The delta-crystallin enhancer-binding protein delta EF1 is a repressor of E2-box-mediated gene activation.

The repressor delta EF1 was discovered by its action on the DC5 fragment of the lens-specific delta 1-crystallin enhancer. C-proximal zinc fingers of delta EF1 were found responsible for binding to the DC5 fragment and had specificity to CACCT as revealed by selection of high-affinity binding sequences from a random oligonucleotide pool. CACCT is present not only in DC5 but also in the E2 box (CACCTG) elements which are the binding sites of various basic helix-loop-helix activators and also the target of an unidentified repressor, raising the possibility that delta EF1 accounts for the E2 box repressor activity. delta EF1 competed with E47 for binding to an E2 box sequence in vitro. In lymphoid cells, endogenous delta EF1 activity as a repressor was detectable, and exogenous delta EF1 repressed immunoglobulin kappa enhancer by binding to the kappa E2 site. Moreover, delta EF1 repressed MyoD-dependent activation of the muscle creatine kinase enhancer and MyoD-induced myogenesis of 10T1/2 cells. Thus, delta EF1 counteracts basic helix-loop-helix activators through binding site competition and fulfills the conditions of the E2 box repressor. In embryonic tissues, the most prominent site of delta EF1 expression is the myotome. Myotomal expression as well as the above results argues for a significant contribution of delta EF1 in regulation of embryonic myogenesis through the modulation of the actions of MyoD family proteins.

Contribution of water molecules in the interior of a protein to the conformational stability.

Water molecules frequently occur in the interior of globular proteins. To elucidate the contribution of buried water molecules to the conformational stability of a protein, we examined the crystal structures and the thermodynamic parameters of denaturation of six Ile to Ala/Gly mutant human lysozymes, in which a cavity is created at each mutation site by the substitution of a smaller side-chain for a larger one. One or two ordered water molecules were found in the cavities created in some mutants (I106A, I59A and I59G). The cavity volumes for these three mutants were bigger than those that remained empty in the other mutants. The stability of the mutant proteins with the newly introduced water molecules was about 8 kJ/mol higher than that expected from the change in hydrophobic surface area (DeltaDeltaASAHP) exposed upon denaturation. It was concluded that a water molecule in a cavity created in the interior of a protein contributes favorably to the stability.

Interaction between Otx2 and Gbx2 defines the organizing center for the optic tectum.

Otx2 is expressed in the mesencephalon and prosencephalon, and Gbx2 is expressed in the rhombencephalon around stage 10. Loss-of-function studies of these genes in mice have revealed that Otx2 is indispensable for the development of the anterior brain segment, and that Gbx2 is required for the development of the isthmus. We carried out gain-of-function experiments of these genes in chick embryos with a newly developed gene transfer system, in ovo electroporation. When Otx2 was ectopically expressed caudally beyond the midbrain-hindbrain boundary (MHB), the alar plate of the metencephalon differentiated into the optic tectum instead of differentiating into the cerebellum. On the other hand, when Gbx2 was ectopically expressed at the mesencephalon, the caudal limit of the tectum shifted rostrally. We looked at the effects of misexpression on the isthmus- and tectum-related molecules. Otx2 and Gbx2 interacted to repress each other's expression. Ectopic Otx2 and Gbx2 repressed endogenous expression of Fgf8 in the isthmus, but induced Fgf8 expression at the interface between Otx2 and Gbx2 expression. Thus, it is suggested that interaction between Otx2 and Gbx2 determines the site of Fgf8 expression and the posterior limit of the tectum.

The structure, stability, and folding process of amyloidogenic mutant human lysozyme.

The physicochemical properties of an amyloidogenic mutant human lysozyme (Ile56Thr) were examined in order to elucidate the mechanism of amyloid formation. The crystal structure of the mutant protein was the same as the wild-type structure, except that the hydroxyl group of the introduced Thr56 formed a hydrogen bond with a water molecule in the interior of the protein. The other physicochemical properties of the mutant protein in the native state were not different from those of the wild-type protein. However, the equilibrium and kinetic stabilities of the mutant protein were remarkably decreased due to the introduction of a polar residue (Thr) in the interior of the molecule. It can be concluded that the amyloid formation of the mutant human lysozyme is due to a tendency to favor (partly or/and completely) denatured structures.

