Leucine Zipper-like Domain Research Articles

Morphogenesis in Aspergillus nidulans requires Dopey (DopA), a member of a novel family of leucine zipper‐like proteins conserved from yeast to humans

DopA is the founding member of a novel protein family required for correct cell morphology and spatiotemporal organization of multicellular structures in the filamentous fungus Aspergillus nidulans. DopA homologues from Saccharomyces cerevisiae (Dop1), Candida albicans, Caenorhabditis elegans, Rattus norvegicus and Homo sapiens have been identified from genome sequencing projects. S. cerevisiae DOP1 is essential for viability and, like DopA, affects cellular morphogenesis. dopA encodes a large protein (207 kDa) containing several putative domains, including three leucine zipper-like domains. Strains with either the temperature-sensitive dopA1(ts) allele, which alters one of the leucine zippers, or the null deltadopA allele, had abnormal morphology of the vegetative hyphae, delayed and asynchronous initiation of asexual development, aberrant morphogenesis of the conidiophore and an early block in the sexual cycle. The expression patterns of key transcriptional regulators of the asexual and sexual cycle (brlA, abaA and steA) are altered in a deltadopA background, suggesting that DopA functions upstream in the developmental pathway. Double mutant analysis showed that dopA interacts genetically with constitutively active and inactive forms of A. nidulans Aras to modulate hyphal morphogenesis and asexual development.

A Carboxy-Terminally Truncated Form of the Vpr Protein of Human Immunodeficiency Virus Type 1 Retards Cell Proliferation Independently of G2 Arrest of the Cell Cycle

Vpr, one of the accessory gene products of HIV-1, is a 96-residue protein with several functions. It is involved in import of the HIV-1 preintegration complex into the nucleus of nondividing cells, in cellular differentiation, inducing cell cycle arrest at the G2/M phase, in immune suppression, and in enhancement of replication of the virus. We found recently that Vpr interferes with the proliferation of mouse NIH3T3 fibroblasts but fails to arrest these cells in the G2 phase. Thus, it seems possible that Vpr might retard cell proliferation via a novel pathway that is distinct from G2 arrest. To elucidate the mechanism by which Vpr induces the retardation of cell growth, we developed a panel of expression vectors that encoded Vpr molecules with deletions of specific putative domains, namely, the first α-helical domain, the second α-helical domain, a leucine zipper-like domain, and an arginine-rich carboxy-terminal domain. These vectors were introduced into HeLa cells since expression of Vpr can induce G2 arrest in such cells. A carboxy-terminally truncated form of Vpr, C81, which failed to induce G2 arrest, led to the G1 arrest and retained the ability to prevent cell proliferation. All the other mutant proteins had completely lost the capacity to induce G2 arrest and to suppress growth. Substitutions of Ile/Leu for Pro at positions 60, 67, 74, and 81 within the leucine zipper-like domain of Vpr or of C81 revealed that Ile60, Leu67, and Ile74 play an important role in the C81-induced suppression of growth, while Ile74 and Ile81 were found to be indispensable for Vpr-induced G2 arrest. Collectively, our results strongly suggest that Vpr can retard cell proliferation independently of G2 arrest of the cell cycle.

The Novel Human HUEL (C4orf1) Gene Maps to Chromosome 4p12–p13 and Encodes a Nuclear Protein Containing the Nuclear Receptor Interaction Motif

