视黄酸诱导人神经母细胞瘤SK-N-SH细胞分化过程中核基质蛋白组成的变化

在鉴定视黄酸(retinoic acid, RA)诱导人神经母细胞瘤SK-N-SH细胞分化的基础上,应用免疫细胞化学、选择性抽提和蛋白质组学分析技术,对SK-N-SH细胞诱导分化过程中核基质蛋白组成变化进行了系统研究.实验结果显示,经1 μmol/L RA处理后SK-N-SH细胞呈极性状,伸出较长的轴突样突起,胞体逐渐变小变圆.免疫细胞化学结果显示,处理后神经细胞特异表达的蛋白synaptophysin、NSE、MAP2的表达量都较对照组有明显增强.双向凝胶电泳分析显示,在RA诱导SK-N-SH细胞分化前后存在52个差异表达的核基质蛋白,经质谱分析,鉴定了其中的41个蛋白.蛋白印迹杂交进一步确证了诱导分化差异表达核基质蛋白中nucleophosmin和prohibitin等的表达变化.研究结果表明,1 μmol/L RA对SK-N-SH细胞具有显著的诱导分化作用,在SK-N-SH细胞分化过程中,其核基质蛋白组成发生了明显变化.这些变化对于揭示人神经母细胞瘤细胞癌变与逆转机制和肿瘤细胞增殖与分化调控机理均有十分重要的意义,从而为研究神经系统正常发育过程及神经系统疾病的发病机理提供科学依据.     

hnRNP A2/B1在人永生化表皮HaCaT细胞凋亡过程中的表达及其调控作用研究

该文以姜黄素诱导人永生化表皮HaCaT细胞凋亡为基础,对hnRNP A2/B1在核基质中的存在、分布及其与细胞凋亡相关基因产物的共定位及相互作用关系进行了研究。蛋白质印迹结果显示,hnRNP A2/B1存在于HaCaT细胞核基质蛋白组分中,在经过姜黄素处理后,表达下调;激光共聚焦显微镜观察显示,hnRNP A2/B1在HaCaT细胞中分别与Fas、p53和Bax等基因产物具有共定位关系,姜黄素处理后其共定位区域出现由核膜或核仁向胞质转移的趋势。GST pull-down实验证实,hnRNPA2/B1分别与Fas、p53和Bax有直接相互作用关系。结果表明,hnRNPA2/B1作为一种核基质蛋白,通过与细胞凋亡相关基因产物的相互作用参与HaCaT细胞的凋亡诱导调控过程,这对深入认识核基质蛋白在细胞凋亡过程中的调控机制具有重要意义。     

姜黄素诱导人成骨肉瘤MG-63细胞凋亡过程中hnRNP A2/B1表达与定位

姜黄素(curcumin)诱导处理的人成骨肉瘤MG-63细胞,在光镜和电镜观察细胞凋亡的基础上,对hnRNP A2/B1在核基质中存在、分布及其与凋亡相关基因产物在MG-63细胞中的共定位关系进行了研究.经姜黄素处理后,细胞出现染色质凝聚、细胞核固缩、凋亡小体等典型的细胞凋亡形态特征;双向凝胶电泳和质谱鉴定结果显示,hnRNP A2/B1存在于MG-63细胞核基质蛋白组分中,在姜黄素处理后细胞核基质蛋白中表达下调.蛋白质印迹杂交结果,证实hnRNP A2/B1在姜黄素处理前后的MG-63细胞核基质蛋白中的存在及其表达下调变化.免疫荧光显微镜观察显示,hnRNP A2/B1定位于MG-63细胞核基质纤维上,经姜黄素处理后出现分布位置与表达水平变化.激光扫描共聚焦显微镜的观察结果显示,hnRNP A2/B1在MG-63细胞凋亡过程中与Bax、Bcl-2、Fas和p53等基因产物具有共定位关系,且其共定位区域发生了变化.研究结果证实了hnRNP A2/B1定位于核基质纤维上,是一种核基质蛋白,在姜黄素诱导人成骨肉瘤MG-63凋亡过程中的表达与分布变化及其与凋亡相关基因的关系显然对MG-63细胞凋亡具有重要影响,这为深入揭示肿瘤细胞凋亡的机制提供了重要科学依据和深入探索的新方向.     

