一种特异性多用途抗人Connexin31抗体的获得与应用

间隙连接蛋白31(Connexin31,Cx31)是间隙连接蛋白(Connexin)家族的一员,目前对于Cx31的功能及其调节方式知之甚少。本实验利用Fmoe固相多肽合成的方法合成Cx31羧基端一个多肽片段(250-266从),经HPLC纯化后偶联到匙孔槭血蓝蛋白,免疫新西兰雄兔后采血检测、并纯化,采用Cx31myc表达蛋白进行Western blotting、细胞免疫荧光染色、免疫沉淀实验,证实得到的抗体为抗间隙连接蛋白31的特异抗体。     

MAP法制备抗人Connexin31抗体

MAP（multiple antigenic peptide）多肽法具有快速、简便、成本低廉的特点．用MAP法,直接在多肽合成仪上合成Connexin31羧基端一个多肽片段（250—266AA）的MAP肽,经超滤器纯化后,免疫新西兰雄兔后采血检测、并纯化．经western blotting、细胞免疫荧光染色、免疫沉淀证实得到的抗体为抗间隙连接蛋白31的特异抗体．     

泛素-蛋白酶体系统参与盐藻鞭毛的解聚

目的:了解泛素-蛋白酶体系统在鞭毛解聚中的作用。方法:用激光共聚焦显微镜观察泛素蛋白在杜氏盐藻鞭毛上的定位;分别诱导鞭毛的组装和解聚,观察鞭毛组装和解聚后泛素化蛋白的变化;采用甘油密度梯度离心法对蛋白酶体进行纯化,荧光多肽底物检测各处理组蛋白酶体活性的变化。结果:所提取的鞭毛蛋白上存在大量泛素化蛋白;与对照组相比,鞭毛解聚组泛素化蛋白增多,蛋白酶体活性升高近2倍。结论:泛素-蛋白酶体参与盐藻鞭毛的解聚,为进一步阐明鞭毛解聚机制提供了依据。     

人间隙连接蛋白 31 相互作用蛋白Annexin Ⅱ的筛选与证实

间隙连接蛋白 31 (connexin31 , Cx31) 是间隙连接蛋白 (connexin) 家族的一员,目前对于 Cx31 的功能及其调节方式知之甚少 . 采用固相多肽合成的方法合成 Cx31 羧基端一个多肽片段 (250~266) ,经 HPLC 纯化后偶联到匙孔血蓝蛋白,免疫新西兰雄兔后采血检测、并纯化、经蛋白质印迹、细胞免疫荧光染色、免疫沉淀证实得到的抗体为特异性抗 Cx31 的抗体 . 运用制备的抗 Cx31 多克隆抗体免疫沉淀, SDS- 聚丙烯酰胺凝胶电泳 (SDS-PAGE) 分离,蛋白质条带回收,蛋白质胶块酶解, Q-TOF 质谱分析,数据库扫描筛选可能相互作用蛋白,运用抗体 pull-down 实验,筛选到可能相互作用蛋白 annexin Ⅱ,经过免疫共沉淀、细胞免疫共定位等实验证实 annexin Ⅱ与 Cx31 相互作用 .     

汉坦病毒结构蛋白的鉴定及其应用

对差速离心纯化的汉坦病毒R84-1毒株进行了SDS-PAGE和免疫印迹试验。发现有67k和43k两条蛋白区带能与汉坦病毒抗体起反应。经单克隆抗体鉴定,67k多肽可能属病毒囊膜蛋白,43k多肽未定。用免疫印迹法对出血热患者进行检测,初步证明野鼠型感染者具有上述两种蛋白抗原的抗体,大鼠型患者仅具有67k蛋白的抗体,这对出血热患者血清学分型有重要意义。     

类泛素化修饰蛋白SUMO1的表达纯化及抗体制备

SUMO是近年发现的类泛素化修饰蛋白,可通过异肽键共价连接到靶蛋白上,影响靶蛋白的细胞内定位、稳定性及与其它生物大分子的相互作用. 为研究蛋白质的SUMO化修饰,本文表达并利用亲和层析的方法纯化了重组的人SUMO1,制备了兔抗hSUMO1的多克隆抗体. 经ELISA和免疫印迹检测,获得了灵敏度高、特异性好的抗体,可用于SUMO化修饰靶蛋白的鉴定及SUMO化修饰的生物学功能研究.     