A 3250-bp novel human cDNA sequence was isolated from the MRC-5 human embryonic lung cell line by the rapid amplification of cDNA ends technique. This gene was designated HUEL and given the symbol C4orf1 by the HUGO Nomenclature Committee. Within HUEL was identified a continuous ORF of 1704 bp encoding a predicted hydrophilic protein of 568 amino acids with a calculated molecular mass of 63,410 Da. The putative protein contains the LXXLL signature motif considered necessary and sufficient for binding of certain coactivators to liganded nuclear receptors, as well as nuclear localization signals, a nuclear export-like signal, a zinc finger-like motif, an acidic region, and two leucine zipper-like domains. Northern blot analysis of human fetal tissues revealed 3.4-kb transcripts, while RT-PCR demonstrated HUEL expression in a wide range of human adult tissues and cancer cell lines. In the SiHa, HT-1080, and G-401 cancer lines was detected an alternative transcript in which a 166-bp segment was excluded by exon skipping, which is predicted to culminate in a protein with a modified and truncated C-terminus. HUEL was localized to chromosome region 4p12–p13 by fluorescence in situ hybridization. In Western blots, affinity-purified antibodies raised against a HUEL-specific synthetic peptide could recognize a distinct protein band of ∼70 kDa. Immunoblotting of subcellular fractions and indirect immunofluorescence of human embryonic lung cells demonstrated the distribution of HUEL predominantly in the cytoplasm, with an apparently cytoskeletal association. However, in smaller or dividing PLC/PRF/5 and TONG liver carcinoma cells, there was a translocation of HUEL from the cytoplasm to the nucleus. Taken together, these data suggest that HUEL plays a role in transcriptional regulation.

T20/DP178, an Ectodomain Peptide of Human Immunodeficiency Virus Type 1 gp41, Is an Activator of Human Phagocyte N-Formyl Peptide Receptor

Human immunodeficiency virus type 1 (HIV-1) envelope protein gp41 mediates viral fusion with human host cells. The peptide segment T20/DP178, located in the C-terminus of the ectodomain of gp41, interacts with the N-terminal leucine zipper-like domain on gp41 to establish the fusogenic conformation of the virus. Synthetic T20/DP178 peptide is highly efficacious in inhibiting HIV-1 infection in vitro by disrupting the transformation of fusogenic status of viral gp41; thus, it has been proposed for clinical trial. We report that synthetic T20/DP178 is a chemoattractant and activator of human peripheral blood phagocytes but not of T lymphocytes. We further demonstrate that T20/DP178 specifically activates a seven-transmembrane, G-protein-coupled phagocyte receptor for N-formylated chemotactic peptides, formyl peptide receptor (FPR). Moreover, synthetic T20/DP178 analogs lacking N-terminal amino acids acted as FPR antagonists. Our results suggest that gp41 peptides regulate phagocyte function via FPR and identify a novel mechanism by which HIV-1 may modulate innate immunity.

T20/DP178, an Ectodomain Peptide of Human Immunodeficiency Virus Type 1 gp41, Is an Activator of Human Phagocyte N-Formyl Peptide Receptor

AbstractHuman immunodeficiency virus type 1 (HIV-1) envelope protein gp41 mediates viral fusion with human host cells. The peptide segment T20/DP178, located in the C-terminus of the ectodomain of gp41, interacts with the N-terminal leucine zipper-like domain on gp41 to establish the fusogenic conformation of the virus. Synthetic T20/DP178 peptide is highly efficacious in inhibiting HIV-1 infection in vitro by disrupting the transformation of fusogenic status of viral gp41; thus, it has been proposed for clinical trial. We report that synthetic T20/DP178 is a chemoattractant and activator of human peripheral blood phagocytes but not of T lymphocytes. We further demonstrate that T20/DP178 specifically activates a seven-transmembrane, G-protein–coupled phagocyte receptor for N-formylated chemotactic peptides, formyl peptide receptor (FPR). Moreover, synthetic T20/DP178 analogs lacking N-terminal amino acids acted as FPR antagonists. Our results suggest that gp41 peptides regulate phagocyte function via FPR and identify a novel mechanism by which HIV-1 may modulate innate immunity.