En-2 regulates the expression of the ligands for Eph type tyrosine kinases in chick embryonic tectum.

The retinotectal projection map is organized in a precise retinotopic order, so that the temporo-nasal axis of the retina corresponds to the rostro-caudal axis of the tectum. en-1 and en-2, homologues of the Drosophila segment polarity gene engrailed, are expressed in a gradient along the rostro-caudal axis of the tectal anlage, and are suggested to confer caudal characteristics as the results of transplantation and ectopic engrailed (en) expression. Recently the ligands for Eph type receptor tyrosine kinases have been shown to be expressed strongly at the caudal tectum and play a role in retinotectal map formation by repulsing the temporal retinal fibers. Using the system of replication competent retroviral vector, en-2 RCAS (A/B), we misexpressed en-2 on the tectum. Elf-1 or RAGS was induced at the ectopic En-2 sites. The present results have shown that En-2 can regulate expression of both Elf-1 and RAGS. This suggests that the cells which express en at the early stage of tectum development acquire positional specificity as 'caudal' tectum, and these cells may later express the ligands for Eph type receptor tyrosine kinases. Therefore the temporal retinal fibers which have the receptors are repelled when they meet the ligands on the tectum.

Axonal growth of the spinal cord interneurons expressing a homophilic adhesion molecule SC1 ectopically.

SC1/DM-GRASP/BEN, a cell adhesion molecule of the immunoglobulin super family, promotes the extension of neurites from neurons that express SC1 in culture, presumably by direct homophilic interactions. SC1 is specifically and transiently expressed on motoneurons during the period of their axonal growth, suggesting that it plays an important role in this growth. To explore this possibility, we expressed SC1 ectopically on the spinal cord interneurons of quail and chick embryos by in ovo electroporation at E3, when the motoneuron axonal growth starts. The axonal growth of the interneurons expressing chick SC1 was analyzed by immunohistochemistry with chick-specific anti-SC1 monoclonal antibody in quail, and by retrograde labeling with a dye in chick and quail embryos at E5. The majority of the axons of SC1-positive interneurons passed through the motor column and extended normally along the spinal cord basement membrane, but a few appeared to grow out from the cord. However, a dye back-labeling of the spinal nerves revealed that none of the interneurons were both SC1 and dye positive. These results suggest that the expression of SC1/DM-GRASP/BEN alone is insufficient for regulating the first step of the selective axonal pathfinding of motoneurons.

Roles of Pax-2 in initiation of the chick tectal development.

Transplantation experiments have shown that the mes-metencephalic boundary (isthmus) acts as an organizer for the development of the optic tectum. We have cloned Pax-2 which is expressed in the isthmus. Previously it was shown that Pax-5, a member of the same Pax subfamily as Pax-2, transformed the diencephalon into a tectum-like structure and induced isthmus- and tectum-related genes both in the mesencephalon and in the diencephalon. In order to define the distinct roles between Pax-2 and Pax-5 in development of the tectum, we expressed Pax-2 ectopically in the mesencephalon and the diencephalon of E2 chick embryos by in ovo electroporation. Histological observation demonstrated that Pax-2 transformed the diencephalon into a tectum-like structure. In Pax-2, transfected embryos the expression of isthmus- and tectum-related genes such as Fgf8 and En-2 was induced in the diencephalon. However, neither Fgf8 nor En-2 expression was induced in the mesencephalon, making a striking contrast with the result of Pax-5 misexpression. In E2 chick embryos, the mesencephalon is committed of its fate to differentiate into the tectum, but the diencephalon has plasticity on its fate. Moreover, Pax-2 expression in the isthmus precedes Pax-5 expression. Taking these results into consideration, it is suggested that Pax-2 plays a crucial role in initiation of the tectal development, and that Pax-5 functions to maintain the state of tectal differentiation.

