Proteomic analysis of ubiquitin-proteasome effects: insight into the function of eukaryotic initiation factor 5A.

The global effect of ubiquitin-proteasome (UP) inhibitors on leukemic cell proteome was analysed. A total of 39 protein spots, affected by UP inhibitors, were identified, including 11 new apoptosis-associated proteins. They are involved in different cellular functions and four were associated with caspase-3 activation. Eukaryotic initiation factor 5A (eIF-5A) was identified in two spots; however, the peptide mass-fingerprinting for the accumulated one included a peptide with lysine50, indicating that hypusine formation was suppressed during UP inhibitor-induced apoptosis. Hypusine modification ensues immediately following translation of eIF-5A precursor, unless cells are treated with the modification inhibitors diaminoheptane. However, UP inhibitors induced a much stronger accumulation of unmodified eIF-5A compared to the effect of diaminoheptane. We further showed the unmodified eIF-5A was regulated in a proteasome-dependent manner. Inhibition of hypusine formation by diaminoheptane triggered apoptosis, but of particular interest is the finding that eIF-5A expression inhibition by antisense oligodeoxynucleotides significantly enhanced the stimulating effect of GM-CSF on cell growth. Therefore, the eIF-5A accumulation played important roles in the apoptosis induced by UP inhibitors. Moreover, hypusine inhibition in apoptosis was further revealed to be associated with the subcellular localization of eIF-5A. Our data pave the way to a better understanding of the mechanisms by which UP system has been linked to apoptosis.

Proteomic analysis of interleukin 6-induced differentiation in mouse myeloid leukemia cells.

Cytokine-induced differentiation of myeloid leukemia cells has important therapeutic implications, but the mechanism remains to be clarified. M1 cell, a mouse acute myeloid leukemia cell line, which underwent growth inhibition, terminal differentiation and apoptosis in response to IL-6, was selected as an experimental model to study on the molecular mechanisms of myeloid cell differentiation on a proteome-wide scale. Cell differentiation was evaluated by cell morphology and CD11b expression. With two-dimensional (2D) gel analyses, 17 protein spots showed obvious changes in quantity during the process of differentiation were found. With matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS) or/and nano-electrospray ionization MS/MS (ESI-MS/MS) analysis, 15 protein spots were identified. The mRNA levels of these 15 proteins during differentiation were also examined using a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. Except two proteins, the mRNA levels demonstrated similar expression patterns to what the proteomic analysis revealed. The identified proteins were known to be involved in different cellular functions, including protein synthesis, transcription, signal transduction, cell cycle control, cell rescue and defense, cellular organization, and metabolism. Notably, seven proteins were not described before to be involved in differentiation. Our data provide novel information for a better understanding of the mechanisms by which terminal differentiation of acute myeloid leukemia cells induced by IL-6.

Proteomic analysis of human acute leukemia cells: insight into their classification.

PURPOSE: French-American-British (FAB) classification of acute leukemia with genetic heterogeneity is important for treatment and prognosis. However, the distinct protein profiles that contribute to the subtypes and facilitate molecular definition of acute leukemia classification are still unclear. EXPERIMENTAL DESIGN: The proteins of leukemic cells from 61 cases of acute leukemia characterized by FAB classification were separated by two-dimensional electrophoresis, and the differentially expressed protein spots were identified by both matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and tandem electrospray ionization MS (ESI-MS/MS). RESULTS: The distinct protein profiles of acute leukemia FAB types or subtypes were successfully explored, including acute myeloid leukemia (AML), its subtypes (M2, M3, and M5) and acute lymphoid leukemia (ALL), which were homogeneous within substantial samples of the respective subgroups but clearly differed from all other subgroups. We found a group of proteins that were highly expressed in M2 and M3, rather than other subtypes. Among them, myeloid-related proteins 8 and 14 were first reported to mark AML differentiation and to differentiate AML from ALL. Heat shock 27 kDa protein 1 and other proteins that are highly expressed in ALL may play important roles in clinically distinguishing AML from ALL. Another set of proteins up-regulated was restricted to granulocytic lineage leukemia. High-level expression of NM23-H1 was found in all but the M3a subtype, with favorable prognosis. CONCLUSIONS: These data have implications in delineating the pathways of aberrant gene expression underlying the pathogenesis of acute leukemia and could facilitate molecular definition of FAB classification. The extension of the present analysis to currently less well-defined acute leukemias will identify additional subgroups.