T21/DP107, A Synthetic Leucine Zipper-Like Domain of the HIV-1 Envelope gp41, Attracts and Activates Human Phagocytes by Using G-Protein-Coupled Formyl Peptide Receptors

A leucine zipper-like domain, T21/DP107, located in the amino terminus of the ectodomain of gp41, is crucial to the formation of fusogenic configuration of the HIV-1 envelope protein gp41. We report that the synthetic T21/DP107 segment is a potent stimulant of migration and calcium mobilization in human monocytes and neutrophils. The activity of T21/DP107 on phagocytes was pertussis toxin-sensitive, suggesting this peptide uses Gi-coupled seven-transmembrane receptor(s). Since the bacterial chemotactic peptide fMLP partially desensitized the calcium-mobilizing activity of T21/DP107 in phagocytes, we postulated that T21/DP107 might preferentially use a lower affinity fMLP receptor. By using cells transfected to express cloned prototype chemotactic N-formyl peptide receptor (FPR) or its variant, FPR-like 1 (FPRL1), we demonstrate that T21/DP107 activates both receptors but has a much higher efficacy for FPRL1. In addition, T21/DP107 at nM concentrations induced migration of FPRL1-transfected human embryonic kidney 293 cells. In contrast, fMLP did not induce significant chemotaxis of the same cells at a concentration as high as 50 microM. Although a lipid metabolite, lipoxin A4, was a high-affinity ligand for FPRL1, it was not reported to induce Ca2+ mobilization or chemotaxis in FPRL1-transfected cells. Therefore, T21/DP107 is a first chemotactic peptide agonist identified thus far for FPRL1. Our results suggest that this peptide domain of the HIV-1 gp41 may have the potential to activate host innate immune response by interacting with FPR and FPRL1 on phagocytes.

Early Assembly Step of a Retroviral Envelope Glycoprotein: Analysis Using a Dominant Negative Assay

As for most integral membrane proteins, the intracellular transport of retroviral envelope glycoproteins depends on proper folding and oligomeric assembly in the ER. In this study, we considered the hypothesis that a panel of 22 transport-defective mutants of the human T cell leukemia virus type 1 envelope glycoprotein might be defective in ER assembly. Upon cell cotransfection with wild-type envelope, however, the vast majority of these transport-defective mutants (21 of 22) exerted a specific trans-dominant negative effect. This effect was due to random dimerization of the mutated and wild-type glycoproteins that prevented the intracellular transport of the latter. This unexpected result suggests that association of glycoprotein monomers precedes the completion of folding. The only mutation that impaired this early assembly was located at the NH2 terminus of the protein. COOH-terminally truncated, soluble forms of the glycoprotein were also trans-dominant negative provided that their NH2 terminus was intact. The leucine zipper-like domain, although involved in oligomerization of the envelope glycoproteins at the cell surface, did not contribute to their intracellular assembly. We propose that, at a step subsequent to translation, but preceding complete folding of the monomers, glycoproteins assemble via their NH2-terminal domains, which, in turn, permits their cooperative folding.

Biophysical Characterization of the Structure of the Amino-terminal Region of gp41 of HIV-1: IMPLICATIONS ON VIRAL FUSION MECHANISM

A peptide of 51 amino acids corresponding to the NH2-terminal region (5-55) of the glycoprotein gp41 of human immunodeficiency virus type 1 was synthesized to study its conformation and assembly. Nuclear magnetic resonance experiments indicated the sequence NH2-terminal to the leucine zipper-like domain of gp41 was induced into helix in the micellar solution, in agreement with circular dichroism data. Light scattering experiment showed that the peptide molecules self-assembled in water into trimeric structure on average. That the peptide molecules oligomerize in aqueous solution was supported by gel filtration and diffusion coefficient experiments. Molecular dynamics simulation based on the NMR data revealed a flexible region adjacent to the hydrophobic NH2 terminus of gp41. The biological significance of the present findings on the conformational flexibility and the propensity of oligomerization of the peptide may be envisioned by a proposed model for the interaction of gp41 with membranes during fusion process.

