Msx2 is a repressor of chondrogenic differentiation in migratory cranial neural crest cells.

During early mouse embryogenesis, cranial neural crest cells (CNCC) emigrate from the posterior midbrain and rhombomeres 1 and 2 of the anterior hindbrain into the first branchial arch-derived maxillary and mandibular processes and there provide cell lineages for several phenotypes, including cartilage, bone, and tooth. Here, we report that Sox9 and Msx2 were coexpressed in a subpopulation of CNCC during their migration. Because Sox9 is a transactivator of chondrogenesis, and Msx genes can act as transcriptional repressors, we hypothesized that Sox9 expression indicates the determination of CNCC-derived chondrogenic cell lineage and that Msx2 represses chondrogenic differentiation until CNCC migration is completed within the mandibular processes. To test whether Msx2 represses chondrogenesis, we designed experiments to inhibit Msx2 function in migratory CNCC in primary cultures through the expression of loss-of-function Msx2 mutants. We showed that infection of migratory CNCC with adenovirus Msx2 mutants accelerated the rate and extent of chondrogenesis, as indicated by the expression level of type II collagen and aggrecan, and the amount of alcian blue staining. Adenovirus infections did not apparently interfere with CNCC proliferation or migration. These findings suggest that an important early event in craniofacial morphogenesis is a transient expression of both Sox9 and Msx2 during emigration into the forming mandibular processes followed by restricted expression of Sox9 within CNCC- derived chondroprogenitor cells. We conclude that Msx2 serves as a repressor of chondrogenic differentiation during CNCC migration.

DACH: genomic characterization, evaluation as a candidate for postaxial polydactyly type A2, and developmental expression pattern of the mouse homologue.

The gene DACH is a human homologue of Drosophila melanogaster dachshund (dac), which encodes a nuclear factor essential for determining cell fates in the eye, leg, and nervous system of the fly. To investigate possible connections between DACH and inherited developmental disorders, we have characterized the human DACH genomic structure and investigated the tissue and cellular distribution of the mouse DACH1 protein during development. DACH spans 400 kb and is encoded by 12 exons. The predominant DACH transcript is 5.2 kb and encodes a 706-amino-acid protein with an observed molecular weight of 97 kDa.DACH mRNA was detected in multiple adult human tissues including kidney and heart. The mouse DACH1 protein was immunolocalized to specific cell types within the developing kidneys, eyes, cochleae, and limb buds. Data suggest genetic linkage of the limb bud patterning defect postaxial polydactyly type A (designated PAP-A2, MIM 602085) to a 28-cM interval on chromosome 13 that includes DACH. However, mutation analysis of DACH in this PAP-A2 pedigree revealed no sequence differences in the coding region, splice sites, or proximal promoter region. The data presented will allow for the analysis of DACH as a candidate for other developmental disorders affecting the limbs, kidneys, eyes, ears, and other sites of DACH expression.

The p400 complex is an essential E1A transformation target.

Here, we report the identification of a new E1A binding protein complex that is essential for E1A-mediated transformation. Its core component is a SWI2/SNF2-related, 400 kDa protein (p400). Other components include the myc- and p/CAF-associated cofactor, TRRAP/PAF400, the DNA helicases TAP54alpha/beta, actin-like proteins, and the human homolog of the Drosophila Enhancer of Polycomb protein. An E1A mutant, defective in p400 binding, is also defective in transformation. Certain p400 fragments partially rescued this phenotype, underscoring the role of E1A-p400 complex formation in the E1A transforming process. Furthermore, E1A and c-myc each alter the subunit composition of p400 complexes, implying that physiological p400 complex formation contributes to transformation suppression.

Equilibrium and kinetic studies on folding of the authentic and recombinant forms of human alpha-lactalbumin by circular dichroism spectroscopy.

