Design of dominant negatives to bHLHZip proteins that inhibit DNA binding.
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C Vinson
Publications and source records attributed to C Vinson.
Phosphorylation destabilizes alpha-helices.
Phosphorylation of threonine destabilizes the leucine zipper of a bZIP protein by 4.6 kcal mol-1 dimer-1, which reduces DNA binding 100-fold. This decrease in stability reflects the low alpha-helix forming propensity of a phosphorylated threonine.
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The alpha-isoform of the CCAAT/enhancer-binding protein is required for mediating cAMP responsiveness of the phosphoenolpyruvate carboxykinase promoter in hepatoma cells.
The gene coding for phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) is expressed in all gluconeogenic tissues, but stimulation of its rate of transcription by cAMP is robust only in liver. Evidence has accumulated which suggests that a liver-enriched transcription factor, likely a member of the CCAAT/enhancer binding protein (C/EBP) family, is required along with other ubiquitously expressed transcription factors to mediate this liver-specific response to cAMP. In this study, we examined the ability of C/EBP to participate in the cAMP-mediated activation of phosphoenolpyruvate carboxykinase (PEPCK) gene transcription in hepatoma cells. Expression of a dominant repressor of C/EBP in hepatoma cells significantly inhibited the protein kinase A-stimulated transcription of the PEPCK promoter, suggesting that a C/EBP family member was required for maximal transcriptional activation by protein kinase A. To provide additional support for this hypothesis, we prepared GAL4 fusion proteins containing C/EBP domains. Both C/EBPalpha and C/EBPbeta GAL4 fusion proteins were capable of stimulating transcription from promoters containing binding sites for the DNA-binding domain of GAL4. However, only the GAL4-C/EBPalpha fusion protein demonstrated the ability to synergize with the other transcription factors bound to the PEPCK promoter which are required to mediate cAMP responsiveness. The DNA-binding domain of C/EBPalpha was not required for this activity in hepatoma cells, although in non-hepatoma cells the basic region leucine zipper domain appeared to inhibit the ability of C/EBPalpha to participate in mediating cAMP responsiveness. These results suggest that the liver-specific nature of the cAMP responsiveness of the PEPCK promoter involves the recruitment of C/EBPalpha to the cAMP response unit.
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Design of a C/EBP-specific, dominant-negative bZIP protein with both inhibitory and gain-of-function properties.
We have developed a bZIP protein, GBF-F, with both dominant-negative (DN) and gain-of-function properties. GBF-F is a chimera consisting of two components: the DNA binding (basic) region from the plant bZIP protein GBF-1 (GBF) and a leucine zipper (F) designed to preferentially heterodimerize with the C/EBP alpha leucine zipper. Biochemical studies show that GBF-F preferentially forms heterodimers with C/EBP alpha and thus binds a chimeric DNA sequence composed of the half-sites recognized by the C/EBP and GBF basic regions. Transient transfections in HepG2 hepatoma cells show that both components of GBF-F are necessary for inhibition of C/EBP alpha transactivation. When the C/EBP alpha leucine zipper is replaced with that of either GCN4 or VBP, the resulting protein can transactivate a C/EBP cis-element but is not inhibited by GBF-F, indicating that the specificity of dominant-negative action is determined by the leucine zipper. All known members of the C/EBP family contain similar leucine zipper regions and are inhibited by GBF-F. GBF-F also exhibits gain-of-function properties, since, with the essential cooperation of a C/EBP family member, it can transactivate a promoter containing the chimeric C/EBP/GBF site. This protein therefore has potential utility both as a dominant-negative inhibitor of C/EBP function and as an activator protein with novel DNA sequence specificity.
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Extending dimerization interfaces: the bZIP basic region can form a coiled coil.
We appended a rationally designed acidic amphipathic protein sequence to the N-terminus of a leucine zipper. Circular dichroism data indicate that this engineered polypeptide sequence can 'zipper' up the basic region of a bZIP monomer into a heterodimeric coiled coil. This propagation of the leucine zipper dimerization interface into the basic region can proceed for up to four heptads and stabilizes the heterodimer complex 2.5 kcal/mol or > 100-fold. The acidic nature of the extension is the most critical component of the design, suggesting that the extension is acting as a DNA mimetic. The dimerization prevents the basic region in this heterodimeric coiled coil structure from binding to DNA. Gel-shift, fluorescence and transient transfection assays indicate that the acidic extension appended to a leucine zipper can inactivate the DNA-binding and transactivation properties of the bZIP protein C/EBP. The three bZIP basic regions examined in this study dimerize with similar stability with the acidic extension, suggesting that this N-terminal extension can be used to develop dominant-negatives to other bZIP transcription factors.
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Clinical implications of intra- and inter-reader agreement in four different automated visual fields.
