亲和素蛋白与含有生物素的支撑平面膜之间相互作用的研究

生物功能分子的二维有序化组装是分子工程学的重要内容。本文通过构建有生物素受体的人工模型膜,对亲和素与生物素这一具有极强亲和力的系统之间的特异性相互作用的某些制约因素进行了探讨．应用ＬＢ膜技术结合激先椭圆偏振术和表面等离激元谱技术,较深入地研究了蛋白质与脂单层膜发生的非特异性吸附,特异性结合以及结合的侧向空间位阻效应．实验结果表明,膜表面电荷对非特异性吸附的速率有很大的影响,而对最终的蛋白吸附量影响不大;非特异性的吸附可以通过二阶阳离子的脱附而去除;受体蛋白质与配体间的特异性结合受到侧向空间位阻效应的制约．     

原子力显微镜单分子力谱研究生物分子间相互作用

原子力显微镜单分子力谱是近年来发展起来的能在单分子水平研究生物分子相互作用的新工具。本文综述了单分子力谱的测定原理、方法及其在研究蛋白．蛋白、蛋白-DNA相互作用,蛋白质去折叠和活细胞上配体／受体结合中的应用进展。     

G-CSF受体胞内区与AK017149的相互作用

为研究G-CSF受体胞内区与AK017149基因之间的相互作用,进而揭示G-CSF受体信号转导通路的可能机制,采用高敏感性的酵母双杂交体系,筛选小鼠胎肝文库,在筛选过程中获得了1个功能未知的基因片段-AK017149,并在酵母和哺乳动物细胞体内验证了该基因片段的表达蛋白与G-CSF受体间的相互作用．实验研究提示,G-CSF受体作为胞内信号转导的起始点,可以与众多蛋白因子相结合,对其中未知蛋白的研究,可以为揭示G-CSF受体信号转导通路起重要的提示作用．     

神经营养蛋白低亲和力受体p75NTR的结合蛋白

神经营养蛋白通过酪氨酸激酶受体Trk和低亲和力受体p75^NTR发挥其多种生理功能。通过蛋白质相互作用的研究技术已确认了十多个与p75^NTR相互作用的蛋白质分子,它们在结构和功能上都存在显著差异,因而可能赋予了p75^NTR的功能多样性。     

人和猴T淋巴细胞表面TRBC受体及其配体不同于E2分子和CD2的配体

T淋巴细胞表面的TRBC受体不同介导E花结形成的E受体(CD2)和E2分子。CD2的配体,人红细胞表面的CD58(LFA-3)和绵羊红细胞表面的T11 TS,S42,S14及S110-220,与TRBC受体的配体无关,TRBC玫瑰花结的形成是通过不同于E花结和人自身玫瑰花结的受体-配体相互作用来实现的,进一步表明,人和猴T淋巴细胞表面和TRBC表面,可能都有独特的蛋白质分子介导TRBC玫瑰花结的形成。     

染色质免疫沉淀技术在研究DNA与蛋白质相互作用中的应用

在后基因组时代,DNA-蛋白质的相互作用是研究基因表达调控的一个重要领域。与其他方法相比,染色质免疫沉淀技术（chromatin immunoprecipitation assay, ChIP）是一种在体内研究DNA-蛋白质相互作用的理想的方法。近年来这种方法与DNA芯片和分子克隆技术相结合,可用于高通量的筛选已知蛋白因子的未知DNA靶点和研究反式作用因子在整个基因组上的分布情况,这将有助于深入理解DNA-蛋白质相互作用的调控网络。总结了染色质免疫沉淀技术的方法,特别介绍了使用这些方法取得的最新进展。     

真核细胞的辅转录激活因子

真核细胞的辅转录激活因子关键词辅转录激活因子真核细胞的增殖与分化主要由细胞外分子激发,这些分子或是自由存在,或是以其它细胞的表面蛋白形式存在,它们都与被激活的细胞的表面受体相互作用。配体─受体相互作用启动了细胞内信号级联,引起精选的基因快速地转录诱导...     

Notch信号转导与调控

Notch是一个进化上十分保守的跨膜受体蛋白家族,它可以通过与表达配体的相邻细胞间的相互作用转导信号,从而决定动物系统发育过程中多种细胞的“命运”.Notch信号转导过程包括Notch受体与配体的结合、Notch受体的酶切活化、可溶性NICD转移至细胞核并与CSL DNA结合蛋白相互作用,从而调控靶基因的表达.Notch活性水平、时间和空间分布受到包括配体、蛋白质转运、泛素化降解等多水平内源性和外源性诱导因素的调节.系统介绍了Notch信号转导通路的分子组成、Notch信号激活的生化机制、Notch信号的多水平调节以及与部分相关疾病的关系.     

