High-throughput screening.
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Journal Article↗
Biomedical subjects
C Pernelle
Publications and source records attributed to C Pernelle.
Analysis of the nucleic acid annealing activities of nucleocapsid protein from HIV-1.
Retroviral nucleocapsid (NC) protein is an integral part of the virion nucleocapsid where it is in tight association with genomic RNA and the tRNA primer. NC protein is necessary for the dimerization and encapsidation of genomic RNA, the annealing of the tRNA primer to the primer binding site (PBS) and the initial strand transfer event. Due to the general nature of NC protein-promoted annealing, its use to improve nucleic acid interactions in various reactions can be envisioned. Parameters affecting NC-promoted nucleic acid annealing of NCp7 from HIV-1 have been analyzed. The promotion of RNA:RNA and RNA:DNA annealing by NCp7 is more sensitive to the concentration of MgCl2 than the promotion of DNA:DNA hybridization. Stimulation of complex formation for all three complexes was efficient at 0-90 mM NaCl, between 23 and 55 degrees C and at pH values between 6.5 and 9.5, inclusive. Parameters affecting NCp7-promoted hybridization of tRNA(Lys,3) to the PBS, which appears to be specific for NC protein, will be discussed. Results implicate the basic regions of NCp7, but not the zinc fingers, in promoting the annealing of complementary nucleic acid sequences. Finally, NCp7 strand transfer activity aids the formation of the most stable nucleic acid complex.
Base Sequence↗
An efficient screening assay for the rapid and precise determination of affinities between leucine zipper domains.
The protein products of the jun and fos oncogenes require a functional protein-protein interaction domain, called the "leucine zipper domain", to exert their transcriptional regulatory activity. A scintillation proximity assay was developed in which the biotinylated leucine zipper domain of the Jun protein (275-315) was immobilized on streptavidin-coated microfluorospheres and in which the leucine zipper domain of the Fos protein (160-200) was used as free, labeled ligand. The Fos leucine zipper peptide specifically bound to the Jun leucine zipper peptide, and for the first time, a dissociation constant (Kd = 110 +/- 12 nM in PBS/0.1% Tween) could be determined. Optimal heterodimer formation was reached at neutral pH. Both acidic and alkaline pH decreased the association of the peptides which was, furthermore, completely abolished by 500 mM NaCl, confirming that charged residues are critical for heterodimerization. A commercially obtained recombinant Jun protein competed as efficiently as the Jun leucine zipper peptide for binding to the Fos peptide, confirming the feasibility of using the two leucine zipper peptides to study the interactions between the two transcription factors. We also injected leucine zipper peptides individually into Xenopus oocytes to study whether they would interfere with the activity of the Fos/Jun heterodimer in vivo. Both peptides blocked selectively insulin-mediated oocyte maturation with an IC50 in the range of 15 ng per oocyte. In conclusion, the scintillation proximity assay described here may be used to investigate protein-protein interactions mediated by leucine zipper structures and to identify compounds that inhibit leucine zipper association.
Amino Acid Sequence↗
Construction, purification, and characterization of a hybrid protein comprising the DNA binding domain of the LexA repressor and the Jun leucine zipper: a circular dichroism and mutagenesis study.
An increasing number of eukaryotic transcription factors interacting specifically with DNA comprise a dimerization motif called the "leucine zipper". These leucine zipper proteins form homodimers and/or heterodimers with another protein containing a leucine zipper motif. The leucine zipper of the oncoprotein Jun is particular in that Jun may form homodimers as well as heterodimers with the oncoprotein Fos, which are however more stable than the Jun-Jun homodimers. Leucine zipper dimerization is thought to occur through a coiled-coil arrangement of parallel alpha-helices, but the rules governing the specificity of homo- and/or heterodimerization are still largely unknown. To address this question in the case of the Jun leucine zipper, we constructed a fusion protein containing the amino-terminal DNA binding domain of the LexA repressor from Escherichia coli fused to the Jun leucine zipper. This hybrid protein (LexA-JunZip) is stable in E. coli and confers much tighter repression in vivo than the DNA binding domain of LexA alone. DNA binding competition experiments with synthetic Jun and Fos leucine zipper peptides in vitro showed that the leucine zipper mediated dimerization of LexA-JunZip is essential for DNA binding of the fusion protein. The purified LexA-JunZip protein dimerizes in vitro with a dimerization constant of 2 x 10(7) M-1 at 5 degrees C. Dimerization is very sensitive to temperature, since the dimerization constant drops at 20 degrees C to 2 x 10(6) M-1 and at 30 degrees C to only 3 x 10(5) M-1.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
An upstream regulatory element of the NCAM promoter contains a binding site for homeodomains.