HIV-1辅助蛋白Vpr多肽抗体的制备与鉴定

预测Vpr蛋白的B细胞抗原表位,并利用合成的B细胞表位肽制备Vpr特异性抗体。应用生物信息学技术获得Vpr蛋白共享氨基酸序列并预测其潜在B细胞抗原表位,与载体蛋白血蓝蛋白(KLH)偶联合成多肽并免疫家兔,鉴定及纯化获得的多肽特异性抗体。软件预测显示,Vpr蛋白N端的第3～19位(N)和C端的第82～95位(C)氨基酸序列为潜在B细胞抗原表位;ELISA检测抗血清中多肽特异性抗体的效价都达到1:105以上;Western-Blotting结果显示,无论对HIV-1B亚型还是CRF07_BC重组型的Vpr蛋白,其多肽N抗体和C抗体均能特异性识别;免疫沉淀结果显示,Vpr多肽N和C抗体也能特异性结合未变性的野生型Vpr或GFP-Vpr融合蛋白。利用生物信息学技术能成功预测Vpr蛋白B细胞抗原表位,免疫所获得的抗体具有较好的特异性和应用性。     

一种从大肠杆菌包涵体中分离纯化GST-TRAF6融合蛋白的方法

利用8 mol/L尿素溶液对表达在大肠杆菌包涵体中的GST-TRAF6融合蛋白进行变性,通过逐级稀释复性的方法对尿素溶解后的GST-TRAF6融合蛋白进行复性,将复性后的GST-TRAF6融合蛋白进一步利用谷胱甘肽琼脂糖树脂亲和层析的方法进行分离纯化,将分离纯化后的蛋白通过Western blot方法进行验证,最后利用体外泛素化反应检测经包涵体变性、复性和纯化后的GST-TRAF6融合蛋白的生物学活性。经过包涵体变性、梯度稀释复性和谷胱甘肽琼脂糖树脂亲和层析3个步骤后纯化得到纯度达90%以上、浓度为396 ng/μL的蛋白质溶液。利用GST蛋白作为对照,经Western blot验证表明,纯化得到的蛋白确为GSTTRAF6融合蛋白。进一步利用体外泛素化反应分析其泛素连接酶活性发现,17 ng/μL浓度的GST-TRAF6融合蛋白能够以泛素分子作为底物在5 min内快速催化自由泛素链的生成。结果表明,表达在大肠杆菌包涵体中的GST-TRAF6融合蛋白经尿素变性溶解后能够成功复性并分离纯化,在溶解性改变的同时恢复了其泛素连接酶活性。为从大肠杆菌包涵体中大规模分离纯化蛋白质提供了一种新的复性方法。     

抗溶葡萄球菌酶N端合成多肽抗体的制备与鉴定

为了制备抗溶葡萄球菌酶N端合成多肽抗体,合成了溶葡萄球菌酶(lysostaphin)分子的3-13位的11个氨基酸的多肽(THEHSAQWLN),并利用戊二醛双功能试剂将人工合成多肤成功地与KLH进行偶联.免疫新西兰兔制备抗lysostaphin合成多肽的抗体,并经亲和层析进行了纯化.对此抗体进行鉴定的结果表明,抗溶葡萄球菌酶合成多肽抗体可与重组溶葡萄球菌酶分子发生特异性反应,并可用于蛋白免疫印迹.该抗体的制备为使用亲和层析纯化溶葡萄球菌酶提供了有用的配基.     

