The Drosophila homolog of human AF10/AF17 leukemia fusion genes (Dalf) encodes a zinc finger/leucine zipper nuclear protein required in the nervous system for maintaining EVE expression and normal growth

Sibling neurons in the embryonic central nervous system (CNS) of Drosophila can adopt distinct states as judged by gene expression and axon projection. In the NB4-2 lineage, two even-skipped (eve)-expressing sibling neuronal cells, RP2 and RP2sib, are formed in each hemineuromere. Throughout embryogenesis, only RP2, but not RP2sib, maintains eve expression. In this report, we describe a P-element induced mutation that alters the expression pattern of EVE in RP2 motoneurons in the Drosophila embryonic CNS. The mutation was mapped to a Drosophila homolog of human AF10/AF17 leukemia fusion genes (alf), and therefore named Dalf. Like its human counterparts, Dalf encodes a zinc finger/leucine zipper nuclear protein that is widely expressed in embryonic and larval tissues including neurons and glia. In Dalf mutant embryos, the RP2 motoneuron no longer maintains EVE expression. The effect of the Dalf mutation on EVE expression is RP2-specific and does not affect other characteristics of the RP2 motoneuron. In addition to the embryonic phenotype, Dalf mutant larvae are retarded in their growth and this defect can be rescued by the ectopic expression of a Dalf transgene under the control of a neuronal GAL4 driver. This indicates a requirement for Dalf function in the nervous system for maintaining gene expression and the facilitation of normal growth.     

Homodimerization via a leucine zipper motif is required for enzymatic activity of quiescent cell proline dipeptidase

Quiescent cell proline dipeptidase (QPP) is an intracellular serine protease that is also secreted upon cellular activation. This enzyme cleaves N-terminal Xaa-Pro dipeptides from proteins, an unusual substrate specificity shared with dipeptidyl peptidase IV (CD26/DPPIV). QPP is a 58-kDa protein that elutes as a 120-130-kDa species from gel filtration, indicating that it forms a homodimer. We analyzed this dimerization with in vivo co-immunoprecipitation assays. The amino acid sequence of QPP revealed a putative leucine zipper motif, and mutational analyses indicated that this leucine zipper is required for homodimerization. The leucine zipper mutants showed a complete lack of enzymatic activity, suggesting that homodimerization is important for QPP function. On the other hand, an enzyme active site mutant retained its ability to homodimerize. These data are the first to demonstrate a role for a leucine zipper motif in a proteolytic enzyme and suggest that leucine zipper motifs play a role in mediating dimerization of a diverse array of proteins.     

Activity of the human centrosomal kinase, Nek2, depends on an unusual leucine zipper dimerization motif.

Nek2 is a human cell cycle-regulated kinase that is structurally related to the mitotic regulator, NIMA, of Aspergillus nidulans. Localization studies have shown that Nek2 is a core component of the centrosome, the microtubule organizing center of the cell, and functional approaches suggest a possible role for Nek2 in centrosome separation at the G2/M transition. Here, we have investigated the importance of an unusual leucine zipper coiled-coil motif present in the C-terminal noncatalytic domain of the Nek2 kinase. Glycerol gradient centrifugation indicated that endogenous Nek2 is present in HeLa cells as a salt-resistant 6 S complex, the predicted size of a Nek2 homodimer. Recombinant Nek2 overexpressed in insect cells also formed a 6 S complex, whereas a Nek2 mutant specifically lacking the leucine zipper motif was monomeric. Using yeast two-hybrid interaction analyses and coprecipitation assays, we found that Nek2 can indeed form homodimers both in vivo and in vitro and that this dimerization specifically required the leucine zipper motif. Moreover, deletion of the leucine zipper prevented a trans-autophosphorylation reaction on the C-terminal domain of Nek2 and strongly reduced Nek2 kinase activity on exogenous substrates. Finally, we emphasize that the Nek2 leucine zipper described here differs from classical leucine zippers in that it exhibits a radically different arrangement of hydrophobic and charged amino acids. Thus, this study reveals not only an important mechanism for the regulation of the Nek2 kinase but, furthermore, highlights an unusual organization of a leucine zipper dimerization motif.     

