Positive autoregulation of the myogenic determination gene MyoD1

Transfection of cDNA expression vectors encoding either MyoD1 or myogenin into 10T1/2 cells converts them to myogenic cells. We show that transfection of 10T1/2 cells with the MyoD1 cDNA activates expression of endogenous MyoD1 mRNA, indicating that MyoD1 is subject to positive autoregulation. This activation of endogenous MyoD1 mRNA was also observed in Swiss 3T6 cells, but not in several other fibroblast or adipoblast cell lines transfected with the MyoD1 cDNA. In addition, transfection of the MyoD1 cDNA leads to activation of myogenin expression, and transfection of the myogenin cDNA leads to activation of MyoD1 expression. Thus, MyoD1 and myogenin appear to function in a positive autoregulatory loop that could either: account for or contribute to the stability of myogenic commitment; or amplify the level of expression of both MyoD1 and myogenin above a critical threshold that is required for activation of the myogenic program.     

Fibroblast growth factor and transforming growth factor beta repress transcription of the myogenic regulatory gene MyoD1.

In this report, we demonstrate that myogenic cultures inhibited from differentiating by treatment with fibroblast growth factor or transforming growth factor beta show reduced levels of MyoD1 mRNA. Although this repression may contribute to the inhibition of myogenesis by growth factors, additional regulatory pathways must be affected, since inhibition still occurs in cultures engineered to constitutively express MyoD1 mRNA.     

Equilibrium dissociation and unfolding of the dimeric human papillomavirus strain-16 E2 DNA-binding domain.

The equilibrium unfolding reaction of the C-terminal 80-amino-acid dimeric DNA-binding domain of human papillomavirus (HPV) strain 16 E2 protein has been investigated using fluorescence, far-UV CD, and equilibrium sedimentation. The stability of the HPV-16 E2 DNA-binding domain is concentration-dependent, and the unfolding reaction is well described as a two-state transition from folded dimer to unfolded monomer. The conformational stability of the protein, delta GH2O, was found to be 9.8 kcal/mol at pH 5.6, with the corresponding equilibrium unfolding/dissociation constant, Ku, being 6.5 x 10(-8) M. Equilibrium sedimentation experiments give a Kd of 3.0 x 10(-8) M, showing an excellent agreement between the two different techniques. Denaturation by temperature followed by the change in ellipticity also shows a concomitant disappearance of secondary and tertiary structures. The Ku changes dramatically at physiologically relevant pH's: with a change in pH from 6.1 to 7.0, it goes from 5.5 x 10(-8) M to 4.4 x 10(10) M. Our results suggest that, at the very low concentration of protein where DNA binding is normally measured (e.g., 10(-11) M), the protein is predominantly monomeric and unfolded. They also stress the importance of the coupling between folding and DNA binding.     

c-myc inhibition of MyoD and myogenin-initiated myogenic differentiation.

In vertebrate development, a prominent feature of several cell lineages is the coupling of cell cycle regulation with terminal differentiation. We have investigated the basis of this relationship in the skeletal muscle lineage by studying the effects of the proliferation-associated regulator, c-myc, on the differentiation of MyoD-initiated myoblasts. Transient cotransfection assays in NIH 3T3 cells using MyoD and c-myc expression vectors demonstrated c-myc suppression of MyoD-initiated differentiation. A stable cell system was also developed in which MyoD expression was constitutive, while myc levels could be elevated conditionally. Induction of this conditional c-myc suppressed myogenesis effectively, even in the presence of MyoD. c-myc suppression also prevented up-regulation of a relative of MyoD, myogenin, which is normally expressed at the onset of differentiation in all muscle cell lines examined and may be essential for differentiation. Additional experiments tested whether failure to differentiate in the presence of myc could be overcome by providing myogenin ectopically. Cotransfection of c-myc with myogenin, MyoD, or a mixture of myogenin and MyoD showed that neither myogenin alone nor myogenin plus MyoD together could bypass the c-myc block. The effects of c-myc were further dissected by showing that c-myc can inhibit differentiation independently of Id, a negative regulator of muscle differentiation. These results lead us to propose that c-myc and Id constitute independent negative regulators of muscle differentiation, while myogenin and any of the other three related myogenic factors (MyoD, Myf-5, and MRF4/herculin/Myf-6) act as positive regulators.     