Localization of Prohibitin in the Nuclear Matrix and Alteration of Its Expression During Differentiation of Human Neuroblastoma SK-N-SH Cells Induced by Retinoic Acid

The nuclear matrix-intermediate filament system of human neuroblastoma SK-N-SH cells before and after retinoic acid (RA) treatment was selectively extracted and the distribution of prohibitin (PHB) in the nuclear matrix, as well as its colocalization with related genes, was observed. Results of two-dimensional gel electrophoresis (2-DE), mass spectrometry (MS) identification, and protein immunoblotting all confirm that PHB was present in the components of SK-N-SH nuclear matrix proteins and was down-regulated after RA treatment. Immunofluorescence microscopy observations show that PHB was localized in the nuclear matrix and its distribution was altered due to RA treatment. Laser confocal microscopy results reveal that PHB colocalized with the expression products of c-myc, c-fos, p53, and Rb, but the colocalization region was altered after RA treatment. Our results prove that PHB is a nuclear matrix protein and is localized in nuclear matrix fibers. The distribution of PHB in SK-N-SH cells and its colocalization with related proto-oncogenes and tumor suppressor genes suggest that PHB plays pivotal roles in the differentiation of SK-N-SH cells and deserves further study.     

HMBA诱导人肝癌SMMC-7721 细胞分化过程中 Nucleophosmin 的表达与定位变化

通过选择性抽提经环六亚甲基双乙酰胺（hexamethylene bisacetamide,HMBA）诱导处理前后的人肝癌SMMC-7721细胞核基质,并运用亚细胞蛋白质组学等分析技术,研究nucleophosmin (NPM)在核基质上的表达和定位变化,及其与相关基因产物的共定位关系,观察研究了nucleophosmin 在诱导分化前后人肝癌SMMC-7721细胞核基质中的存在、分布及其与相关基因产物的共定位关系.双向凝胶电泳和质谱鉴定结果显示,nucleophosmin 存在于 SMMC-7721 细胞核基质蛋白组分中,在 HMBA 处理后细胞核基质中表达下调.蛋白质印迹杂交实验结果确证了 nucleophosmin 在核基质中的存在及其在诱导处理后细胞核基质中表达下调的变化.免疫荧光显微镜观察显示,nucleophosmin 定位在 SMMC-7721细胞核基质上,经 HMBA处理后出现分布位置与表达水平的变化.激光扫描共聚焦显微镜观察结果显示,SMMC-7721细胞中,nucleophosmin与 c-fos、c-myc、rb、p53 等基因产物具有共定位关系,但在诱导处理后细胞内的共定位区域发生了改变.研究结果证实,nucleophosmin 是一种核基质蛋白,定位于核基质纤维上,nucleophosmin 在人肝癌 SMMC-7721 细胞诱导分化过程中的表达分布,及其与相关癌基因、抑癌基因产物的关系对 SMMC-7721 细胞分化具有重要影响.     

Proteomic analysis of MOLT-4 cells treated by valproic acid

The effect of valproic acid (VA) on protein expression in human T-lymphocytic leukemia cells MOLT-4 was studied. VA is an inhibitor of histonedeacetylases and has a potential use as antitumor agent in leukemia treatment. The authors in this work prove that 4 h long incubation with 2 mmol/l VA causes phosphorylation of histone H2A.X and its colocalization with 53BP1 in nuclear foci. Their co-localization is typical for DSB signaling machinery. These foci were detected in cells after 4 h exposure without increase of Annexin V positive apoptotic cells. Slight increase in apoptosis (Annexin V positivity) after 24 h is accompanied by more intensive increase in phosphorylation of H2A.X and also by formation of nuclear foci containing γH2A.X and 53BP1. Treatment of cells with 2 mmol/l VA resulted in induction of apoptosis affecting about 30% of cells after incubation for 72 h. The changes in protein expression were examined after cell incubation with 2 mmol/l VA for 4 h. Proteins were separated by two-dimensional electrophoresis and quantified using image evaluation system. Those exhibiting significant VA-induced abundance alterations were identified by mass spectrometry. Changes in expression of 22 proteins were detected, of which 15 proteins were down-regulated. Proteomic analysis resulted in successful identification of three proteins involving alfa-tubulin 3, tubulin-specific chaperone and heterogeneous nuclear ribonucloprotein F. Expression of seven proteins was up-regulated, including heterogeneous nuclear ribonucloprotein A/B. Identified proteins are related to microtubular system and hnRNP family. Suppression of microtubular proteins and changes of balance among hnRNPs can contribute to proliferation arrest and apoptosis induction.     