泛素连接酶LNX1的表达、活性测定和功能初探

目的:在大肠杆菌中表达重组人泛素连接酶LNX1,并研究其对钾离子通道蛋白Kv1.4的泛素化作用。方法:构建人LNX1重组蛋白原核表达载体pGEX-LNX1,在大肠杆菌中通过IPTG诱导表达重组蛋白,表达产物经GSTrap FF纯化,并通过体外泛素化(in vitroubiquitination)方法测定其泛素连接酶活性,同样用体外泛素化方法研究其对含有Kv1.4的C端的人工底物的泛素化。结果:获得了纯化的有泛素连接酶活性的重组人LNX1蛋白,重组LNX1可以在体外泛素化体系中泛素化含有Kv1.4的C端的人工底物。结论:重组人LNX1原核表达成功,具有泛素连接酶活性,并催化Kv1.4的泛素化。     

Chronic overexpression of the calcineurin inhibitory gene DSCR1 (Adapt78) is associated with Alzheimer's disease

The DSCR1 (Adapt78) gene was independently discovered as a resident of the "Down syndrome candidate region"and as an "adaptive response"shock or stress gene that is transiently induced during oxidative stress. Recently the DSCR1 (Adapt78) gene product was discovered to be an inhibitor of the serine/threonine phosphatase, calcineurin, and its signaling pathways. We hypothesized that DSCR1 (Adapt78) might also be involved in the development of Alzheimer's disease. To address this question we first studied DSCR1 (Adapt78) in multiple human tissues and found significant expression in brain, spinal cord, kidney, liver, mammary gland, skeletal muscle, and heart. Within the brain DSCR1 (Adapt78) is predominantly expressed in neurons within the cerebral cortex, hippocampus, substantia nigra, thalamus, and medulla oblongata. When we compared DSCR1 (Adapt78) mRNA expression in post-mortem brain samples from Alzheimer's disease patients and individuals who had died with no Alzheimer's diagnosis, we found that DSCR1 (Adapt78) mRNA levels were about twice as high in age-matched Alzheimer's patients as in controls. DSCR1 (Adapt78) mRNA levels were actually three times higher in patients with extensive neurofibrillary tangles (a hallmark of Alzheimer's disease) than in controls. In comparison, post-mortem brain samples from Down syndrome patients (who suffer Alzheimer's symptoms) also exhibited DSCR1 (Adapt78) mRNA levels two to three times higher than controls. Using a cell culture model we discovered that the amyloid beta(1-42) peptide, which is a major component of senile plaques in Alzheimer's, can directly induce increased expression of DSCR1 (Adapt78). Our findings associate DSCR1 (Adapt78) with such major hallmarks of Alzheimer's disease as amyloid protein, senile plaques, and neurofibrillary tangles.     

NF-kappaB-inducing kinase phosphorylates and blocks the degradation of Down syndrome candidate region 1

Down syndrome, the most frequent genetic disorder, is characterized by an extra copy of all or part of chromosome 21. Down syndrome candidate region 1 (DSCR1) gene, which is located on chromosome 21, is highly expressed in the brain of Down syndrome patients. Although its cellular function remains unknown, DSCR1 expression is linked to inflammation, angiogenesis, and cardiac development. To explore the functional role of DSCR1 and the regulation of its expression, we searched for novel DSCR1-interacting proteins using a yeast two-hybrid assay. Using a human fetal brain library, we found that DSCR1 interacts with NF-kappaB-inducing kinase (NIK). Furthermore, we demonstrate that NIK specifically interacts with and phosphorylates the C-terminal region of DSCR1 in immortalized hippocampal cells as well as in primary cortical neurons. This NIK-mediated phosphorylation of DSCR1 increases its protein stability and blocks its proteasomal degradation, the effects of which lead to an increase in soluble and insoluble DSCR1 levels. We show that an increase in insoluble DSCR1 levels results in the formation of cytosolic aggregates. Interestingly, we found that whereas the formation of these inclusions does not significantly alter the viability of neuronal cells, the overexpression of DSCR1 without the formation of aggregates is cytotoxic.     

Over-Expression of DSCR1 Protects against Post-Ischemic Neuronal Injury

Background and Purpose

The Down syndrome candidate region 1 (DSCR1) gene is located on human chromosome 21 and its protein is over-expressed in brains of Down syndrome individuals. DSCR1 can modulate the activity of calcineurin, a phosphatase abundant in the brain, but its influence on stroke outcome is not clear. We compared stroke outcome in wildtype (WT) and transgenic (DSCR1-TG) mice which over-express isoform 1 of human DSCR1.