The equilibrium and kinetics of the unfolding and refolding of authentic and recombinant human alpha-lactalbumin, the latter of which had an extra methionine residue at the N-terminus, were studied by circular dichroism spectroscopy, and the results were compared with the results for bovine and goat alpha-lactalbumins obtained in our previous studies. As observed in the bovine and goat proteins, the presence of the extra methionine residue in the recombinant protein remarkably destabilized the native state, and the destabilization was entirely ascribed to an increase in the rate of unfolding. The thermodynamic stability of the native state against the unfolded state was lower, and the thermodynamic stability of the molten globule state against the unfolded state was higher for the human protein than for the other alpha-lactalbumins previously studied. Thus, the population of the molten globule intermediate was higher during the equilibrium unfolding of human alpha-lactalbumin by guanidine hydrochloride. Unlike the molten globule states of the bovine and goat proteins, the human alpha-lactalbumin molten globule showed remarkably more intense circular dichroism ellipticity than the native state in the far-ultraviolet region below 225 nm. During refolding from the unfolded state, human alpha-lactalbumin thus exhibited overshoot kinetics, in which the alpha-helical peptide ellipticity exceeded the native value when the molten globule folding intermediate was formed in the burst phase. The subsequent folding involved reorganization of nonnative secondary structures. It should be noted that the rate constant of the major refolding phase was approximately the same among the three types of alpha-lactalbumin and that the rate constant of unfolding was accelerated 18-600 times in the human protein, and these results interpreted the lower thermodynamic stability of this protein.

Involvement of the TIP60 histone acetylase complex in DNA repair and apoptosis.

It is well known that histone acetylases are important chromatin modifiers and that they play a central role in chromatin transcription. Here, we present evidence for novel roles of histone acetylases. The TIP60 histone acetylase purifies as a multimeric protein complex. Besides histone acetylase activity on chromatin, the TIP60 complex possesses ATPase, DNA helicase, and structural DNA binding activities. Ectopic expression of mutated TIP60 lacking histone acetylase activity results in cells with defective double-strand DNA break repair. Importantly, the resulting cells lose their apoptotic competence, suggesting a defect in the cells' ability to signal the existence of DNA damage to the apoptotic machinery. These results indicate that the histone acetylase TIP60-containing complex plays a role in DNA repair and apoptosis.

Fast folding of Escherichia coli cyclophilin A: a hypothesis of a unique hydrophobic core with a phenylalanine cluster.

Escherichia coli cyclophilin A, a 164 residue globular protein, shows fast and slow phases of refolding kinetics from the urea-induced unfolded state at pH 7.0. Given that the slow phases are independent of the denaturant concentration and may be rate-limited by cis/trans isomerizations of prolyl peptide bonds, the fast phase represents the true folding reaction. The extrapolation of the fast-phase rate constant to 0 M urea indicates that the folding reaction of cyclophilin A is extraordinarily fast and has about 700 s(-1) of the rate constant. Interrupted refolding experiments showed that the protein molecules formed in the fast phase had already been fully folded to the native state. This finding overthrows the accepted view that the fast folding is observed only in small proteins of fewer than 100 amino acid residues. Examination of the X-ray structure of cyclophilin A has shown that this protein has only one unique hydrophobic core (phenylalanine cluster) formed by evolutionarily conserved phenylalanine residues, and suggests that this architecture of the molecule may be responsible for the fast folding behavior.

Expression of a synthetic gene encoding canine milk lysozyme in Escherichia coli and characterization of the expressed protein.

A high-expression plasmid of the canine milk lysozyme, which belongs to the family of calcium-binding lysozymes, was constructed in order to study its physico-chemical properties. Because the cDNA sequence of the protein has not yet been determined, a 400 base-pair gene encoding canine milk lysozyme was first designed on the basis of the known amino acid sequence. The gene was constructed by an enzymatic assembly of 21 chemically synthesized oligonucleotides and inserted into an Escherichia coli expression vector by stepwise ligation. The expression plasmid thus constructed was transformed into BL21(DE3)/pLysS cells. The gene product accumulated as inclusion bodies in an insoluble fraction. Recombinant canine milk lysozyme was obtained by purification and refolding of the product and showed the same characteristics in terms of bacteriolytic activity and far- and near-UV circular dichroism spectra as the authentic protein. The NMR spectra of refolded lysozyme were also characteristic of a native globular protein. It was concluded that recombinant canine milk lysozyme was folded into the correct native structure. Moreover, the thermal unfolding profiles of the refolded recombinant lysozyme showed a stable equilibrium intermediate, indicating that the molten globule state of this protein was extraordinarily stable. This expression system of canine milk lysozyme will enable biophysical and structural studies of this protein to be extended.