Folding mechanism of FIS, the intertwined, dimeric factor for inversion stimulation

FIS, the factor for inversion stimulation, from Escherichia coli and other enteric bacteria, is an interwined alpha-helical homodimer. Size exclusion chromatography and static light scattering measurements demonstrated that FIS is predominately a stable dimer at the concentrations (1-10 microM monomer) and buffer conditions employed in this study. The folding and unfolding of FIS were studied with both equilibrium and kinetic methods by circular dichroism using urea and guanidinium chloride (GdmCl) as the perturbants. The equilibrium folding is reversible and well-described by a two-state folding model, with stabilities at 10 degrees C of 15.2 kcal mol(-1) in urea and 13.5 kcal mol(-1) in GdmCl. The kinetic data are consistent with a two-step folding reaction where the two unfolded monomers associate to a dimeric intermediate within the mixing time for the stopped-flow instrument (<5 ms), and a slower, subsequent folding of the dimeric intermediate to the native dimer. Fits of the burst phase amplitudes as a function of denaturant showed that the free energy for the formation of the dimeric intermediate constitutes the majority of the stability of the folding (9.6 kcal mol(-1) in urea and 10.5 kcal mol(-1) in GdmCl). Folding-to-unfolding double jump kinetic experiments were also performed to monitor the formation of native dimer as a function of folding delay times. The data here demonstrate that the dimeric intermediate is obligatory and on-pathway. The folding mechanism of FIS, when compared to other intertwined, alpha-helical, homodimers, suggests that a transient kinetic dimeric intermediate may be a common feature of the folding of intertwined, segment-swapped, alpha-helical dimers.     

Thrombin-binding affinities of different disulfide-bonded isomers of the fifth EGF-like domain of thrombomodulin.

The fifth EGF-like domain of thrombomodulin (TM), both with and without the amino acids that connect the fifth domain to the sixth domain, has been synthesized and refolded to form several different disulfide-bonded isomers. The domain without the connecting region formed three disulfide-bonded isomers upon refolding under redox conditions. Of these three isomers, the (1-2,3-4,5-6) bonded isomer was the best inhibitor of fibrinogen clotting and also of the thrombin-TM interaction that results in protein C activation, but all the isomers were inhibitors in both assays. The isomer containing an EGF-like disulfide-bonding pattern (1-3,2-4,5-6) was not found among the oxidation products. The domain with the connecting region amino acids (DIDE) at the C-terminus formed two isolable products upon refolding in redox buffer. These products had the same two disulfide-bonding patterns as the earliest and latest eluting isomers of the domain without the DIDE. In order to compare the thrombin-binding affinities of these isomers to the isomer with the EGF-like disulfide bonds, acetamidomethyl protection of the second and fourth cysteines was used to force the disulfide bonds into the EGF-like pattern. Thrombin-binding affinity, measured as inhibition of fibrinogen clotting and as inhibition of protein C activation correlated inversely with the number of crossed disulfide bonds. As was found for the domain without the connecting region, the isomer that was the best inhibitor of fibrinogen clotting and of protein C activation was the isomer with no crossing disulfide bonds (1-2,3-4,5-6).(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetically driven refolding of the hyperstable EBNA1 origin DNA-binding dimeric beta-barrel domain into amyloid-like spherical oligomers

The Epstein-Barr nuclear antigen 1 (EBNA1) is essential for DNA replication and episome segregation of the viral genome, and participates in other gene regulatory processes of the Epstein-Barr virus in benign and malignant diseases related to this virus. Despite the participation of other regions of the protein in evading immune response, its DNA binding, dimeric beta-barrel domain (residues 452-641) is necessary and sufficient for the main functions. This domain has an unusual topology only shared by another viral origin binding protein (OBP), the E2 DNA binding domain of papillomaviruses. Both the amino acid and DNA target sequences are completely different for these two proteins, indicating a link between fold conservation and function. In this work we investigated the folding and stability of the DNA binding domain of EBNA1 OBP and found it is extremely resistant to chemical, temperature, and pH denaturation. The thiocyanate salt of guanidine is required for obtaining a complete transition to a monomeric unfolded state. The unfolding reaction is extremely slow and shows a marked uncoupling between tertiary and secondary structure, indicating the presence of intermediate species. The Gdm.SCN unfolded protein refolds to fully soluble and spherical oligomeric species of 1.2 MDa molecular weight, with identical fluorescence centre of spectral mass but different intensity and different secondary structure. The refolded spherical oligomers are substantially less stable than the native recombinant dimer. In keeping with the substantial structural rearrangement in the oligomers, the spherical oligomers do not bind DNA, indicating that the DNA binding site is either disrupted or participates in the oligomerization interface. The puzzling extreme stability of a dimeric DNA binding domain from a protein from a human infecting virus in addition to a remarkable kinetically driven folding where all molecules do not return to the most stable original species suggests a co-translational and directional folding of EBNA1 in vivo, possibly assisted by folding accessory proteins. Finally, the oligomers bind Congo red and thioflavin-T, both characteristic of repetitive beta-sheet elements of structure found in amyloids and their soluble precursors. The stable nature of the "kinetically trapped" oligomers suggest their value as models for understanding amyloid intermediates, their toxic nature, and the progress to amyloid fibers in misfolding diseases. The possible role of the EBNA1 spherical oligomers in the virus biology is discussed.     

