Heterogeneous Nuclear Ribonucleoprotein D0 Research Articles

Identification and Verification of Two Novel Differentially Expressed Proteins from Non-neoplastic Mucosa and Colorectal Carcinoma Via iTRAQ Combined with Liquid Chromatography-Mass Spectrometry.

Recurrence or metastasis of colorectal cancer (CRC) is common following surgery and/or adjuvant therapy, particularly in patients with an advanced stage of the cancer. Identifying key molecular markers of CRC is beneficial for early diagnosis and early treatment, which may eventually improve the prognosis of patients with CRC. Isobaric mass tags for relative and absolute quantification (iTRAQ) in combination with multidimensional liquid chromatography and tandem mass spectrometry (LC-MS/MS) were used to identify differentially expressed proteins between CRC tissues and paired adjacent normal mucosa. Among the 105 patients, adenocarcinoma was the most common CRC subtype, stage III was the most common Tumor-Node-Metastasis stage and high levels of Ki-67 indicated the rapid proliferation of tumor cells in the samples. The LC-MS/MS-based iTRAQ technology identified 271 differentially expressed proteins, with 130 upregulated proteins and 141 downregulated proteins. Bioinformatics analysis revealed that golgin subfamily A member 2 (GOLGA2) and heterogeneous nuclear ribonucleoprotein D0 (hnRNPD) were located in the center of the upregulated protein network, and were closely associated with the development of CRC. The upregulation of GOLGA2 and hnRNPD was further verified in human tissues using western blotting and immunohistochemistry. GOLGA2 and hnRNPD were identified as two novels differentially expressed proteins in human CRC. Furthermore, the LC-MS/MS-based iTRAQ proteomic approach is a useful tool for searching and identifying differentially expressed proteins, and may be used to provide a comprehensive understanding of the processes that mediate the development of CRC.

Quantitative proteomics of forebrain subcellular fractions in fragile X mental retardation 1 knockout mice following acute treatment with 2-Methyl-6-(phenylethynyl)pyridine: Relevance to developmental study of schizophrenia.

The fragile X mental retardation 1 knockout (Fmr1 KO) mouse replicates behavioral deficits associated with autism, fragile X syndrome, and schizophrenia. Less is known whether protein expression changes are consistent with findings in subjects with schizophrenia. In the current study, we used liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomics to determine the protein expression of four subcellular fractions in the forebrains of Fmr1 KO mice vs. C57BL/6J mice and the effect of a negative allosteric modulator of mGluR5-2-Methyl-6-(phenylethynyl)pyridine (MPEP)-on protein expression. Strain- and treatment-specific differential expression of proteins was observed, many of which have previously been observed in the brains of subjects with schizophrenia. Western blotting verified the direction and magnitude of change for several proteins in different subcellular fractions as follows: neurofilament light protein (NEFL) and 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNP) in the total homogenate; heterogeneous nuclear ribonucleoproteins C1/C2 (HNRNPC) and heterogeneous nuclear ribonucleoprotein D0 (HNRNPD) in the nuclear fraction; excitatory amino acid transporter 2 (EAAT2) and ras-related protein rab 3a (RAB3A) in the synaptic fraction; and ras-related protein rab 35 (RAB35) and neuromodulin (GAP43) in the rough endoplasmic reticulum fraction. Individuals with FXS do not display symptoms of schizophrenia. However, the biomarkers that have been identified suggest that the Fmr1 KO model could potentially be useful in the study of schizophrenia.

Human Obesity Associated with an Intronic SNP in the Brain-Derived Neurotrophic Factor Locus.

Brain-derived neurotrophic factor (BDNF) plays a key role in energy balance. In population studies, SNPs of the BDNF locus have been linked to obesity, but the mechanism by which these variants cause weight gain is unknown. Here, we examined human hypothalamic BDNF expression in association with 44 BDNF SNPs. We observed that the minor C allele of rs12291063 is associated with lower human ventromedial hypothalamic BDNF expression (p < 0.001) and greater adiposity in both adult and pediatric cohorts (p values < 0.05). We further demonstrated that the major T allele for rs12291063 possesses a binding capacity for the transcriptional regulator, heterogeneous nuclear ribonucleoprotein D0B, knockdown of which disrupts transactivation by the T allele. Binding and transactivation functions are both disrupted by substituting C for T. These findings provide a rationale for BDNF augmentation as a targeted treatment for obesity in individuals who have the rs12291063 CC genotype.