Effects of proline mutations on the folding of staphylococcal nuclease.

Effects of proline isomerizations on the equilibrium unfolding and kinetic refolding of staphylococcal nuclease were studied by circular dichroism in the peptide region (225 nm) and fluorescence spectra of a tryptophan residue. For this purpose, four single mutants (P11A, P31A, P42A, and P56A) and four multiple mutants (P11A/P47T/P117G, P11A/P31A/P47T/P117G, P11A/P31A/P42A/P47T/P117G, and P11A/P31A/P42A/P47T/P56A/P117G) were constructed. These mutants, together with the single and double mutants for Pro47 and Pro117 constructed in our previous study, cover all six proline sites of the nuclease. The P11A, P31A, and P42A mutations did not change the stability of the protein remarkably, while the P56A mutation increased protein stability to a small extent by 0.5 kcal/mol. The refolding kinetics of the protein were, however, affected remarkably by three of the mutations, namely, P11A, P31A, and P56A. Most notably, the amplitude of the slow phase of the triphasic refolding kinetics of the nuclease observed by stopped-flow circular dichroism decreased by increasing the number of the proline mutations; the slow phase disappeared completely in the proline-free mutant (P11A/P31A/P42A/P47T/P56A/P117G). The kinetic refolding reactions of the wild-type protein assessed in the presence of Escherichia coli cyclophilin A showed that the slow phase was accelerated by cyclophilin, indicating that the slow phase was rate-limited by cis-trans isomerization of the proline residues. Although the fast and middle phases of the refolding kinetics were not affected by cyclophilin, the amplitude of the middle phase decreased when the number of the proline mutations increased; the percent amplitudes for the wild-type protein and the proline-free mutants were 43 and 13%, respectively. In addition to these three phases detected with stopped-flow circular dichroism, a very fast phase of refolding was observed with stopped-flow fluorescence, which had a shorter dead time (3.6 ms) than the stopped-flow circular dichroism. The following conclusions were drawn. (1) The effects of the P11A, P31A, and P56A mutations on the refolding kinetics indicate that the isomerizations of the three proline residues are rate-limiting, suggesting that the structures around these residues (Pro11, Pro31, and Pro56) may be organized at an early stage of refolding. (2) The fast phase corresponds to the refolding of the native proline isomer, and the middle phase whose amplitude has decreased when the number of proline mutations was increased may correspond to the slow refolding of non-native proline isomers. The occurrence of the fast- and slow-refolding reactions together with the slow phase rate-limited by the proline isomerization suggests that there are parallel folding pathways for the native and non-native proline isomers. (3) The middle phase did not completely disappear in the proline-free mutant. This suggests that the slow-folding isomer is produced not only by the proline isomerizations but also by another conformational event that is not related to the prolines. (4) The very fast phase detected with the fluorescent measurements suggests that there is an intermediate at a very early stage of kinetic refolding.

Effect of the extra n-terminal methionine residue on the stability and folding of recombinant alpha-lactalbumin expressed in Escherichia coli.

The structure, stability, and unfolding-refolding kinetics of Escherichia coli-expressed recombinant goat alpha-lactalbumin were studied by circular dichroism spectroscopy, X-ray crystallography, and stopped-flow measurements, and the results were compared with those of the authentic protein prepared from goat milk. The electric properties of the two proteins were also studied by gel electrophoresis and ion-exchange chromatography. Although the overall structures of the authentic and recombinant proteins are the same, the extra methionine residue at the N terminus of the recombinant protein remarkably affects the native-state stability and the electric properties. The native state of the recombinant protein was 3.5 kcal/mol less stable than the authentic protein, and the recombinant protein was more negatively charged than the authentic one. The recombinant protein unfolded 5.7 times faster than the authentic one, although there were no significant differences in the refolding rates of the two proteins. The destabilization of the recombinant protein can be fully interpreted in terms of the increased unfolding rate of the protein, indicating that the N-terminal region remains unorganized in the transition state of refolding, and hence is not involved in the folding initiation site of the protein. A comparison of the X-ray structures of recombinant alpha-lactalbumin determined here with that of the authentic protein shows that the structural differences between the proteins are confined to the N-terminal region. Theoretical considerations for the differences in the conformational and solvation free energies between the proteins show that the destabilization of the recombinant protein is primarily due to excess conformational entropy of the N-terminal methionine residue in the unfolded state, and also due to less exposure of hydrophobic surface on unfolding. The results suggest that when the N-terminal region of a protein has a rigid structure, expression of the protein by E. coli, which adds the extra methionine residue, destabilizes the native state through a conformational entropy effect. It also shows that differences in the electrostatic interactions of the N-terminal amino group with the side-chain atoms of Thr38, Asp37, and Asp83 bring about a difference in the pKa value of the N-terminal amino group between the proteins, resulting in a greater negative net charge of the recombinant protein at neutral pH.

