Younsang Oh

Systematic evaluation of transcellular activities of secretory phospholipases A2. High activity of group V phospholipases A2 to induce eicosanoid biosynthesis in neighboring inflammatory cells.

The mechanisms by which secretory phospholipase A2 (PLA2) exerts cellular effects are not fully understood. To elucidate these mechanisms, we systematically and quantitatively assessed the activities of human group IIA, V, and X PLA2s on originating and neighboring cells using orthogonal fluorogenic substrates in various mixed cell systems. When HEK293 cells stably expressing each of these PLA2s were mixed with non-transfected HEK293 cells, group V and X PLA2s showed strong transcellular lipolytic activity, whereas group IIA PLA2 exhibited much lower transcellular activity. The transcellular activity of group V PLA2 was highly dependent on the presence of cell surface heparan sulfate proteoglycans of acceptor cells. Activation of RBL-2H3 and DLD-1 cells that express endogenous group V PLA2 led to the secretion of group V PLA2 and its transcellular action on neighboring human neutrophils and eosinophils, respectively. Similarly, activation of human bronchial epithelial cells, BEAS-2B, caused large increases in arachidonic acid and leukotriene C4 release from neighboring human eosinophils. Collectively, these studies show that group V and X PLA2s can act transcellularly on mammalian cells and suggest that group V PLA2 released from neighboring cells may function in triggering the activation of inflammatory cells under physiological conditions.

Molecular characterization of a gene encoding the Drosophila melanogaster phospholipase A2.

A gene encoding Drosophila melanogaster secretory phospholipase A2 (sPLA2) has been cloned and characterized(.) The coding region of the sPLA2 gene was interrupted by a short intron, and codes for a signal peptide of 18 amino acids, followed by a mature protein of 168 amino acids, containing the structural features of group III sPLA2. From a Northern blot analysis, about a 1.0-kb Drosophila sPLA2 transcript was found to be expressed throughout its development and in both the adult bodies and heads. The recombinant Drosophila sPLA2 expressed and purified in Escherichia coli was found to be Ca(+2)-dependent and maximally active at pH 5.

The DNA replication-related element (DRE)–DRE-binding factor (DREF) system may be involved in the expression of the Drosophila melanogaster TBP gene

The TATA box binding protein (TBP) is a general transcription factor required for initiation by all three eukaryotic RNA polymerases. Previously, we found that the promoter region of the Drosophila melanogaster TBP gene contains three sequences similar to the DNA replication‐related element (DRE) (5′‐TATCGATA). In the present study, we found that the DRE‐like sequences are also present in the promoter of the Drosophila virilis TBP gene, suggesting a role for these sequences in TBP expression. Band mobility shift assays revealed that oligonucleotides containing sequences similar to the DRE of D. melanogaster TBP gene promoter form specific complexes with a factor in a Kc cell nuclear extract and with recombinant DRE‐binding factor (DREF). Furthermore, these complexes were either supershifted or diminished by monoclonal antibodies to DREF. Transient luciferase assays demonstrated that induction of mutations in two DRE‐related sequences at positions −223 and −63 resulted in an extensive reduction of promoter activity. Thus, the DRE–DREF system appears to be involved in the expression of the D. melanogaster TBP gene.

Brain proteins interacting with the tetramerization region of non-erythroid alpha spectrin

The N-terminal region of non-erythroid alpha spectrin (SpαII) is responsible for interacting with its binding partner, beta spectrin, to form functional spectrin tetramers. We used a yeast-two-hybrid system, with an N-terminal segment of alpha spectrin representing the functional tetramerization site, as a bait to screen human brain c-DNA library for proteins that interact with the alpha spectrin segment. In addition to several beta spectrin isoforms, we identified 14 proteins that interact with SpαII. Seven of the 14 were matched to 6 known proteins: Duo protein, Lysyl-tRNA synthetase, TBP associated factor 1, two isoforms (b and c) of a protein kinase A interacting protein and Zinc finger protein 333 (2 different segments). Four of the 6 proteins are located primarily in the nucleus, suggesting that spectrin plays important roles in nuclear functions. The remaining 7 proteins were unknown to the protein data base. Structural predictions show that many of the 14 proteins consist of a large portion of unstructured regions, suggesting that many of these proteins fold into a rather flexible conformation. It is interesting to note that all but 3 of the 14 proteins are predicted to consist of one to four coiled coils (amphiphilic helices). A mutation in SpαII, V22D, which interferes with the coiled coil bundling of SpαII with beta spectrin, also affects SpαII interaction with Duo protein, TBP associated factor 1 and Lysyl-tRNA synthetase, suggesting that they may compete with beta spectrin for interaction with SpαII. Future structural and functional studies of these proteins to provide interaction mechanisms will no doubt lead to a better understanding of brain physiology and pathophysiology.

Molecular characterization of the Drosophila melanogaster gene encoding the pterin 4α-carbinolamine dehydratase

We have isolated and characterized the cDNA and the genomic DNA encoding Drosophila melanogaster pterin 4alpha-carbinolamine dehydratase (PCD). The amino acid sequence deduced from the cDNA sequence was very similar to those of PCDs previously reported in other species (19-57% identity). The protein coding region of the cDNA was expressed in E. coli as a histidine fusion protein, and the expressed protein proved to have PCD activity. The characterization of the Drosophila genomic clone revealed that the Drosophila PCD gene is interrupted by two introns. The potential promoter region, deduced from the determination of the transcription start point (tsp), lacks the distinct TATAAA box consensus sequence.

Isolation and characterization of the gene encoding the Drosophila melanogaster transcriptional elongation factor, TFIIS.

We have characterized a genomic clone encoding the Drosophila melanogaster transcriptional elongation factor, TFIIS. The coding region of the TFIIS gene is interrupted by a short intron. The potential promoter region, deduced from the determination of the transcription start point (tsp), lacks distinct TATAAA or CCAAT box consensus sequences. Southern analysis and the in situ hybridization to chromosomes suggests that it is single-copy gene which is localized to the 35B region on the left arm of the second chromosome.