人源性基因calsenilin/KChIP3/DREAM单克隆抗体的制备及鉴定

Calsenilin/KChIP3/DREAM, 是脑中高表达蛋白,最初发现是因其与presenilin 和钙离子结合而得名.作为转录因子抑制因子,该基因在细胞核内具有多种功能.该基因在钙离子作用下细胞核内常常与c-fos、prodynorphin等基因的启动子下游的特异性DRE位点相结合,调节这些基因的表达.另一方面,作为钾离子通道结合蛋白,该基因具有4种isoforms,其中KChIP1广泛存在于各种组织中而KChIP2只在心脏中特异表达,KChIP3和 KChIP4则在脑中显示较高的表达.4种基因在C-端结构非常相象,N-端则显示多样性.除此之外,和许多基因相似,calsenilin经PKC、CKI、PKA等激酶作用可产生多位点的磷酸化,其中主要位点Ser63的磷酸化可以阻止caspase-3对该基因的降解作用.另一方面,Calsenilin作为转录因子激动因子结合于维生素D和视黄酸效应因子启动子上游促进转录的进行. 到目前为止,Calsenilin/KChIP3/DREAM在细胞核内具有双重基因表达调控作用,即当结合于启动子上游时显示正调控而当结合在启动子下游时显示负调控.为了更加深入研究calsenilin的功能及寻找新的受其调控的基因,首先制备可特异性识别的单克隆抗体.利用RT-PCR 技术,从人脑中提取RNA扩增calsenilin全基因,克隆于pGEX-4T-2原核细胞表达载体中,经IPTG诱导表达、Gluthathion Sepharose 4B纯化得到GST-calsenilin/DREAM/KChIP3重组蛋白,并免疫小鼠.通过PEG细胞融合得到单克隆抗体.经细胞免疫染色及Western blotting检测显示说明本实验得到单克隆抗体可以用来进行细胞免疫染色及Western blotting等检测.该抗体的成功制备,为今后对calsenilin/DREAM/KChIP3调控基因表达的更深入研究提供了有效工具,也填补了国内尚无该基因单克隆抗体资源的空白.     

DREAM/Calsenilin/KChIP3:神经系统的多功能蛋白

新近发现的转录因子DREAM(downstreamregulatoryelementantagonistmodulator)可结合到基因(包括PPD、Hrk、cfos等)的DRE(downstreamregulatoryelement)位点,抑制基因转录。它是第一个已知的可以直接与DNA结合发挥转录抑制作用的Ca2 结合蛋白,为Ca2 调节基因表达除蛋白激酶/磷酸酶这条主要通路外提供了另一条通路。由于DREAM与另两个研究小组发现的蛋白calsenilin和KChIP3实为同一种物质,所以DREAM具有PS(presenilin)作用蛋白、Kv4通道调节蛋白和转录因子的多重功能特性。本文将就DREAM的分布、功能及其调控、DREAM与疼痛的关系作简要综述。     

GLI3、EN1基因在单纯性马蹄内翻足发生中的功能研究

为了探讨人类GLI3基因在单纯性马蹄内翻足(Idiopathic congenital talipes equinovarus, ICTEV)发生时所起的作用, 文章构建了大鼠Gli3基因启动子区域荧光素酶报告基因表达载体来分析Gli3基因启动子的活性, 并利用P-Match软件预测大鼠Gli3基因启动子区域可能的调控元件, 应用ChIP实验加以验证。并利用RT-PCR、免疫组化和Western blotting的方法分析大鼠En1与ICTEV的相关性。经P-Match软件预测, Gli3基因启动子区域有３个转录因子En1的可能结合位点, 经ChIP实验证实位点1是真正结合位点。RT-PCR、免疫组化和Western blotting方法都证实En1基因在马蹄内翻足模型鼠中表达下降。结果提示大鼠的转录因子 En1可能是Gli3基因的上游负调控元件。在ICTEV患者中, 很可能是由于EN1基因表达水平的下降导致了GLI3基因表达水平的上升, 最终导致ICTEV的发生。     

一个功能丰富的转录调控分子——下游调控元件拮抗分子

下游调控元件拮抗分子(downstream regulatory element antagonist modulator,DREAM)与钙衰蛋白(Calsenilin)和钾通道辅助亚基(potassium channel interacting protein 3,KCh IP3),三者同属于神经钙感受器蛋白(neuronal calcium sensor,NCS)家族,由同一基因编码,但亚细胞定位不同且执行不同功能,其中DREAM定位于细胞核,有4个EF手型结构域(EF-hand-like motifs)能与钙离子可逆结合,诱导蛋白空间结构变化,结合到多种基因的下游调控元件(downstream regulatory element,DRE)位点发挥基因转录调节作用。DREAM在中枢神经系统(central nervous system,CNS)尤其是小脑皮层中高表达,通过调控N-甲基-D-天冬氨酸受体(N-methyl-D-aspartic acid receptor,NMDAR)影响学习和记忆,也参与阿尔兹海默症发病、炎症反应、血栓形成。随着更多DREAM新功能的发现,其在CNS中的生物功能受到更多关注。本文回顾DREAM的发现历史,分析该蛋白的结构功能特点、组织分布,讨论了近些年来在DREAM入核的调控、以及特有的钙依赖的基因调控机理方面研究进展,重点关注了DREAM-强啡肽原(prodynorphin,PDYN)-强啡肽(dynorphin,DYN)通路调节慢性疼痛的可能机制。     