Misexpression of genes in brain vesicles by in ovo electroporation.

Transfection to living chick embryos in ovo by electroporation has been recently developed. In this mini-review, misexpression in brain vesicles is introduced. To transfect, expression plasmid is inserted in the brain vesicle, and the square pulse of 25 V, 50 ms was charged five times. The translation product of the transfected gene is detected 2 h after electroporation, and reaches the peak at 24 h after electroporation. Transfection is so effective that this method is contributing greatly to the study of the molecular mechanisms of morphogenesis.

The stability and folding process of amyloidogenic mutant human lysozymes.

Amyloid deposits are frequently formed by mutant proteins that have a lower stability than the wild-type proteins. Some reports, however, have shown that mutant-induced thermodynamic destabilization is not always a general mechanism of amyloid formation. To obtain a better understanding of the mechanism of amyloid fibril formation, we show in this study that equilibrium and kinetic refolding-unfolding reaction experiments with two amyloidogenic mutant human lysozymes (I56T and D67H) yield folding pathways that can be drawn as Gibbs energy diagrams. The equilibrium stabilities between the native and denatured states of both mutant proteins were decreased, but the degrees of instability were different. The Gibbs energy diagrams of the folding process reveal that the Gibbs energy change between the native and folding intermediate states was similar for both proteins, and also that the activation Gibbs energy change from the native state to the transition state decreased. Our results confirm that the tendency to favor the intermediate of denaturation facilitates amyloid formation by the mutant human lysozymes more than equilibrium destabilization between the native and completely denatured states does.

Antagonizing activity of chick Grg4 against tectum-organizing activity.

Alar plate of chick mesencephalon differentiates into the optic tectum. It has been shown that factors expressed in the mes-metencephalic boundary induce the tectum and give positional specificity. Chick Grg4 is expressed at first in the anterior neural fold. The expression localizes from the posterior diencephalon to the mesencephalon by stage 10. To investigate the function of Grg4 in mesencephalic development, Grg4 overexpression was carried out by in ovo electroporation. After Grg4 overexpression, expression of En-2, Pax5, Fgf8, and EphrinA2 was repressed, and Pax6 was upregulated in the mesencephalic region. Grg4 overexpression caused the morphological change; mesencephalic swelling became smaller and the di-mesencephalic boundary shifted posteriorly, that is, the anterior limit of tectum shifted posteriorly. Importantly, cotransfection of Grg4 with Pax5 canceled the tectum-inducing activity of Pax5. These results suggest that Grg4 works as an antagonist against tectum-organizing activity. It was also shown that transfected N-terminal domains of Grg4 induced En-2 expression. Since N-terminal domains were transported to the nucleus in the neuroepithelium, they could act as dominant negative for endogenous Grg4. These results indicate that Grg4 has repressing activity against the organizing molecules and suggest that Grg4 plays important roles in formation of anterior tectal boundary and polarity.

Are the parameters of various stabilization factors estimated from mutant human lysozymes compatible with other proteins?

The various factors which contribute to protein stability have been extensively examined using mutant proteins, but the same kinds of substitutions have given different results depending on the substitution sites. Recently, the contributions of some stabilization factors have been quantitatively derived as parameters by a unique equation, considering the conformational changes due to the mutations using mutant human lysozymes [Funahashi et al. (1999) Protein ENG: 12, 841-850]. To evaluate these parameters estimated from the mutant human lysozymes, stability-structure datasets for the mutant T4 lysozymes were selected. The stabilities for the mutant T4 lysozymes could be roughly estimated using these parameters. Notable differences between the estimated and experimental stabilities were caused by the uncertainty in part of the structures due to some Arg and Lys residues fluctuating on the surface of the T4 lysozyme. Excluding these atoms from the estimation gave a good correlation between the estimated and experimental stabilities. These results suggest that the parameters of the various stabilization factors derived from the mutant human lysozymes are compatible with the mutant T4 lysozymes, although they should be improved with respect to some points using more information.

