/Significance: These data establish a mechanism for the observed DNA damage-induced negative regulation of MDM4 and potentially provide a novel means to manipulate MDM4 expression without introducing DNA damage. [ABSTRACT FROM AUTHOR]

Rad9, Rad1, and Hus1 (9-1-1) are part of the DNA integrity checkpoint control system. It was shown previously that the Cterminal end of the human Rad9 protein, which contains a nuclear localization sequence (NLS) nearby, is critical for the nuclear transport of Rad1 and Hus1. In this study, we show that in Drosophila, Hus1 is found in the cytoplasm, Rad1 is found throughout the entire cell and that Rad9 (DmRad9) is a nuclear protein. More specifically, DmRad9 exists in two alternatively spliced forms, DmRad9A and DmRad9B, where DmRad9B is localized at the cell nucleus, and DmRad9A is found on the nuclear membrane both in Drosophila tissues and also when expressed in mammalian cells. Whereas both alternatively spliced forms of DmRad9 contain a common NLS near the C terminus, the 32 C-terminal residues of DmRad9A, specific to this alternative splice form, are required for targeting the protein to the nuclear membrane. We further show that activation of a meiotic checkpoint by a DNA repair gene defect but not defects in the anchoring of meiotic chromosomes to the oocyte nuclear envelope upon ectopic expression of non-phosphorylatable Barrier to Autointegration Factor (BAF) dramatically affects DmRad9A localization. Thus, by studying the localization pattern of DmRad9, our study reveals that the DmRad9A C-terminal region targets the protein to the nuclear membrane, where it might play a role in response to the activation of the meiotic checkpoint. [ABSTRACT FROM AUTHOR]

The Multiple Sequence Alignment (MSA) is a computational abstraction that represents a partial summary either of indel history, or of structural similarity. Taking the former view (indel history), it is possible to use formal automata theory to generalize the phylogenetic likelihood framework for finite substitution models (Dayhoff's probability matrices and Felsenstein's pruning algorithm) to arbitrary-length sequences. In this paper, we report results of a simulation-based benchmark of several methods for reconstruction of indel history. The methods tested include a relatively new algorithm for statistical marginalization of MSAs that sums over a stochastically-sampled ensemble of the most probable evolutionary histories. For mammalian evolutionary parameters on several different trees, the single most likely history sampled by our algorithm appears less biased than histories reconstructed by other MSA methods. The algorithm can also be used for alignment-free inference, where the MSA is explicitly summed out of the analysis. As an illustration of our method, we discuss reconstruction of the evolutionary histories of human protein-coding genes. [ABSTRACT FROM AUTHOR]

Identification of potential viral-host protein interactions is a vital and useful approach towards development of new drugs targeting those interactions. In recent days, computational tools are being utilized for predicting viral-host interactions. Recently a database containing records of experimentally validated interactions between a set of HIV-1 proteins and a set of human proteins has been published. The problem of predicting new interactions based on this database is usually posed as a classification problem. However, posing the problem as a classification one suffers from the lack of biologically validated negative interactions. Therefore it will be beneficial to use the existing database for predicting new viral-host interactions without the need of negative samples. Motivated by this, in this article, the HIV-1-human protein interaction database has been analyzed using association rule mining. The main objective is to identify a set of association rules both among the HIV-1 proteins and among the human proteins, and use these rules for predicting new interactions. In this regard, a novel association rule mining technique based on biclustering has been proposed for discovering frequent closed itemsets followed by the association rules from the adjacency matrix of the HIV-1-human interaction network. Novel HIV-1-human interactions have been predicted based on the discovered association rules and tested for biological significance. For validation of the predicted new interactions, gene ontology-based and pathway-based studies have been performed. These studies show that the human proteins which are predicted to interact with a particular viral protein share many common biological activities. Moreover, literature survey has been used for validation purpose to identify some predicted interactions that are already validated experimentally but not present in the database. Comparison with other prediction methods is also discussed. [ABSTRACT FROM AUTHOR]