Dissection of antibacterial and toxic activity of melittin: a leucine zipper motif plays a crucial role in determining its hemolytic activity but not antibacterial activity

Melittin, a naturally occurring antimicrobial peptide, exhibits strong lytic activity against both eukaryotic and prokaryotic cells. Despite a tremendous amount of work done, very little is known about the amino acid sequence, which regulates its toxic activity. With the goal of understanding the basis of toxic activity and poor cell selectivity in melittin, a leucine zipper motif has been identified. To evaluate the possible structural and functional roles of this motif, melittin and its two analogs, after substituting the heptadic leucine by alanine, were synthesized and characterized. Functional studies indicated that alanine substitution in the leucine zipper motif resulted in a drastic reduction of the hemolytic activity of melittin. However, interestingly, both the designed analogs exhibited antibacterial activity comparable to melittin. Mutations caused a significant decrease in the membrane permeability of melittin in zwitterionic but not in negatively charged lipid vesicles. Although both the analogs exhibited similar secondary structures in the presence of negatively charged lipid vesicles as melittin, they failed to adopt a significant helical structure in the presence of zwitterionic lipid vesicles. Results suggest that the substitution of heptadic leucine by alanine impaired the assembly of melittin in an aqueous environment and its localization only in zwitterionic but not in negatively charged membrane. Altogether, the results suggest the identification of a structural element in melittin, which probably plays a prominent role in regulating its toxicity but not antibacterial activity. The results indicate that cell selectivity in some antimicrobial peptides can probably be introduced by modulating their assembly in an aqueous environment.     

Genetic and molecular properties of human and rat renin-binding proteins with reference to the function of the leucine zipper motif.

The presence of a leucine zipper motif was recognized in the deduced amino acid sequences of human and rat renin-binding proteins (RnBPs) on cloning and sequence analysis of the RnBP cDNAs. The in vitro synthesized RnBPs, with the respective cDNAs, formed heterodimers with porcine renin and homodimers. On comparison of these properties with those of porcine RnBP, the leucine zipper motif was suggested to be a functional domain common to animal RnBPs. In addition to the motif, a hydrophobic domain adjacent to the motif and 10 cysteine residues were also well conserved in the three RnBPs. Moreover, about 85% of their amino acid sequences were identical. The RnBP mRNAs were expressed in the kidneys as the same size of 1.5-kb and the genes are suggested to exist as single copies in the genomes. Despite the high similarities in genetic and molecular properties, the molecular weights of human and rat RnBPs were 43,000, which is 1,000 larger than that of porcine RnBP. The immunoreactivities of human and rat RnBPs toward anti-porcine RnBP antiserum were 88 and 8% that of porcine RnBP, respectively, and the affinities of the two RnBPs for porcine renin were remarkably less than that of porcine RnBP. Moreover, the human and rat RnBP homodimers were partly dissociated under the conditions under which porcine RnBP existed as a dimer. These results indicate distinct differences in the molecular properties among the three RnBPs, in spite of their being highly similar structurally and functionally.     

The Drosophila homolog of human AF10/AF17 leukemia fusion genes (Dalf) encodes a zinc finger/leucine zipper nuclear protein required in the nervous system for maintaining EVE expression and normal growth

Sibling neurons in the embryonic central nervous system (CNS) of Drosophila can adopt distinct states as judged by gene expression and axon projection. In the NB4-2 lineage, two even-skipped (eve)-expressing sibling neuronal cells, RP2 and RP2sib, are formed in each hemineuromere. Throughout embryogenesis, only RP2, but not RP2sib, maintains eve expression. In this report, we describe a P-element induced mutation that alters the expression pattern of EVE in RP2 motoneurons in the Drosophila embryonic CNS. The mutation was mapped to a Drosophila homolog of human AF10/AF17 leukemia fusion genes (alf), and therefore named Dalf. Like its human counterparts, Dalf encodes a zinc finger/leucine zipper nuclear protein that is widely expressed in embryonic and larval tissues including neurons and glia. In Dalf mutant embryos, the RP2 motoneuron no longer maintains EVE expression. The effect of the Dalf mutation on EVE expression is RP2-specific and does not affect other characteristics of the RP2 motoneuron. In addition to the embryonic phenotype, Dalf mutant larvae are retarded in their growth and this defect can be rescued by the ectopic expression of a Dalf transgene under the control of a neuronal GAL4 driver. This indicates a requirement for Dalf function in the nervous system for maintaining gene expression and the facilitation of normal growth.     

