Handong Wei

Proteome Analysis of Hepatocellular Carcinoma by Two-dimensional Difference Gel Electrophoresis Novel Protein Markers in Hepatocellular Carcinoma Tissues

Hepatocellular carcinoma (HCC) is a highly malignant tumor, and chronic infection with hepatitis B virus is one of its major risk factors. To identify the proteins involved in HCC carcinogenesis, we used two-dimensional fluorescence DIGE to study the differentially expressed proteins in tumor and adjacent nontumor tissue samples. Samples from 12 hepatitis B virus-associated HCC patients were analyzed. A total of 61 spots were significantly up-regulated (ratio ≥ 2, p ≤ 0.01) in tumor samples, whereas 158 spots were down-regulated (ratio ≤ −2, p ≤ 0.01). Seventy-one gene products were identified among these spots. Members of the heat shock protein 70 and 90 families were simultaneously up-regulated, whereas metabolism-associated proteins were decreased in HCC samples. The down-regulation of mitochondrial and peroxisomal proteins in these results suggested loss of special organelle functions during HCC carcinogenesis. Four metabolic enzymes involved in the methylation cycle in the liver were down-regulated in HCC tissues, indicating S-adenosylmethionine deficiency in HCC. Two gene products, glyceraldehyde-3-phosphate dehydrogenase and formimidoyltransferase-cyclodeaminase, were identified from inversely altered spots, suggesting that different isoforms or post-translational modifications of these two proteins might play different roles in HCC. For the first time, the overexpression of Hcp70/Hsp90-organizing protein and heterogeneous nuclear ribonucleoproteins C1/C2 in HCC tissues was confirmed by Western blot and then by immunohistochemistry staining in 70 HCC samples, suggesting their potential as protein tumor markers. In summary, we profiled proteome alterations in HCC tissues, and these results may provide useful insights for understanding the mechanism involved in the process of HCC carcinogenesis.

Characterization and expression of three novel differentiation-related genes belong to the human NDRG gene family.

NDRG1(N-Myc downstream regulated) is upregulated during cell differentiation, repressed by N-myc and c-myc in embryonic cells, and suppressed in several tumor cells. A nonsense mutation in the NDRG1 gene has been reported to be causative for hereditary motor and sensory neuropathy-Lom (HMSNL), indicating that NDRG1 functions in the peripheral nervous system necessary for axonal survival. Here, we cloned three human cDNAs encoding NDRG2 (371aa), NDRG3 (375aa) and NDRG4 (339aa), which are homologous to NDRG1. These three genes, together with NDRG1, constitute the NDRG gene family. The phylogenetic analysis of the family demonstrated that human NDRG1 and NDRG3 belong to a subfamily, and NDRG2 and NDRG4 to another. At amino acid (aa) level, the four members share 53–65% identity. Each of the four proteins contains an α/β hydrolase fold as in human lysosomal acid lipase. Expression of the fusion proteins NDRG2/GFP, NDRG3/GFP and NDRG4/GFP in COS-7 cells showed that all of them are cytosolic proteins. Based on UniGene cluster analysis, the genes NDRG2, NDRG3 and NDRG4 are located at chromosome 14q11.1–11.2, 20q12–11.23 and 16q21–22.1, respectively. Northern and dot blot analysis shows that all of the three genes are highly expressed in adult brain and almost not detected in the eight human cancer lines. In addition, in contrast to the relatively ubiquitous expression of NDRG1, NDRG2 is highly expressed in adult skeletal muscle and brain, NDRG3 highly expressed in brain and testis, and NDRG4 specifically expressed in brain and heart, suggesting that they might display different specific functions in distinct tissues.

Proteomic analysis on structural proteins of Severe Acute Respiratory Syndrome coronavirus