老年痴呆症相关基因Nicastrin的转录调控

APP蛋白经过降解,形成老年痴呆症患者脑内老年斑的主要成分．由PS(早老素),NCT, PEN-2和APH-1 4种膜蛋白组成的γ分泌酶催化该降解过程．为了了解人类nicastrin( NCT )基因的转录调控机制,确定了其在人脑中的转录起始位点以及其编码区上游大小不等片段的转录起始活性．EMSA分析证实NCT启动子区的4个AP-1结合位点和2个NFAT结合位点能够与相应的转录因子结合,能够改变转录因子调控能力的定点突变和PDTC诱导使得NCT启动子在HeLa细胞和大鼠皮质神经元中的启动活性都有所改变．以上结果说明：AP-1和NFAT确实参与了人类NCT基因的转录调控．     

PC-1增强受体酪氨酸激酶家族成员EphA3在前列腺癌细胞中的转录

为了研究前列腺癌相关基因(prostate and colon gene 1, PC-1）对受体酪氨酸激酶家族分子EphA3表达的影响,用RT-PCR、实时PCR和Western印迹检测表达不同水平PC-1的前列腺癌细胞系LNCaP和C4-2中EphA3的表达情况. 发现PC-1可诱导EphA3基因表达上调. 采用荧光素酶实验检测PC-1对于EphA3启动子转录活性的影响,结果显示,PC-1对转录起始位点上游916 bp的启动子活性没有影响,而可增强转录起始位点上游2011 bp启动子的活性.对EphA3启动子－916 bp～－2　011 bp区域进行生物信息学分析,结果显示,此区域包含HSF、NF-1、Nkx-2、SP1和GATA-1等多种转录因子结合位点.实验结果表明,PC-1可通过影响EphA3启动子诱导EphA3基因高表达,其调控区域位于转录起始位点上游－2　011　bp至－916 bp之间,提示PC-1可能通过影响一些结合于此区域的转录因子来影响EphA3启动子的转录活性.     

沙眼衣原体质粒调控基因的上游序列有广泛的ChxR结合位点

目的:利用凝胶阻滞实验分析沙眼衣原体质粒调控基因的上游DNA序列与ChxR 的相互作用;方法:利用表达载体p ET-21b在大肠杆菌BL21(DE3)中重组表达ChxR ,重组蛋白的N端有T7标签、C端有6×His标签,用TALON金属亲和介质纯化重组蛋白;PCR扩增沙眼衣原体质粒调控基因的上游区约150 bp,连接p CR2.1载体,重组载体经单酶切,制备3'端生物素标记探针;用引物延伸实验确定glgA的转录起始位点,并合成5条glgA上游区的30 bp重叠探针;用凝胶阻滞实验分析ChxR 与以上探针的相互作用。结果:4个质粒调控基因的上游均有多个ChxR 结合位点,特别是glgA上游区至少有6个结合位点;glgA上游的ChxR 结合位点多位于启动子和启动子下游区。结论:ChxR 可能参与质粒调控基因的转录;在glgA转录中,ChxR 可能起转录抑制子的作用。     

Myocardin调控心肌H9C2细胞Ca2+通道机制研究

目的：研究同样在维持心脏正常结构和功能过程中发挥着重要作用的转录因子心肌素Myocardin对L型Ca²⁺通道Cav1.2的转录调控作用及分子机制。方法：全细胞膜片钳技术记录心肌细胞膜Ca²⁺电流,慢病毒包装技术制备Myocardin-GFP慢病毒用于感染心肌细胞以过表达Myocardin,Real-time PCR定量检测Cav1.2基因mRNA水平,Western blotting检测Cav1.2蛋白表达水平。PCR介导的定点突变技术得到Ca²⁺通道启动子区特定CarGbox位点突变的突变体。荧光素酶报告系统检测野生型WT和突变体MU启动子活性,以确定Myocardin在Cav1.2基因启动子区的作用位点。结果：全细胞膜片钳技术表明Myocardin激活Cav1.2而增加心肌细胞膜Ca²⁺电流,real-time PCR和Western blotting结果表明,Myocardin激活Cav1.2基因的转录和表达,荧光素酶报告系统检测突变体启动子活性,发现Myocardin激活Cav1.2基因的转录依赖其启动子区的CarGbox。结论：Myocardin通过与Cav1.2基因启动子区CarGbox结合进而激活其转录和表达,促进Ca²⁺通道蛋白装配到心肌细胞膜上,加强Ca²⁺内流,增强膜电流。     

Pro-apoptotic function of calsenilin/DREAM/KChIP3.