Cellular DExD/H-box RNA-helicases perform essential functions during mRNA biogenesis. The closely related human proteins UAP56 and URH49 are members of this protein family and play an essential role for cellular mRNA export by recruiting the adaptor protein REF to spliced and unspliced mRNAs. In order to gain insight into their mode of action, we aimed to characterize these RNA-helicases in more detail. Here, we demonstrate that UAP56 and URH49 exhibit an intrinsic CRM1-independent nucleocytoplasmic shuttling activity. Extensive mapping studies identified distinct regions within UAP56 or URH49 required for (i) intranuclear localization (UAP56 aa81-381) and (ii) interaction with REF (UAP56 aa51-428). Moreover, the region conferring nucleocytoplasmic shuttling activity was mapped to the C-terminus of UAP56, comprising the amino acids 195-428. Interestingly, this region coincides with a domain within Uap56p of S. pombe that has been reported to be required for both Rae1p-interaction and nucleocytoplasmic shuttling. However, in contrast to this finding we report that human UAP56 shuttles independently from Rae1. In summary, our results reveal nucleocytoplasmic shuttling as a conserved feature of yeast and human UAP56, while their export receptor seems to have diverged during evolution. [ABSTRACT FROM AUTHOR]

Protein-domains play an important role in mediating protein-protein interactions. Furthermore, the same domain-pairs mediate different interactions in different contexts and in various organisms, and therefore domain-pairs are considered as the building blocks of interactome networks. Here we extend these principles to the host-virus interface and find the domain-pairs that potentially mediate human-herpesvirus interactions. Notably, we find that the same domain-pairs used by other organisms for mediating their interactions underlie statistically significant fractions of human-virus protein interinteraction networks. Our analysis shows that viral domains tend to interact with human domains that are hubs in the human domain-domain interaction network. This may enable the virus to easily interfere with a variety of mechanisms and processes involving various and different human proteins carrying the relevant hub domain. Comparative genomics analysis provides hints at a molecular mechanism by which the virus acquired some of its interacting domains from its human host. [ABSTRACT FROM AUTHOR]

The Enhancer of Zeste 2 (EZH2) protein has been reported to stimulate cell growth in some cancers and is therefore considered to represent an interesting new target for therapeutic intervention. Here, we investigated a possible role of EZH2 for the growth control of colon cancer cells. RNA interference (RNAi)-mediated intracellular EZH2 depletion led to cell cycle arrest of colon carcinoma cells at the G1/S transition. This was associated with a reduction of cell numbers upon transient transfection of synthetic EZH2-targeting siRNAs and with inhibition of their colony formation capacity upon stable expression of vectorborne siRNAs. We furthermore tested whether EZH2 may repress the growth-inhibitory p27 gene, as reported for pancreatic cancer. However, expression analyses of colon cancer cell lines and colon cancer biopsies did not reveal a consistent correlation between EZH2 and p27 levels. Moreover, EZH2 depletion did not re-induce p27 expression in colon cancer cells, indicating that p27 repression by EZH2 may be cell- or tissue-specific. Whole genome transcriptome analyses identified cellular genes affected by EZH2 depletion in colon cancer cell lines. They included several cancer-associated genes linked to cellular proliferation or invasion, such as Dag1, MageD1, SDC1, Timp2, and Tob1. In conclusion, our results demonstrate that EZH2 depletion blocks the growth of colon cancer cells. These findings might provide benefits for the treatment of colon cancer. [ABSTRACT FROM AUTHOR]

Background: Oxidative damage to DNA, if not repaired, can be both miscoding and blocking. These genetic alterations can lead to mutations and/or cell death, which in turn cause cancer and aging. Oxidized DNA bases are substrates for two overlapping repair pathways: base excision (BER) and nucleotide incision repair (NIR). Hydantoin derivatives such as 5- hydroxyhydantoin (5OH-Hyd) and 5-methyl-5-hydroxyhydantoin (5OH-5Me-Hyd), major products of cytosine and thymine oxidative degradation pathways, respectively, have been detected in cancer cells and ancient DNA. Hydantoins are blocking lesions for DNA polymerases and excised by bacterial and yeast DNA glycosylases in the BER pathway. However little is known about repair of pyrimidine-derived hydantoins in human cells. Methodology/Principal Findings: Here, using both denaturing PAGE and MALDI-TOF MS analyses we report that the bacterial, yeast and human AP endonucleases can incise duplex DNA 59 next to 5OH-Hyd and 5OH-5Me-Hyd thus initiating the NIR pathway. We have fully reconstituted the NIR pathway for these lesions in vitro using purified human proteins. Depletion of Nfo in E. coli and APE1 in HeLa cells abolishes the NIR activity in cell-free extracts. Importantly, a number of redundant DNA glycosylase activities can excise hydantoin residues, including human NTH1, NEIL1 and NEIL2 and the former protein being a major DNA glycosylase activity in HeLa cells extracts. Conclusions/Significance: This study demonstrates that both BER and NIR pathways can compete and/or back-up each other to remove hydantoin DNA lesions in vivo. [ABSTRACT FROM AUTHOR]