HMBA诱导人成骨肉瘤MG-63细胞分化过程中prohibitin的表达与定位变化

本文以HMBA诱导处理前后的人成骨肉瘤MG-63细胞为对象,对prohibitin在核基质中存在、分布及其与相关基因产物在HMBA处理前后MG-63细胞中的共定位关系进行观察研究.蛋白印迹杂交结果确证prohibitin存在于人成骨肉瘤MG-63细胞核基质蛋白组分中,并在HMBA处理后细胞核基质中表达下调,免疫荧光显微镜观察显示prohibitin定位在核基质上,经HMBA处理后出现分布位置与表达水平的变化.激光共聚焦显微镜观察可见prohibitin与MG-63细胞中c-fos、c-myc、p53和rb基因产物均存在共定位关系,但在HMBA处理后细胞中其共定位分布区域出现变化.研究结果证实prohibitin是一种核基质蛋白,定位于核基质上,prohibitin在人成骨肉瘤MG-63细胞诱导分化过程中的表达分布及其与相关癌基因、抑癌基因产物的共定位现象值得进一步探索和研究.     

Kallikrein 1 is overexpressed by astrocytes in the hippocampus of patients with refractory temporal lobe epilepsy, associated with hippocampal sclerosis

Kallikrein 1 (hK1) is a tissue enzyme responsible for kinin release in inflammatory cascade. This study was delineated to study the distribution and the co-localization of hK1 and kinin B1 and B2 receptors with glial and/or neuronal proteins markers, in the hippocampus of patients with refractory temporal lobe epilepsy, associated with hippocampal sclerosis (TLE-HS), comparing with control tissues. Hippocampal levels of KLK1 mRNA were also measured. hK1, kinin B1 and B2 receptors, NeuN and GFAP were analyzed using immunohistochemistry and confocal microscopy and KLK1 mRNA was quantified with real time PCR. Increased expression of hK1 by astrocytes co-localized with GFAP was found, contrasting with kinin B1 and B2 receptors, which were co-localized with NeuN in the sclerotic hippocampus. In addition, KLK1 mRNA was also up-regulated in same tissues. These data suggest an overexpression of kallikrein-kinin system and a neuron-glia interaction in the inflammatory process present in refractory TLE-HS.     

Localization of nucleophosmin in nuclear matrix and changes in its expression during the differentiation of human neuroblastoma induced by retinoic acid

In this article, we selectively extracted the nuclear matrix and intermediate filament system of human neuroblastoma SK‐N‐SH cells pre‐ and post‐treated with retinoic acid (RA). The distribution of nucleophosmin (NPM) in the nuclear matrix and its colocalization with several products of related genes were investigated. Results from two‐dimensional gel electrophoresis and MALDI‐TOF showed that NPM was a component of the nuclear matrix and its expression in SK‐N‐SH cells post‐treated with RA was down‐regulated. Immunofluorescent microscopy observations further showed that NPM was localized in the nuclear matrix of SK‐N‐SH cells, and its expression level and distribution were altered after treatment with RA. The colocalization of NPM with c‐myc, c‐fos, p53, and Rb in SK‐N‐SH cells was observed under a laser scanning confocal microscope, but the colocalization region was changed by RA. Our results prove that NPM is a nuclear matrix protein, which is localized in nuclear matrix fibers. The colocalization of NPM with its related genes and oncogenes affect the differentiation of SK‐N‐SH cells. The expression of NPM and its distribution in the process of cell differentiation deserve more intensive investigation. J. Cell. Biochem. 111: 67–74, 2010. © 2010 Wiley‐Liss, Inc.     