Alpha-helix 1 in the DNA-binding domain of heat shock factor 1 regulates its heat-induced trimerization and DNA-binding

Heat shock factor 1 (HSF1) primarily regulates various cellular stress responses. The role of α-helix1 (H1) in its DNA-binding domain (DBD) during HSF1 activation remains unknown. Here, HSF1 lacking H1 loses its heat-induced activity, suggesting the importance of the latter. Furthermore, the CD spectra and AMBER prediction show that this H1 deficiency does not change the structure of HSF1 monomer, but does impact its heat-induced trimerization. Point mutation showed that Phe18 in H1 interacts with Tyr60, and that Trp23 interacts with Phe104 by an aromatic-aromatic interaction. Thus, the presence of H1 stabilizes the DBD structure, which facilitates the heat-induced trimerization and DNA-binding of HSF1.     

Backbone dynamics of the glucocorticoid receptor DNA-binding domain.

The extent of rapid (picosecond) backbone motions within the glucocorticoid receptor DNA-binding domain (GR DBD) has been investigated using proton-detected heteronuclear NMR spectroscopy on uniformly 15N-labeled protein fragments containing the GR DBD. Sequence-specific 15N resonance assignments, based on two- and three-dimensional heteronuclear NMR spectra, are reported for 65 of 69 backbone amides within the segment C440-A509 of the rat GR in a protein fragment containing a total of 82 residues (MW = 9200). Individual backbone 15N spin-lattice relaxation times (T1), rotating-frame spin-lattice relaxation times (T1 rho), and steady-state (1H)-15N nuclear Overhauser effects (NOEs) have been measured at 11.74 T for a majority of the backbone amide nitrogens within the segment C440-N506. T1 relaxation times and NOEs are interpreted in terms of a generalized order parameter (S2) and an effective correlation time (tau e) characterizing internal motions in each backbone amide using an optimized value for the correlation time for isotropic rotational motions of the protein (tau R = 6.3 ns). Average S2 order parameters are found to be similar (approximately 0.86 +/- 0.07) for various functional domains of the DBD. Qualitative inspection as well as quantitative analysis of the relaxation and NOE data suggests that the picosecond flexibility of the DBD backbone is limited and uniform over the entire protein, with the possible exception of residues S448-H451 of the first zinc domain and a few residues for which relaxation and NOE parameters were not obtained. in particular, we find no evidence for extensive rapid backbone motions within the second zinc domain. Our results therefore suggest that the second zinc domain is not disordered in the uncomplexed state of DBD, although the possibility of slowly exchanging (ordered) conformational states cannot be excluded in the present analysis.     

Folding of the protein domain hbSBD

The folding of the alpha-helix domain hbSBD of the mammalian mitochondrial branched-chain alpha-ketoacid dehydrogenase complex is studied by the circular dichroism technique in absence of urea. Thermal denaturation is used to evaluate various thermodynamic parameters defining the equilibrium unfolding, which is well described by the two-state model with the folding temperature T(F) = 317.8 +/- 1.95 K and the enthalpy change DeltaH(G) = 19.67 +/- 2.67 kcal/mol. The folding is also studied numerically using the off-lattice coarse-grained Go model and the Langevin dynamics. The obtained results, including the population of the native basin, the free-energy landscape as a function of the number of native contacts, and the folding kinetics, also suggest that the hbSBD domain is a two-state folder. These results are consistent with the biological function of hbSBD in branched-chain alpha-ketoacid dehydrogenase.     

Folding and structural characterization of highly disulfide-bonded beetle antifreeze protein produced in bacteria

The hyperactive antifreeze protein from the beetle, Tenebrio molitor, is an 8.5-kDa, threonine-rich protein containing 16 Cys residues, all of which are involved in disulfide bonds. When produced by Escherichia coli, the protein accumulated in the supernatant in an inactive, unfolded state. Its correct folding required days or weeks of oxidation at 22 or 4 degrees C, respectively, and its purification included the removal of imperfectly folded forms by reversed-phase HPLC. NMR spectroscopy was used to assess the degree of folding of each preparation. One-dimensional (1)H and two-dimensional (1)H total correlation spectroscopy spectra were particularly helpful in establishing the characteristics of the fully folded antifreeze in comparison to less well-folded forms. The recombinant antifreeze had no free -SH groups and was rapidly and completely inactivated by 10 mM DTT. It had a thermal hysteresis activity of 2.5 degrees C at a concentration of 1 mg/ml, whereas fish antifreeze proteins typically show a thermal hysteresis of approximately 1.0 degrees C at 10-20 mg/ml. The circular dichroism spectra of the beetle antifreeze had a superficial resemblance to those of alpha-helical proteins, but deconvolution of the spectra indicated the absence of alpha-helix and the presence of beta-structure and coil. NMR analysis and secondary structure predictions agree with the CD data and are consistent with a beta-helix model proposed for the antifreeze on the basis of its 12-amino-acid repeating structure and presumptive disulfide bond arrangement.