人脑中硒蛋白W与鞘脂激活蛋白原相互作用的筛选与验证

硒蛋白W(SelW)是人脑中一种重要的硒蛋白.在缺硒的条件下,SelW在脑中具有优先储备的特性,但其具体机制和在脑中的功能至今尚不清楚.本文以SelW突变体SelW′为"诱饵",采用酵母双杂交系统对人胎脑文库进行筛选,获得与SelW相互作用的蛋白,其中一种为鞘脂激活蛋白原(PSAP).采用荧光共振能量转移技术中的受体漂白和敏化发射两种方法,验证了SelW与PSAP的相互作用.构建表达载体在大肠杆菌中成功表达出SelW′,利用Pull-down技术验证了SelW′与PSAP在细胞外的直接相互作用.采用免疫共沉淀的方法验证了上述两种蛋白在昆明小鼠脑组织中的内源性相互作用.基于SelW和PSAP的已知生物功能,推测SelW可能在脑部发育和神经退行性疾病形成过程中发挥着重要作用.     

Mdfic与Rhox5蛋白相互作用的关键结构域鉴定

Mdfic（MyoD family inhibitor domain containing）是一个新发现的含有MyoD抑制素结构域（I-mfa domain）的转录调控因子,可能在肌细胞的分化过程中发挥重要作用. 小鼠Rhox5为同源异型框基因,隶属于Rhox基因簇（reproductive homeobox on the X chromosome genes cluster）β亚簇.在前期证实Mdifc能结合Rhox5蛋白的基础上,进一步鉴定两者相互作用的关键结构域.生物信息学分析Mdfic 的氨基酸序列,PCR方法扩增Mdfic A截短型片段（第72～247位氨基酸残基）,含保守的I-mfa结构域; 双向酵母双杂交和体外GST-Pull down结果表明,该截短型片段可以与Rhox5蛋白结合,且结合力度较完整的Mdfic蛋白强; 将Mdfic A片段划分为两段: Mdfic B（72～191 aa, 不含I-mfa结构域）和Mdfic C（191～247 aa, 含I-mfa结构域）.结果表明,含保守I-mfa结构域的Mdfic C截短型片段丧失了与Rhox5蛋白结合的能力,而不含I-mfa结构域的Mdfic B截短型片段可以结合Rhox5蛋白. 鉴于Mdfic蛋白的非I-mfa结构域在Rhox5/Mdfic结合中发挥关键作用, Rhox5与Mdfic的结合可能进一步调控由Mdfic的I-mfa结构域参与的其他转录因子（如MyoD）的调控,三者形成一个复杂的调控网络,共同参与肌细胞发生及分化的调控.     

鞘脂激活蛋白原:一种新型神经营养因子

鞘脂激活蛋白 (ｓｐｈｉｎｇｏｌｉｐｉｄａｃｔｉｖａｔｏｒｐｒｏ ｔｅｉｎ ,ｓａｐｏｓｉｎ)是包含鞘脂激活蛋白Ａ、Ｂ、Ｃ、Ｄ的一组热稳定糖蛋白 ,它们在溶酶体酶解鞘脂的过程中发挥着重要作用[1] 。鞘脂激活蛋白由其前体鞘脂激活蛋白原 (ｐｒｏｓａｐｏｓｉｎ)水解产生。最近的研究表明 ,除作为前体外 ,鞘脂激活蛋白原还具有较强的神经营养活性。本文就这一方面的研究进展作一综述。1 .鞘脂激活蛋白原的神经营养作用及其分布1 .1　作用的发现　　鞘脂激活蛋白原是在对鞘脂激活蛋白的研究中发现的。Ｋｏｎｄｏｎ等用免疫组织法发现 ,脑…     