[Nano-ESI-MS/MS identification on differentiation-associated proteins in M1 mouse myeloid leukemia cells induced by IL-6].

OBJECTIVE: To identify two differentiation-associated proteins induced by rhIL-6 in M1 mouse myeloid leukemia cells. METHODS: Protein spots were excised from 2-D gels and digested in-gel with trypsin. The trypsin lysis products were first analyzed by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) through peptide mass fingerprinting and then performed peptide sequencing by nano-electrospray ionization mass spectrometry/mass spectrometry (nano-ESI-MS/MS). The database search was finished with the Mascot search engine (http://www.matrixscience.co.uk) using the data processed through MaxEnt3 and MasSeq. RESULTS: The two proteins were not revealed by peptide mass fingerprint using MALDI-TOF-MS, while they were respectively identified as Destrin and Putative protein after the sequence of their trypic peptides were obtained by the nano-ESI-MS/MS techniques. CONCLUSION: Nano-ESI-MS/MS technique can successfully identify the two differentiation-associated proteins induced by rhIL-6 and has great advantage in protein analysis.

[Immunoregulation effects in vitro of the xenoprotein in combination with recombinant human granulocyte-macrophage colony stimulating factor and bacillus Calmette-Guerin].

This study was aimed to investigate the effects of xenogeneic antigen neu-Fc in combination with the recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) and Bacillus Calmette-Guerin (BCG) on the regulation of Th1 and Th2 immune response in vitro. The rat neu L2-S2 domain was engineered as a chimeric protein with human IgG Fc. The eukaryotic expression vector was constructed. The recombinant protein was stably expressed in CHO cells and purified by rProtein A Sepharose Fast Flow column. The recombinant protein was identified by SDS-PAGE and Western blot. Peripheral blood mononuclear cells (PBMNCs) were obtained by means of standard Ficoll separation from the blood of healthy donors. Neu-Fc-induced PBMNC proliferation was tested by MTT. The production of IL-12 and IL-10 was measured by ELISA. The results showed that the level of IL-12 decreased and IL-10 increased after PBMNCs were incubated with MCF-7 cultural supernatant. 10 nmol/L neu-Fc strongly induced the cell proliferation. Compared with neu-Fc or GM-CSF or BCG treatment alone, neu-Fc in combination with GM-CSF and BCG significantly stimulated IL-12 production and inhibited IL-10 production (p < 0.01). It is concluded that the neu-Fc can stimulate the proliferation activity of PBMNCs. neu-Fc, GM-CSF and BCG costimulation efficiently induces Th1 immune response.

Autoantibody profiling of Chinese patients with autoimmune hepatitis using immunoproteomic analysis.

In the present study, immunoproteomic analysis was utilized to systemically characterize global autoantibody profiles in autoimmune hepatitis (AIH). Sera from 21 patients with AIH and 15 healthy controls were analyzed for the antibody reactivity against the protein antigens of HepG2, a human hepatoma cell line. The lysates of HepG2 cells were separated by two-dimensional electrophoresis and then immunoblotted with each serum sample. Matrix-assisted laser desorption/ionization mass spectrometry or/and nanoelectrospray ionization MS/MS were then used to identify antigens, among which a bifunctional enzyme in mitochondrial, fumarate hydratase (FH), was further analyzed by ELISA using recombinant FH as a coating antigen. A total of 18 immunoreactive spots were identified as 13 proteins, 8 of which have not been reported in AIH. Immune reactivity to FH was detected in 66.67% of patients with AIH, 19.35% of patients with primary biliary cirrhosis (PBC), 12.31% of patients with chronic hepatitis B (CHB), 6.35% of patients with chronic hepatitis C (CHC), 11.32% of patients with systemic lupus erythematosus (SLE), and 3.57% of normal individuals. The differences of prevalence between AIH patients and healthy controls as well as other diseases were of statistical significance (P<0.001). These data demonstrate the serological heterogeneity in AIH and suggest the diversity of the mechanisms underlying AIH. FH, recognized mainly in AIH rather than in viral hepatitis and other autoimmune diseases, may have utility in improved diagnosis of AIH.