酵母表面展示技术应用的研究进展

酵母菌的蛋白表达方式与高等真核生物细胞十分相似．酵母表面展示技术是研究真核生物,尤其是人类蛋白质表达的理想方法之一。酵母茵表面标记可溶性配基,通过流式细胞技术对酵母菌展示文库进行快速、精确的筛选,其已广泛用于研究天然蛋白质一蛋白质的相互作用、抗体Fab片段的亲和力,成熟、抗体的检测与筛选,以及功能蛋白质抗原表位图谱绘制等。     

EphB4/ephrinB2逆向信号对RAW264．7破骨细胞分化中PDZ结构域蛋白表达变化的研究

Eph受体是酪氨酸蛋白激酶受体家族中最大的亚家族,ephrin(Eph受体相互作用蛋白)是其配体,它们是膜结合蛋白,相互依赖进行信号转导.内居蛋白(syntenin)与Pick1属于PDZ结构域(PSD-95/Dlg-/Zo-1 domain)蛋白,报道称能与ephrinB配体结合,但是否受Eph受体调控尚未见报道.以RAW264.7细胞株为研究对象,通过蛋白质印迹及/或免疫荧光分析显示RAW264.7细胞经RANKL诱导的破骨细胞表达ephrinB2、内居蛋白(syntenin)和Pick1三个蛋白质.将提前成簇的可溶性EphB4蛋白加入培养液,与ephrinB2配体结合,用来研究EphB4/ephrinB2逆向信号对syntenin和Pick1表达水平变化的影响.免疫印迹及Real-time RT-PCR分析结果显示,在EphB4-Fc实验组中Pick1的蛋白质及mRNA水平都有明显增加,然而在EphB4-Fc实验组与Fc对照组别间syntenin的蛋白质及mRNA水平未见明显变化.免疫共沉淀结果显示,syntenin和Pick1不能与ephrinB2共沉淀.以上结果初步探索了体外破骨细胞分化过程中,EphB4/ephrinB2逆向信号对PDZ结构域蛋白(ephrinB2配体潜在的下游信号分子)表达变化的调控.     

On-chip Escherichia coli culture, purification, and detection of expressed proteins

In a recent study, we reported the results of a rapid high-throughput expression analysis of the affinity-tagged proteins present in total cell lysates, using a surface plasmon resonance (SPR) imaging protein chip system. In this paper, we describe a novel method, which is able to sequentially carry out a recombinant Escherichia coli culture, as well as the detection and purification of the expressed proteins on a single microwell chip, fabricated on a two-dimensional thin gold film. Following the induction of the protein on the microwell chip, the E. coli cells were lysed on the chip via the addition of lysozymes, and the expressed glutathione S-transferase-fused green fluorescent protein (GST–GFP) was then purified on the chip via affinity interaction with the glutathionylated gold surface of the chip. Finally, the expressed protein was directly detected using the surface plasmon resonance (SPR) imaging system. This system saves a substantial amount of time, experimental resources, and labor, by allowing for the complicated and labor-intensive procedures inherent to the production of recombinant proteins to be conducted on a single microwell chip, simply and economically.M. Kim and S. Y. Lee contributed equally to this work.     

GABA<Subscript>A</Subscript> receptor-associated protein (GABARAP) induces apoptosis by interacting with DEAD (Asp-Glu-Ala-Asp/His) box polypeptide 47 (DDX 47)

GABA_A receptor-associated protein (GABARAP) is a 14-kDa cytoplasmic protein initially identified as a molecular chaperone for GABA_A receptor in cortical neurons. However, evidence indicates that the function of GABARAP is much broader than a specific role in neuronal cells. As an initial step to define the biological role of GABARAP, we searched for binding partners of GABARAP using co-immunoprecipitation coupled with LC-MS/MS analysis. As a result, DEAD (Asp-Glu-Ala-Asp/His) box polypeptide 47 (DDX 47), recently identified as RNA helicase, is identified as a binding partner of GABARAP. An interaction between GABARAP and DDX 47 was further confirmed by yeast two-hybrid systems. Further, co-transfection of GABARAP and DDX47 cDNA into a tumor cell line induces apoptosis. This result may be the first evidence showing that GABARAP and DDX 47 are involved in the apoptotic process.     

