HNRNPC as a candidate biomarker for chemoresistance in gastric cancer.

Chemoresistance is a major cause of treatment failure and high mortality in advanced gastric cancer (AGC). Currently, the mechanism of chemoresistance remains unclear, and there is no biomarker to accurately predict the efficacy of chemotherapy. In the present study, we established human gastric cancer (GC) cell lines resistant to 5-fluorouracil (5FU), paclitaxel (TA), or cisplatin (DDP) by gradient drug treatment and generated a novel monoclonal antibody 5B2 targeting heterogeneous nuclear ribonucleoproteins C1/C2 (HNRNPC) overexpressed in chemoresistant GC cells. Overexpressing HNRNPC in GC cells promoted chemoresistance, and knockdown of HNRNPC by small interfering RNA (siRNA) reversed chemoresistance. By utilizing available datasets, we demonstrated that high level of HNRNPC transcript indicated poor overall survival (OS) and free of progression (FP). HNRNPC expression was negatively correlated with OS of GC patients treated with 5FU-based drugs and with time to progression (TTP) of GC patients treated with CF regimen. These data suggest the potential usefulness of HNRNPC as a prognostic and therapeutic marker of GC.

Online nanoflow RP-RP-MS reveals dynamics of multicomponent Ku complex in response to DNA damage.

Tandem affinity purification (TAP) coupled with mass spectrometry has become the technique of choice for characterization of multicomponent protein complexes. While current TAP protocols routinely provide high yield and specificity for proteins expressed under physiologically relevant conditions, analytical figures of merit required for efficient and in-depth LC-MS analysis remain unresolved. Here we implement a multidimensional chromatography platform, based on two stages of reversed-phase (RP) separation operated at high and low pH, respectively. We compare performance metrics for RP-RP and SCX-RP for the analysis of complex peptide mixtures derived from cell lysate, as well as protein complexes purified via TAP. Our data reveal that RP-RP fractionation outperforms SCX-RP primarily due to increased peak capacity in the first dimension separation. We integrate this system with miniaturized LC assemblies to achieve true online fractionation at low (≤5 nL/min) effluent flow rates. Stable isotope labeling is used to monitor the dynamics of the multicomponent Ku protein complex in response to DNA damage induced by γ radiation.

Heterogeneous ribonucleoprotein C displays a repressor activity mediated by T-cell intracellular antigen-1-related/like protein to modulate Fas exon 6 splicing through a mechanism involving Hu antigen R.

T-cell intracellular antigen (TIA)-proteins are known regulators of alternative pre-mRNA splicing. In this study, pull-down experiments and mass spectrometry indicate that TIAR/TIAL1 and hnRNP C1/C2 are associated in HeLa nuclear extracts. Co-immunoprecipitation and GST-pull-down assays confirmed this interaction. Interestingly, binding requires the glutamine-rich (Q-rich) C-terminal domain of TIAR and the leucine-rich plus acidic residues-rich C-terminal domains of hnRNP C1/C2. This interaction also occurs in an RNA-dependent manner. Recombinant GFP-TIAR and RFP-hnRNP C1 proteins display partial nuclear co-localization when overexpressed in HeLa cells, and this requires the Q-rich domain of TIAR. hnRNP C1 overexpression in the presence of rate-limiting amounts of TIAR in HeLa and HEK293 cells affects alternative splicing of Fas and FGFR2 minigenes, promoting Fas exon 6 and FGFR2 exon K-SAM skipping, respectively. The repressor activity of hnRNP C1 on Fas exon 6 splicing is mediated by Hu antigen R (HuR). Experiments involving tethering approaches showed that the repressor capacity of hnRNP C1 is associated with an exonic splicing silencer in Fas exon 6. This effect was reversed by splice-site strengthening and is linked to its basic leucine zipper-like motif. These results suggest that hnRNP C1/C2 acts as a bridge between HuR and TIAR to modulate alternative Fas splicing.

Analysis of PTEN complex assembly and identification of heterogeneous nuclear ribonucleoprotein C as a component of the PTEN-associated complex.

