Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P51532 (transcriptional activator)
 6,546 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We measure the effects of low concentrations of helix-stabilizing cosolvents, including 2,2,2-trifluoroethanol (TFE), on the thermodynamics and kinetics of folding of the dimeric alpha-helical coiled coil derived from the leucine zipper region of bZIP transcriptional activator GCN4. The change in kinetic behavior upon addition of 5% (v/v) TFE indicates that it stabilizes the transition state to the same degree as the fully helical native state. However, folding rates are largely insensitive to alanine to glycine mutagenesis, indicating that the majority of helical structure is formed after the transition state. Equilibrium hydrogen isotope partitioning measurements indicate that intramolecular hydrogen bonds are not strengthened by TFE and that amide hydrogen bonds in the transition state are nearly the same strength as those in the unfolded state. Thus, the mechanism by which TFE exerts its helix-stabilizing effects can be divorced from helix formation and does not depend on the strengthening of intrahelical hydrogen bonds. Rather, TFE increases the structure of the binary alcohol/water solvent, thereby increasing the energetic cost associated with solvation of the polypeptide backbone. At low concentrations, TFE destabilizes the unfolded species and thereby indirectly enhances the kinetics and thermodynamics of folding of the coiled coil. A high degree of polypeptide backbone desolvation, and not the formation of regular helical structure and native strength hydrogen bonds, is the critical feature of the transition state for folding of this small dimeric protein.
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PMID:Trifluoroethanol promotes helix formation by destabilizing backbone exposure: desolvation rather than native hydrogen bonding defines the kinetic pathway of dimeric coiled coil folding. 977 90

bHLH and bHLHZip are highly conserved structural domains mediating DNA binding and specific protein-protein interactions. They are present in a family of transcription factors, acting as dimers, and their selective dimerization is utilized to switch on and off cell proliferation, differentiation or apoptosis. Myc is a bHLHZip protein involved in growth control and cancer, which operates in a network with the structurally related proteins Max, Mad and Mnt. It does not form homodimers, working as a heterodimer with Max; Max, instead, forms homodimers and heterodimers with Mad and Mnt. Myc/Max dimers activate gene transcription, while Mad/Max and Mnt/Max complexes are Myc/Max antagonists and act as repressors. Modifying the molecular recognition of dimers may provide a tool for interfering with Myc function and, in general, for directing the molecular switches operated via bHLH(Zip) proteins. By molecular modelling and mutagenesis, we analysed the contribution of single amino acids to the molecular recognition of Myc, creating bHLHZip domains with altered dimerization specificity. We report that Myc recognition specificity is encoded in a short region within the leucine zipper; mutation of four amino acids generates a protein, Omomyc, that homodimerizes efficiently and can still heterodimerize with wild type Myc and Max. Omomyc sequestered Myc in complexes with low DNA binding efficiency, preventing binding to Max and inhibiting Myc transcriptional activator function. Consistently with these results, Omomyc produced a proliferation arrest in NIH3T3 cells. These data demonstrate the feasibility of interfering with fundamental biological processes, such as proliferation, by modifying the dimerization selectivity of a bHLHZip protein; this may facilitate the design of peptides of potential pharmacological interest.
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PMID:Design and properties of a Myc derivative that efficiently homodimerizes. 982 57

We report here the cloning, characterisation and developmental expression profile of the Xenopus laevis CCAAT-enhancer binding protein beta (xC/EBPbeta) gene. The protein synthesised from the xC/EBPbeta gene interacts specifically with a C/EBP-recognition sequence and acts as a transcriptional activator. Several conserved regions are present in the xC/EBPbeta sequence, including the basic region, leucine zipper, activation domains, three in-frame AUG codons, and a consensus site for mitogen activated protein kinase. The corresponding mRNA is present at high levels in the kidney, liver, lung, muscle and adipose tissue, and at low levels in the ovary, brain and heart. Although the xC/EBPbeta mRNA and protein are present throughout embryogenesis, there is a biphasic increase in their expression levels during development. Whole-mount in situ hybridisation shows a restricted spatial expression profile of the xC/EBPbeta gene during early embryogenesis, with transcripts present around the blastopore lip and in the endodermal cells at the mid-gastrula stage, and, the whole dorsal side at the neurula and early tailbud stage. The expression domain becomes almost ubiquitous during later embryonic development, and includes the brain, spinal cord, somites and regions that give rise to the liver and the heart.
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PMID:Characterisation and developmental regulation of the Xenopus laevis CCAAT-enhancer binding protein beta gene. 983 41