Trafficking of HIV-1 RNA is mediated by heterogeneous nuclear ribonucleoprotein A2 expression and impacts on viral assembly

Few details are known about how the human immunodeficiency virus type 1 (HIV-1) genomic RNA is trafficked in the cytoplasm. Part of this process is controlled by the activity of heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2). The role of hnRNP A2 during the expression of a bona fide provirus in HeLa cells is investigated in this study. Using immunofluorescence and fluorescence in situ hybridization techniques, we show that knockdown of hnRNP A2 expression in HIV-1-expressing cells results in the rapid accumulation of HIV-1 genomic RNA in a distinct, cytoplasmic space that corresponds to the microtubule-organizing center (MTOC). The RNA exits in the nucleus and accumulates at the MTOC region as a result of hnRNP A2 knockdown even during the expression of a provirus harboring mutations in the hnRNP A2-response element (A2RE), the expression of which results in nuclear retention of genomic RNA. We also demonstrate that hnRNP A2 expression is required for downstream trafficking of genomic RNA from the MTOC in the cytoplasm. Genomic RNA localization at the MTOC that was both the result of hnRNP A2 knockdown and the overexpression of Rab7-interacting lysosomal protein had little effect on pr55Gag synthesis but negatively influenced virus production and infectivity. These data indicate that altered HIV-1 genomic RNA localization modulates viral assembly and that the MTOC serves as a central site to which HIV-1 genomic RNA converges following its exit from the nucleus, with the host protein, hnRNP A2, playing a central role in taking it to and from this site in the cell.     

人成骨肉瘤MG-63细胞在HMBA诱导分化后核基质蛋白表达的变化

HMBA诱导处理人成骨肉瘤MG-63细胞分化后,选择性抽提核基质蛋白,经双向凝胶电泳技术分析．共有12个蛋白点表达发生变化,经肽指纹图谱分析鉴定了9个蛋白质,其中,MHCⅡ类抗原、IFN刺激的基因因子3α蛋白、DKFZp434M2221．1蛋白、8-羟基-鸟嘌呤糖基化酶同功酶oggl和波形蛋白表达上调,hnRNP A2/B1和肌动蛋白表达下调,60S核糖体蛋白L21和ST2蛋白仅在分化的MG-63细胞中表达。研究结果表明肿瘤细胞增殖分化过程中伴随核基质蛋白表达的变化,并为深入揭示核基质蛋白与细胞癌变和逆转的关系以及阐明细胞增殖分化的基因表达调控原理提供了依据。     

Necdin interacts with the ribonucleoprotein hnRNP U in the nuclear matrix

Necdin is expressed predominantly in terminally differentiated neurons, and its ectopic expression suppresses cell proliferation. We screened a cDNA library from neurally differentiated embryonal carcinoma P19 cells for necdin-binding proteins by the yeast two-hybrid assay. One of the positive clones contained cDNA encoding a carboxyl-terminal portion of heterogeneous nuclear ribonucleoprotein U (hnRNP U), a nuclear matrix-associated protein that interacts with chromosomal DNA. We isolated cDNA encoding full-length mouse hnRNP U to analyze its physical and functional interactions with necdin. The necdin-binding site of hnRNP U was located near a carboxyl-terminal region that mediated the association between hnRNP U and the nuclear matrix. In postmitotic neurons, endogenously expressed necdin and hnRNP U were detected in the nuclear matrix and formed a stable complex. Ectopically expressed necdin was concentrated in the nucleoli, but coexpressed hnRNP U recruited necdin to the nucleoplasmic compartment of the nuclear matrix. Furthermore, under the same conditions necdin and hnRNP U cooperatively suppressed the colony formation of transfected SAOS-2 cells. These results suggest that necdin suppresses cell proliferation through its interaction with hnRNP U in the specific subnuclear structure.     

The aberrant expression and localization of prohibitin during apoptosis of human cholangiocarcinoma Mz-ChA-1 cells

In this study, we aimed to investigate the aberrant expression and shift in localization of prohibitin (PHB) during apoptosis of human cholangiocarcinoma cells. Our study demonstrated that PHB was expressed primarily in the cytoplasm and only a little in the nucleus. However, PHB expression significantly decreased, and its localization shifted from the cytoplasm to the nucleus during apoptosis. PHB co-localized with AIF, Rb, p53, and c-Fos, but the region of co-localization was altered after treatment. Meanwhile, we detected a direct interaction between PHB and both p53 and Rb in Mz-ChA-1 cells. These results suggest that the altered localization and expression of PHB, as well as its co-localization with related oncogenes and tumor suppressor genes, can affect the apoptosis of Mz-ChA-1 cells.     