Identification of nuclear factor delta EF1 and its binding site essential for lens-specific activity of the delta 1-crystallin enhancer.

The lens-specific reglatory element of the delta 1-crystallin enhancer lies within the core segment (Goto et al., (1990) Mol. Cell. Biol. 10, 935-964). The element was allocated within the 55 bp long HN fragment of the core. Block-wise base substitutions were introduced to the 55 bp and their effect on the enhancer activity of the multimers in lens cells was examined. By base sequence alteration of either of the contiguous blocks 5 and 6, with their original sequence of TTGCT and CACCT, respectively, enhancer activity was totally lost. A lens nuclear factor delta EF1 was found which bound specifically to the base sequences defined by the blocks. DNA binding activity very similar to delta EF1 was also found in extracts of tissues other than lens, suggesting that delta EF1 participates in lens-specific regulation through tissue-dependent modification or interaction with other factors.

Crossregulation between En-2 and Wnt-1 in chick tectal development.

En-1, En-2 and Wnt-1 are proposed to be essential signals for the development of the optic tectum in chick embryos. Drosophila engrailed and wingless, homologs of En (En-1 and En-2) and Wnt-1, respectively, have been shown to crossregulate each other. In the present paper, it is reported that crossregulation between En-2 and Wnt-1 is preserved in the development of the chick optic tectum. When En-2 is overexpressed by the replication competent retroviral vector, Wnt-1 is expressed ectopically at the dorsal midline of the diencephalon. When Wnt-1 is introduced extrinsically either by ectopic transplantation of mesencephalon, or by implantation of Wnt-1 producing cells, En-2 is induced ectopically at the dorsal midline of the tel-diencephalic border. Thus, ectopic expression of En-2 and Wnt-1 leads to crossregulation of each other in the chick brain. As diencephalon transdifferentiates into the optic tectum by an appropriate signal, the crossregulation of En-2 and Wnt-1 in the diencephalon may mimic the relationship required for early development in the tectum.

Contribution of amino acid substitutions at two different interior positions to the conformational stability of human lysozyme.

To elucidate correlative relationships between structural change and thermodynamic stability in proteins, a series of mutant human lysozymes modified at two buried positions (Ile56 and Ile59) were examined. Their thermodynamic parameters of denaturation and crystal structures were studied by calorimetry and X-ray crystallography. The mutants at positions 56 and 59 exhibited different responses to a series of amino acid substitutions. The changes in stability due to substitutions showed a linear correlation with changes in hydrophobicity of substituted residues, having different slopes at each mutation site. However, the stability of each mutant was found to be represented by a unique equation involving physical properties calculated from mutant structures. By fitting present and previous stability data for mutant human lysozymes substituted at various positions to the equation, the magnitudes of the hydrophobicity of a carbon atom and the hydrophobicity of nitrogen and neutral oxygen atoms were found to be 0.178 and -0.013 kJ/mol.A(2), respectively. It was also found that the contribution of a hydrogen bond with a length of 3.0 A to protein stability was 5.1 kJ/mol and the entropy loss of newly introduction of a water molecules was 7.8 kJ/mol.

Role of surface hydrophobic residues in the conformational stability of human lysozyme at three different positions.

To evaluate the contribution of the amino acid residues on the surface of a protein to its stability, a series of hydrophobic mutant human lysozymes (Val to Gly, Ala, Leu, Ile, Met, and Phe) modified at three different positions on the surface, which are located in the alpha-helix (Val 110), the beta-sheet (Val 2), and the loop (Val 74), were constructed. Their thermodynamic parameters of denaturation and crystal structures were examined by calorimetry and by X-ray crystallography at 100 K, respectively. Differences in the denaturation Gibbs energy change between the wild-type and the hydrophobic mutant proteins ranged from 4.6 to -9.6 kJ/mol, 2.7 to -1.5 kJ/mol, and 3.6 to -0.2 kJ/mol at positions 2, 74, and 110, respectively. The identical substitution at different positions and different substitutions at the same position resulted in different degrees of stabilization. Changes in the stability of the mutant proteins could be evaluated by a unique equation considering the conformational changes due to the substitutions [Funahashi et al. (1999) Protein Eng. 12, 841-850]. For this calculation, secondary structural propensities were newly considered. However, some mutant proteins were not adapted to the equation. The hydration structures around the mutation sites of the exceptional mutant proteins were affected due to the substitutions. The stability changes in the exceptional mutant proteins could be explained by the formation or destruction of the hydration structures. These results suggest that the hydration structure mediated via hydrogen bonds covering the protein surface plays an important role in the conformational stability of the protein.