Recently, a new coronavirus was isolated from the lung tissue of autopsy sample and nasal/throat swabs of the patients with Severe Acute Respiratory Syndrome (SARS) and the causative association with SARS was determined. To reveal further the characteristics of the virus and to provide insight about the molecular mechanism of SARS etiology, a proteomic strategy was utilized to identify the structural proteins of SARS coronavirus (SARS‐CoV) isolated from Vero E6 cells infected with the BJ‐01 strain of the virus. At first, Western blotting with the convalescent sera from SARS patients demonstrated that there were various structural proteins of SARS‐CoV in the cultured supernatant of virus infected‐Vero E6 cells and that nucleocaspid (N) protein had a prominent immunogenicity to the convalescent sera from the patients with SARS, while the immune response of spike (S) protein probably binding with membrane (M) glycoprotein was much weaker. Then, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) was used to separate the complex protein constituents, and the strategy of continuous slicing from loading well to the bottom of the gels was utilized to search thoroughly the structural proteins of the virus. The proteins in sliced slots were trypsinized in‐gel and identified by mass spectrometry. Three structural proteins named S, N and M proteins of SARS‐CoV were uncovered with the sequence coverage of 38.9, 93.1 and 28.1% respectively. Glycosylation modification in S protein was also analyzed and four glycosylation sites were discovered by comparing the mass spectra before and after deglycosylation of the peptides with PNGase F digestion. Matrix‐assisted laser desorption/ionization‐mass spectrometry determination showed that relative molecular weight of intact N protein is 45 929 Da, which is very close to its theoretically calculated molecular weight 45 935 Da based on the amino acid sequence deduced from the genome with the first amino acid methionine at the N‐terminus depleted and second, serine, acetylated, indicating that phosphorylation does not happen at all in the predicted phosphorylation sites within infected cells nor in virus particles. Intriguingly, a series of shorter isoforms of N protein was observed by SDS‐PAGE and identified by mass spectrometry characterization. For further confirmation of this phenomenon and its related mechanism, recombinant N protein of SARS‐CoV was cleaved in vitro by caspase‐3 and ‐6 respectively. The results demonstrated that these shorter isoforms could be the products from cleavage of caspase‐3 rather than that of caspase‐6. Further, the relationship between the caspase cleavage and the viral infection to the host cell is discussed.

Intracrine hepatopoietin potentiates AP-1 activity through JAB1 independent of MAPK pathway

Many growth factors and cytokines are involved in liver regeneration. Of them, only hepatopoietin (HPO)/ALR (augmenter of liver regeneration) is a specifically hepatotrophic factor originally identified from the cytosol of regenerating or hyperplastic hepatic cells. Previous reports indicate that extracellular HPO triggers the MAPK pathway by binding its specific receptor on the cell surface. However, its function in the cytosol of hepatocytes is unclear. Here we identified that JAB1 (Jun activation domain‐binding protein 1), a co‐activator of AP‐1, which is essential for liver regeneration, specifically interacts with intracellular HPO. JAB1 colocalizes with HPO in nuclei of hepatic cells or COS‐7 cells. As an intracrine factor, the intracellular function of HPO is to increase c‐Jun phosphorylation independent of c‐Jun amino‐terminal kinase (JNK), extracellular signal‐regulated kinase (ERK) ‐1 and ‐2, and leads to potentiation of JAB1‐mediated AP‐1 activation. Amino acids 1‐63 of HPO molecule are sufficient to bind to JAB1, but the full‐length HPO is necessary for its intracellular signaling. Taken together, these results elucidate a novel mechanism of intracrine cytokine signaling by specifically modulating the AP‐1 pathway through JAB1, in a MAPK‐independent fashion.

Identification and Characterization of Receptor for Mammalian Hepatopoietin That Is Homologous to Yeast ERV1

Hepatopoietin (HPO) is a novel polypeptide mitogen specific for hepatocytes and hepatoma cell lines, which is derived from liver and supports its regeneration. To determine whether HPO acts via a receptor-based signal transduction, recombinant human hepatopoietin was labeled by iodination and used to characterize its binding activity by specific displacement test and Scatchard analysis in primarily cultured rat hepatocytes and human hepatoma Hep-G2 cells. The binding was saturable and specific because it was replaceable by HPO but not by epidermal growth factor, transforming growth factor-α, or insulin. Scatchard analysis indicated the presence of a single class of high affinity receptor with dissociation constant (K d ) of 2 and 0.7 pm, and a receptor density of about 10,000 sites/cell and 55,000 sites/cell in the rat hepatocytes and human hepatoma cells, respectively. TheK d values were consistent with the half-maximum dose of HPO activity. Affinity cross-linking of the receptor with125I-HPO revealed a polypeptide of molecular mass approximately 90 kDa by SDS-polyacrylamide gel electrophoresis. Thus, the molecular mass of the HPO receptor was calculated to be about 75 kDa. These data demonstrated the existence of an HPO receptor in hepatocytes and hepatoma cells, which may account for biological effect.

Role for the pleckstrin homology domain- containing protein CKIP-1 in AP-1 regulation and apoptosis

The oncogenic transcription factor c‐Jun plays an important role in cell proliferation, transformation and differentiation. All identified c‐Jun‐interacting proteins are localized to the nucleus or cytoplasm and function in their intact forms. Here we show that the pleckstrin homology domain‐containing protein CKIP‐1 (casein kinase 2‐interacting protein‐1) functions as a plasma membrane‐bound AP‐1 regulator. During apoptosis, CKIP‐1 is cleaved by caspase‐3 and translocated to the cytoplasm and then to the nucleus. C‐terminal fragments of cleaved CKIP‐1 strongly repress AP‐1 activity. Importantly, CKIP‐1 overexpression promotes apoptosis by forming a positive feedback loop between CKIP‐1 and caspase‐3. RNA interference of CKIP‐1 or overexpression of c‐Jun attenuates the sensitivity to apoptosis, indicating a novel role of CKIP‐1 in apoptosis. CKIP‐1 is the first case of a c‐Jun‐interacting protein that regulates AP‐1 activity via caspase‐3‐dependent cleavage and translocation.