Apoptotic cell death and increased production of amyloid b peptide (Ab) are pathological features of Alzheimer's disease (AD), although the exact contribution of apoptosis to the pathogenesis of the disease remains unclear. Here we describe a novel pro-apoptotic function of calsenilin/DREAM/KChIP3. By antisense oligonucleotide-induced inhibition of calsenilin/DREAM/KChIP3 synthesis, apoptosis induced by Fas, Ca2+-ionophore, or thapsigargin is attenuated. Conversely, calsenilin/DREAM/KChIP3 expression induced the morphological and biochemical features of apoptosis, including cell shrinkage, DNA laddering, and caspase activation. Calsenilin/DREAM/KChIP3-induced apoptosis was suppressed by caspase inhibitor Z-VAD and by Bcl-XL, and was potentiated by increasing cytosolic Ca2+, expression of Swedish amyloid precursor protein mutant (APPSW) or presenilin 2 (PS2), but not by a PS2 deletion lacking its C-terminus (PS2/411stop). In addition, calsenilin/DREAM/KChIP3 expression increased Ab42 production in cells expressing APPsw, which was potentiated by PS2, but not by PS2/411stop, which suggests a role for apoptosis-associated Ab42 production of calsenilin/DREAM/KChIP3.     

A role for calsenilin and related proteins in multiple aspects of neuronal function

The protein variously called calsenilin, DREAM, or KChIP3 has been independently discovered three times, accounting for the three different names currently in use. Calsenilin appears to have three, perhaps independent, roles. Calsenilin binds and modulates some of the effects of the Alzheimer disease-related protein presenilin, while presenilin can modulate some of the effects of calsenilin. Calsenilin binds the dynorphin response element and regulates dynorphin expression, hence regulating nociception. Calsenilin binds Kv channels and modulates potassium conductance, playing a role in long-term potentiation as well as in other important plastic pathways. Other members of the calsenilin family share at least some of the roles. For example, KChIP1, KChIP2, and CALP can all bind presenilins and can all modulate A-type potassium channels. Further functional dissection of this family of proteins will provide insight into numerous aspects of neuronal function and will illuminate the role of the calsenilin family of proteins in disease.     

Contribution of presenilin/gamma-secretase to calsenilin-mediated apoptosis

Mutant presenilins cause early-onset of familial Alzheimer's disease and render cells vulnerable to apoptosis. Calsenilin/DREAM/KChIP3 is a multifunctional calcium-binding protein that interacts with presenilin and mediates calcium-mediated apoptosis. In the present study, we report that the calsenilin-mediated apoptosis is regulated by presenilin. The expression of calsenilin was highly up-regulated in neuronal cells undergoing Abeta42-triggered cell death. The incidence of calsenilin-mediated apoptosis was diminished in presenilin-1(-/-) mouse embryonic fibroblast cells or neuronal cells stably expressing a loss-of-function presenilin-1 mutant. On the contrary, an array of familial Alzheimer's disease-associated presenilin mutants (gain-of-function) increased calsenilin-induced cell death. Moreover, gamma-secretase inhibitors, including compound E and DAPT, decreased the calsenilin-induced cell death. These results suggest that the pro-apoptotic activity of calsenilin coordinates with presenilin/gamma-secretase activity to play a crucial role in the neuronal death of Alzheimer's disease.     

Hyperglycemia induces early upregulation of the calcium sensor KChIP3/DREAM/calsenilin in the rat retina

Hyperglycemia alters the tight control of intracellular calcium dynamics in retinal cells and may lead to the development of diabetic retinopathy. The potassium channel interacting protein 3 (KChIP3) also known as DREAM (Downstream Regulatory Element Antagonist Modulator) or calsenilin (KChIP3/DREAM/calsenilin), a member of the neuronal calcium sensor protein family, is expressed in Müller glial cells and upregulated under high glucose experimental culture conditions. Here, we analyzed the expression and function of KChIP3 in the retina of streptozotocin induced diabetic Long Evans rats by immunofluorescence confocal microscopy, western blot, co-immunoprecipitation, whole cell patch clamp recording on isolated cells and KChIP3 gene silencing by RNA interference. Three weeks after streptozotocin application, KChIP3 was increased throughout the different retinal layers and this process was not linked to augmented apoptosis. KChIP3 co-immunoprecipitated with voltage gated K(+) channels of the K(V)4.2-4.3 subtype in retinal extracts from control and hyperglycemic rats. Electrophysiological analysis showed that control cells did not express A type (K(V)4-mediated) K(+) currents but most of the cells from streptozotocin treated retinas displayed macroscopic currents with an inactivating component sensitive to 4-AP, suggesting the persistence of the A type currents at early times after treatment. siRNA analysis in Müller cells cultures grown under high glucose experimental conditions corroborated that, when the expression of KChIP3 is 50% reduced, the number of cells expressing A type currents decreases significantly. Together these data suggest an altered expression and function of KChIP3 after streptozotocin induced hyperglycemia that might help explain some pathological alterations in early diabetic retinopathy.