Various endocrine and neuronal functions are governed by the cAMP-dependent signaling pathway. In eukaryotes, transcriptional regulation upon stimulation of the adenylyl cyclase signaling pathway is mediated by a family of cAMP-responsive nuclear factors. This family consists of a large number of members which may act as activators or repressors. These factors contain the basic domain/leucine zipper motifs and bind as dimers to cAMP-response elements (CRE). The function of CRE-binding proteins (CREBs) is modulated by phosphorylation by several kinases. Direct activation of gene expression by CREBs requires phosphorylation by the cAMP-dependent protein kinase A to the serine-133 residue. The gene CREM encodes various transcription factors which play key physiological and developmental roles within the hypothalamic-pituitary-gonadal axis. We have previously shown that the transcriptional activator CREMtau is highly expressed in postmeiotic cells. Spermiogenesis is a complex process by which postmeiotic male germ cells differentiate into mature spermatozoa. This process involves remarkable structural and biochemical changes which are under the hormonal control of the hypothalamic-pituitary axis. We have addressed the specific role of CREM in spermiogenesis using CREM-mutant mice generated by homologous recombination. Analysis of the seminiferous epithelium from mutant male mice reveals that spermatogenesis stops at the first step of spermiogenesis. Late spermatids are completely absent while there is a significant increase in apoptotic germ cells. A series of postmeiotic germ cell-specific genes are not expressed. Mutant male mice completely lack spermatozoa. This phenotype is reminiscent of cases of human infertility.
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PMID:Regulating the balance between differentiation and apoptosis: role of CREM in the male germ cells. 984 51

We describe the construction of a number of vectors that can be used in yeast genetic selection systems for cloning of transcription factors or other DNA-binding proteins and for identification of the target sites recognized by transcription factors. For transcription factor cloning we have designed an integration vector with two HIS3 reporter gene cassettes to stably integrate reporter gene constructs at the non-essential yeast PDC6 locus. This set of plasmids was tested in a one-hybrid assay with the rice transcription factor Oshox1, a member of the class of homeodomain leucine zipper proteins. A hybrid protein of Oshox1 and the Gal4 transcriptional activation domain was shown to specifically activate a reporter gene construct with upstream Oshox1 binding sites, which had been integrated at the PDC6 locus using the described vector system. Target site identification by genetic selection in yeast employs a transcriptional activator construct and a library of genomic or random DNA fragments upstream of a reporter gene. We have constructed two variants of a bacteriophage lambda vector which facilitates the construction of the required reporter gene library because of high cloning efficiency and easy conversion into a yeast/Escherichia coli shuttle vector library by Cre-loxP-mediated automatic subcloning. Tests with Oxhox1 as transcriptional activator demonstrated the usefulness of the deprived reporter gene vector.
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PMID:Vectors for transcription factor cloning and target site identification by means of genetic selection in yeast. 984 32

ToxR is a transmembrane regulatory protein that controls virulence gene expression in Vibrio cholerae. Previous experiments using lambda repressor-ToxR chimeric proteins and a lambda repressor-controlled reporter system (OR1 PR-lacZY) established that ToxR sequences can effectively dimerize the amino-terminal domain of lambda repressor in Escherichia coli. However, in E. coli, ToxR does not respond to environmental signals that control virulence gene expression in V. cholerae. Here, we report the results of experiments designed to test whether environmental signals that modulate virulence gene expression in V. cholerae also modulate a monomer to dimerization transition of lambda-ToxR chimeras. When the OR1 PR-lacZY reporter fusion and chimeric proteins were transferred to V. cholerae, we unexpectedly found that lambda-ToxR chimeras did not dimerize significantly. Interestingly, experiments evaluating the ability of lambda-ToxR proteins to form tetramers in E. coli suggested that lambda-ToxR dimers could act co-operatively. Using a redesigned reporter system containing multiple lambda operator sites (OR1 OR2 OR3 PR-lacZY), we found that lambda-ToxR could dimerize quite efficiently in V. cholerae. These data imply that multiple DNA binding sites might enhance the ability of ToxR to dimerize in V. cholerae and suggest that ToxR dimers might be capable of co-operative interactions. However, we falled to correlate a monomer-dimer transition of the lambda-ToxR chimeras with changes in virulence gene expression in response to environmental signals in V. cholerae. Finally, because of conflicting results in the literature, the importance of membrane localization of ToxR and dimerization of the ToxR periplasmic domain was re-evaluated. This was accomplished by measuring the ability of various chimeric proteins to activate toxin gene expression in both E. coli and V. cholerae. These assays suggest that, in V. cholerae, deletion of the transmembrane domain has a profound effect on ToxR activity, although it is not an absolute requirement when ToxR is dimerized by a heterologous domain. In addition, we noted differences in chimeric protein activity when expressed in E. coli and V. cholerae. A construct substituting the monomeric MalE domain for the periplasmic domain of ToxR was unable to activate a ctx::lacZ reporter fusion in E. coli. Although the addition of leucine zipper sequences to this construct resulted in enhanced activity of the chimera in E. coli, both chimeras were able to produce wild-type levels of toxin in V. cholerae. These data support the notion that dimerization of ToxR stimulates its activity as a transcriptional activator in E. coli. In V. cholerae, however, we present data that do not demonstrate a correlation between dimerization of the periplasmic domain and ToxR activity.
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PMID:ToxR co-operative interactions are not modulated by environmental conditions or periplasmic domain conformation. 998 31