天花粉蛋白与膜脂相互作用的研究

天花粉蛋白（ＴＣＳ）在≥２．２２μｍｏｌ／Ｌ时能引起大豆磷脂脂质体内含物释放,Ｃａ＾２＋对这种释放有一定的促进作用,低ＰＨ值也能促进ＴＣＳ与脂质体的作用;将ＴＣＳ与细细胞一起保温;当ＴＣＳ终浓度达１４．７μｍｏｌ／Ｌ时能损伤红细胞膜,产生溶血作用;ＴＣＳ还能作用于红细胞血影膜,改变其脂双层的不对称性。     

脂滴与线粒体相互作用的研究进展

肥胖和多种代谢类疾病的发生有着密切的关系,而导致肥胖的脂肪多以中性脂的形式储存于细胞的一种细胞器——脂滴中。越来越多的研究表明,脂滴能够和其它细胞器发生相互作用,而它和线粒体的相互作用可能与Ⅱ型糖尿病的形成密切相关:非正常的脂滴和线粒体的相互作用有可能是导致细胞胰岛素抵抗的重要原因。我们通过对脂滴表面蛋白质组学、脂滴与线粒体的空间位置,以及相关蛋白等研究的总结,结合本实验室的研究结果,对脂滴与线粒体相互作用的物质基础及可能方式、受骨骼肌有氧运动的影响,及其与骨骼肌胰岛素抵抗发生的关系等,进行了讨论。     

生物间高阶相互作用研究进展

生物间的相互作用是物种共存和生物多样性维持的关键。传统的物种共存研究主要关注配对物种之间的直接相互作用, 而忽略了更为复杂的间接相互作用。本文首先介绍了两种间接相互作用: 链式相互作用(本质上仍是两两物种之间的相互作用)和高阶相互作用。在此基础上, 我们回顾了高阶相互作用定义的演变历史(包括狭义的高阶相互作用和广义的高阶相互作用)及其检验方法, 并介绍了高阶相互作用在多营养级之间和同一营养级内的研究概况。目前, 生态学家主要对多营养级之间(如食物网)的高阶相互作用的特征、发生机制、作用途径及实验证据等方面进行了详尽的研究。近年来, 同一营养级内的高阶相互作用也开始受到关注, 因此我们进一步介绍了同一营养级内个体水平高阶相互作用的重要意义和度量方法。从个体水平上研究高阶相互作用, 既能统一狭义和广义高阶相互作用在定义上的争议, 又可以将个体间的差异(如个体大小、个体的空间分布等信息)考虑进来。最后, 本文对高阶相互作用一些可能的重要研究方向进行了展望: 在自然群落中(尤其同一营养级内)检验高阶相互作用的普遍性与相对重要性, 探讨高阶相互作用的发生机制以及如何将高阶相互作用整合到现有的理论体系中等。高阶相互作用的研究有助于我们全面深刻地理解物种共存和生物多样性的维持机制, 丰富和完善群落生态学的理论框架, 为人类世背景下的生物多样性保护和生态系统功能维持与提升提供基础。     

根系间的相互作用——竞争与互利

植物根系间的相互作用分为竞争和互利两种形式 ,它是决定植物群落动态变化和群落结构的重要因素。根系间的竞争包括植株个体自身根系的竞争以及个体与个体根系间 (同种或异种 )的竞争两方面 ,前者的发生在农林系统中是不可避免的并且很难调控 ,后者可以分为种间植物根系的竞争和种内植物根系的竞争。还阐述了根系的竞争能力和与其密切相关的根系生长率、根组织的新陈代谢、植物的生长形式和根系的空间结构等植物特性 ,同时介绍了根系对水和养分的竞争机理、形式、影响以及竞争强度计算方法。接着具体分析包括根系错位在内的各种根系互利现象和相关机理。影响根系间相互作用的限制性因子有土壤营养的异质性、大气 CO2 浓度、地下草食生物、根系生产力和生物量、根系结构、形态和生理调节、土壤养分的扩散性以及植物间距等。随着科技的进步和各门学科的发展 ,未来根系的研究方向主要体现在结合实践优化农林系统中不同物种间的作用关系、预测根系竞争在全球气候变化下的发展规律、更新实验研究方法及手段研究作用机理等 3个方面。     