Convergence of the BMP and EGF signaling pathways on Smad1 in the regulation of chondrogenesis.

Bone morphogenetic protein 4 (BMP4) induces, whereas epidermal growth factor (EGF) inhibits chondrogenesis. We hypothesize that BMP4 and EGF mediated intracellular signals are both coupled in the regulation of Meckel's cartilage development. Two chondrogenic experimental model systems were employed to test the hypothesis: (1) an ex vivo, serum-free, organ culture system for mouse embryonic mandibular processes, and (2) a micromass culture system for chicken embryonic mandibular processes. Chondrogenesis was assayed by alcian blue staining and expression of Sox9 and type II collagen. Exogenous EGF inhibited and BMP4 induced ectopic cartilage in a dose-dependent manner. When BMP4- and EGF-soaked beads were implanted in juxtaposition within embryonic day 10 mouse mandibular processes, the incidence and amount of ectopic cartilage, and Sox9 and type II collagen expression induced by BMP4, were significantly reduced as the concentration of EGF was increased. Similarly, in chicken serum-free micromass cultures, expression of a constitutively active BMP receptor type IB by replication competent avian retrovirus system promoted the rate and extent of chondrogenesis; however, exogenous EGF attenuated this effect. In micromass cultures, BMP signaling resulted in nuclear translocation and accumulation of the signaling molecule Smad1, whereas the addition of EGF inhibited this event. Our results suggest that BMP4 and EGF function antagonistically, yet are coupled in the regulation of initial chondrogenesis. Smad1 serves as a point of convergence for the integration of two different growth factor signaling pathways during chondrogenesis.

Refolding kinetics of staphylococcal nuclease and its mutants in the presence of the chaperonin GroEL.

We have analyzed the effect of the chaperonin GroEL on the refolding kinetics of staphylococcal nuclease and its three mutants by stopped-flow fluorescence measurements. It was found that a transient folding intermediate of staphylococcal nuclease was tightly bound to GroEL and refolded in the GroEL-bound state without releasing the non-native protein in solution, and the refolding rate in the GroEL-bound state was 0.01 s-1. The GroEL-affected refolding of the nuclease appears to be in decided contrast to that of apo-alpha-lactalbumin reported in our previous study, wherein alpha-lactalbumin was shown to be more weakly bound by GroEL and to refold in the free state in solution. In spite of the apparent difference between the proteins, the GroEL-affected refolding reactions of both the proteins can be represented by a common unified reaction scheme. On the basis of this scheme, the binding constant between the nuclease intermediate and GroEL was estimated to be larger than 10(9) M-1. The stoichiometry of binding of the nuclease and its mutants to GroEL was found to be two (nuclease/GroEL 14-mer). The increase in ionic strength resulted in a weakening of the interaction between the nuclease and GroEL, which was attributed to a weakening of the electrostatic attraction between the two proteins as a result of electrostatic screening by ions. Although ATP was found to accelerate the GroEL-affected refolding of the nuclease, the refolding rate was still far from the rate of the free refolding. The free refolding behavior of the nuclease and its mutants was restored in the presence of the cochaperonin GroES and ATP.