Integration of surface plasmon resonance with mass spectrometry: automated ligand fishing and sample preparation for MALDI MS using a Biacore 3000 biosensor.

Integrating surface plasmon resonance analysis with mass spectrometry allows detection and characterization of molecular interactions to be complemented with identification of interaction partners. We have developed a procedure for Biacore 3000 that automatically performs all steps from ligand fishing and recovery to sample preparation for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry including on-target digestion. In the model system used in this study a signal transduction protein, calmodulin, was selectively captured from brain extract by one of its interaction partners immobilized on a sensor chip. The bound material was eluted, deposited directly onto a MALDI target, and analyzed by mass spectrometry both as an intact protein and after on-target tryptic digestion. The procedure with direct deposition of recovered material on the MALDI target reduces sample losses and, in combination with automatic sample processing, increases the throughput of surface plasmon resonance mass spectrometry analysis.     

Characterization of the Molecular Architecture of Human Caveolin-3 and Interaction with the Skeletal Muscle Ryanodine Receptor

Caveolin-3 (cav-3), an integral membrane protein, is a building block of caveolae as well as a regulator of a number of physiological processes by facilitating the formation of multiprotein signaling complexes. We report that the expression of cav-3 in insect (Sf9) cells induces caveola formation, comparable in size with those observed in native tissue. We have also purified the recombinant cav-3 determining that it forms an oligomer of ∼220 kDa. We present the first three-dimensional structure for cav-3 (using transmission electron microscopy and single particle analysis methods) and show that nine cav-3 monomers assemble to form a complex that is toroidal in shape, ∼16.5 nm in diameter and ∼ 5.5 nm in height. Labeling experiments and reconstitution of the purified cav-3 into liposomes have allowed a proposal for the orientation of the protein with respect to the membrane. We have identified multiple caveolin-binding motifs within the ryanodine receptor (RyR1) sequence employing a bioinformatic analysis. We have then shown experimentally that there is a direct interaction between recombinant cav-3 nonamers and purified RyR1 homotetramers that would imply that at least one of the predicted cav-3-binding sites is exposed within the fully assembled RyR1 structure. The cav-3 three-dimensional model provides new insights as to how a cav-3 oligomer can bind multiple partners in close proximity to form signaling complexes. Furthermore, a direct interaction with RyR1 suggests a possible role for cav-3 as a modifier of muscle excitation-contraction coupling and/or for localization of the receptor to regions of the sarcoplasmic reticulum.     

SARS-CoV核蛋白和宿主细胞相互作用的初步研究

寻找与SARS-CoV核蛋白相互作用的宿主细胞蛋白,从而探索SARS-CoV的致病机理。可溶性表达SARS-CoV核蛋白,利用His标签和离子交换层析对表达的蛋白进行了纯化,获得较纯的可溶性核蛋白。再将SPR/BIA技术与MALDI-TOF MS技术结合起来,使用SPR生物传感芯片作为亲和吸附的表面,分别捕获2BS细胞和A549细胞裂解液中与SARS-CoV核蛋白相互作用的细胞蛋白,收集足够量的相互作用蛋白,再利用MALDI-TOF-MS分析获得蛋白的性质。结果鉴定出与SARS-CoV核蛋白相互作用的蛋白:26S蛋白酶调节亚单位S10B(蛋白酶体亚单位p42)(蛋白酶体26S亚单位ATPase 6)(P62333),属于泛素/蛋白酶体系统;目前国内外尚未见类似报道。此研究初步发现了一种与SARS-CoV核蛋白在细胞外相互作用的蛋白,但这种相互作用在SARS-CoV感染及SARS的发生发展中发挥的作用还有待于深入研究和探索。     

Surface plasmon resonance imaging protein arrays for analysis of triple protein interactions of HPV, E6, E6AP, and p53

We have developed a surface plasmon resonance (SPR)-based protein microarray to study protein-protein interactions in a high-throughput mode. As a model system, triple protein interactions have been explored with human papillomaviral E6 protein, tumor suppressor p53, and ubiquitin ligase E6AP. Human papillomavirus (HPV) is known to be a causative agent of cervical cancer. Upon infection, the viral E6 protein forms a heterotrimeric protein complex with p53 and E6AP. The formation of the complex eventually results in the degradation of p53. In the present study, a GST-fused E6AP protein was layered onto a glutathione (GSH)-modified gold chip surface. The specific binding of GST-E6AP protein onto the gold chip surface was facilitated through the affinity of GST to its specific ligand GSH. The interacting proteins (E6 and/or p53) were then spotted. Detection of the interaction was performed using a SPR imaging (SPRI) technique. The resulting SPRI intensity data showed that the protein-protein interactions of E6AP, E6, and p53 were detected in a concentration-dependent manner, suggesting that the SPRI-based microarray system can be an effective tool to study protein-protein interactions where multiple proteins are involved.     

