规模化蛋白质相互作用的研究技术

蛋白质相互作用既是蛋白质执行功能的主要方式,也是细胞功能调控网络的结构基础。蛋白质间异常的相互作用及其连锁网络的紊乱是引起许多病理改变的原因。作为功能基因组和蛋白质组研究的重要内容,规模化蛋白质相互作用研究已成为近年国际上研究的热点之一。文章综述了当前规模化蛋白质相互作用研究中的常用技术和常用蛋白质相互作用数据库,研究者可根据研究需要和技术特点利用这些资源。     

Glutamate Receptor-interacting Protein 1 Protein Binds to the Microtubule-associated Protein

To identify the interaction proteins for the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunit glutamate receptor-interacting protein 1 (GRIP1), GRIP1 interactions with microtubule-associated protein (MAP)-1B light chain (LC) were investigated. GRIP1 interacts with MAP-1A and MAP-1B in the yeast two-hybrid assay, as is indicated also by glutathione S-transferase (GST) pull-down and coimmunoprecipitation with MAP-1B LC antibody in brain fractions. These results suggest a novel mechanism for localizing AMPA receptors to synaptic sites.     

SARS冠状病毒核壳蛋白对蛋白翻译的影响

目的:研究SARS冠状病毒核壳蛋白(N蛋白)对蛋白翻译的影响。方法:构建N蛋白表达载体FLAG-pcDNA3-N,分别与FLAG-pcDNA3和表达荧光素酶的质粒共转染293T人胚肾细胞,通过检测荧光素酶的活性来判断N蛋白对细胞内蛋白翻译的影响;在体外翻译体系中检测N蛋白对体外翻译的影响。结果:构建了载体FLAG-pcDNA3-N,在293T人胚肾细胞内表达后,荧光素酶活性被抑制;在体外翻译体系中加入N蛋白,体外翻译被抑制。结论:SARS冠状病毒N蛋白抑制蛋白翻译。     

Protein Solubility and Protein Homeostasis: A Generic View of Protein Misfolding Disorders

According to the “generic view” of protein aggregation, the ability to self-assemble into stable and highly organized structures such as amyloid fibrils is not an unusual feature exhibited by a small group of peptides and proteins with special sequence or structural properties, but rather a property shared by most proteins. At the same time, through a wide variety of techniques, many of which were originally devised for applications in other disciplines, it has also been established that the maintenance of proteins in a soluble state is a fundamental aspect of protein homeostasis. Taken together, these advances offer a unified framework for understanding the molecular basis of protein aggregation and for the rational development of therapeutic strategies based on the biological and chemical regulation of protein solubility.Virtually every complex biochemical process taking place in living cells depends on the ability of the molecules involved to self-assemble into functional structures (Dobson 2003; Robinson et al. 2007; Russel et al. 2009), and a sophisticated quality control system is responsible for regulating the reactions leading to this organization within the cellular environment (Dobson 2003; Balch et al. 2008; Hartl and Hayer-Hartl 2009; Powers et al. 2009; Vendruscolo and Dobson 2009). Proteins are the molecules that are essential for enabling, regulating, and controlling almost all the tasks necessary to maintain such a balance. To function, the majority of our proteins need to fold into specific three-dimensional structures following their biosynthesis in the ribosome (Hartl and Hayer-Hartl 2002). The wide variety of highly specific structures that results from protein folding, and which serve to bring key functional groups into close proximity, has enabled living systems to develop an astonishing diversity and selectivity in their underlying chemical processes by using a common set of just 20 basic molecular components, the amino acids (Dobson 2003). Given the central importance of protein folding, it is not surprising that the failure of proteins to fold correctly, or to remain correctly folded, is at the origin of a wide variety of pathological conditions, including late-onset diabetes, cystic fibrosis, and Alzheimer’s and Parkinson’s diseases (Dobson 2003; Chiti and Dobson 2006; Haass and Selkoe 2007). In many of these disorders proteins self-assemble in an aberrant manner into large molecular aggregates, notably amyloid fibrils (Chiti and Dobson 2006; Ramirez-Alvarado et al. 2010).     

