Subcellular localization of GTPase of immunity-associated protein 2 / 北京大学学报(医学版)

OBJECTIVE@#To analyze the subcellular localization of GTPase of immunity-associated protein 2 (GIMAP2) for the further functional study.@*METHODS@#In the study, we first obtained the protein sequences of GTPase of immunity-associated protein 2 (GIMAP2) from National Center for Biotechnology Information (NCBI) database, and then performed a prediction analysis of its transmembrane structure, nuclear localization signal (NLS), nuclear export signal (NES) and subcellular localization through bioinformatics online tools. GIMAP2 gene amplified by PCR was inserted into the expression vector pQCXIP-mCherry-N1 and positive clones were selected by ampicillin resistance. After using methods to extract and purify, the sequenced recombinant plasmid pQCXIP-GIMAP2-mCherry, together with the retroviral packaging plasmids VSVG and Gag/pol, was transferred into HEK293FT cells by liposomes for virus packaging. The virus supernatant was collected 48 h after transfection and directly infected the human breast cancer cell line MDA-MB-436. Immunofluorescence staining was constructed to detect the localization of endogenous and exogenous GIMAP2 in MDA-MB-436 cells. Meanwhile, green fluorescent chemical dyes were used to label mitochondria, endoplasmic reticulum, and lipid droplets in living MDA-MB-436 cells stably expressing the GIMAP2-mCherry fusion protein. Images for the three dye-labeled organelles and GIMAP2-mCherry fusion protein were captured by super-resolution microscope N-SIM.@*RESULTS@#Bioinformatics analysis data showed that GIMAP2 protein composed of 337 amino acids might contain two transmembrane helix (TM) structures at the carboxyl terminus, of which TMs were estimated to contain 40-41 expected amino acids, followed by the residual protein structures toward the cytoplasmic side. NES was located at the 279-281 amino acids of the carboxyl terminus whereas NLS was not found. GIMAP2 might locate in the lumen of the endoplasmic reticulum. Sequencing results indicated that the expression vector pQCXIP-GIMAP2-mCherry was successfully constructed. Fluorescent staining confirmed that GIMAP2-mCherry fusion protein, co-localized well with endogenous GIMAP2, expressed successfully in the endoplasmic reticulum and on the surface of lipid droplets in MDA-MB-436 cells.@*CONCLUSION@#GIMAP2 localizes in the endoplasmic reticulum and on the surface of LDs, suggesting potential involvement of GIMAP2 in lipid metabolism.

Definition and function identification of nucleus export signal of BRD7 / 中南大学学报(医学版)

OBJECTIVE@#To localize and define the region of nucleus export signal (NES) on BRD7, and determine the role of this region in nucleus export of the external protein.@*METHODS@#Based on an in vitro expressed model of green fluorescence protein (GFP), we performed DNA walking analysis to set BRD7 into several sections according to the structural characteristics of BRD7, investigated the effect of different sections of BRD7 on nucleus export of GFP, defined the region of nucleus export signal sequence of BRD7, and further ascertained the content of amino acids in BRD7 and potential localization of BRD7 NES by bioinformatics.@*RESULTS@#B7C1 fragments ranged from aa219 to aa450 in BRD7 were found to target the external protein GFP into the cytoplasm detected by GFP direct fluorescence, which could be inhibited by NES inhibitor Leptomycin B (LMB). This region was rich in hydrophobic amino acid residues but no typical NES with characteristics of leucine-rich sequence by bioinformatics.@*CONCLUSION@#The region from aa219 to aa450 is primarily defined as an atypical NES in BRD7.

Analysis of the nuclear localization signal of TRF1 in non-small cell lung cancer

Several studies revealed a similar down-regulation of telomeric repeat binding factor 1 (TRF1) in tumors. We have previously reported the TRFl expression levels were down-regulation in non-small cell lung cancer (NSCLC). The regulation of TRFl localization is proposed to be important for the function and expression. The nuclear localization signal (NLS) and nuclear export signal (NES) are often important clues to localization of protein. The objective of the present study was to investigate the NLS and NES of TRFl in NSCLC patients. Thirty (30) patients with NSCLCs had undergone radical operations in The First Affiliated Hospital, College of Medicine, Zhejiang University. DNA sequences of NLSs and NESs were amplified by PCR. The PCR products were analyzed by DNA sequencing. There were four NLSs of the TRFl protein, including two monopartite and two bipartite NLSs. The NLSs sequences were included in 337KKERRVGTPQSTKKKKESRR356. The exon 8 and exon 9 of TRFl DNA were covered the NLS sequences. The sequences of predicted NESs were 11WMLDFLCLSL86 and 174NLLKLQALAV183, respectively. The exon 1, exon 3 and exon 4 of TRFl were covered the NES sequences. In NSCLCs, there was no a mutation, deletion, or substitution in NLS and NES of TRFl. We conclude that the NLS and NES sequences in NSCLCs patients did not have mutations. Down-expression of TRFl does not indicate gene mutation of NLS and NES in NSCLCs.

Expression of nuclear export factor CRM1 and p27 in glioma / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the expression of nuclear export factor CRM1, Ser10-phosphorylated p27 and p27 in human gliomas.</p><p><b>METHODS</b>The expression of CRM1, Ser10-phosphorylated p27 and p27 were investigated in 70 cases of human gliomas and 10 specimens of the normal brain tissue by immunohistochemical technique and Western blot.</p><p><b>RESULTS</b>There were significant differences on the expression levels of CRM1, Ser10-phosphorylated p27 and p27 among normal brain tissue, gliomas of grades II and gliomas of grades III plus IV (P < 0.01). The expression of CRM1 in gliomas was inversely correlated with the expression of p27 (r(s) = -0.727, P < 0.01) and positively correlated with the expression of Ser10-phosphorylated p27 (r(s) = 0.954, P < 0.01) and Ki-67 (r(s) = 0.799, P < 0.01). Moreover, the expression of Ser10-phosphorylated p27 was inversely correlated with p27 (r(s) = -0.744, P < 0.01) and positively correlated with Ki-67 (r(s) = 0.785, P < 0.01).</p><p><b>CONCLUSIONS</b>CRM1, through recognizing and binding with Ser10-phosphorylated p27, may promote moving of p27CRM1 from its original locating sites; act as a critical signaling component in the proliferative process of glioma cells and then, plays an important role in the development of gliomas.</p>

Expression and Packaging of a Human Endogenous Retrovirus-K Genomic DNA Clone

Human contains large number of human endogenous retroviruses (HERVs) in its genome. One of the HERV families, HERV-K, entered human genome most recently and includes many members with full-length intact proviruses. Normally, these proviruses do not express but infrequently they seem to express in cancers or autoimmune disease patients. To investigate expression mechanisms of these endogenous retroviruses, a DNA copy of HERV-K was cloned and its expression was studied. The transfection of the full-length clone into human cell lines did not produce any detectable viral capsid protein, Gag, and the transcription from its own promoter in LTR was extremely poor. The transcription was less than 10 percent compare to the exogenous retrovirus. However, when the Gag coding region was cloned under CMV promoter, Gag could be expressed efficiently and secreted as particles, probably virus like particles. The efficient expression also required a nuclear export signal. The expressed Gag could also package its own genomic RNA. These results indicate that the LTR of HERV-K is normally not active but its genes have a potential to express and possibly produce infectious particles.