Human proteins that specifically bind to 8-oxoguanine-containing RNA and their responses to oxidative stress

Exposure of cells to oxygen radicals damage various biologically important molecules. Among the oxidized bases produced in nucleic acids, 8-oxo-7,8-dihydroguanine (8-oxoguanine) is particularly important since it causes base mispairing. To ensure accurate gene expression, organisms must have a mechanism to discriminate 8-oxoguanine-containing RNA from normal transcripts. We searched for proteins that specifically bind to 8-oxoguanine-containing RNA from human HeLa cell extracts, and the candidate proteins were identified using mass spectrometry. Among the identified candidates, splicing isoform 1 of heterogeneous nuclear ribonucleoprotein D0 (HNRNPD) and splicing isoform C1 of heterogeneous nuclear ribonucleoprotein C1/C2 (HNRNPC) exhibited strong abilities to bind to oxidized RNA. The amount of HNRNPD protein rapidly decreased when cells were exposed to hydrogen peroxide, an agent that enhances oxidative stress. Moreover, the suppression of HNRNPD expression by siRNA caused cells to exhibit an increased sensitivity to hydrogen peroxide. The application of siRNA against HNRNPC also caused an increase in sensitivity to hydrogen peroxide. Since no additive effect was observed with a combined addition of siRNAs for HNRNPD and HNRNPC, we concluded that the two proteins may function in the same mechanism for the accurate gene expression.

LC/MS identification of 12 intracellular cytoskeletal and inflammatory proteins from monocytes adherent on surface‐adsorbed fibronectin‐derived peptides

The extent and duration of the host response determines device efficacy, yet the mechanism is poorly understood. U937 promonocytic cells were cultured on peptide-adsorbed tissue-culture polystyrene to better understand surface-modulated intracellular events. Phosphotyrosine proteins were enriched by immunoprecipitation and analyzed by nanospray HPLC-coupled tandem mass spectrometry (LC/MS). Tyrosine-phosphorylated proteins were chosen based on physiological significance and previous densitometry results, which identified a set of proteins ranging from approximately 200 to approximately 23 kDa showing altered phosphorylation levels in response to various surface-adsorbed ligands and phosphorylation inhibitor AG18. Although LC/MS has been used for nearly a decade, its application to the field of biomaterials is relatively novel. Twelve intracellular proteins identified by nanospray LC/MS are potentially related to the host response. Eight of the twelve proteins are related to the cytoskeleton including: moesin, heat shock protein 90beta, alpha-tubulin, elongation factor 1alpha, beta actin, vimentin, plasminogen activator inhibitor 2, and heterogeneous ribonuclear protein A2. The remaining four proteins: high mobility group box 1, caspase recruitment domain 5, glycoprotein 96, and heterogeneous nuclear ribonucleoprotein D0 modulate inflammation. The specific effect each peptide has upon modulating the phosphorylation state of these proteins cannot be determined from this work; however, 12 viable targets have been identified for further investigation into the role each plays in the surface-mediated monocyte response.