生长因子和细胞因子与糖复合物间的相互作用

生长因子和细胞因子与糖复合物间的相互作用王克夷（中国科学院上海生物化学研究所,２０００３１）关键词生长因子,细胞因子,糖类,相互作用生长因子和细胞因子是目前生物学中的研究热点,它们和细胞生物学、免疫学以及神经生物学都有密切关系,其中不少可以用于临床。...     

蛋白质间相互作用技术的研究近况

蛋白质间相互作用技术的研究近况黄翠芬叶棋浓（军事医学科学院生物工程研究所,北京１００８５０关键词：蛋白质,相互作用,技术ＲｅｃｅｎｔＡｄｖａｎｃｅｓｉｎｔｈｅＴｅｃｈｎｉｑｕｅｓｏｆＰｒｏｔｅｉｎ┐ＰｒｏｔｅｉｎＩｎｔｅｒａｃｔｉｏｎｓＨｕａｎｇＣｕ...     

High-level expression, polyclonal antibody preparation and sub-cellular localization analysis of mouse Rhox5 protein

Mouse reproductive homeobox on the X chromosome (Rhox) is a novel homeobox gene cluster. Rhox5, also called Pem, belongs to the beta subcluster of Rhox. Codon analysis indicated that the cDNA contains 16% of codons rarely used in Escherichia coli. To achieve high-level expression of Rhox5, the coding sequence of Rhox5 was amplified and subcloned into the prokaryotic expression vector pET22b (+) in order to produce 6His-tagged fusion protein in the modified BL21 (DE3) cells, namely Rosetta2 (DE3) cells. The 6His-tagged Rhox5 was expressed efficiently in Rosetta2 (DE3), compared with marginal expression in BL21 (DE3). The fusion protein amounted to 16% of the total bacterial proteins after induction with 0.4mM IPTG for 1.5h at 37 degrees C. After purification, Rhox5-6His was used to immunize New Zealand white rabbits following standard protocol. The homemade antiserum could detect both endogenous Rhox5 protein expressed in eukaryotic cells (Cos-7) and exogenous GFP-Rhox5 protein. Furthermore, the antiserum was used to determine the localization of Rhox5 in NIH3T3 cells using an immunofluorescence technique. The results demonstrated that Rhox5 was localized predominantly in the nucleus. Preparation of the anti-Rhox5 polyclonal antibody will facilitate further functional study of Rhox5.     

Rhox5基因真核表达质粒的构建及其在NIH3T3细胞中的稳定表达

目的:构建同源异性框基因Rhox5的真核表达质粒,转染NIH3T3细胞,建立稳定过表达Rhox5的细胞系。方法:PCR方法扩增Rhox5的全长cDNA序列,PCR产物双酶切后和人工合成的HA抗原表位标签共同克隆至pcDNA3.1(-)哺乳动物细胞表达载体中,构建pcDNA-Rhox5-HA融合表达质粒。脂质体法将经过测序成功的pcDNA-Rhox5-HA融合质粒和pcDNA3.1空载体分别转染NIH3T3细胞,潮霉素B筛选后建立阴性对照pcDNA3.1 in NIH3T3和稳定过表达Rhox5的Rhox5-HA in NIH3T3细胞系。RT-PCR和western blotting方法检测Rhox5-HA在稳定转染细胞系中的表达情况。结果:成功构建了pcDNA-Rhox5-Myc重组质粒,获得稳定过表达Rhox5的NIH3T3细胞系。RT-PCR和Western blotting结果表明,构建的稳定细胞系中成功表达Rhox5-HA融合蛋白。结论:Rhox5基因真核表达质粒的构建及其在NIH3T3细胞中的稳定表达为进一步体外研究Rhox5蛋白单独的功能及其与其他分子间功能性相互作用奠定了实验基础。     