BACKGROUND: This study was designed and conducted to examine the degree of inter- and intra-reader agreement when four readers evaluate visual fields in patients known to have glaucoma and patients known to not have glaucoma. METHODS: Fifty-three patients known to have glaucoma and 60 patients known not to have glaucoma were selected randomly from a population at the University of Alabama at Birmingham School of Optometry. Four visual fields were performed on each patient, non-dilated, with the proper correction in place. The fields were performed on three different instruments by technicians who were unaware of the diagnostic status of the patient. Four independent (blinded) clinicians read the visual fields and judged them as: "glaucoma field defect," "no glaucoma field defect," and "poor reliability-cannot judge." RESULTS: Statistical analysis revealed that a single reader's interpretation of four different types of visual fields are very different from, are poorer than, and are more consistent than are the four readers' interpretations of a single type of visual field. The analysis also shows that the readers consistently underestimated the number of glaucoma patients with one instrument and overestimated the number with another. CONCLUSIONS: This results suggest that a single visual field will discriminate patients with glaucoma with an accuracy in the 70 to 75 percent range. For one of the instruments, the shorter protocol was almost as effective in discriminating glaucomatous fields as the longer protocol. This study also suggests that repeating the visual fields or analysis of a single field by two readers improves reliability.
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A thermodynamic scale for leucine zipper stability and dimerization specificity: e and g interhelical interactions.
The leucine zipper is a dimeric coiled-coil protein structure composed of two amphipathic alpha-helices with the hydrophobic surfaces interacting to create the dimer interface. This structure has been found to mediate the dimerization of two abundant classes of DNA binding proteins: the bZIP and bHLH-Zip proteins. Several workers have reported that amino acids in the e and g positions of the coiled coil can modulate dimerization stability and specificity. Using the bZIP protein VBP as a host molecule, we report a thermodynamic scale (delta delta G) for 27 interhelical interactions in 35 proteins between amino acids in the g and the following e positions (g<==>e') of a leucine zipper coiled coil. We have examined the four commonly occurring amino acids in the e and g positions of bZIP proteins, lysine (K), arginine (R), glutamine (Q), glutamic acid (E), as well as the only other remaining charged amino acid aspartic acid (D), and finally alanine (A) as a reference amino acid. These results indicate that E<==>R is the most stable interhelical pair, being 0.35 kcal/mol more stable than E<==>K. A thermodynamic cycle analysis shows that the E<==>R pair is 1.33 kcal/mol more stable than A<==>A with -1.14 kcal/mol of coupling energy (delta delta Gint) coming from the interaction of E with R. The E<==>K coupling energy is only -0.14 kcal/mol. E interacts with more specificity than Q. The R<==>R pair is less stable than the K<==>K by 0.24 kcal/mol. R interacts with more specificity than K. Q forms more stable pairs with the basic amino acids K and R rather than with E. Changing amino acids in the e position to A creates bZIP proteins that form tetramers.
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Administrative knowledge and skills needed by physician executives.
A survey was mailed to 100 physician executives identified through the 1991 American College of Physicians Executives directory. The subjects were asked to rate 17 managerial areas on their value to the subjects' current work, on the subjects' preparation in the areas, and on the need for training in the areas. In addition, the subjects were asked how best to accomplish training in the areas and for a list of areas of greatest importance in the future for physician executives. The subjects rated communication skills, quality assurance, utilization review, and personnel management as being of primary value in their current roles. Preparation was most adequate in communication skills and most inadequate in the areas of finance and organizational management. Training was deemed desirable in all areas, but was thought to be most necessary in communication skills, negotiations, strategic planning, and organizational management. There was least desire for training in the areas of labor law and employment law. The most popular means of training were doing a fellowship in administrative medicine, receiving continuing education through seminars or workshops, or getting a degree in management.
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Molecular structure of frizzled, a Drosophila tissue polarity gene.
The function of the frizzled (fz) locus is required to coordinate the cytoskeletons of pupal epidermal cells so that a parallel array of cuticular hairs and bristles is produced. We report here the molecular cloning and characterization of the fz locus. The locus is very large. Mutations that inactivate the gene are spread over 100 kb of genomic DNA. The major mRNA product of the gene is a 4-kb RNA that is encoded by 5 exons spread over more than 90 kb of genomic DNA. Conceptual translation of this mRNA indicates that it encodes an integral membrane protein that is likely to contain both extracellular and cytoplasmic domains.
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Infections caused by Pseudomonas maltophilia. Expanding clinical spectrum.
Pseudomonas maltophilia (Xanthomonas maltophilia) is a frequently isolated commensal that is gaining increasing recognition as an opportunistic pathogen in debilitated hosts. We report three unusual infections due to P maltophilia that illustrate the ability of the organism to cause life-threatening illness. We describe a case of postoperative meningitis, a case of recurrent bacteremia complicated by ecthyma gangrenosum, and a case of native valve endocarditis in a drug addict. Because of frequent isolation from noninfected sites, the pathogenic potential of P maltophilia may be overlooked. The notable resistance of this organism is commonly used beta-lactam and aminoglycoside antibiotics may complicate therapy.
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