repE--the Dictyostelium homolog of the human xeroderma pigmentosum group E gene is developmentally regulated and contains a leucine zipper motif.

We have cloned and characterized the Dictyostelium discoideum repE gene, a homolog of the human xeroderma pigmentosum (XP) group E gene which encodes a UV-damaged DNA binding protein. The repE gene maps to chromosome 4 and it is the first gene identified in Dictyostelium that is homologous to those involved in nucleotide excision repair and their related XP diseases in humans. The predicted protein encodes a leucine zipper motif. The repE gene is not expressed by mitotically dividing cells, and repE mRNA is first detected during the aggregation phase of development when the cells have ceased dividing and replicating genomic DNA. The mRNA level plateaus by the time the developing cells have entered multicellular aggregates and remains at the same steady-state level for the remainder of development. In addition, we have demonstrated that the level of mRNA is very low in developing cells. These observations suggest that repE may play a regulatory role in development. The data indicate that potential developmental roles for XP-related genes can be profitably studied in this system.     

The Drosophila homolog of the human AF10 is an HP1-interacting suppressor of position effect variegation

In chromosomal rearrangements of acute myeloid leukaemia patients the mixed lineage leukaemia (MLL) gene, a human homolog of the Drosophila gene trithorax, is frequently fused to AF10. Here we describe the identification and a functional characterization of the Drosophila homolog dAF10. We show that dAF10 functions in heterochromatin-dependent genomic silencing of position effect variegation, a phenomenon associated with chromosomal rearrangements that cause mosaic expression of euchromatic genes when relocated next to heterochromatin. We also demonstrate that dAF10 can associate with the heterochromatin protein 1 (HP1) in vitro and in vivo. The results indicate that dAF10 is an HP1-interacting component of the heterochromatin-dependent gene silencing pathway, which either contributes to the stability of the heterochromatin complex or to its function.     

The leucine zipper motif of the envelope glycoprotein ectodomain of human immunodeficiency virus type 1 contains conformationally flexible regions as revealed by NMR and circular dichroism studies in different media.

A 43-mer peptide derived from the coiled coil domain of the transmembrane glycoprotein, gp41, of human immunodeficiency virus type 1, was synthesized. Light scattering measurements suggested that the peptide molecules likely exist in the aqueous solution in trimeric form. Circular dichroism experiments showed a moderate helix population enhancement for the peptide in 80% methanol solution relative to helicity in sodium dodecyl sulfate micellar suspension. NMR spectroscopy indicated that the N-terminal section of the peptide was conformationally more sensitive to the medium. The conformationally labile regions contain residues implicated in gp41-gp120 association. Our data support the idea that the coiled coil region is responsible for oligomerization of the gp41 ectodomain and suggest a site of conformational isomerization following receptor binding-induced gp120 dissociation from gp41.     

A human homologue of the Drosophila melanogaster diaphanous gene is disrupted in a patient with premature ovarian failure: evidence for conserved function in oogenesis and implications for human sterility.

Premature ovarian failure (POF) is a defect of ovarian development and is characterized by primary or secondary amenorrhea, with elevated levels of serum gonadotropins, or by early menopause. The disorder has been attributed to various causes, including rearrangements of a large "critical region" in the long arm of the X chromosome. Here we report identification, in a family with POF, of a gene that is disrupted by a breakpoint. The gene is the human homologue of the Drosophila melanogaster diaphanous gene; mutated alleles of this gene affect spermatogenesis or oogenesis and lead to sterility. The protein (DIA) encoded by the human gene (DIA) is the first human member of the growing FH1/FH2 protein family. Members of this protein family affect cytokinesis and other actin-mediated morphogenetic processes that are required in early steps of development. We propose that the human DIA gene is one of the genes responsible for POF and that it affects the cell divisions that lead to ovarian follicle formation.     

