酵母双杂交系统筛选与RapGAP相互作用的蛋白质

目的筛选与Rap GAP相互作用的蛋白质,为进一步研究人源Rap1GAP介导的信号转导通路、揭示其与肿瘤的关系提供实验依据。方法选用与Rap1GAP同源的来自美丽线虫的Rap GAP作为饵蛋白,以来源于美丽线虫的c DNA文库作为靶蛋白,应用p PC97、p PC86组成的酵母双杂交系统筛选c DNA文库中与Rap GAP相互作用的蛋白质。结果通过营养缺陷平板(-LTH)筛选出63个拟似阳性菌落。经过Lac Z鉴定,19个菌落为阳性,其中7个为强阳性。提取来自19个酵母菌落中的重组DNA,经PCR扩增,12个菌落出现阳性结果。将该19个重组DNA分别电转化入DH5α细菌,涂板培养后,每板挑取4~10个克隆,通过Sal I和Not I双酶切鉴定进行阳性克隆筛选。将阳性克隆的重组DNA进行序列测定。测序结果与Gen Bank比较,其中4个克隆的DNA片段为Y39b6a基因片段、2个为Rap GAP、1个为苯丙氨酸-4-羟化酶、1个为细胞色素C氧化酶,还有1个DNA片段编码美丽线虫特有的小分子蛋白的基因片段,其余11个DNA片段不编码已知蛋白质。结论初步筛选出与Rap GAP相互作用的蛋白质,特别是其中有2个克隆为Rap GAP,提示Rap GAP可能以二聚体的方式存在。     

Down-regulation of the ubiquitin�Cproteasome proteolysis system by amino acids and insulin involves the adenosine monophosphate-activated protein kinase and mammalian target of rapamycin pathways in rat hepatocytes

The purpose of this work was to examine whether changes in dietary protein levels could elicit differential responses of tissue proteolysis and the pathway involved in this response. In rats fed with a high protein diet (55%) for 14?days, the liver was the main organ where adaptations occurred, characterized by an increased protein pool and a strong, meal-induced inhibition of the protein breakdown rate when compared to the normal protein diet (14%). This was associated with a decrease in the key-proteins involved in expression of the ubiquitin-proteasome and autophagy pathway gene and a reduction in the level of hepatic ubiquitinated protein. In hepatocytes, we demonstrated that the increase in amino acid (AA) levels was sufficient to down-regulate the ubiquitin proteasome pathway, but this inhibition was more potent in the presence of insulin. Interestingly, AICAR, an adenosine monophosphate-activated protein kinase (AMPK) activator, reversed the inhibition of protein ubiquination induced by insulin at high AA concentrations. Rapamycin, an mammalian target of rapamycin (mTOR) inhibitor, reversed the inhibition of protein ubiquination induced by a rise in insulin levels with both high and low AA concentrations. Moreover, in both low and high AA concentrations in the presence of insulin, AICAR decreased the mTOR phosphorylation, and in the presence of both AICAR and rapamycin, AICAR reversed the effects of rapamycin. These results demonstrate that the inhibition of AMPK and the activation of mTOR transduction pathways, are required for the down-regulation of protein ubiquitination in response to high amino acid and insulin concentrations.     

CD40L co-stimulation from CD8+ to CD4+ effector memory T cells supports CD4+ expansion

Effector memory T cells (T(EM)) have an important role in immunity against infection. However, little is known about the factors regulating T(EM) maintenance and proliferation. In this study, we investigated the role of direct interactions between CD4(+) and CD8(+) T cells (TC) for human T(EM) expansion. Proliferation of separated or mixed CD4(+) and CD8(+)T(EM) populations was analyzed after polyclonal stimulation in vitro. Compared to each isolated subset mixed T(EM) populations showed increased proliferation and expansion of both CD4(+) and CD8(+)T(EM) subpopulations. Combined activation of CD4(+) and CD8(+) memory T cells (Tmem) induced an increased expression of CD40L and CD40 on both populations. Subsequently, CD40/CD40L caused a bi-directional stimulation of CD40(+)CD4(+)T(EM) by CD40L(+)CD8(+)T(EM) and of CD40(+)CD8(+)T(EM) by CD40L(+)CD4(+)T(EM). Blocking of CD40L on activated CD8(+)T(EM) selectively inhibited proliferation of CD4(+)T(EM), while blocking of CD40L on CD4(+)T(EM) abrogated proliferation of CD8(+)T(EM). Taken together, we demonstrate for the first time that the expression of CD40L is exploited on the one hand by CD8(+)T(EM) to increase the proliferation of activated CD4(+)T(EM) and on the other hand by CD4(+)T(EM) to support the expansion of activated CD8(+)T(EM). Thus, efficient T(EM) expansion requires bi-directional interactions between CD4(+) and CD8(+)T(EM) cells.     

