Zinc finger structure-function in Ikaros

The zinc finger motif was used as a vehicle for the initial discovery of Ikaros in the context of T-cell differentiation and has been central to all subsequent analyses of Ikaros function. The Ikaros gene is alternately spliced to produce several isoforms that confer diversity of function and consequently have complicated analysis of the function of Ikaros in vivo. Key features of Ikaros in vivo function are associated with six C2H2 zinc fingers; four of which are alternately incorporated in the production of the various Ikaros isoforms. Although no complete structures are available for the Ikaros protein or any of its family members, considerable evidence has accumulated about the structure of zinc fingers and the role that this structure plays in the functions of the Ikaros family of proteins. This review summarizes the structural aspects of Ikaros zinc fingers, individually, and in tandem to provide a structural context for Ikaros function and to provide a structural basis to inform the design of future experiments with Ikaros and its family members.     

A novel Zinc finger protein, ZCCHC11, interacts with TIFA and modulates TLR signaling

Toll-like receptors (TLRs) play an important role as a sensor of microbial pathogens in the innate immune response. TLRs transmit signals through the recruitment of adaptor proteins including tumor necrosis factor-associated factor 6 (TRAF6), which mediates the activation of IkappaB kinase (IKK). TIFA (TRAF-interacting protein with a forkhead-associated (FHA) domain) has been shown to bind to TRAF6 and activate IKK by promoting the oligomerization and ubiquitin-ligase activity of TRAF6. FHA domains preferentially bind to phospho-threonine residues in their targets. Here, we identified a novel zinc finger protein, ZCCHC11, that interacts with TIFA from phosphoproteins of a macrophage cell line, RAW 264.7, by using affinity purification with GST-TIFA and mass spectrometric analysis. By a search of the EST database, we found a 200kDa full-length form (ZCCHC11L). ZCCHC11L was mostly located to the nucleus, but translocated into the cytoplasm in response to LPS and bound to TIFA. Overexpression and knockdown by siRNA indicated that ZCCHC11 functions as a negative regulator of TLR-mediated NF-kappaB activation. The N-terminal region (ZCCHC11S) including C2H2-type [corrected] Zn-finger motif was sufficient for suppression of NF-kappaB. We propose that ZCCHC11 is a unique TLR signal regulator, which interacts with TIFA after LPS treatment and suppresses the TRAF6-dependent activation of NF-kappaB.     

Re-programming DNA-binding specificity in zinc finger proteins for targeting unique address in a genome

Recent studies provide a glimpse of future potential therapeutic applications of custom-designed zinc finger proteins in achieving highly specific genomic manipulation. Custom-design of zinc finger proteins with tailor-made specificity is currently limited by the availability of information on recognition helices for all possible DNA targets. However, recent advances suggest that a combination of design and selection method is best suited to identify custom zinc finger DNA-binding proteins for known genome target sites. Design of functionally self-contained zinc finger proteins can be achieved by (a) modular protein engineering and (b) computational prediction. Here, we explore the novel functionality obtained by engineered zinc finger proteins and the computational approaches for prediction of recognition helices of zinc finger proteins that can raise our ability to re-program zinc finger proteins with desired novel DNA-binding specificities.     

逆境相关植物锌指蛋白的研究进展

锌是植物必需的营养元素,锌指蛋白因其具有指状结构特征且能结合Zn2 而得名,植物锌指蛋白包含特有的QALGGH保守结构,可能涉及调控植物特有的生物学功能。人们已经从拟南芥(Arabidopsis thaliana)、矮牵牛(Petunia hybrida Vilm)、水稻(Oryza sativa)、大豆(Glycine max)、棉花(Gossypium hirsutum)等植物中克隆了许多编码锌指蛋白的基因,并对其结构及功能进行了研究。利用转基因技术,将一些与逆境胁迫相关的锌指蛋白基因在目标植物中过量表达后,能对植物起到增强抗逆性的作用,说明锌指蛋白在增强植物逆境抗性方面有着广阔的应用前景。     

Electrophoretic mobility shift assay of zinc finger proteins: competition for Zn(2+) bound to Sp1 in protocols including EDTA

The electrophoretic mobility shift assay (EMSA) offers a principal method to detect specific DNA-protein interactions. As commonly conducted, the reaction and electrophoresis running buffers contain large concentrations of EDTA. EDTA has large affinity for Zn²⁺ and readily competes with zinc finger peptides for Zn²⁺ resulting in protein unfolding. Nevertheless, EMSA is routinely used to detect zinc finger protein-DNA adducts. This paper examines the chemistry that permits the detection of zinc finger-DNA complexes in the presence of EDTA, using Zn₃-Sp1 and a cognate DNA binding site, GC1. Twice as much adduct was detected when the reaction was conducted in the absence than in the presence of EDTA. The observation of Zn-Sp1-GC1 was shown to depend on three properties: the inertness of Zn-Sp1-GC1 to reaction with EDTA and the comparatively similar rates of reaction of EDTA and GC1 with Zn₃-Sp1 under the conditions of the assay that permit some Zn₃-Sp1-GC1 to form. Inquiring about the mechanism of stabilization of Zn₃-Sp1 by GC1, EDTA readily reacted with Zn₃-Sp1 bound to a non-specific DNA, (polydI-dC). Two structurally similar but oppositely charged chelators, nitrilotriacetate (NTA) and tris-(2-ethylaminoethyl) amine (TREN), that react with free Zn₃-Sp1 failed to compete for zinc bound in the Zn₃-Sp1-GC-1 adduct. On the basis of these, other results indicated that the stability of Zn₃-Sp1-GC-1 has a thermodynamic, not a kinetic origin. It is concluded that the observation of zinc finger proteins in the EMSA rests on a fortuitous set of chemical properties that may vary depending on the structures involved.     