Sample preparation method for isolation of single‐cell types from mouse liver for proteomic studies

It becomes increasingly clear that separation of pure cell populations provides a uniquely sensitive and accurate approach to protein profiling in biological systems and opens up a new area for proteomic analysis. The method we described could simultaneously isolate population of hepatocytes (HCs), hepatic stellate cells (HSCs), Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs) by a combination of collagenase‐based density gradient centrifugation and magnetic activated cell sorting with high purity and yield for the first time. More than 98% of the isolated HCs were positive for cytokeratin 18, with a viability of 91%. Approximately 97% of the isolated HSCs expressed glial fibrillary acidic protein with a viability of 95%. Nearly 98% of isolated KCs expressed F4/80 with a viability of 94%. And the purity of LSECs reached up to 91% with a viability of 94%. And yield for HCs, HSCs, LSECs and KCs were 6.3, 1.3, 2.6 and 5.0 million per mouse. This systematic isolation method enables us to study the proteome profiling of different types of liver cells with high purity and yield, which is especially useful for sample preparation of Human Liver Proteome Project.

A Dataset of Human Fetal Liver Proteome Identified by Subcellular Fractionation and Multiple Protein Separation and Identification Technology

A high throughput process including subcellular fractionation and multiple protein separation and identification technology allowed us to establish the protein expression profile of human fetal liver, which was composed of at least 2,495 distinct proteins and 568 non-isoform groups identified from 64,960 peptides and 24,454 distinct peptides. In addition to the basic protein identification mentioned above, the MS data were used for complementary identification and novel protein mining. By doing the analysis with integrated protein, expressed sequence tag, and genome datasets, 223 proteins and 15 peptides were complementarily identified with high quality MS/MS data.

Identification, Characterization, and Functional Study of the Two Novel Human Members of the Semaphorin Gene Family

We cloned two novel human transmembrane semaphorins, (HSA)SEMA6C and (HSA)SEMA6D, that belong to the class VI subgroup of the semaphorin family. The genes for SEMA6C and SEMA6D are mapped on chromosome 1q12–21.1 and 15q21.1, respectively. Among the adult tissues, SEMA6C is expressed only in skeletal muscle, whereas SEMA6D is expressed abundantly in kidney, brain, and placenta and moderately in the heart and skeletal muscles. During murine development, neither SEMA6C nor SEMA6D was expressed in embryonic day 10.5 (E10.5) embryos, but both were highly expressed in the areas of the lateral ventricle, the striatum, the wall of the midbrain, the pons/midbrain junction, and the choroid plexus of E13 embryos. Were neurons, neither axons nor astrocytes, highly expressed both semaphorins. Three isoforms of SEMA6C and five isoforms of SEMA6D derived from alternative splicing were identified, and their expression was regulated in a tissue- and development-dependent manner. Deletion analysis indicated that a sema domain and a PSI domain are integrally necessary for correct post-translation modification and subcellular localization. The extracellular domain of SEMA6C inhibited axonal extension of nerve growth factor-differentiated PC12 cells and induced the growth cone collapse of chicken dorsal root ganglion, rat hippocampal neurons, and rat cortical neurons in a dose-responsive manner. SEMA6D acted like SEMA6C except it had no significant effect on the growth cones of rat cortical neurons.

Quantitative proteome analysis of HCC cell lines with different metastatic potentials by SILAC.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and metastasis is the main cause for treatment failure and high fatality of HCC. In order to make further exploration into the mechanism of HCC metastasis and to search for the candidates of diagnostic marker and therapeutic target, stable‐isotope labeling by amino acids in cell culture (SILAC) technique was employed to conduct differential proteome analysis on HCC cell lines – MHCC97L and HCCLM6 with low and high metastatic potentials. In total, 2335 reliable proteins were identified using LTQ‐FT mass spectrum, among which 91 proteins were upregulated and 61 proteins were downregulated in HCCLM6. Most of the upregulated proteins were involved in adherence, morphogenesis, and lipid synthesis, while lots of the downregulated proteins were involved in electron transport, which might be crucial for HCC metastasis. Six dysregulated proteins were validated by Western blotting in the cell lines. Interestingly, the upregulation of solute carrier family 12 member 2 (SLC 12A2) and protein disulfide‐isomerase A4 (PDIA4) were further confirmed in the culture supernatants by Western blotting and in the sera of HCC patients with different metastatic potentials by ELISA. Our study provided not only the valuable insights into the HCC metastasis mechanisms but also the potential candidate biomarkers for prediction of HCC metastasis.