绿色荧光蛋白在蛋白质研究中的应用

绿色荧光蛋白（green fluorescent protein,GFP）自发现以来,由于具有自发荧光等特性,在分子生物学和细胞生物学领域得到广泛应用。GFP作为一种报道分子,在研究蛋白质相互作用和构象变化、检测蛋白质表达、蛋白质和细胞荧光示踪中,起到了重要的作用。该文通过对绿色荧光蛋白特性的分析．介绍其作为荧光标记在蛋白质研究中的应用,并展望进一步的研究前景。     

Inhibition of Protein Synthesis Alters Protein Degradation through Activation of Protein Kinase B (AKT)

The homeostasis of protein metabolism is maintained and regulated by the rates of protein biosynthesis and degradation in living systems. Alterations of protein degradation may regulate protein biosynthesis through a feedback mechanism. Whether a change in protein biosynthesis modulates protein degradation has not been reported. In this study, we found that inhibition of protein biosynthesis induced phosphorylation/activation of AKT and led to phosphorylation of AKT target substrates, including FoxO1, GSK3α/β, p70S6K, AS160, and the E3 ubiquitin ligase MDM2. Phosphorylation of ribosomal protein S6 was also modulated by inhibition of protein biosynthesis. The AKT phosphorylation/activation was mediated mainly through the PI3K pathway because it was blocked by the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. The activated AKT phosphorylated MDM2 at Ser¹⁶⁶ and promoted degradation of the tumor suppressor p53. These findings suggest that inhibition of protein biosynthesis can alter degradation of some proteins through activation of AKT. This study reveals a novel regulation of protein degradation and calls for caution in blocking protein biosynthesis to study the half-life of proteins.     

蛋白质二硫键异构酶相关蛋白A的表达及性质的研究

克隆了Aspergillus niger T21中的蛋白质二硫键异构酶相关蛋白A（PRPA）基因,并将它插入pET23b表达载体。在E. coli中表达时,PRPA占菌体总蛋白的34%。经过超声破细胞、硫酸铵分级沉淀和离子交换层析获得了纯度大于90%的重组蛋白。PRPA有二硫键异构酶活性。在PRPA存在下,变性和还原的溶菌酶复性率和复性速度降低,电泳结果表明溶菌酶聚集增多。荧光结果表明PRPA表面有较多的疏水基团。     

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...     

Protein Purification with C-Terminal Fusion of Maltose Binding Protein

For affinity-chromatography-based purification of proteins that are prone to abnormal termination of translation or that may not be modified at their N-termini, affinity tags are needed which can be fused to the C-terminus. In this publication we describe that maltose binding protein (MBP) fused to the C-terminus of the plant photoreceptor phytochrome B allows purification of the fusion protein via amylose affinity chromatography. After overexpression in yeast a 125-fold enrichment could be achieved. The spectral properties of phytochrome B were not impaired by the fusion and purification. These results demonstrate that not only the widely used N-terminal fusions of MBP but also C-terminal fusions can be employed for protein purification.     

植物磷胁迫蛋白和铁胁迫蛋白研究进展

综述了近十年来国内外有关研究植物磷胁迫蛋白和铁胁迫蛋白的文献。着重阐述了磷胁迫和铁胁迫条件下的植物蛋白质变化,如新的蛋白和新的多肽的特异产生,以及相关的分子生物学进展。     

丝裂原活化蛋白激酶激活蛋白激酶(MK)研究进展

丝裂原活化蛋白激酶(MAPK)信号通路介导多种重要的细胞生理反应.对下游蛋白激酶的磷酸化是MAPK家族成员发挥生理作用的重要方式.在MAPK的下游存在3个结构上相关的MAPK激活蛋白激酶(MAPKAPKorMK),即MK2,MK3和MK5.在被MAPK激活后,MK可将信号传递至细胞内不同靶标,从而在转录和翻译水平调节基因表达,调控细胞骨架和细胞周期,介导细胞迁移和胚胎发育.最近,在基因敲除研究的基础上,不同MK亚族成员之间的功能区分已经逐渐明晰,使我们对于MK的认识有了长足的进步.     