[Specific cytotoxicity of CTL which was induced by DC pulsed with mannosylated antigen].

AIM: To investigate the role of mannosylated tumor antigen in the course of anti-tumor response. METHODS: L2 domain of HER-2/neu (ErbB2) ectodomain was expressed in E.coli, purified and mannosylated. Dendritic cells(DCs) were induced from human peripheral blood mononuclear cell by GM-CSF and IL-4. The maturation and functional capacity of DCs pulsed with mannosylated L2 (mL2) protein was investigated. RESULTS: L2 protein could induce DC maturation, which was accompanied by elevated expression of MHC and costimulatory molecules. The effect of mL2 protein on DC maturation was more remarkable than that of non-mannosylated L2 protein. Furthermore, DCs pulsed with mL2 could stimulate high tumor cell lysis by CTL. CONCLUSION: Our experiment provide the foundation for the study of new tumor vaccine by mannosylation.

[Cloning and expression of soluble B lymphocyte stimulator].

AIM: To clone and express soluble B lymphocyte stimulator (sBLyS). METHODS: Total RNA was isolated from peripheral blood mononuclear cells, and used to synthesize cDNA by reverse transcription. sBLyS cDNA was amplified by PCR with specific primers and inserted into a prokaryotic expression vector pET-30a. Recombinant plasmid was transformed into E.coli strain BL21(DE3). sBLyS was expressed in E.coli, purified in vitro, and analyzed with peptide mass fingerprinting and Daudi cell proliferation assay. RESULTS: sBLyS cDNA was cloned. Peptide mass fingerprinting of purified BLyS matched with that of BLyS proteins. Purified sBLyS could stimulate Daudi cell proliferation in vitro. CONCLUSION: sBLyS with biological activity was successfully expressed and purified.

[The expression, renaturation, purification and preliminary identification of human IgE Cepsilon3-Cepsilon4].

AIM: To gain recombinant protein Cepsilon3-Cepsilon4 of IgE Fc (E34). METHODS: We cloned the gene coding human IgE Cepsilon3-Cepsilon4 (E34) and constructed an expression vector pET28a(+)-E34. The target protein was expressed as inclusion body in E.coli BL-21. Following renaturation and purification through a CM sephorose FF column, the soluble protein was acquired, and its binding ability to murine anti-hIgE mAb was identified by Western blot and ELISA. RESULTS: The cloned E34 gene was sequenced and proved by SDS-PAGE to be the same as reported sequence. SDS-PAGE analysis showed the relative molecular mass of E34 protein obtained was correct as predicted. Western blot and ELISA data revealed that it owned the epitope of binding to murine anti-hIgE mAb. CONCLUSION: The expression vector pET28a(+)-E34 has been successfully constructed and the target protein E34 recognized specifically by murine anti-hIgE mAb is obtained.

[Fusion expression and purification of recombinant ricin A-chain].

