Expression of a Zinc-Binding Domain of Boar Spermatidal Transition Protein 2 in Escherichia coli

Transition protein 2 (TP2; 137 amino acid residues) from boar late spermatid nuclei has three potential zinc finger motifs in the N-terminal region. Gel shift assays revealed that boar TP2 recognized a CpG island sequence in a zinc-dependent manner. However, there was some nonspecific recognition of the oligonucleotide. Then, we constructed the expression system of zinc-binding domain of TP2 (TP2Z) (residues 1–103) in Escherichia coli. Double-stranded DNA fragments encoding TP2Z were synthesized as 18 fragments with 103 residues, annealed, and cloned into the expression plasmid pET11d. TP2Z was expressed upon induction with 1 mM isopropylthiogalactoside and extracted with acid including 0.71 M 2-mercaptoethanol. TP2Z was purified by ion-exchange chromatography on Fractogel EMD SO⁻₃ and HPLC on Nucleosil 300 7C18 and on Diol-120. Atomic absorption and CD spectroscopy showed that TP2Z bound three atoms of zinc per molecule of the protein and underwent a zinc-dependent conformational change in a manner similar to that for intact TP2. Gel shift assays indicated that TP2Z recognized a CpG island sequence more specifically than intact TP2 and that the specificity is dependent on zinc.     

Interactions of CCCH zinc finger proteins with mRNA. Binding of tristetraprolin-related zinc finger proteins to Au-rich elements and destabilization of mRNA

Macrophages derived from tristetraprolin (TTP)-deficient mice exhibited increased tumor necrosis factor alpha (TNFalpha) release as a consequence of increased stability of TNFalpha mRNA. TTP was then shown to destabilize TNFalpha mRNA after binding directly to the AU-rich region (ARE) of the 3'-untranslated region of the TNFalpha mRNA. In mammals and in Xenopus, TTP is the prototype of a small family of three known zinc finger proteins containing two CCCH zinc fingers spaced 18 amino acids apart; a fourth more distantly related family member has been identified in Xenopus and fish. We show here that representatives of all four family members were able to bind to the TNFalpha ARE in a cell-free system and, in most cases, promote the breakdown of TNFalpha mRNA in intact cells. Because the primary sequences of these CCCH proteins are most closely related in their tandem zinc finger domains, we tested whether various fragments of TTP that contained both zinc fingers resembled the intact protein in these assays. We found that amino- and carboxyl-terminal truncated forms of TTP, as well as a 77 amino acid fragment that contained both zinc fingers, could bind to the TNFalpha ARE in cell-free cross-linking and gel shift assays. In addition, these truncated forms of TTP could also stimulate the apparent deadenylation and/or breakdown of TNFalpha mRNA in intact cells. Alignments of the tandem zinc finger domains from all four groups of homologous proteins have identified invariant residues as well as group-specific signature amino acids that presumably contribute to ARE binding and protein-specific activities, respectively.     

NRF-2 transcription factor is essential in promoting human Tomm70 gene expression

TOMM70 is a subunit of the outer mitochondrial membrane translocase that plays a major role as a receptor of hydrophobic pre-proteins targeted to the mitochondria. We report the presence of two nuclear respiratory factor 2 (NRF-2) binding motifs in the 5'-flanking region of the human Tomm70 gene and establish their essential role for promoter activity by using reporter assays in HeLa cells. We show that both NRF-2 binding sites are functional and present evidence that interactions between these sites and a CpG island contribute to expression. Mobility shift assays show that these NRF-2 sites are specifically recognized by NRF-2 present in HeLa nuclear extracts.     