Assessing high affinity binding to HLA-DQ2.5 by a novel peptide library based approach

Here we report on a novel peptide library based method for HLA class II binding motif identification. The approach is based on water soluble HLA class II molecules and soluble dedicated peptide libraries. A high number of different synthetic peptides are competing to interact with a limited amount of HLA molecules, giving a selective force in the binding. The peptide libraries can be designed so that the sequence length, the alignment of binding registers, the numbers and composition of random positions are controlled, and also modified amino acids can be included. Selected library peptides bound to HLA are then isolated by size exclusion chromatography and sequenced by tandem mass spectrometry online coupled to liquid chromatography. The MS/MS data are subsequently searched against a library defined database using a search engine such as Mascot, followed by manual inspection of the results. We used two dodecamer and two decamer peptide libraries and HLA-DQ2.5 to test possibilities and limits of this method. The selected sequences which we identified in the fraction eluted from HLA-DQ2.5 showed a higher average of their predicted binding affinity values compared to the original peptide library. The eluted sequences fit very well with the previously described HLA-DQ2.5 peptide binding motif. This novel method, limited by library complexity and sensitivity of mass spectrometry, allows the analysis of several thousand synthetic sequences concomitantly in a simple water soluble format.     

Zn(II)-bis(cyclen) complexes and the imaging of apoptosis/necrosis

In vivo cell-death imaging is still a challenging issue. Until now, only (99m)Tc-labeled HYNIC-rh-annexin A5 has been extensively studied in clinical trials. In the ongoing search for an alternative imaging agent, we synthesized a series of fluorescent zinc-cyclen complexes as annexin A5 mimics and studied structural variations on the uptake behavior of cells undergoing apoptosis/necrosis. The number of cyclen chelators was varied and the spacer separating cyclen from the central scaffold was modified. Five zinc-cyclen complexes were labeled with fluorescein for flow cytometric studies and one was labeled with (18)F for in vivo applications. Jurkat cells were treated with staurosporine to induce apoptosis/necrosis, incubated with the fluorescein-labeled zinc complexes and analyzed them by flow cytometry. Fluorescent annexin A5 and propidium iodide were applied as reference dyes. Flow cytometry revealed greater accumulation of zinc-cyclen complexes in staurosporine treated cells. The uptake was contingent on the presence of zinc and the fluorescence intensity was dependent on the number of zinc-cyclen groups. Confocal laser scanning microscopy showed the {bis[Zn(cyclen)]}(4+) complex distributed throughout the cytosol different to annexin A5. Owing to the structural similarity of the bis-cyclen ligands with CXCR4 binding bis-cyclam derivatives the zinc-cyclen complex uptake was challenged with the meta derivative of AMD3100. Lack of uptake depletion in staurosporine treated cells ruled out measurable CXCR4 interaction. PET imaging using the (18)F labeled zinc-cyclen complex revealed significantly higher uptake in an irradiated Dunning R3327-AT1 prostate tumor as compared to the contralateral control tumor. PET imaging of a HelaMatu tumor model additionally showed an increased uptake after taxol treatment. It could be demonstrated that the fluorescent zinc-cyclen complexes offer potential as new agents for flow cytometry and microscopic imaging of cell death. In addition, the (18)F labeled analogue holds promise for in vivo applications providing informations about cell death after radiation therapy and cytostatic drug treatment.     

Comparative transcriptome profiling of amyloid precursor protein family members in the adult cortex

Background

SOX2 is a key gene implicated in maintaining the stemness of embryonic and adult stem cells. SOX2 appears to re-activate in several human cancers including glioblastoma multiforme (GBM), however, the detailed response program of SOX2 in GBM has not yet been defined.

Results

We show that knockdown of the SOX2 gene in LN229 GBM cells reduces cell proliferation and colony formation. We then comprehensively characterize the SOX2 response program by an integrated analysis using several advanced genomic technologies including ChIP-seq, microarray profiling, and microRNA sequencing. Using ChIP-seq technology, we identified 4883 SOX2 binding regions in the GBM cancer genome. SOX2 binding regions contain the consensus sequence wwTGnwTw that occurred 3931 instances in 2312 SOX2 binding regions. Microarray analysis identified 489 genes whose expression altered in response to SOX2 knockdown. Interesting findings include that SOX2 regulates the expression of SOX family proteins SOX1 and SOX18, and that SOX2 down regulates BEX1 (brain expressed X-linked 1) and BEX2 (brain expressed X-linked 2), two genes with tumor suppressor activity in GBM. Using next generation sequencing, we identified 105 precursor microRNAs (corresponding to 95 mature miRNAs) regulated by SOX2, including down regulation of miR-143, -145, -253-5p and miR-452. We also show that miR-145 and SOX2 form a double negative feedback loop in GBM cells, potentially creating a bistable system in GBM cells.

Conclusions

We present an integrated dataset of ChIP-seq, expression microarrays and microRNA sequencing representing the SOX2 response program in LN229 GBM cells. The insights gained from our integrated analysis further our understanding of the potential actions of SOX2 in carcinogenesis and serves as a useful resource for the research community.