AIM: To prepare recombinant ricin A-chain(RTA) protein with high biological activity. METHODS: RTA gene containing KDEL sequence at the carboxyl terminal was cloned in pET32a vector, which was fused with thioredoxin. Furthermore, the constructed recombinant plasmid was transformed into the competent cell BL21, and induced with low concentration of IPTG (0.4 mmol/L) under low temperature (20 degrees Celsius). After binding to Co2+ chelating column, the expressed supernatants were eluted by applying imidazole solutions with the concentration from 20 to 100 mmol/L. The purified protein was identified with SDS-PAGE and Western blot analysis and was used to cleave supercoiled dsDNA. RESULTS: About 60 mg fusion proteins were obtained from 1,000 mL cultures , with purity above 90% and M(r) 45,000. The cleavage of supercoiled dsDNA demonstrated that RTA-Trx fusion proteins could significantly cleave supercoiled dsDNA as native RTA. CONCLUSION: The pET32a vector expression system can be used to produce a mass of soluble RTA-Trx fusion proteins with high biological activity.

A novel neutralizing monoclonal antibody against cell-binding polypeptide of ricin.

One strain of neutralizing monoclonal antibody (MAb) against cell-binding polypeptide of ricin, named 3E1, was generated efficiently. The antibody recognized the linearity epitope of RTB located in a toxin structure domain characterized by Western blotting. The safe period of mice for intraperitoneal injection of 100 microg of antibody was 20 min after intraperitoneal injection of 2 microg of Ricin (10 times LD50). The neutralizing MAb we obtained could be developed into an immunotherapeutic agent to counteract the use of ricin as a terrorist or biological warfare weapon. It might be useful, as well, for antibody-based prophylaxis.

Two-dimensional electrophoresis protein profiling as an analytical tool for human acute leukemia classification.

Two-dimensional electrophoresis (2-DE) was used to profile the proteins of leukemic cells from 61 cases of akute leukemia (AL) characterized by the French-American-British (FAB) classification. The differentially expressed protein spots were identified by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and electrospray ionization-tandem MS (ESI-MS/MS). The distinct protein profiles (DPPs) of AL FAB subtypes were explored successfully, including acute myeloid leukemia (AML), its subtypes (M2, M3, and M5), and acute lymphoid leukemia (ALL), which were homogeneous within different samples of the same subgroup but clearly differed from all other subgroups. We also found a group of proteins differentially expressed between AL cells and normal white blood cells. Among the DPPs of AL subtypes, some proteins have been reported, but most of them were first reported here to mark AML differentiation and to discriminate AML from ALL. These data show that 2-DE protein profiling could be used as an analytical tool for facilitating molecular definition of human AL classification and understanding the mechanism of leukemogensis, and the extension of the present analysis to the currently less well-defined AL will identify additional subgroups and may promote the identification of new targets for specific treatment approaches.

[Development of an anti-rTNF-alpha chimeric antibody].

AIM: To develop an human-mouse chimeric antibody against rTNF-alpha. METHODS: Expression vector of human-mouse chimeric antibody CZ12 gene was constructed by using VL and VH genes of mAb Z12 with neutralizing activity, and then COS7 cells were transfected. Supernatant containing CZ12 was detected by RT-PCR, ELISA, Western blot and in-vitro neutralization assay, respectively. RESULTS: The RT-PCR analysis showed that there was the transcription of human-mouse chimeric antibody mRNA in the transfected COS7 cells. ELISA and Western blot detection showed that the CZ12 could recognize specifically TNF-alpha. The in-vitro neutralization assay proved that CZ12 could neutralize the cytoxicity of TNF-alpha to L929 cells. CONCLUSION: human-mouse chimeric antibody CZ12 with neutralizing activity was successfully expressed in eukaryotic cells.

[The effect of eIF-5A on the G1-S in cell cycle regulation].