Expression profile and interactions of hnRNP A3 within hnRNP/mRNP complexes in mammals

The hnRNP A/B family contains abundant nuclear proteins with major roles in alternative splicing and the ability for nucleo-cytoplasmic shuttling. Compared to the best known members of this family (hnRNP A1, A2/B1), hnRNP A3 is a relatively less known protein. We report herein immunochemical studies with the hnRNP A3 isoforms (A3a and A3b) that provided evidence for species-specific expression. The unspliced A3a was found in human and murine cells, while the spliced A3b was a unique and abundant isoform in mouse/rat. In addition, a tissue-specific variation was observed in mice, as the brain was the only tissue found to overexpress hnRNP A3a. Both hnRNP A3a and A3b were able to stably associate with immunoselected hnRNP and mRNP complexes. Use of the auxiliary domain of hnRNP A3 in pull-down assays on human cell extracts revealed its unique ability to form a network of interactions not only with other A/B proteins but also with additional hnRNPs. All interactions, except those of hnRNP A1, were highly enhanced by previous RNase A digestion of the extracts. Our findings revealed novel characteristics of hnRNP A3 and supported its extensive involvement in the many aspects of mRNA maturation processes along with the other hnRNP A/B proteins.     

Structural Insight Into hnRNP A2/B1 Homodimerization and DNA Recognition

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) has been identified as a nuclear DNA sensor. Upon viral infection, hnRNP A2/B1 recognizes pathogen-derived DNA as a homodimer, which is a prerequisite for its translocation to the cytoplasm to activate the interferon response. However, the DNA binding mechanism inducing hnRNP A2/B1 homodimerization is unknown. Here, we show the crystal structure of the RNA recognition motif (RRM) of hnRNP A2/B1 in complex with a U-shaped ssDNA, which mediates the formation of a newly observed protein dimer. Our biochemical assays and mutagenesis studies confirm that the hnRNP A2/B1 homodimer forms in solution by binding to pre-generated ssDNA or dsDNA with a U-shaped bulge. These results depict a potential functional state of hnRNP A2/B1 in antiviral immunity and other cellular processes.     

Heterogeneous ribonucleoprotein C displays a repressor activity mediated by T-cell intracellular antigen-1-related/like protein to modulate Fas exon 6 splicing through a mechanism involving Hu antigen R

T-cell intracellular antigen (TIA)-proteins are known regulators of alternative pre-mRNA splicing. In this study, pull-down experiments and mass spectrometry indicate that TIAR/TIAL1 and hnRNP C1/C2 are associated in HeLa nuclear extracts. Co-immunoprecipitation and GST-pull-down assays confirmed this interaction. Interestingly, binding requires the glutamine-rich (Q-rich) C-terminal domain of TIAR and the leucine-rich plus acidic residues-rich C-terminal domains of hnRNP C1/C2. This interaction also occurs in an RNA-dependent manner. Recombinant GFP-TIAR and RFP-hnRNP C1 proteins display partial nuclear co-localization when overexpressed in HeLa cells, and this requires the Q-rich domain of TIAR. hnRNP C1 overexpression in the presence of rate-limiting amounts of TIAR in HeLa and HEK293 cells affects alternative splicing of Fas and FGFR2 minigenes, promoting Fas exon 6 and FGFR2 exon K-SAM skipping, respectively. The repressor activity of hnRNP C1 on Fas exon 6 splicing is mediated by Hu antigen R (HuR). Experiments involving tethering approaches showed that the repressor capacity of hnRNP C1 is associated with an exonic splicing silencer in Fas exon 6. This effect was reversed by splice-site strengthening and is linked to its basic leucine zipper-like motif. These results suggest that hnRNP C1/C2 acts as a bridge between HuR and TIAR to modulate alternative Fas splicing.