Methods

Transient cerebral ischemia was produced by occlusion of the middle cerebral artery for 0.5 h. After 23.5 h reperfusion, we assessed neurological impairment, brain infarct and edema volume, leukocyte infiltration and markers of inflammation. Intrinsic resistance to apoptosis following glucose deprivation was also assessed in primary cultures of WT and DSCR1-TG neurons.

Results

In contrast to WT, DSCR1-TG mice had an improved neurological deficit score, greater grip strength, attenuated infarct volume and brain swelling, and lacked hippocampal lesions after stroke. Expression of mouse DSCR1-1, but not DSCR1-4, mRNA and protein was increased by ischemia in both WT and DSCR1-TG. Brain calcineurin activity was increased to a similar degree after ischemia in each genotype. DSCR1-TG mice had fewer infiltrating neutrophils and activated microglia compared with WT, in association with an attenuated upregulation of several pro-inflammatory genes. Neurons from DSCR1-TG mice were more resistant than WT neurons to apoptotic cell death following 24 h of glucose deprivation.

Conclusions

Over-expression of DSCR1 in mice improves outcome following stroke. Mechanisms underlying this protection may involve calcineurin-independent, anti-inflammatory and anti-apoptotic effects mediated by DSCR1 in neurons.     

DSCR1 interacts with FMRP and is required for spine morphogenesis and local protein synthesis

Most common genetic factors known to cause intellectual disability are Down syndrome and Fragile X syndrome. However, the underlying cellular and molecular mechanisms of intellectual disability remain unclear. Recently, dendritic spine dysmorphogenesis and impaired local protein synthesis are posited to contribute to the cellular mechanisms of intellectual disability. Here, we show that Down syndrome critical region1 (DSCR1) interacts with Fragile X mental retardation protein (FMRP) and regulates both dendritic spine morphogenesis and local protein synthesis. Interestingly, decreasing the level of FMRP restores the DSCR1-induced changes in dendritic spine morphology. Our results imply that DSCR1 is a novel regulator of FMRP and that Fragile X syndrome and Down syndrome may share disturbances in common pathways that regulate dendritic spine morphology and local protein synthesis.     

Mutational analyses of the signals involved in the subcellular location of DSCR1

Background  

Down syndrome is the most frequent genetic disorder in humans. Rare cases involving partial trisomy of chromosome 21 allowed a small chromosomal region common to all carriers, called Down Syndrome Critical Region (DSCR), to be determined. The DSCR1 gene was identified in this region and is expressed preferentially in the brain, heart and skeletal muscle. Recent studies have shown that DSCR1 belongs to a family of proteins that binds and inhibits calcineurin, a serine-threonine phosphatase. The work reported on herein consisted of a study of the subcellular location of DSCR1 and DSCR1-mutated forms by fusion with a green fluorescent protein, using various cell lines, including human.     

Pituitary Adenylate Cyclase-activating Polypeptide (PACAP) Targets Down Syndrome Candidate Region 1 (DSCR1/RCAN1) to control Neuronal Differentiation

Pituitary adenylate cyclase-activating peptide (PACAP) is a neurotrophic peptide involved in a wide range of nervous functions, including development, differentiation, and survival, and various aspects of learning and memory. Here we report that PACAP induces the expression of regulator of calcineurin 1 (RCAN1, also known as DSCR1), which is abnormally expressed in the brains of Down syndrome patients. Increased RCAN1 expression is accompanied by activation of the PKA-cAMP response element-binding protein pathways. EMSA and ChIP analyses demonstrate the presence of a functional cAMP response element in the RCAN1 promoter. Moreover, we show that PACAP-dependent neuronal differentiation is significantly disturbed by improper RCAN1 expression. Our data provide the first evidence of RCAN1, a Down syndrome-related gene, as a novel target for control of the neurotrophic function of PACAP.     