Delta-crystallin enhancer binding protein delta EF1 is a zinc finger-homeodomain protein implicated in postgastrulation embryogenesis.

We investigated nuclear factors that bind to delta 1-crystallin enhancer core and regulate lens-specific transcription. A nuclear factor delta EF1, which binds to the essential element of the delta 1-crystallin enhancer core, was molecularly cloned from the chicken by a southwestern method. The protein organization of delta EF1 deduced from the cDNA sequence indicated that it has heterogeneous domains for DNA-binding, two widely separated zinc fingers and a homeodomain, analogous to Drosophila ZFH-1 protein. The C-terminal zinc fingers were found to be responsible for binding to the delta 1-crystallin enhancer core sequence. delta EF1 had proline-rich and acidic domains common to various transcriptional activators. During embryogenesis, delta EF1 expression was observed in the postgastrulation period in mesodermal tissues; initially, in the notochord, followed by somites, nephrotomes and other components. The expression level changed dynamically in a tissue, possibly reflecting the differentiation states of the constituent cells. Besides mesoderm, delta EF1 was expressed in the nervous system and the lens, but other ectodermal tissues and endoderm remained very low in delta EF1 expression. Cotransfection experiments indicated that this factor acts as a repressor of delta 1-crystallin enhancer. Possession of heterogeneous DNA-binding domains and its dynamic change of expression in embryogenesis strongly suggest that delta EF1 acts in multiple ways depending on the cell type and the gene under its regulation.

Contribution of intra- and intermolecular hydrogen bonds to the conformational stability of human lysozyme(,).

In globular proteins, there are intermolecular hydrogen bonds between protein and water molecules, and between water molecules, which are bound with the proteins, in addition to intramolecular hydrogen bonds. To estimate the contribution of these hydrogen bonds to the conformational stability of a protein, the thermodynamic parameters for denaturation and the crystal structures of five Thr to Val and five Thr to Ala mutant human lysozymes were determined. The denaturation Gibbs energy (DeltaG) of Thr to Val and Thr to Ala mutant proteins was changed from 4.0 to -5.6 kJ/mol and from 1.6 to -6.3 kJ/mol, respectively, compared with that of the wild-type protein. The contribution of hydrogen bonds to the stability (DeltaDeltaG(HB)) of the Thr and other mutant human lysozymes previously reported was extracted from the observed stability changes (DeltaDeltaG) with correction for changes in hydrophobicity and side chain conformational entropy between the wild-type and mutant structures. The estimation of the DeltaDeltaG(HB) values of all mutant proteins after removal of hydrogen bonds, including protein-water hydrogen bonds, indicates a favorable contribution of the intra- and intermolecular hydrogen bonds to the protein stability. The net contribution of an intramolecular hydrogen bond (DeltaG(HB[pp])), an intermolecular one between protein and ordered water molecules (DeltaG(HB[pw])), and an intermolecular one between ordered water molecules (DeltaG(HB[ww])) could be estimated to be 8. 5, 5.2, and 5.0 kJ/mol, respectively, for a 3 A long hydrogen bond. This result shows the different contributions to protein stability of intra- and intermolecular hydrogen bonds. The entropic cost due to the introduction of a water molecule (DeltaG(H)()2(O)) could be also estimated to be about 8 kJ/mol.