Inhibition of lytic activity of Escherichia coli toxin hemolysin E against human red blood cells by a leucine zipper peptide and understanding the underlying mechanism

To investigate as to whether a peptide derived from hemolysin E (HlyE) can inhibit the cytotoxic activity of this protein or not, several peptides were examined for their efficacy to inhibit the lytic activity of the protein against human red blood cells (hRBCs). It was found that a wild-type peptide, H-205, derived from an amphipathic leucine zipper motif, located in the amino acid region 205-234, inhibited the lytic activity of hemolysin E against hRBCs. To understand the basis of this inhibition, several functional and structural studies were performed. Western blotting analysis indicated that the preincubation of HlyE with H-205 did not inhibit its binding to hRBC. The results indicated that H-205 but not its mutant inhibited the hemolysin E-induced depolarization of hRBCs. Flow cytometric studies with annexin V-FITC staining of hRBCs after incubation with either protein or protein/peptide complex suggested that H-205 prevented the hemolysin E-induced damage of the membrane organization of hRBCs. Tryptophan fluorescence and circular dichroism studies showed that H-205 induced a conformational change in HlyE, which was accompanied by the enhancement of an appreciable helical structure. Fluorescence studies with rhodamine-labeled peptides showed that H-205 reversibly self-assembled in aqueous environment, which raised a possibility that the H-205 peptide could interact with its counterpart in the protein and thus disturb the proper conformation of HlyE, resulting in the inhibition of its cytotoxic activity. The peptides derived from the homologous segments of other members of this toxin family may also act as inhibitors of the corresponding toxin.     

Inhibition of microglial egress in excised ganglia by human interleukin 10: implications for its activity in invertebrates

We studied the effects of recombinant human interleukin-10 (IL-10) on invertebrate immunocytes and microglia. The present report demonstrates that the spontaneous activation of invertebrate immunocytes can be specifically inhibited by recombinant human IL-10. Induced immunocyte activation by fMLP can also be significantly diminished by IL-10. This inhibition becomes apparent over hours and causes ameboid cells to become round and nonmobile. Furthermore, Mytilus edulis pedal ganglia maintained in culture, over the course of 24 hours, emit microglia. IL-10 significantly reduces this microglial egress, an action that can be diminished by concomitant exposure of the excised ganglia to an antibody specific to IL-10 as well as IL-10. The anti-IL-10 alone is without effect. Active-ameboid microglia that egress become round and inactive following IL-10 exposure, an action prevented by anti-IL-10. Lastly, a substance immunoreactively similar to human IL-10 can be detected in pedal ganglia homogenates. Taken together, and since the immunocytes and microglia are responding to IL-10, it implies that an IL-10-like substance could be present in invertebrates. In conclusion, the study demonstrates that both invertebrate immunocytes and microglia respond to IL-10, suggesting an early evolution of this generally inhibitory cytokine.     

The use of promoter fusions in Drosophila genetics: isolation of mutations affecting the heat shock response

We have constructed a gene fusion using the promoter of Drosophila hsp70 and the structural gene for Drosophila alcohol dehydrogenase (Adh) and used this construct to transform Adh-deficient flies. In these transformants, Adh is expressed only after heat shock. Like hsp70 itself, this heat-shock-inducible Adh (Adhhs) is induced in a wide variety of tissues. It fails to be induced in primary spermatocytes. Although the tissue distribution of Adh activity is very different from wild type, this does not appear to be deleterious. Indeed, the induction of Adhhs allows flies to survive exposure to ethanol. We have used this latter characteristic to select dominant, trans-acting mutations that alter the response of flies to heat shock.