植物磷胁迫蛋白和铁胁迫蛋白研究进展

综述了近十年来国内外有关研究植物磷胁迫蛋白和铁胁迫蛋白的文献,着重阐述了磷胁迫和铁胁迫条件下的植物蛋白质变化,如新的蛋白和新的多肽的特异产生,以及相关的分子生物学进展。     

运动神经元生存蛋白SMN与Sm蛋白的结合作用

脊髓性肌萎缩症（spinal muscular atrophy,SMA）是一类与运动神经元存活基因（survival of motor neurons gene,SMN gene）突变有关的神经系统变性疾病,而SMN基因的转录产物即为SMN蛋白（survival of motorneurons protein,SMN protein）。SMN蛋白与多种蛋白结合后发挥作用,如SMN-Sm蛋白的相互作用在富含尿嘧啶的小核核糖核蛋白体（uridine—richsmallribonucleo—proteins,UsnRNPs）转运装配中有重要意义。SMN蛋白是通过其Tudor结构域与剪接体sm蛋白的二甲基化修饰的富含精氨酸一氨基乙酸域（ar—ginineandglycine—rich,RG）结合。     

The Influence of Codon Usage,Protein Abundance,and Protein Stability on Protein Evolution Vary by Evolutionary Distance and the Type of Protein

The Protein Journal - In general, the evolutionary rate of proteins is not primarily related to protein and amino acid functions, and factors such as protein abundance, codon usage, and...     

Evolution of Robustness to Protein Mistranslation by Accelerated Protein Turnover

Translational errors occur at high rates, and they influence organism viability and the onset of genetic diseases. To investigate how organisms mitigate the deleterious effects of protein synthesis errors during evolution, a mutant yeast strain was engineered to translate a codon ambiguously (mistranslation). It thereby overloads the protein quality-control pathways and disrupts cellular protein homeostasis. This strain was used to study the capacity of the yeast genome to compensate the deleterious effects of protein mistranslation. Laboratory evolutionary experiments revealed that fitness loss due to mistranslation can rapidly be mitigated. Genomic analysis demonstrated that adaptation was primarily mediated by large-scale chromosomal duplication and deletion events, suggesting that errors during protein synthesis promote the evolution of genome architecture. By altering the dosages of numerous, functionally related proteins simultaneously, these genetic changes introduced large phenotypic leaps that enabled rapid adaptation to mistranslation. Evolution increased the level of tolerance to mistranslation through acceleration of ubiquitin-proteasome–mediated protein degradation and protein synthesis. As a consequence of rapid elimination of erroneous protein products, evolution reduced the extent of toxic protein aggregation in mistranslating cells. However, there was a strong evolutionary trade-off between adaptation to mistranslation and survival upon starvation: the evolved lines showed fitness defects and impaired capacity to degrade mature ribosomes upon nutrient limitation. Moreover, as a response to an enhanced energy demand of accelerated protein turnover, the evolved lines exhibited increased glucose uptake by selective duplication of hexose transporter genes. We conclude that adjustment of proteome homeostasis to mistranslation evolves rapidly, but this adaptation has several side effects on cellular physiology. Our work also indicates that translational fidelity and the ubiquitin-proteasome system are functionally linked to each other and may, therefore, co-evolve in nature.     

IEX-1相互作用蛋白的筛选与鉴定

骨肉瘤即原发于骨的恶性肿瘤,易发生早期肺转移且预后差,恶性程度高.本研究小组前期研究发现,IEX-1在骨肉瘤中具有重要作用.为了阐明其作用机制,本研究运用酵母双杂交技术筛选其相互作用蛋白,共鉴定出12个IEX-1相互作用蛋白,包括生物氧化相关酶类、分子伴侣、信号转导相关蛋白等.并首次证实clusterin(CLU,又名载脂蛋白J)与IEX-1存在相互作用,两者在细胞中具有很好的共定位.采用RNAi干扰减低骨肉瘤细胞中内源性CLU表达水平,显著抑制了细胞增殖与细胞侵袭能力.为阐明IEX-1在骨肉瘤发生发展中的作用机制提供了重要的线索,为骨肉瘤的早期诊治及预后监测提供了新的靶点.