Kinetic refolding of beta-lactoglobulin. Studies by synchrotron X-ray scattering, and circular dichroism, absorption and fluorescence spectroscopy.

beta-Lactoglobulin (beta LG) is a predominantly beta-sheet protein with a markedly high helical propensity and forms non-native alpha-helical intermediate in the refolding process. We measured the refolding reaction of beta LG with various techniques and characterized the folding kinetics and the structure of the intermediate formed within the burst phase of measurements, i.e. the burst-phase intermediate. Time-resolved stopped-flow X-ray scattering measurements using the integral intensity of scattering show that beta LG forms a compact, globular structure within 30 ms of refolding. The averaged radius of gyration within 100 ms is only 1.1 times larger than that in the native state, ensuring that the burst-phase intermediate is compact. The presence of a maximum peak in a Kratky plot shows a globular shape attained within 100 ms of refolding. Stopped-flow circular dichroism, tryptophan absorption and fluorescence spectroscopy show that pronounced secondary structure regains rapidly in the burst phase with concurrent non-native alpha-helix formation, and that the subsequent compaction process is accompanied by annealing of non-native secondary structure and slow acquisition of tertiary structure. These findings strongly suggest that both compaction and secondary structure formation in protein folding are quite rapid processes, taking place within a millisecond time-scale. The structure of the burst-phase intermediate in beta LG refolding was characterized as having a compact size, a globular shape, a hydrophobic core, substantial beta-sheets and remarkable non-native alpha-helical structure, but little tertiary structure. These results suggest that both local interactions and non-local hydrophobic interactions are dominant forces early in protein folding. The interplay of local and non-local interactions throughout folding processes is important in understanding the mechanisms of protein folding.

Kinetic folding and cis/trans prolyl isomerization of staphylococcal nuclease. A study by stopped-flow absorption, stopped-flow circular dichroism, and molecular dynamics simulations.

We studied the urea-induced unfolding transition of staphylococcal nuclease (SNase) and its five proline mutants (P47A, P47T, P117G, P47T/P117G, and P47A/P117G) [corrected] by peptide and aromatic circular dichroism and aromatic absorption spectroscopy at equilibrium and the refolding-unfolding kinetics of the proteins by stopped-flow circular dichroism and stopped-flow absorption techniques. Recent studies have revealed that the cis/trans isomerizations about the Pro47 and Pro117 peptide bonds of SNase occur not only in the unfolded state but also in the native state. The mutational effects on the stability and the refolding-unfolding kinetics of SNase were, however, remarkably different between the two sites. The substitution of Ala or Thr for Pro47 neither changed the stability nor affected the refolding-unfolding kinetics of SNase, whereas the substitution of Gly for Pro117 increased the protein stability by 1.2 kcal/mol (pH 7.0 and 20 degrees C) and affected the kinetics. These results have been attributed to the high flexibility of the loop around Pro47, which has been revealed by molecular dynamics simulations of native SNase. Under every condition studied, cooperative refolding-unfolding kinetics of SNase were observed. Refolding of wild-type SNase was represented by two urea concentration-dependent fast phases and a urea concentration-independent slow phase. The double mutant (P47T/P117G) [corrected] of SNase still showed multiphasic refolding kinetics that involved two urea concentration-independent slow phases, suggesting that isomerization of proline residues other than Pro47 and Pro117 may occur in the unfolded state of the mutant. Two phases were observed in the unfolding of the wild-type and mutant proteins that contained Pro117, a fast phase corresponding to the unfolding of the trans isomer and a slow phase corresponding to that of the cis isomer. On the basis of these results, the folding scheme of SNase is discussed.

Protein globularization during folding. A study by synchrotron small-angle X-ray scattering.