OsBP-5与OsEBP-89两蛋白之间相互作用区域的确定

OsBP-5(MYC类转录因子)与OsEBP-89(AP2/EREBP类转录因子)两个蛋白之间可以相互作用,并能协同调控水稻waxy基因的表达.将OsBP-5与OsEBP-89 cDNA的不同限制性片段分别克隆到酵母双杂交系统的载体上,利用酵母双杂交的方法确定了OsEBP-89中与OsBP-5相互作用的区域位于AP2/EREBP保守域的RAYD元件内;OsBP-5中与OsEBP-89相互作用的区域则有两个区段,它们分别位于Pro68与Val171之间和Leu284与Gly335之间,而不是位于HLH保守域内.酵母系统中的实验还表明,OsEBP-89的3′端部分氨基酸在一定程度上防碍了它与OsBP-5蛋白的相互作用.     

A Stretch of 17 Amino Acids in the Prosaposin C Terminus Is Critical for Its Binding to Sortilin and Targeting to Lysosomes

Prosaposin, the precursor of four lysosomal cofactors required for the hydrolysis of sphingolipids, is transported to the lysosomes via the alternative receptor, sortilin. In this study, we identified a specific domain of 17 amino acids within the C terminus of prosaposin involved in binding to this sorting receptor. We generated six prosaposin deletion constructs and examined the effect of truncation by coimmunoprecipitation and confocal microscopy. The experiments revealed that the first half of the prosaposin C terminus (aa 524–540), containing a saposin-like motif, was required and necessary to bind sortilin and to transport it to the lysosomes. Based on this result, we introduced twelve site-directed point mutations within the first half of the C terminus. Although the interaction of prosaposin with sortilin was pH dependent, the mutation of hydrophilic amino acids that usually modulate pH-dependent protein interactions did not affect the binding of prosaposin to sortilin. Conversely, a tryptophan (W530) and two cysteines (C528 and C536) were essential for its interaction with sortilin and for its transport to the lysosomes. In conclusion, our investigation demonstrates that a saposin-like motif within the first half of the prosaposin C terminus contains the sortilin recognition site. (J Histochem Cytochem 58:287–300, 2010)     

Identification of the Ubiquitin-like Domain of Midnolin as a New Glucokinase Interaction Partner

Glucokinase acts as a glucose sensor in pancreatic beta cells. Its posttranslational regulation is important but not yet fully understood. Therefore, a pancreatic islet yeast two-hybrid library was produced and searched for glucokinase-binding proteins. A protein sequence containing a full-length ubiquitin-like domain was identified to interact with glucokinase. Mammalian two-hybrid and fluorescence resonance energy transfer analyses confirmed the interaction between glucokinase and the ubiquitin-like domain in insulin-secreting MIN6 cells and revealed the highest binding affinity at low glucose. Overexpression of parkin, an ubiquitin E3 ligase exhibiting an ubiquitin-like domain with high homology to the identified, diminished insulin secretion in MIN6 cells but had only some effect on glucokinase activity. Overexpression of the elucidated ubiquitin-like domain or midnolin, containing exactly this ubiquitin-like domain, significantly reduced both intrinsic glucokinase activity and glucose-induced insulin secretion. Midnolin has been to date classified as a nucleolar protein regulating mouse development. However, we could not confirm localization of midnolin in nucleoli. Fluorescence microscopy analyses revealed localization of midnolin in nucleus and cytoplasm and co-localization with glucokinase in pancreatic beta cells. In addition we could show that midnolin gene expression in pancreatic islets is up-regulated at low glucose and that the midnolin protein is highly expressed in pancreatic beta cells and also in liver, muscle, and brain of the adult mouse and cell lines of human and rat origin. Thus, the results of our study suggest that midnolin plays a role in cellular signaling of adult tissues and regulates glucokinase enzyme activity in pancreatic beta cells.     