Autologous expression of recombinant human proteins in human cells for biomedical research and product development is often hampered by low expression yields limiting subsequent structural and functional analyses. Following RNA and codon optimization, 50 candidate genes representing five classes of human proteins - transcription factors, ribosomal and polymerase subunits, protein kinases, membrane proteins and immunomodulators - all showed reliable, and 86% even elevated expression. Analysis of three representative examples showed no detrimental effect on protein solubility while unaltered functionality was demonstrated for JNK1, JNK3 and CDC2 using optimized constructs. Molecular analysis of a sequence-optimized transgene revealed positive effects at transcriptional, translational, and mRNA stability levels. Since improved expression was consistent in HEK293T, CHO and insect cells, it was not restricted to distinct mammalian cell systems. Additionally, optimized genes represent powerful tools in functional genomics, as demonstrated by the successful rescue of an siRNA-mediated knockdown using a sequence-optimized counterpart. This is the first large-scale study addressing the influence of multiparameter optimization on autologous human protein expression. [ABSTRACT FROM AUTHOR]

Proteins can move from blood circulation into salivary glands through active transportation, passive diffusion or ultrafiltration, some of which are then released into saliva and hence can potentially serve as biomarkers for diseases if accurately identified. We present a novel computational method for predicting salivary proteins that come from circulation. The basis for the prediction is a set of physiochemical and sequence features we found to be discerning between human proteins known to be movable from circulation to saliva and proteins deemed to be not in saliva. A classifier was trained based on these features using a support-vector machine to predict protein secretion into saliva. The classifier achieved 88.56% average recall and 90.76% average precision in 10-fold cross-validation on the training data, indicating that the selected features are informative. Considering the possibility that our negative training data may not be highly reliable (i.e., proteins predicted to be not in saliva), we have also trained a ranking method, aiming to rank the known salivary proteins from circulation as the highest among the proteins in the general background, based on the same features. This prediction capability can be used to predict potential biomarker proteins for specific human diseases when coupled with the information of differentially expressed proteins in diseased versus healthy control tissues and a prediction capability for blood-secretory proteins. Using such integrated information, we predicted 31 candidate biomarker proteins in saliva for breast cancer. [ABSTRACT FROM AUTHOR]

The RNA binding protein HuR/ELAVL1 binds to AU-rich elements (AREs) promoting the stabilization and translation of a number of mRNAs into the cytoplasm, dictating their fate. We applied the AlphaScreen technology using purified human HuR protein, expressed in a mammalian cell-based system, to characterize in vitro its binding performance towards a ssRNA probe whose sequence corresponds to the are present in TNFα 3’ untranslated region. We optimized the method to titrate ligands and analyzed the kinetic in saturation binding and time course experiments, including competition assays. The method revealed to be a successful tool for determination of HuR binding kinetic parameters in the nanomolar range, with calculated Kd of 2.5±0.60 nM, kon of 2.76±0.56*106 M-1 min-1, and koff of 0.007±0.005 min-1. We also tested the HuR-RNA complex formation by fluorescent probe-based RNA-EMSA. Moreover, in a 384-well plate format we obtained a Z-factor of 0.84 and an averaged coefficient of variation between controls of 8%, indicating that this biochemical assay fulfills criteria of robustness for a targeted screening approach. After a screening with 2000 small molecules and secondary verification with RNA-EMSA we identified mitoxantrone as an interfering compound with rHuR and TNFα probe complex formation. Notably, this tool has a large versatility and could be applied to other RNA Binding Proteins recognizing different RNA, DNA, or protein species. In addition, it opens new perspectives in the identification of small-molecule modulators of RNA binding proteins activity. [ABSTRACT FROM AUTHOR]