Various conformational states of polypeptide chains were investigated by synchrotron small-angle X-ray scattering (SAXS). SAXS patterns of proteins and model polypeptides in globular states (native and "molten globule") and in non-globular states (unfolded protein as well as randomly coiled, partially alpha-helical and partially beta-structural synthetic polypeptides) were analyzed in terms of Guinier and Kratky plots. Large differences in the SAXS pattern have been found between globular and non-globular conformations of the polypeptide chains, and they have been interpreted in terms of differences in the shape and size of the globular and non-globular scatterers with the same molecular mass. The equilibrium and time-resolved unfolding curves of bovine carbonic anhydrase and yeast phosphoglycerate kinase were monitored by integrated SAXS intensity, and were found to be coincident with the curves measured by other physicochemical techniques, such as tryptophan fluorescence and peptide circular dichroism spectra. The intermolecular association of the protein "molten globule"-like intermediates accumulated during the guanidine hydrochloride-induced unfolding of bovine carbonic anhydrase has been investigated by various SAXS parameters. It has been shown that the integrated SAXS intensity is much less sensitive to the protein intermolecular association than the zero angle intensity and the radius of gyration. We propose the integrated SAXS intensity as a global parameter which is particularly appropriate for fast kinetic studies of protein coil to globule transitions. Time-resolved refolding curves of the above proteins were monitored by the integrated SAXS intensity to investigate the globularization process in protein folding. Two fast kinetic processes for bovine carbonic anhydrase and two fast (each within two seconds) as well as two slow (within 500 seconds) kinetic processes for yeast phosphoglycerate kinase have been recorded. The kinetic processes reflect both protein intramolecular globularization and its intermolecular association.

Human endothelin converting enzyme gene (ECE1) mapped to chromosomal region 1p36.1.

The chromosomal localization of the human endothelin converting enzyme gene (ECE1) has been identified. Southern blot analysis of human genomic DNA from human x mouse somatic cell hybrids demonstrated that ECE1 maps to chromosome 1. Fluorescence in situ hybridization of a digoxigenin-labeled human ECE1 probe to normal human metaphase chromosomes showed that the gene is located within chromosome band 1p36.1.

Solution X-ray scattering study on the chaperonin GroEL from Escherichia coli.

The molecular architecture of native GroEL has been studied by solution X-ray scattering. The radius of gyration for the native molecule was estimated to be 66.0 A in 50 mM Tris-HCl, 100 mM KCl at pH 7.5 and 25 degrees C. The maximum dimension was estimated to be 170 A, based on the pair distance distribution function. A cylindrical structure or two heptameric rings was found to be the best for native GroEL among structures examined by using a multi-sphere model analysis in which the radius of constituent sphere was 6 A. The results of the model analysis show that the radius of GroEL is 68.0 A and the height is 150.7 A. Unexpectedly, the central penetrating hole through GroEL was not confirmed in the best-fit structure.

Genetic transfer of endothelin converting enzyme activity to CHO-K1 cells: detection of positive cells by reverse hemolytic plaque assay.

We have established a novel method of molecular cloning of endothelin converting enzyme, a key enzyme in the production of a potent vasoconstrictor endothelin-1, by modification of the reverse hemolytic plaque assay. Also, we demonstrated that a cell line, CHO-K1, showed no detectable activity of endothelin converting enzyme. This cell line was transfected with a cDNA library of bovine endothelial cells. The modified reverse hemolytic plaque assay was shown to detect even a single CHO-K1 cell that was changed to produce mature ET-1 by transfection. Thus, this novel method is suggested to be useful for the molecular cloning of other secreted antigens and their processing enzyme.

cDNA cloning and expression of bovine endothelin converting enzyme.

We have cloned cDNA encoding bovine endothelin converting enzyme (ECE). The predicted amino acid sequence of bovine ECE consisted of 758 amino acid residues. Northern blot analysis revealed that ECE mRNA was abundantly expressed in lung. Co-expression of the cloned cDNA of bovine ECE with human preproET-1 cDNA in CHO-K1 cells resulted in the production of mature ET-1 and this production was inhibited by phosphoramidon.

The chaperonin GroEL does not recognize apo-alpha-lactalbumin in the molten globule state.

We investigate here the interaction between GroEL and two kinds of non-native alpha-lactalbumin. alpha-Lactalbumin is a Ca(2+)-binding protein which assumes a molten globule state in the absence of Ca2+ (apo-alpha-lactalbumin) at neutral pH. Our results, obtained by molecular-sieve chromatography and hydrogen-exchange measurements, show that apo-alpha-lactalbumin in this molten globule state is not bound to GroEL either in the absence or in the presence of KCl. On the other hand, we show by molecular-sieve chromatography that alpha-lactalbumin, in which the four disulphide bonds are fully reduced, is bound to GroEL when 50 mM KCl is present. The results demonstrate that the protein state recognized by GroEL is more unfolded and expanded than the typical molten globule state of alpha-lactalbumin.