人Pokemon蛋白锌指结构域的表达与纯化

旨在原核表达Pokemon基因的锌指结构域,纯化获得GST-Zinc finger的融合蛋白。以人胶质瘤T98G细胞的c DNA为模板,利用PCR扩增带有Bam H I和Sal I酶切位点的人Pokemon基因的锌指结构域,然后将其克隆到p GEX-4T-1原核表达载体中。将正确的重组载体转入大肠杆菌BL21(DE3),用IPTG诱导表达,再利用Magne GST particles亲和纯化Zinc finger融合蛋白,最后通过Western blot鉴定此融合蛋白。结果显示,成功构建p GEX-4T-1-Zinc finger原核表达载体;30℃条件下,0.2 mmol/L的IPTG能诱导出大量的可溶性GST-Zinc finger蛋白;经Magne GST particles纯化的GST-Zinc finger蛋白可被识别Pokemon锌指结构域的抗体特异识别。纯化的GST-Zinc finger蛋白可用于后续的生物学研究。     

Zinc ions stimulate the cooperative RNA binding of hordeiviral gammab protein

A small regulatory gammab protein of the Poa semilatent hordeivirus (PSLV) contains two zinc finger-like motifs separated by a basic motif in the N-terminal part and a C-terminal coiled-coil motif. Interactions of the recombinant PSLV gammab protein and its mutants with various RNAs (ssRNA, dsRNA, ssRNA oligonucleotides) and ssDNA were studied in gel-shift assays. The results demonstrated that zinc ions are essential for effective nucleic-acid-binding activity of the gammab protein, suggesting the important role of zinc finger motifs in these interactions. Deletion of the C-proximal coiled-coil region did not affect highly cooperative RNA-protein binding, indicating that the N-terminal part of the protein contributes to the protein-protein interactions needed for the protein-RNA cooperativity.     

Unidirectional cloning by cleaving heterogeneous sites with a single sandwiched zinc finger nuclease

We previously developed a novel type of zinc finger nucleases (ZFNs), sandwiched ZFNs that can discriminate DNA substrates from cleavage products and thus cleave DNA much more efficiently than conventional ZFNs as well as perform with multiple turnovers like restriction endonucleases. In the present study, we used the sandwiched ZFN to unidirectionally clone exogenous genes into target vectors by cleaving heterogeneous sites that contained heterogeneous spacer DNAs between two zinc-finger protein binding sites with a single sandwiched ZFN. We demonstrated that the sandwiched ZFN cleaved a 40-fold excess of both insert and vector plasmids within 1 h and confirmed by sequencing that the resulting recombinants harbored the inserted DNA fragment in the desired orientation. Because sandwiched ZFNs can recognize and cleave a variety of long (?26-bp) target DNAs, they may not only expand the utility of ZFNs for construction of recombinant plasmids, but also serve as useful meganucleases for synthesis of artificial genomes.     

Zinc finger protein binding to DNA: an energy perspective using molecular dynamics simulation and free energy calculations on mutants of both zinc finger domains and their specific DNA bases

Energy calculations based on MM-GBSA were employed to study various zinc finger protein (ZF) motifs binding to DNA. Mutants of both the DNA bound to their specific amino acids were studied. Calculated energies gave evidence for a relationship between binding energy and affinity of ZF motifs to their sites on DNA. ΔG values were ?15.82(12), ?3.66(12), and ?12.14(11.6) kcal/mol for finger one, finger two, and finger three, respectively. The mutations in the DNA bases reduced the value of the negative energies of binding (maximum value for ΔΔG = 42Kcal/mol for F1 when GCG mutated to GGG, and ΔΔG = 22 kcal/mol for F2, the loss in total energy of binding originated in the loss in electrostatic energies upon mutation (r = .98). The mutations in key amino acids in the ZF motif in positions-1, 2, 3, and 6 showed reduced binding energies to DNA with correlation coefficients between total free energy and electrostatic was .99 and with Van der Waal was .93. Results agree with experimentally found selectivity which showed that Arginine in position-1 is specific to G, while Aspartic acid (D) in position 2 plays a complicated role in binding. There is a correlation between the MD calculated free energies of binding and those obtained experimentally for prepared ZF motifs bound to triplet bases in other reports (), our results may help in the design of ZF motifs based on the established recognition codes based on energies and contributing energies to the total energy.     

锌指蛋白与心脏发育

锌指是最大的DNA结合蛋白家族,是最普遍的核酸识别元件.近年来发现锌指参与生物体的基因转录,复制及蛋白质的合成等各种基因调节和控制过程,心脏发育过程中涉及大量锌指基因.综述了心脏发育过程中起重要调控作用的锌指蛋白以及它们的作用机制.     

重组PLZF蛋白锌指结构域的表达、提纯和活性分析

PLZF(promyelocytic leukaemia zinc finger protein)是一种重要的转录抑制因子,它由位于N端的BTB结构域和C端的锌指结构域构成。鉴于目前对于锌指结构域的立体结构还不是十分清楚,对其进行了高效表达和提纯。为了表达PLZF蛋白的锌指结构域,在其编码序列的5'端加上起始密码ATG后插入到表达载体PET-11a的多克隆位点。构建好的表达质粒转化到BL21 (DE3)大肠杆菌内并用IPTG诱导表达,发现重组蛋白主要以不溶性的包涵体形式在胞内表达。用含有SDS变性剂的缓冲液溶解包涵体后,采用凝胶过滤方法将重组蛋白纯化到纯度达96%以上。对纯化后的蛋白质用反透析的方法进行复性,然后用DNA结合实验进行活性分析,发现复性后的蛋白质具有特异的DNA结合活性,这为进一步研究PLZF蛋白锌指结构域的立体结构打下了重要基础。