Spectroscopic characterization of the interaction between calmodulin-dependent protein kinase I and calmodulin

Calmodulin-dependent protein kinase I (CaM kinase I) is a member of the expanding class of protein kinases that are regulated by calmodulin (CaM). Its putative CaM-binding region is believed to occur within a 22-residue sequence (amino acids 299-320). This sequence was chemically synthesized and utilized for CaM interaction studies. Gel band shift assays and densitometry experiments with intact CaM kinase I and the CaM-binding domain peptide (CaMKIp) reveal that they bind in an analogous manner, giving rise to 1:1 complexes. Fluorescence analysis using dansyl-CaM showed that conformational changes in CaM on binding CaM kinase I or CaMKIp were nearly identical, suggesting that the peptide mimicked the CaM-binding ability of the intact protein. In the presence of CaM, the peptide displays an enhancement of its unique Trp fluorescence as well as a marked blue shift of the emission maximum, reflecting a transfer to a more rigid, less polar environment. Quenching studies, using acrylamide, confirmed that the Trp in the peptide on binding CaM is no longer freely exposed to solvent as is the case for the free peptide. Studies with a series of Met mutants of CaM showed that the Trp-containing N-terminal end of CaMKIp was bound to the C-terminal lobe of CaM. Near-UV CD spectra also indicate that the Trp of the peptide and Phe residues of the protein are involved in the binding. These results show that the CaM-binding domain of CaM kinase I binds to CaM in a manner analogous to that of myosin light chain kinase.     

Zinc-binding sites in the N terminus of Mycoplasma arthritidis-derived mitogen permit the dimer formation required for high affinity binding to HLA-DR and for T cell activation

Zinc-dependent superantigens can be divided into two subfamilies based on how they use zinc ions for interactions with major histocompatibility complex (MHC) class II molecules. Members of the first subfamily use zinc ions for interactions with histidine 81 on the beta-chain of MHC class II molecules, whereas members of the second subfamily use zinc ions for dimer formation. The zinc-binding motif is located in the C terminus of the molecule in both subfamilies. While our recent studies with Mycoplasma arthritidis-derived mitogen (MAM) have provided the first direct evidence demonstrating the binding to MHC class II molecules in a zinc-dependent manner, it still not known how zinc coordinates the interaction. Data presented here show that the zinc ion is mainly required to induce MAM/MAM dimer formation. Residues in the N terminus of MAM are involved in dimer formation and MHC class II binding, while histidine 14 and aspartic acid 31 of the MAM sequence are the major residues mediating MAM/MAM dimerization. Zinc-induced dimer formation is necessary for MAM binding, MHC class II-induced cell-cell adhesion, and efficient T cell activation. Together these results depict the unique mode of interaction of MAM in comparison with other superantigens.     

Domain structure and antiparallel dimers of microtubule-associated protein 2 (MAP2).

We have studied the microtubule-associated protein MAP2 from porcine brain and its subfragments by limited proteolysis, antibody labeling, and electron microscopy. Two major chymotryptic fragments start at lys 1528 and arg 1664, generating microtubule-binding fragments of Mr 36 kDa (303 residues, analogous to the "assembly domain" of Vallee, 1980) and 18 kDa (167 residues). These fragments can be labeled with the antibody 2-4 which recognizes the last internal repeat of MAP2 (Dingus et al., 1991). The epitope of another monoclonal antibody, AP18 (Binder et al., 1986), was mapped to the first 151 residues of MAP2. The interaction with AP18 is phosphorylation dependent; dephosphorylated MAP2 is not recognized. Intact MAP2 forms rod-like particles of 97 nm mean length, similar to Gottlieb and Murphy's (1985) observations. Both antibodies bind near an end of the rod, suggesting that the sequence and the structure are approximately colinear. There is a pronounced tendency for MAP2 to form dimers whose components are nearly in register but of opposite polarity. MAP2 can also fold in a hairpin-like fashion, generating 50-nm rods, and it can self-associate into oligomers and fibers. The 36-kDa microtubule-binding fragment also has a rod-like shape; its mean length is 49 nm, half of the intact molecule, even though the fragment contains only one-sixth of the mass. The antibody 2-4 decorates one end of the rod, similar to the intact protein. The fragment also forms antiparallel dimers, but its tendency for higher self-assembly forms is much lower than with intact MAP2.     