PTEN (phosphatase and tensin homolog deleted on chromosome 10) is well characterized for its role in antagonizing the phosphoinositide 3-kinase pathway. Previous studies using size-exclusion chromatography demonstrated PTEN recruitment into high molecular mass complexes and hypothesized that PTEN phosphorylation status and PDZ binding domain may be required for such complex formation. In this study, we set out to test the structural requirements for PTEN complex assembly and identify the component(s) of the PTEN complex(es). Our results demonstrated that the PTEN catalytic function and PDZ binding domain are not absolutely required for its complex formation. On the other hand, PTEN phosphorylation status has a significant impact on its complex assembly. Our results further demonstrate enrichment of the PTEN complex in nuclear lysates, suggesting a mechanism through which PTEN phosphorylation may regulate its complex assembly. These results prompted further characterization of other protein components within the PTEN complex(es). Using size-exclusion chromatography and two-dimensional difference gel electrophoresis followed by mass spectrometry analysis, we identified heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a novel protein recruited to higher molecular mass fractions in the presence of PTEN. Further analysis indicates that endogenous hnRNP C and PTEN interact and co-localize within the nucleus, suggesting a potential role for PTEN, alongside hnRNP C, in RNA regulation.

Early osteogenic differential protein profile detected by proteomic analysis in human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Human periodontal ligament cells play a pivotal role in maintaining periodontal ligament space, contain progenitors that are able to differentiate into cementoblasts/osteoblasts and have a tremendous potential to regenerate periodontal tissue. However, the exact molecular mechanisms governing the differentiation mechanisms of progenitors in periodontal ligament cells remain largely unknown. This study was carried out to investigate the differentially expressed proteins involved in the osteogenic differentiation of progenitors presented in periodontal ligament cells. MATERIAL AND METHODS: Using two-dimensional gel electrophoresis, mass spectrometry and peptide mass fingerprinting, we analyzed the differential protein profiles of periodontal ligament cells undergoing mineralization. RESULTS: Compared with undifferentiated periodontal ligament cells, 61 proteins in periodontal ligament cells undergoing differentiation showed at least a 1.5-fold change in intensity, of which 29 differentially expressed proteins were successfully identified by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry. The expression of some of the identified proteins was further confirmed by western blotting and reverse transcription-polymerase chain reaction analysis. The identified proteins were cytoskeleton proteins and cytoskeleton-associated proteins, nuclear proteins and cell membrane-bound molecules. CONCLUSION: Our results suggest that the proteins identified in this study may be associated with the unique function of periodontal ligament cells in maintaining periodontal tissue homeostasis, thus providing a comprehensive reference for understanding and investigating in greater detail the molecular mechanisms of periodontal ligament cells involved in periodontal regeneration.

Up-regulation of a hydrogen peroxide-responsive pre-mRNA binding protein in atherosclerosis and intimal hyperplasia.

BACKGROUND: Multiple lines of investigation have implicated hydrogen peroxide (H(2)O(2)) as an important endogenous mediator of cell proliferation in the vessel wall. Heterogeneous nuclear ribonucleoprotein C (hnRNP-C), a nuclear pre-mRNA binding protein that plays roles in vertebrate cell proliferation and differentiation, has been identified as a component of a vascular cell signaling pathway activated by low physiologic levels of H(2)O(2). The expression of hnRNP-C in human arteries has not previously been assessed. METHODS: Segments of human proximal internal carotid arteries were evaluated for the expression of hnRNP-C by immunohistochemistry. RESULTS: In normal proximal internal carotid arteries, hnRNP-C is expressed predominantly by the endothelium, with significantly lower expression by medial smooth muscle. In preatherosclerotic intimal hyperplasia, hnRNP-C is up-regulated in the artery wall, due to the robust expression by the intimal smooth muscle cells, without up-regulation in the medial smooth muscle cells. In arteries with atherosclerotic lesions, there is strong expression of hnRNP-C not only by intimal cells but also by medial smooth muscle cells. CONCLUSIONS: The H(2)O(2) responsive pre-mRNA binding protein hnRNP-C is up-regulated in atherosclerosis and in preatherosclerotic intimal hyperplasia in humans, supporting the hypothesis that H(2)O(2) is a regulator of vascular cell proliferation in these conditions. These data also suggest that hnRNP-C may be useful as a marker of vascular cell activation.

Identification of human hnRNP C1/C2 as a dengue virus NS1-interacting protein.

Dengue virus nonstructural protein 1 (NS1) is a key glycoprotein involved in the production of infectious virus and the pathogenesis of dengue diseases. Very little is known how NS1 interacts with host cellular proteins and functions in dengue virus-infected cells. This study aimed at identifying NS1-interacting host cellular proteins in dengue virus-infected cells by employing co-immunoprecipitation, two-dimensional gel electrophoresis, and mass spectrometry. Using lysates of dengue virus-infected human embryonic kidney cells (HEK 293T), immunoprecipitation with an anti-NS1 monoclonal antibody revealed eight isoforms of dengue virus NS1 and a 40-kDa protein, which was subsequently identified by quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) as human heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Further investigation by co-immunoprecipitation and co-localization confirmed the association of hnRNP C1/C2 and dengue virus NS1 proteins in dengue virus-infected cells. Their interaction may have implications in virus replication and/or cellular responses favorable to survival of the virus in host cells.

