SAK-HV Decreases the Self-Ubiquitination of MEKK1 to Promote Macrophage Proliferation via MAPK/ERK and JNK Pathways

SAK-HV is an anti-atherosclerosis recombinant fusion protein developed by our lab. Our study determined that SAK-HV promoted macrophage proliferation, of which the mechanism was explored by both RAW264.7 cells and primary macrophages. Mass spectrometric analysis and co-immunoprecipitation were combined to screen the SAK-HV-interacting proteins in RAW264.7 cells. Confocal microscopy was adopted to detect the localization of SAK-HV in cells. The results indicated that SAK-HV triggered macrophage proliferation via the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK) pathways by its SAK-mutant functional domain. We screened out Uba1 as the SAK-HV-interacting protein in the RAW264.7 cells and discovered their co-localization in the cytoplasm and nucleus. Inhibiting Uba1 significantly decreased the SAK-HV-induced macrophage proliferation. Thus, we postulated an attractive model of ubiquitination, in which the interactions between Uba1 and specific E2 enzymes are blocked by its interaction with SAK-HV. Based on this model, we detected the decreased self-ubiquitination of MEKK1 after SAK-HV treatment and concluded that SAK-HV inhibits the self-ubiquitination of MEKK1 via its SAK-mutant functional domain to activate MAPK/ERK and JNK pathways, promoting macrophage proliferation. This conclusion highly supported our hypothesized model of ubiquitination at the level of Uba1, which may represent a novel paradigm to promote macrophage proliferation by using the E1 enzyme (Uba1) as a switch

Identification and characterization of MT-1X as a novel FHL3-binding partner.

Four and a half LIM domain protein 3 (FHL3) is a member of the FHL protein family that plays roles in the regulation of cell survival, cell adhesion and signal transduction. However, the mechanism of action for FHL3 is not yet clear. The aim of present study was to identify novel binding partner of FHL3 and to explore the underlying mechanism. With the use of yeast two-hybrid screening system, FHL3 was used as the bait to screen human fetal hepatic cDNA library for interacting proteins. Methionine-1X was identified as a novel FHL3 binding partner. The interaction between FHL3 and the full length MT-1X was further confirmed by yeast two-hybrid assay, co-immunoprecipitation and GST pull-down assays. Furthermore,the result demonstrated that MT-1X knockdown promoted the FHL3-induced inhibitory effect on HepG2 cells by regulating FHL3-mediated Smad signaling and involving in the modulation the expression of G2/M phase-related proteins through interaction with FHL3. These findings suggest that functional interactions between FHL3 and MT-1X may provide some clues to the mechanisms of FHL3-regulated cell proliferation

Angiogenin promotes U87MG cell proliferation by activating NF-κB signaling pathway and downregulating its binding partner FHL3.

Angiogenin (Ang) is known to induce cell proliferation and inhibit apoptosis by cellular signaling pathways and its direct nuclear functions, but the mechanism of action for Ang in astrocytoma is not yet clear. Astrocytoma is the most frequent one among various neurogliomas, of which a subtype known as glioblastoma multiforme (GBM) is the most malignant brain glioma and seriously influences the life quality of the patients. The expression of Ang and Bcl-xL were detected in 28 cases of various grades of astrocytoma and 6 cases of normal human tissues by quantitative real-time PCR. The results showed that the expression of Ang and Bcl-xL positively correlated with the malignant grades. Cytological experiments indicated that Ang facilitated human glioblastoma U87MG cell proliferation and knock-down of endogenous Ang promoted cell apoptosis. Furthermore, Ang activated NF-κB pathway and entered the U87MG cell nuclei, and blocking NF-κB pathway or inhibiting Ang nuclear translocation partially suppressed Ang-induced cell proliferation. The results suggested that Ang participated in the regulation of evolution process of astrocytoma by interfering NF-κB pathway and its nucleus function. In addition, four and a half LIM domains 3 (FHL3), a novel Ang binding partner, was required for Ang-mediated HeLa cell proliferation in our previous study. We also found that knockdown of FHL3 enhanced IκBα phosphorylation and overexpression of Ang inhibited FHL3 expression in U87MG cells. Together our findings suggested that Ang could activate NF-κB pathway by regulating the expression of FHL3. In conclusion, the present study established a link between Ang and FHL3 proteins and identifies a new pathway for regulating astrocytoma progression

Additional file 1: Table S1. of Recombinase polymerase amplification combined with a lateral flow dipstick for rapid and visual detection of Schistosoma japonicum

Assessed the diagnostic validity of LFD-RPA assay with 45 clinical samples and compared with that of IHA and ELISA assays. (DOCX 15 kb

The interaction between full length MT-1X and FHL3 by yeast two hybrid assay.

<p>(A) Identification of recombinant plasmid pAS2-1/FHL3d4 by restriction analysis. M:DNA marker; Lane1: Digested pAS2-1/FHL3d4 plasmid with <i>Nde</i>I and <i>Eco</i>RI. (B) Identification of recombinant plasmid pACT2/MT-1X by restriction analysis. M:DNA marker; Lane1-4: Digested pACT2/MT-1X plasmid with <i>Bam</i>HI and <i>Eco</i>RI. (C) Detection of MT-1X auto-activation by yeast two hybrid analysis. Line1:1,2: positive control; Line1: 3,4: negative control; Line2:1-5:transform pACT2/MT-1X. (D) Coloration analysis to verify the interaction between full length MT-1X and FHL3. Line1:1,2:positive control; Line1: 3,4: negative control. Line2:1–7: Co-transform pAS2-1/FHL3d4 and pACT2/MT-1X.</p

GST pull-down assay demonstrating <i>in vitro</i> binding between MT-1X and FHL3.

<p>(A) Purification of GST-FHL3 protein. Purification of GST protein (Lane1) and GST-FHL3 protein (Lane2) by Western blot analysis using anti-GST monoclonal antibody. (B) GST pull-down assay. Lane1 demonstrated the signal from purified His-MT-1X. GST protein and GST-FHL3 fusion protein were mixed with purified His-MT-1X (Lane2 and 3) and precipitated with Glutathione Agarose followed by Western blot analysis.</p

MT-1X regulated expression of cell cycle-related proteins.

<p>(A) Extracts from HepG2 cells transfected with MT-1X siRNA were used for Western blot analysis with the indicated antibodies. (B) Extracts from HepG2 cells transfected with MT-1X expression vector were used for Western blot analysis with the indicated antibodies.</p

The interaction between FHL3 and MT-1X <i>in vivo</i>.

<p>293T cells were transfected by Myc-MT-1X with HA-FHL3. Lane 1: lysate of cells transfected with Myc-MT-1X and HA-FHL3. 2: IP of cells with anti-IgG monoclonal antibody as negative control. 3: IP of cells with anti-Myc monoclonal antibody; After 30 h, total cell lysates were analyzed before co-immunoprecipitation to verify expression of Myc-MT-1X and HA-FHL3 (lane 1). After co-immunoprecipitation with anti-Myc tag antibody, MT-1X was detected only in the presence of immune complexes of Myc-FHL3 (lane 3).</p