Role of Pax-5 in the regulation of a mid-hindbrain organizer's activity.

The mes-metencephalic boundary (isthmus) has been suggested to act as an organizer in the development of the optic tectum. Pax-5 was cloned as a candidate for regulator of the organizing center. Isthmus-specific expression of Pax-5 and analogy with the genetic cascade in Drosophila suggest that Pax-5 may be at a higher hierarchical position in the gene regulation cascade of tectum development. To examine this possibility, a gain-of-function experiment on Pax-5 was carried out. In ovo electroporation on E2 chick brain with the eucaryotic expression vector that encodes chick Pax-5 cDNA was used. Not only was a considerable amount of Pax-5 expressed ectopically in the transfected brain, but irregular bulging of the neuroepithelium was induced in the diencephalon and mesencephalon. At Pax-5 misexpressing sites, uptake of BrdU was increased. Histological examination of E7 transfected brain revealed that Pax-5 caused transdifferentiation of diencephalon into the tectum-like structure. In the bulges of the E7 mesencephalon, differentiation of laminar structure was repressed when compared to the normal side. In transfected embryos, En-2, Wnt-1 and Fgf8 were up-regulated ectopically, and Otx2 was down-regulated in the diencephalon to mesencephalon. Moreover, Ephrin-A2, which is expressed specifically in the tectum with a gradient highest at the caudal end, is suggested to be involved in pathfinding of the retinal fibers, and was induced in the bulges. When the mouse Fgf8 expression vector was electroporated, Pax-5 and chick Fgf8 were also induced ectopically. These results suggest that Pax-5, together with Fgf8, hold a higher position in the genetic hierarchy of the isthmus organizing center and regulate its activity.

Contribution of hydrogen bonds to the conformational stability of human lysozyme: calorimetry and X-ray analysis of six Ser --> Ala mutants.

To further examine the contribution of hydrogen bonds to the conformational stability of the human lysozyme, six Ser to Ala mutants were constructed. The thermodynamic parameters for denaturation of these six Ser mutant proteins were investigated by differential scanning calorimetry (DSC), and the crystal structures were determined by X-ray analysis. The denaturation Gibbs energy (DeltaG) of the Ser mutant proteins was changed from 2.0 to -5.7 kJ/mol, compared to that of the wild-type protein. With an analysis in which some factors that affected the stability due to mutation were considered, the contribution of hydrogen bonds to the stability (Delta DeltaGHB) was extracted on the basis of the structures of the mutant proteins. The results showed that hydrogen bonds between protein atoms and between a protein atom and a water bound with the protein molecule favorably contribute to the protein stability. The net contribution of one intramolecular hydrogen bond to protein stability (DeltaGHB) was 8.9 +/- 2.6 kJ/mol on average. However, the contribution to the protein stability of hydrogen bonds between a protein atom and a bound water molecule was smaller than that for a bond between protein atoms.

Contribution of hydrogen bonds to the conformational stability of human lysozyme: calorimetry and X-ray analysis of six tyrosine --> phenylalanine mutants.

The contribution of hydrogen bonds to the conformational stability of human lysozyme was investigated by the combination of calorimetric and X-ray analyses of six Tyr --> Phe mutants. Unfolding Delta G and unfolding Delta H values of the Tyr --> Phe mutant proteins were changed by from +0.3 to -4.0 kJ/mol and from 0 to -16 kJ/mol, respectively, compared to those of the wild-type protein. The net contribution of a hydrogen bond at a specific site to stability (Delta Gwild/HB), considering factors affected by substitutions, was evaluated on the basis of X-ray structures of the mutant proteins. In the present study, one of six mutant proteins was suitable for evaluating the strength of the hydrogen bond. Delta Gwild/HB for the intramolecular hydrogen bond at Tyr124 was evaluated to be 7.5 kJ/mol. Results of the analysis of other mutants also suggest that hydrogen bonds of the hydroxyl group of Tyr, including the hydrogen bond with a water molecule, contribute to the stabilization of the human lysozyme.