In the present study, we have analyzed an upstream regulatory element of the neural cell adhesion molecule (NCAM) promoter which is required for full promoter activity. It contains an ATTATTA motif that resembles the core recognition sequence of homeodomain (HD) proteins of the Antennapedia (Antp) and related types. Electrophoretic mobility shift (EMSA) and DNase I footprinting analyses revealed that the Drosophila HDs coded by the Antp and the zerknüllt (zen) genes bind this site in vitro. In contrast, the engrailed (en) protein did not produce a detectable footprint. The functional relevance of the ATTATTA motif was demonstrated by showing that a two-nucleotide exchange curtailed stimulation of an heterologous promoter. An oligonucleotide known to be recognized with high affinity by Antp-like HDs efficiently competed for endogenous factor binding. These results suggest that the NCAM gene may be a target for HD proteins.
Animals↗
Antennapedia homeobox peptide regulates neural morphogenesis.
We synthesized the 60-amino acid polypeptide corresponding to the sequence of the Drosophila antennapedia gene homeobox. This peptide (pAntp) recognized the consensus motif for binding to the promoter region of Hox-1.3. pAntp mechanically introduced into mammalian nerve cells provoked a dramatic morphological differentiation of the neuronal cultures. Moreover, pAntp directly added to already differentiated neuronal cultures penetrated the cells and further augmented their morphological differentiation. Examination of live and fixed neurons in classical and confocal fluorescence microscopy demonstrated that pAntp was captured at all regions of the nerve cells and accumulated in the nuclei. In addition, the effect of pAntp on neurite extension was blocked in the presence of the protein synthesis inhibitor cycloheximide. Thus, our results demonstrate that neurons possess an efficient uptake system for the antennapedia homeobox peptide and suggest that binding of pAntp to consensus motifs present in nerve cell nuclei influences neuronal morphogenetic programs.
Animals↗
alpha-2,8-Polysialic acid is the neuronal surface receptor of antennapedia homeobox peptide.
A synthetic peptide that is 60 amino acids in length and corresponds to the homeobox sequence of antennapedia protein (pAntp) is specifically and efficiently captured by neurons in culture and conveyed to their nuclei. The internalization process is followed by a strong induction of neuronal morphological differentiation. In the study described here, all treatments masking or removing the alpha-2,8-polysialic acid (PSA) chains specific to the neuronal cell adhesion molecule (NCAM) were found to block the penetration of pAntp and abolish its morphogenetic effects. Structural comparison between PSA and double-stranded DNA suggests that a sequence of eight sialic acid residues can mimic one large groove of the DNA. We propose that this structural similarity is the basis for the property of NCAM polysialic acid to participate in the internalization of the homebox polypeptide.
Animals↗
Monoclonal antibodies against synovial collagenase: use for immunopurification and characterization of the latent and active enzyme.
Monoclonal antibodies have been produced against porcine synovial collagenase which recognize both the active enzyme and its inactive precursor. These antibodies inhibited the collagenolytic activity of collagenase, but not its activity with a synthetic peptide substrate. The antibodies were also able to recognize human synovial, human skin fibroblast and human chondrocyte collagenase but not the enzyme from human granulocytes. One of the monoclonal antibodies was successfully used for the immunopurification of the porcine enzyme and these experiments led to the demonstration of an endogenous activator of procollagenase in the synovial cell culture medium.
Animals↗