Heterogeneous nuclear ribonuclear protein C is increased in the celecoxib-induced growth inhibition of human oral squamous cell carcinoma.

Celecoxib is a selective inhibitor of cyclooxygenase-2 (COX-2) that is a critical factor in carcinogenesis, but precise mechanism of its action remains to be elucidated. Here we evaluated the inhibitory effect of celecoxib on cell growth of human oral squamous cell carcinoma (OSCC) YD-10(B), which was established to be used as in vitro OSCC model, and identified celecoxib-regulated protein by proteomics techniques. Celecoxib (IC(50)=37 microM) inhibited the growth of YD-10(B) cells with the decrease of COX-2 protein expression. Its inhibition could be linked in the arrest of G(1) phase with increased levels of p(27) protein, a specific CDK inhibitor. Using proteomics, the 10- to 20-fold increase of heterogeneous nuclear ribonuclear protein C (hnRNP C), which has been suggested to be related with the translation of p(27) mRNA, was observed in celecoxib-treated YD-10(B) cells. In summary, celecoxib has a potential to induce the protein expression of hnRNP C and its increase subsequently induce the translation of p(27) mRNA, which trigger the inhibition of cell growth via p(27)-regulated cell cycle arrest in YD-10(B) cells. In addition, YD-10(B) cells could be useful to study the pathological mechanism of OSCC.

Decreased brain levels of Lupus La protein and increased U5 small ribonucleoprotein-specific 40 kDa protein in fetal Down syndrome.

RNA-binding proteins have important role in the post-transcriptional regulation of gene expression. They are involved in events such as mRNA processing, transport, stability and translation. Studies in different species indicate that mutants with defect in RNA-binding proteins are defective in cell growth and differentiation. Expression of various RNA-binding proteins in prenatal life was analyzed by the highly sensitive two-dimensional electrophoresis coupled to matrix-assisted laser desorption ionization mass spectroscopy. No apparent change was obtained in levels of heterogeneous nuclear ribonucleoproteins (A3, C1-C2, L and M), nucleolin, polyadenylate binding protein-1, nuclear factor associated with double stranded RNA-2 and RNA-binding motif protein-4 between control and Down syndrome fetuses. By contrast, U5 small nuclear ribonucleoprotein-specific 40 kDa protein (p < 0.05) and Lupus La protein (p < 0.01) were significantly elevated and reduced, respectively in fetal DS. As a conclusion we can say U5 small nuclear ribonucleoprotein-specific 40 kDa protein appears to play important role in spliceosome assembly and disassembly, whereas La protein is involved in small nuclear riboncleoprotein complex biogenesis and transfer RNA maturation. Aberrant expression of these proteins points to the fact that dysregulation of the splicing and translation processes is apparent early in prenatal life, and may contribute to the defective growth and differentiation in Down syndrome.

Basal and hydrogen peroxide stimulated sites of phosphorylation in heterogeneous nuclear ribonucleoprotein C1/C2.

Hydrogen peroxide (H2O2) is a recently recognized second messenger, which regulates mammalian cell proliferation and migration. The biochemical mechanisms by which mammalian cells sense and respond to low concentrations of H2O2 are poorly understood. Recently, heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNP-C1/C2) was found to be rapidly phosphorylated in response to the application of low concentrations of H2O2 to human endothelial cells. Here, using tandem mass spectrometry, four sites of phosphorylation are identified in hnRNP-C1/C2, all of which are in the acidic C-terminal domain of the protein. Under resting conditions, the protein is phosphorylated at S247 and S286. In response to low concentrations of H2O2, there is increased phosphorylation at S240 and at one of the four contiguous serine residues from S225-S228. Studies using a recombinant acidic C-terminal domain of hnRNP-C overexpressed in Escherichia coli demonstrate that protein kinase CK2 phosphorylates hnRNP-C1/C2 at S247, while protein kinase A and several protein kinase C isoforms fail to phosphorylate the isolated domain. These findings demonstrate that the acidic C-terminal domain of hnRNP-C1/C2 serves as the site for both basal and stimulated phosphorylation, indicating that this domain may play an important role in the regulation of mRNA binding by hnRNP-C1/C2.