Identification of two novel zinc finger modules and nuclear localization signal in rat spermatidal protein TP2 by site-directed mutagenesis

Spermatidal protein TP2, which appears transiently during stages 12-16 of mammalian spermiogenesis, is a DNA condensing zinc metalloprotein with a preference to GC-rich DNA. We have carried out a detailed site-directed mutagenesis analysis of rat spermatidal protein TP2 to delineate the amino acid residues involved in coordination with two atoms of zinc. Two zinc fingers modules have been identified involving 4 histidine and 4 cysteine residues, respectively. The modular structure of the two zinc fingers identified in TP2 define a new class of zinc finger proteins that do not fall into any of the known classes of zinc fingers. Transfection experiments with COS-7 cells using wild type and the two zinc finger pocket mutants have shown that TP2 preferentially localizes to nucleolus. The nuclear localization signal in TP2 was identified to be (87)GKVSKRKAV(95) present in the C-terminal third of TP2 as a part of an extended NoLS sequence.     

DNA motifs associated with aberrant CpG island methylation

Epigenetic silencing involving the aberrant methylation of promoter region CpG islands is widely recognized as a tumor suppressor silencing mechanism in cancer. However, the molecular pathways underlying aberrant DNA methylation remain elusive. Recently we showed that, on a genome-wide level, CpG island loci differ in their intrinsic susceptibility to aberrant methylation and that this susceptibility can be predicted based on underlying sequence context. These data suggest that there are sequence/structural features that contribute to the protection from or susceptibility to aberrant methylation. Here we use motif elicitation coupled with classification techniques to identify DNA sequence motifs that selectively define methylation-prone or methylation-resistant CpG islands. Motifs common to 28 methylation-prone or 47 methylation-resistant CpG island-containing genomic fragments were determined using the MEME and MAST algorithms (). The five most discriminatory motifs derived from methylation-prone sequences were found to be associated with CpG islands in general and were nonrandomly distributed throughout the genome. In contrast, the eight most discriminatory motifs derived from the methylation-resistant CpG islands were randomly distributed throughout the genome. Interestingly, this latter group tended to associate with Alu and other repetitive sequences. Used together, the frequency of occurrence of these motifs successfully discriminated methylation-prone and methylation-resistant CpG island groups with an accuracy of 87% after 10-fold cross-validation. The motifs identified here are candidate methylation-targeting or methylation-protection DNA sequences.     

Diversity of human insulin-like growth factor (IGF) binding protein-2 fragments in plasma: primary structure, IGF-binding properties, and disulfide bonding pattern

The insulin-like growth factor binding proteins (IGFBPs) play a major role in the regulation of the effects and the bioavailability of the insulin-like growth factors (IGFs). IGFs are released from IGFBP-IGF complexes by proteolysis of IGFBPs generating fragments with reduced ligand-binding properties. To identify naturally occurring fragments of IGFBP-2, a peptide library generated from human hemofiltrate was immunologically screened. Purification of immunoreactive IGFBP-2 fragments was performed by consecutive chromatographic steps. A total of 18 different IGFBP-2 fragments was isolated and characterized. The peptides exhibited different N-terminal amino acid residues that were located in the variable midregion of IGFBP-2. Four major cleavage sites were determined to be between Tyr103 and Gly104, Leu152 and Ala153, Arg156 and Glu157, and Gln165 and Met166. The resulting fragments were further processed by amino and/or carboxy peptidases and comprised 37-185 amino acid residues. Ligand blotting, solution binding assays, and BIAcore analyses revealed that all tested fragments retained low IGF-binding capacity. The most abundant fragment IGFBP-2 (167-279) showed 10% of IGF-II binding compared to recombinant human (rh)IGFBP-2. Furthermore, the disulfide bonding pattern of the C-terminal domain of rhIGFBP-2 was defined, indicating linkages between cysteine residues 191-225, 236-247, and 249-270. This study provides the most comprehensive molecular characterization of human IGFBP-2 fragments formed in vivo, exhibiting both residual IGF-binding capacities and the integrin-binding sequence.     

The isolation of CpG islands from human chromosomal regions 11q13 and Xp22 by segregation of partlymelted molecules.