DSCR2, a Down syndrome critical region protein, is localized to the endoplasmic reticulum of mammalian cells

We used immunocytochemical and fluorescence assays to investigate the subcellular location of the protein encoded by Down syndrome critical region gene 2 (DSCR2) in transfected cells. It was previously suggested that DSCR2 is located in the plasma membrane as an integral membrane protein. Interestingly, we observed this protein in the endoplasmic reticulum (ER) of cells. We also studied whether the truncated forms of DSCR2 showed different subcellular distributions. Our observations indicate that DSCR2 probably is not inserted into the membrane of the endoplasmic reticulum since the fragments lacking the predicted transmembrane (TM) helices remained associated with the ER. Our analyses suggest that, although DSCR2 is associated with the endoplasmic reticulum, it is not an integral membrane protein and it is maintained on the cytoplasmic side of the ER by indirect interaction with the ER membrane or with another protein.     

DSCR1 gene expression is dependent on NFATc1 during cardiac valve formation and colocalizes with anomalous organ development in trisomy 16 mice

The Down syndrome critical region 1 (DSCR1) gene is present in the region of human chromosome 21 and the syntenic region of mouse chromosome 16, trisomy of which is associated with congenital heart defects observed in Down syndrome. DSCR1 encodes a regulatory protein in the calcineurin/NFAT signal transduction pathway. During valvuloseptal development in the heart, DSCR1 is expressed in the endocardium of the developing atrioventricular and semilunar valves, the muscular interventricular septum, and the ventricular myocardium. Human DSCR1 contains an NFAT-rich calcineurin-responsive element adjacent to exon 4. Transgenic mice generated with a homologous regulatory region of the mouse DSCR1 gene linked to lacZ (DSCR1(e4)/lacZ) show gene activation in the endocardium of the developing valves and aorticopulmonary septum of the heart, recapitulating a specific subdomain of endogenous DSCR1 cardiac expression. DSCR1(e4)/lacZ expression in the developing valve endocardium colocalizes with NFATc1 and, endocardial DSCR1(e4)/lacZ, is notably reduced or absent in NFATc1(-/-) embryos. Furthermore, expression of the endogenous DSCR1(e4) isoform is decreased in the outflow tract of NFATc1(-/-) hearts, and the DSCR1(e4) intragenic element is trans-activated by NFATc1 in cell culture. In trisomy 16 (Ts16) mice, expression of endogenous DSCR1 and DSCR1(e4)/lacZ colocalizes with anomalous valvuloseptal development, and transgenic Ts16 hearts have increased beta-galactosidase activity. DSCR1 and DSCR1(e4)/lacZ also are expressed in other organ systems affected by trisomy 16 in mice or trisomy 21 in humans including the brain, eye, ear, face, and limbs. Together, these results show that DSCR1(e4) expression in the developing valve endocardium is dependent on NFATc1 and support a role for DSCR1 in normal cardiac valvuloseptal formation as well as the abnormal development of several organ systems affected in individuals with Down syndrome.     

Down syndrome candidate region-1 protein interacts with Tollip and positively modulates interleukin-1 receptor-mediated signaling

Background

The Down syndrome candidate region-1 gene (DSCR1, also known as RCAN1) is situated close to the Down Syndrome Critical Region (DSCR), which contains genes responsible for many features of Down syndrome. DSCR1 modulates calcineurin phosphatase activity, though its functional role is incompletely understood.

Methods

Here we investigated the role of DSCR1-1S isoform in IL-1 receptor (IL-1R)-mediated signaling by analyzing interaction between DSCR1-1S and the IL-1R pathway components Tollip, IRAK-1, and TRAF6.

Results

Co-immunoprecipitation analyses of HEK293 cells revealed that DSCR1-1S interacted with Tollip, an IRAK-1 inhibitor, leading to the dissociation of IRAK-1 from Tollip. Similarly, both DSCR1-1S and Tollip interacted with TRAF6, with DSCR1 reducing interaction between Tollip and TRAF6. DSCR1-1S also stimulated IL-1R-mediated signaling pathways, TAK1 activation, NF-κB transactivation, and IL-8 production, all downstream consequences of IL-1R activation.

General significance

Together, these results suggest that DSCR1-1S isoform positively modulates IL-1R-mediated signaling pathways by regulating Tollip/IRAK-1/TRAF6 complex formation.