与PRRSV nsp11互作的宿主细胞蛋白鉴定及生物信息学分析

【目的】研究猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)nsp11与宿主细胞蛋白之间的相互作用,对于揭示nsp11在病毒复制过程中发挥的功能至关重要。【方法】在病毒感染细胞的基础上,利用nsp11的单克隆抗体,采用免疫沉淀结合串联质谱的方法,筛选与PRRSV nsp11相互作用的宿主细胞蛋白,并对所筛选出的宿主细胞蛋白进行了GO注释、COG注释和KEGG代谢通路注释;选取筛选出的宿主细胞蛋白IRAK1,利用免疫共沉淀技术和激光共聚焦技术鉴定其与nsp11之间的相互作用。【结果】与空白对照组相比,病毒感染组中出现3条差异带;经质谱分析共筛选得到了201个与nsp11相互作用的宿主细胞蛋白,分别与蛋白质代谢、细胞信号通路转导以及病原致病性等密切相关;在生物信息学分析的基础上,实验验证了nsp11确与宿主细胞蛋白IRAK1进行相互作用。【结论】鉴定出与PRRSV nsp11相互作用的宿主细胞蛋白,生物信息学分析显示它们在病毒的复制和致病过程中发挥重要作用。研究结果为探究nsp11的生物学功能指明了方向,也为研究宿主细胞蛋白与病毒蛋白间的相互作用及其调控病毒复制和致病性的分子机制奠定了基础。     

One-step, non-denaturing isolation of an RNA polymerase enzyme complex using an improved multi-use affinity probe resin

The rapid isolation of protein complexes is critical to the goal of establishing protein interaction networks. High-throughput methods for identifying protein binding partners in a way suitable for mass spectrometric identification and structural analysis are required and small molecule/peptide interactions provide the key. We have now shown that a redesigned resin derivatized with a bisarsenical dye can be used to isolate the Shewanella oneidensis RNA polymerase core enzyme with a tetracysteine-tagged RNA polymerase A as bait protein. A critical advantage of this method is the ability to release the intact complex using a mild, one-step procedure with a competing dithiol. In addition to the identification of the core complex, additional interaction partners, including universal stress protein, were identified. These results provide a path forward to identifying how changes in critical protein complexes over time modulate cell function.     

Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity

Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction K_m. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome.     

The Mitosis and Neurodevelopment Proteins NDE1 and NDEL1 Form Dimers, Tetramers, and Polymers with a Folded Back Structure in Solution

Paralogs NDE1 (nuclear distribution element 1) and NDEL1 (NDE-like 1) are essential for mitosis and neurodevelopment. Both proteins are predicted to have similar structures, based upon high sequence similarity, and they co-complex in mammalian cells. X-ray diffraction studies and homology modeling suggest that their N-terminal regions (residues 8–167) adopt continuous, extended α-helical coiled-coil structures, but no experimentally derived information on the structure of their C-terminal regions or the architecture of the full-length proteins is available. In the case of NDE1, no biophysical data exists. Here we characterize the structural architecture of both full-length proteins utilizing negative stain electron microscopy along with our established paradigm of chemical cross-linking followed by tryptic digestion, mass spectrometry, and database searching, which we enhance using isotope labeling for mixed NDE1-NDEL1. We determined that full-length NDE1 forms needle-like dimers and tetramers in solution, similar to crystal structures of NDEL1, as well as chain-like end-to-end polymers. The C-terminal domain of each protein, required for interaction with key protein partners dynein and DISC1 (disrupted-in-schizophrenia 1), includes a predicted disordered region that allows a bent back structure. This facilitates interaction of the C-terminal region with the N-terminal coiled-coil domain and is in agreement with previous results showing N- and C-terminal regions of NDEL1 and NDE1 cooperating in dynein interaction. It sheds light on recently identified mutations in the NDE1 gene that cause truncation of the encoded protein. Additionally, analysis of mixed NDE1-NDEL1 complexes demonstrates that NDE1 and NDEL1 can interact directly.