We isolated fragments containing parts of CpG islands from human chromosomal regions chosen for expected differences in gene density by segregation of partly melted molecules. Restriction fragments of P1 bacteriophage clones covering a region of 11q13 and those of cosmid clones derived from Xp22 were recovered from bands in denaturing gradient gels that were retained following prolonged exposure to electric field. Forty-five independent fragments derived from 11q13 and five from Xp22 were isolated. Nucleotide sequence analysis revealed that 11 of the 45 fragments from 11q13 contained CpG islands including four derived from known genes in 11q13. None of the five fragments derived from Xp22 resembled CpG islands. The number of CpG island fragments obtained was consistent with the expectation based on the number of Not I restriction endonuclease sites present at these regions. Adjustment of parameters in our quasi-theoretical approach to the rate of fragment dissociation improves the discrimination between retention and non-retention. The results support probable identification of CpG island fragments by their reduced rate of strand dissociation when retarded in a denaturing gradient gel.     

YY1's longer DNA-binding motifs

The DNA-binding sites of YY1 located within two newly identified downstream genes of YY1, Peg3 (GGCGCCATnTT) and Xist (CCGCCATnTT), are longer than the known motif of YY1 (CGCCATnTT). Gel shift assays indicated that these DNA-binding sites are previously unnoticed, longer motifs of YY1. Independent DNA-binding motif studies further confirmed that YY1 recognizes a longer sequence (GCCGCCATTTTG) as its consensus motif. This longer motif exhibited higher affinity to the YY1 protein than the known motif. Another DNA-binding motif study was also performed using a protein containing three amino acid substitutions in the first zinc finger unit of YY1, mimicking the zinc finger domain of pho (a drosophila homologue of YY1). The substitutions cause the weakening of DNA-binding specificity at both 5'- and 3'-sides of the longer motif, yielding a much shorter sequence (GCCAT) as a consensus motif. This indicates that the intact first finger unit is required for recognition of the longer motif of YY1. Also, this shortening suggests that the DNA recognition by YY1 is mediated through the concerted, but not modular, contribution by its four zinc finger units.     

Interconversion between serine and aspartic acid in the alpha helix of the N-terminal zinc finger of Sp1: implication for general recognition code and for design of novel zinc finger peptide recognizing complementary strand

In the typical base recognition mode of the C(2)H(2)-type zinc finger, the amino acid residues at alpha-helical positions -1, 3, and 6 make a contact with the base in one strand (the primary strand), and the residue at position 2 interacts with the base in a complementary strand (the secondary strand). The N-terminal zinc finger of the three-zinc-finger domain of Sp1 has inherently a unique five-base-pair binding mode in which the guanine bases are recognized in both strands. To clarify the effect of the amino acid at position 2 on DNA binding affinity and base specificity, we have created a library of the mutants by the interconversion between serine and aspartic acid in the N-terminal zinc finger of Sp1 and recombinant variants of finger order. Gel mobility shift and methylation interference assays showed that the combination of arginine and serine at positions -1 and 2, respectively, provides a newly strong guanine contact in the secondary strand and a higher binding affinity than that of wild-type Sp1. Of special interest are the facts that the mutant with lysine and aspartic acid at positions -1 and 2 in the alpha helix predominantly recognizes the bases in the secondary strand and that its DNA binding affinity is higher than that of the wild-type. The aspartic acid or serine at position 2 independently contributes to the DNA binding affinity and base specificity. The present results provide useful information for the design of a novel zinc finger protein with priority for the bases in the secondary strand.     

Isolation and amino-acid sequence analysis of human sperm protamines P1 and P2. Occurrence of two forms of protamine P2

The two protamines of human sperm cell nuclei, P1 and P2, were isolated in pure form after extraction with 6M guanidine/5% mercaptoethanol and alkylation with vinyl pyridine by reversed-phase high-performance liquid chromatography. The amino-acid sequence of protamine P1 was determined by analysing the intact protein and the fragments obtained by cyanogen bromide cleavage. Out of the 50 amino-acid residues 24 are arginines and 6 are cysteines. The sequence of protamine P2 was determined by analysing the intact protein and the fragments resulting from cleavage with endoproteinase Lys-C and thermolysin. Protamine P2 was found to occur in two forms which only differ in their N-terminal regions. The form P2' is three amino-acid residues longer at the N-terminus than the form P2'. Out of the 57 amino-acid residues in the longer form 27 are arginines and 5 are cysteines. Human protamine P1 is highly homologous with the protamines isolated from bull, boar, ram and mouse sperm cells, but human protamine P2 shows a novel type of structure, although also here the dominant amino acids are arginine and cysteine.