We measured whether solvent viscosity, and hence chain diffusion, plays a role in the rate-limiting step of the folding reactions of GCN4-p2', a simple alpha-helical coiled coil derived from the leucine zipper region of bZIP transcriptional activator GCN4. To deconvolute the dual effects of viscosogenic solvents on both viscosity, eta, and stability, earlier attempts assumed that the cosolvent and denaturant interact to the same degree in the transition state. Applying this analysis to GCN4-p2' yielded a nearly 1/eta dependence between folding rates and viscosity for both the dimeric and the cross-linked, monomeric versions of the coiled coil, but it revealed no such coherent relationship for cytochrome c. We also developed a method to determine the relative viscosity dependence of the dimeric and monomeric forms of the coiled coil independent of the assumption concerning the transition state's relative interaction with cosolvents and denaturants. Application of this method indicated that the effect of viscosity on both the folding and the unfolding rates was the same for the dimeric and monomeric versions, further supporting the view that the folding of the dimeric version is folding-limited rather than encounter-limited. The finding that GCN4-p2' folding appears to exhibit a 1/eta viscosity dependence implies that the rate-limiting step in folding is opposed predominantly by solvent-derived rather than internal frictional forces. These results are interpreted in relation to various models for protein folding.
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PMID:Viscosity dependence of the folding kinetics of a dimeric and monomeric coiled coil. 1002 55

The hepatitis B virus pX protein is a potent transcriptional activator of viral and cellular genes whose mechanism of action is poorly understood. Here we show that pX dramatically stimulates in vitro DNA binding of a variety of cellular proteins that contain basic region/leucine zipper (bZIP) DNA binding domains. The basis for increased DNA binding is a direct interaction between pX and the conserved bZIP basic region, which promotes bZIP dimerization and the increased concentration of the bZIP homodimer then drives the DNA binding reaction. Unexpectedly, we found that the DNA binding specificity of various pX-bZIP complexes differs from one another and from that of the bZIP itself. Thus, through recognition of the conserved basic region, pX promotes dimerization, increases DNA binding, and alters DNA recognition. These properties of pX are remarkably similar to those of the human T-cell lymphotrophic virus type I Tax protein. Although Tax and pX are not homologous, we show that the regions of the two proteins that stimulate bZIP binding contain apparent metal binding sites. Finally, consistent with this in vitro activity, we provide evidence that both Tax and pX activate transcription in vivo, at least in part, by facilitating occupancy of bZIPs on target promoters.
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PMID:The hepatitis B pX protein promotes dimerization and DNA binding of cellular basic region/leucine zipper proteins by targeting the conserved basic region. 1031 8

The Epstein-Barr virus (EBV) immediate-early protein BZLF1 (Z) is a key regulator of the EBV latent-to-lytic switch. Z is a transcriptional activator which induces EBV early gene expression. We demonstrate here that Z interacts with CREB-binding protein (CBP), a histone acetylase and transcriptional coactivator. This interaction requires the amino-terminal region of CBP as well as the transactivation and leucine zipper domains of Z. We show that CBP enhances Z-mediated transactivation of EBV early promoters, in reporter gene assays and in the context of the endogenous genome. We also demonstrate that Z decreases CREB transactivation function and that this inhibitory effect is reversed by overexpression of CBP. We show that Z also interacts directly with CREB. However, mutational analysis indicates that Z inhibition of CREB activity requires the direct interaction between Z and CBP but not the direct interaction between Z and CREB. We propose that Z interacts with CBP to enhance viral early gene transcription. In addition, the Z-CBP interaction may control host cellular transcription factor activity through competition for limiting amounts of cellular CBP.
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PMID:The Epstein-Barr virus BZLF1 protein interacts physically and functionally with the histone acetylase CREB-binding protein. 1040 Jul 51

We have investigated the folding behavior of dimeric and covalently crosslinked versions of the 33-residue alpha-helical GCN4-p1 coiled coil derived from the leucine zipper region of the transcriptional activator GCN4. The effects of multisite substitutions indicate that folding occurs along multiple routes with nucleation sites located throughout the protein. The similarity in activation energies of the different routes together with an analysis of intrinsic helical propensities indicate that minimal helix is present before a productive collision of the two chains. However, approximately one-third to one-half of the total helical structure is formed in the postcollision transition state ensemble. For the crosslinked, monomeric version, folding occurs along a single robust pathway. Here, the region nearest the crosslink, with the least helical propensity, is structured in the transition state whereas the region farthest from the tether, with the most propensity, is completely unstructured. Hence, the existence of transition state heterogeneity and the selection of folding routes critically depend on chain topology.
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PMID:Transition state heterogeneity in GCN4 coiled coil folding studied by using multisite mutations and crosslinking. 1048 89


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