BRPF1及其新型转录本BRPF2与RHOX5蛋白间的相互作用

RHOX5基因是最早发现的小鼠RHOX基因簇(reproductive homeobox on the X chromosome)成员,可特异性地在生殖系统中表达.RHOX5蛋白在胚胎发育、生殖组织的发育、精子的生成和成熟等多个环节发挥作用,但其功能的发挥途径尚不明确.在前期筛选与RHOX5蛋白相互作用的分子中初步获得一个BRPF1的新型转录本BRPF2.进一步构建pGBKT7-BRPF2质粒,酵母双杂交实验确定其与RHOX5蛋白的相互作用,GST-pull down实验确定其在体外的直接结合;PCR扩增BRPF1基因,构建pGBKT7-BRPF1和pGADT7-BRPF1质粒,酵母双杂交实验和GST-pull down实验证明RHOX5蛋白亦可以直接结合BRPF1蛋白.BRPF1及其新型转录本BRPF2与RHOX5蛋白间的相互作用证实暗示了BRPF2极有可能与BRPF1竞争性结合RHOX5蛋白,为三种蛋白功能的研究提供了新的思路.     

Identification and Characterization of MT-1X as a Novel FHL3-Binding Partner

Four and a half LIM domain protein 3 (FHL3) is a member of the FHL protein family that plays roles in the regulation of cell survival, cell adhesion and signal transduction. However, the mechanism of action for FHL3 is not yet clear. The aim of present study was to identify novel binding partner of FHL3 and to explore the underlying mechanism. With the use of yeast two-hybrid screening system, FHL3 was used as the bait to screen human fetal hepatic cDNA library for interacting proteins. Methionine-1X was identified as a novel FHL3 binding partner. The interaction between FHL3 and the full length MT-1X was further confirmed by yeast two-hybrid assay, co-immunoprecipitation and GST pull-down assays. Furthermore,the result demonstrated that MT-1X knockdown promoted the FHL3-induced inhibitory effect on HepG2 cells by regulating FHL3-mediated Smad signaling and involving in the modulation the expression of G2/M phase-related proteins through interaction with FHL3. These findings suggest that functional interactions between FHL3 and MT-1X may provide some clues to the mechanisms of FHL3-regulated cell proliferation.     

Identification of tyrosine hydroxylase as a physiological substrate for Cdk5

Cyclin-dependent kinase 5 (Cdk5) is emerging as a neuronal protein kinase involved in multiple aspects of neurotransmission in both post- and presynaptic compartments. Within the reward/motor circuitry of the basal ganglia, Cdk5 regulates dopamine neurotransmission via phosphorylation of the postsynaptic signal transduction pathway integrator, DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, M(r) 32,000). Cdk5 has also been implicated in regulating various steps in the presynaptic vesicle cycle. Here we report that Cdk5 phosphorylates tyrosine hydroxylase (TH), the key enzyme for synthesis of dopamine. Using phosphopeptide mapping, site-directed mutagenesis, and phosphorylation state-specific antibodies, the site was identified as Ser31, a previously defined extracellular signal-regulated kinases 1/2 (ERK1/2) site. The phosphorylation of Ser31 by Cdk5 versus ERK1/2 was investigated in intact mouse striatal tissue using a pharmacological approach. The results indicated that Cdk5 phosphorylates TH directly and also regulates ERK1/2-dependent phosphorylation of TH through the phosphorylation of mitogen-activated protein kinase kinase 1 (MEK1). Finally, phospho-Ser31 TH levels were increased in dopaminergic neurons of rats trained to chronically self-administer cocaine. These results demonstrate direct and indirect regulation of the phosphorylation state of a Cdk5/ERK1/2 site on TH and suggest a role for these pathways in the neuroadaptive changes associated with chronic cocaine exposure.