Selective dimerization of a C2H2 zinc finger subfamily

The C2H2 zinc finger is the most prevalent protein motif in the mammalian proteome. Two C2H2 fingers in Ikaros are dedicated to homotypic interactions between family members. We show here that these fingers comprise a bona fide dimerization domain. Dimerization is highly selective, however, as homologous domains from the TRPS-1 and Drosophila Hunchback proteins support homodimerization, but not heterodimerization with Ikaros. Ikaros-Hunchback selectivity is determined by 11 residues concentrated within the alpha-helical regions typically involved in base recognition. Preferential homodimerization of one chimeric protein predicts a parallel dimer interface and establishes the feasibility of creating novel dimer specificities. These results demonstrate that the C2H2 motif provides a versatile platform for both sequence-specific protein-nucleic acid interactions and highly specific dimerization.     

Specific RNA binding by a single C2H2 zinc finger

Zinc finger proteins with high affinity for human immunodeficiency virus Rev responsive element stem loop IIB (RRE-IIB) were previously isolated from a phage display zinc finger library. Zinc fingers from one of these proteins, RR1, were expressed individually and assayed for RRE-IIB affinity. The C-terminal zinc finger retained much of the binding affinity of the two-finger parent and was disrupted by mutations predicted to narrow the RRE-IIB major groove and which disrupt Rev binding. In contrast, the N-terminal zinc finger has a calculated affinity at least 1000-fold lower. Despite the high affinity and specificity of RR1 for RRE-IIB, binding affinity for a 234-nucleotide human immunodeficiency virus Rev responsive element (RRE234) was significantly lower. Therefore, zinc finger proteins that bind specifically to RRE234 were constructed using an in vitro selection and recombination approach. These zinc fingers bound RRE234 with subnanomolar dissociation constants and bound the isolated RRE-IIB stem loop with an affinity 2 orders of magnitude lower but similar to the affinity of an arginine-rich peptide derived from Rev. These data show that single C2H2 zinc fingers can bind RNA specifically and suggest that their binding to stem loop IIB is similar to that of Rev peptide. However, binding to RRE234 is either different from stem loop IIB binding or the tertiary structure of stem loop IIB is changed within the Rev responsive element.     

Quantitative electrospray ionization mass spectrometry of zinc finger oxidation: the reaction of XPA zinc finger with H(2)O(2)

Oxidation plays an important role in the functioning of zinc fingers (ZFs). Electrospray ionization mass spectrometry (ESI-MS) is a very useful technique to study products of ZF oxidation, but its application has been limited largely to qualitative analysis of reaction products. On the other hand, ESI-MS has been applied successfully on several occasions to determine binding constants in metalloproteins. We used a synthetic 37-residue peptide acetyl-DYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHK-amide (XPAzf), which corresponds to the Cys4 ZF sequence of human nucleotide excision repair protein XPA, to find out whether ESI-MS might be used quantitatively to study ZF reaction kinetics. For this purpose, we studied oxidation of the Zn(II) complex of XPAzf (ZnXPAzf) by H(2)O(2) using three techniques in parallel: high-performance liquid chromatography (HPLC) of covalent reaction products, 4-(2-pyridylazo)-resorcinol monosodium salt (PAR)-based spectrophotometric zinc release assay, and ESI-MS. Single and double intrapeptide disulfides were detected by ESI-MS to be the sole reaction products. All three techniques yielded independently the same reaction rate, thereby demonstrating that ESI-MS may indeed be used in quantitative kinetic studies of ZF reactions. The comparison of experimental information demonstrated that the formation of the Cys5-Cys8 single disulfide was responsible for zinc release.     

烟草C2H2锌指蛋白转录因子家族成员的鉴定与表达分析

C2H2锌指蛋白转录因子家族在真核生物中具有重要的生物学功能,广泛参与植物叶的发生、花器官的调控、侧枝的形成及逆境胁迫等生命过程。植物C2H2锌指蛋白不仅结合DNA和RNA,而且与蛋白质之间相互作用。本研究利用普通烟草(Nicotiana tabacum)基因组数据库,运用Blastp比对,结合Pfam和SMART分析,鉴定了118条普通烟草C2H2锌指蛋白家族成员;对烟草C2H2锌指蛋白家族进行了进化树分析、结构域分析、物理化学性质分析、染色体定位、基因结构分析、三维结构分析及组织表达分析等。结果表明：不同成员的氨基酸长度差异较大;系统进化及结构域分析显示,所有C2H2家族成员可以被分为5个亚家族,同一亚家族成员之间在结构域和理化性质上呈现较高一致性;每个成员都含有C2H2结构域,在数量上存在较大差异;将所有基因家族成员定位在22条染色体上;组织表达分析表明,每个C2H2亚家族都有成员在不同组织中表达,在叶及根中有些基因的表达量较高。     

Expression analysis of an Arabidopsis C2H2 zinc finger protein gene

C₂H₂ zinc finger protein genes encode nucleic acid-binding proteins involved in the regulation of gene activity. AtZFP1 (Arabidopsis thaliana zinc finger protein 1) is one member of a small family of C₂H₂ zinc finger-encoding sequences previously characterized from Arabidopsis. The genomic sequence corresponding to the AtZFP1 cDNA has been determined. Molecular analysis demonstrates that AtZFP1 is a unique, intronless gene which encodes a 1100 nucleotides mRNA highly expressed in roots and stems. A construct in which 2.5 kb of AtZFP1 upstream sequences is linked to the -glucuronidase gene was introduced into Arabidopsis by Agrobacterium-mediated transformation of roots. Histochemical analysis of transgenic Arabidopsis carrying the AtZFP1 promotor:-glucuronidase fusion shows good correlation with RNA blot hybridization analysis. This transgenic line will be a useful tool for analyzing the regulation of AtZFP1 to further our understanding of its function.     

The histone H4 acetyltransferase MOF uses a C2HC zinc finger for substrate recognition

Site-specific acetylation of histone H4 by MOF is central to establishing the hyperactive male X chromosome in Drosophila. MOF belongs to the MYST family of histone acetyltransferases (HATs) characterized by an unusual C₂HC-type zinc finger close to their HAT domains. The function of these rare zinc fingers is unknown. We found that this domain is essential for HAT activity, in addition to the established catalytic domain. MOF uses its zinc finger to contact the globular part of the nucleosome as well as the histone H4 N-terminal tail substrate. Point mutations that leave the zinc-finger structure intact nevertheless abolish its interaction with the nucleosome. Our data document a novel role of the C₂HC-type finger in nucleosome binding and HAT activity.     

The zinc finger protein A20 targets TRAF2 to the lysosomes for degradation

The zinc finger-containing protein A20 is a negative regulator of TNF-induced JNK (c-Jun-N-terminal kinase) and NFkappaB (nuclear factor kappaB) signaling. A20 is an unusual enzyme that contains both ubiquitinating and deubiquitinating activities. Although A20 is mostly localized in the cytosol, our recent studies reveal that a fraction of A20 can associate with a lysosome-interacting compartment in a manner that requires its carboxy terminal zinc fingers, but independent of its ubiquitin modifying activities. Whether the lysosome-associated A20 has a function in cellular signaling is unclear. Here, we demonstrate that A20 is capable of targeting an associated signaling molecule such as TRAF2 to the lysosomes for degradation. This process is dependent on the membrane tethering zinc finger domains of A20, but does not require A20 ubiquitin modifying activity. Our findings suggest a novel mode of A20 action that involves lysosomal targeting of signal molecules bound to A20.