Search for Cancer Markers from Endometrial Tissues Using Differentially Labeled Tags iTRAQ and cICAT with Multidimensional Liquid Chromatography and Tandem Mass Spectrometry

A total of nine potential markers for endometrial cancer (EmCa) have been discovered and identified from endometrial tissue homogenates using a combination of differentially labeled tags, iTRAQ and cICAT, with multidimensional liquid chromatography and tandem mass spectrometry. The tissues were snap frozen in liquid nitrogen within 15-20 min after devitalization. Samples for proteomic analysis were treated with protease inhibitors before processing. Marker proteins that were overexpressed in EmCa are chaperonin 10, pyruvate kinase M1 or M2 isozyme, calgizzarin, heterogeneous nuclear ribonucleoprotein D0, macrophage migratory inhibitory factor, and polymeric immunoglobulin receptor precursor; those that were underexpressed are alpha-1-antitrypsin precursor, creatine kinase B, and transgelin. The chaperonin 10 result confirms our earlier observation of overexpression in EmCa tissues using surface-enhanced laser desorption/ionization mass spectrometry, verified by Western analysis and immunohistochemistry [Yang, E. C. C. et al. J. Proteome Res. 2004, 3, 636-643]. Pyruvate kinase was observed to be overexpressed using both iTRAQ and cICAT labeling. All nine markers have been found to be associated with various forms of cancer. A panel of these plus other markers may confer sufficient selectivity for diagnosing and screening of EmCa. The use of cICAT led to identification of a higher proportion of lower-abundance signaling proteins; conversely, iTRAQ resulted in a higher percentage of the more abundant ribosomal proteins and transcription factors.

Molecular Basis for the Loss of CD28 Expression in Senescent T Cells

CD28(null) T cells are the most consistent biological indicator of the aging immune system in humans and are predictors of immunoincompetence in the elderly. The loss of CD28 is the result of an inoperative transcriptional initiator (INR), which consists of two nonoverlapping alpha and beta motifs that have distinct protein binding profiles but function as a unit. In CD28(null) T cells, there is a coordinate loss of alpha-/beta-bound complexes, hence the alphabeta-INR is inactive. In the present work therefore, studies were conducted to identify the components of such complexes that may account for the trans-activation of the alphabeta-INR. By affinity chromatography and tandem mass spectrometry, two proteins, namely, nucleolin and the A isoform of heterogeneous nuclear ribonucleoprotein-D0 (hnRNP-D0A), were identified to be among the key components of the site alpha complex. In DNA binding assays, specific antibodies indicated their antigenic presence in alpha-bound complexes. Transcription assays showed that they are both required in the trans-activation of alphabeta-INR-driven DNA templates. Because CD28 is T cell-restricted, and nucleolin and hnRNP-D0A are ubiquitous proteins, these results support the notion that cell-specific functions can be regulated by commonly expressed proteins. The present data also provide evidence for INR-regulated transcription that is independent of the known components of the basal transcription complex.

Heterogeneous nuclear ribonucleoprotein D0 contains transactivator and DNA-binding domains

Heterogeneous nuclear ribonucleoprotein D0 contains transactivator and DNA-binding domains

Heterogeneous nuclear ribonucleoprotein D0 contains transactivator and DNA-binding domains

Heterogeneous nuclear ribonucleoprotein D0 (hnRNP D0) is an abundant, ubiquitous protein that binds RNA and DNA sequences specifically, and has been implicated in the transcriptional regulation of the human complement receptor 2 gene. We found that in vivo expression of hnRNP D0-GAL4 fusion proteins increased the transcriptional activity of a GAL4-driven reporter gene, providing direct proof that hnRNP D0 possesses a transactivator domain. We found, using truncated hnRNP D0 proteins fused to GAL4, that 29 amino acids in the N-terminal region are critical for transactivation. We established, using a series of recombinant truncated hnRNP D0 proteins, that the tandem RNA-binding domains alone were not able to bind double-stranded DNA. Nevertheless, 24 additional amino acids of the C-terminus imparted sequence-specific DNA binding. Experiments using peptide-specific antisera supported the importance of the 24-amino-acid region in DNA binding, and suggested the involvement of the 19-amino-acid alternative insert which is present in isoforms B and D. The N-terminus had an inhibitory effect on binding of hnRNP D0 to single-stranded, but not to double-stranded, DNA. Although both recombinant hnRNP D0B and D0D bound DNA, only the B isoform recognized DNA in vivo. We propose that the B isoform of hnRNP D0 functions in the nucleus as a DNA-binding transactivator and has distinct transactivator and DNA-binding domains.