Eukaryotic initiation factor 5A (eIF-5A) contains an unusual amino acid, hypusine, which is formed post-translationally. Although eIF-5A and its hypusine modification are essential for eukaryotic cell viability, the precise physiological function of it has remained elusive. The aim of the study is to investigate how hypusine formation modulate the proliferation, cell cycle and apoptosis in leukaemia cells. The effects of 1,7-diaminoheptane (DAH), a potent inhibitor of deoxyhypusine synthase, on proliferation and cell viability of leukemia cell lines (Mo7e, TF-1 and THP-1) and MCF-7 cells, were investigated. eIF-5A expression level was detected after cell synchronization. The results showed that inhibition of cell proliferation by DAH was in a concentration-dependent manner while apoptosis was also induced at the same time. Upon treatment of the cell lines with DAH, cell growth was inhibited. Cell cycle analysis showed that DAH induced cell growth arrest at the G(1)-S boundary of the cell cycle. In synchronized MCF-7 cells, the expression level of eIF-5A peaked at G(1) phase but very low at S and G(2)/M phases. It is concluded that hypusine formation of eIF-5A exits in the regulation of cell cycle and the results suggest that eIF-5A is involved in the expression of proteins regulating transition of G(1)-S phase of cell cycle.

[Induction of Apoptosis in Leukemic Cells by Inhibiting the Ubiquitin-Proteasome Pathway and Its Possible Mechanism]

The ubiquitin-proteasome pathway is the principal mechanism for the degradation of short-lived proteins in eukaryotic cells. Recently, proteasome inhibitors have been shown to induce apoptosis in many kinds of human malignant cells. In this study, the mechanism of apoptosis induced by proteasome inhibitor in leukemic cells was examined. Evaluated by MTT assay, treatment of leukemic cells with Z-LLL-CHO, a reversible proteasome inhibitor, induced cell death in a dose-dependent manner. Appearance of the sub G(0)/G(1) fraction of cell cycle observed in flow cytometry assay suggested the induction of apoptosis, which was further proved by typical DNA ladder and morphological study. Western blot displayed the cleavage of bcl-2 into a shortened 22 kD fragment and the decrease in the levels of caspase-3 precursor. A highly sensitive colorimetric assay was employed and the elevation of caspase-3 activity was detected in both cell lines after treatment with Z-LLL-CHO. By comparison, these results showed that the leukemic cell line M-07e and KG-1a, which both express bcl-2 at a relative high level, had different susceptibility to undergo apoptosis induced by Z-LLL-CHO, which possibly due to their different levels of expression and activation of caspase-3 precursor, as well as their different degree of bcl-2 cleavage after treated by Z-LLL-CHO.

A novel eIF5A complex functions as a regulator of p53 and p53-dependent apoptosis.

Although eukaryotic translation initiation factor 5A (eIF5A) was originally designated as an "initiation factor," recent data have shown it to be also involved in apoptosis. However, the actual function of eIF5A in apoptosis is still unknown. In this study, we performed yeast two-hybrid screens to identify eIF5A-interacting proteins to help us understand the mechanisms of eIF5A. Our results demonstrated that eIF5A and syntenin could engage in a specific interaction both in vitro and in vivo and functioned collaboratively to regulate p53 activity. Our findings, for the first time, revealed a new biological activity for eIF5A as the regulator of p53. Overexpression of eIF5A or its EFP domain resulted in up-regulation of p53, and silencing eIF5A by small interfering RNA reduced the p53 protein level. Further analysis by reverse transcription PCR showed eIF5A-activated p53 transcription. The effect of eIF5A on p53 transcriptional activity was further demonstrated by the increasing expressions of p21 and Bax, well known target genes of p53. In contrast, a point mutant of eIF5A, hypusination being abolished, was revealed to be functionally defective in p53 up-regulation. Overexpression of eIF5A led to a p53-dependent apoptosis or sensitized cells to induction of apoptosis by chemotherapeutic agents. However, when eIF5A interacted with its novel partner, syntenin, the eIF5A-induced increase in p53 protein level was significantly inhibited. Therefore, eIF5A seems to be a previously unrecognized regulator of p53 that may define a new pathway for p53-dependent apoptosis, and syntenin might regulate p53 by balancing the regulation of eIF5A signaling to p53 for apoptosis.