HSK4, a member of a novel subfamily of calcium-activated potassium channels

The gene for hSK4, a novel human small conductance calcium-activated potassium channel, or SK channel, has been identified and expressed in Chinese hamster ovary cells. In physiological saline hSK4 generates a conductance of approximately 12 pS, a value in close agreement with that of other cloned SK channels. Like other members of this family, the polypeptide encoded by hSK4 contains a previously unnoted leucine zipper-like domain in its C terminus of unknown function. hSK4 appears unique, however, in its very high affinity for Ca2+ (EC50 of 95 nM) and its predominant expression in nonexcitable tissues of adult animals. Together with the relatively low homology of hSK4 to other SK channel polypeptides (approximately 40% identical), these data suggest that hSK4 belongs to a novel subfamily of SK channels.

Specific Interactions of the Autoantigen L7 with Multi-zinc Finger Protein ZNF7 and Ribosomal Protein S7

The eucaryotic protein L7, which associates with the large subunit of ribosomes, has been shown to be a major autoantigen in systemic autoimmune arthritis. The N terminus carries a sequence motif that is similar to the leucine zipper domain of eucaryotic transcription factors. This domain promotes the homodimerization of protein L7 through alpha-helical coiled-coil formation and binds to distinct mRNAs, thereby inhibiting their cell-free translation. Using a yeast two-hybrid selection, we have identified from a Jurkat T lymphoma cDNA library ribosomal protein S7 and the multi-zinc finger protein ZNF7 as proteins that interact with protein L7. A fragment of L7 carrying the leucine zipper-like domain is fully sufficient to mediate these interactions. Their potential biological significance is indicated by low apparent dissociation constants of S7-L7 (15 x 10(-9) M) and, respectively, ZNF7-L7 (2 x 10(-9) M) complexes and co-immunoprecipitation of proteins S7, ZNF7, and L7 from a cell lysate with an anti-L7 antibody. We also show that ZNF7-like L7 and S7 can exist in a ribosome-bound form. This study provides further evidence suggesting that L7 is involved in translational regulation through interactions with components of the translational apparatus.

Specific interactions of the autoantigen L7 with multi zinc finger protein ZNF7 and ribosomal protein S7

Abstract The eucaryotic protein L7, which associates with the large subunit of ribosomes, has been shown to be a major autoantigen in systemic autoimmune arthritis. The N terminus carries a sequence motif that is similar to the leucine zipper domain of eucaryotic transcription factors. This domain promotes the homodimerization of protein L7 through α-helical coiled-coil formation and binds to distinct mRNAs, thereby inhibiting their cell-free translation. Using a yeast two-hybrid selection, we have identified from a Jurkat T lymphoma cDNA library ribosomal protein S7 and the multi-zinc finger protein ZNF7 as proteins that interact with protein L7. A fragment of L7 carrying the leucine zipper-like domain is fully sufficient to mediate these interactions. Their potential biological significance is indicated by low apparent dissociation constants of S7-L7 (15 × 10−9 m) and, respectively, ZNF7-L7 (2 × 10−9 m) complexes and co-immunoprecipitation of proteins S7, ZNF7, and L7 from a cell lysate with an anti-L7 antibody. We also show that ZNF7-like L7 and S7 can exist in a ribosome-bound form. This study provides further evidence suggesting that L7 is involved in translational regulation through interactions with components of the translational apparatus.

Redox Regulation of GA-binding Protein-α DNA Binding Activity

We have investigated the reduction/oxidation (redox) regulation of the heteromeric transcription factor GA-binding protein (GABP). GABP, also known as nuclear respiratory factor 2, regulates the expression of nuclear encoded mitochondrial proteins involved in oxidative phosphorylation, including cytochrome c oxidase subunits IV and Vb, as well as the expression of mitochondrial transcription factor 1. GABP is composed of two subunits, the Ets-related GABP-alpha, which mediates specific DNA binding, and GABP-beta, which forms heterodimers and heterotetramers on DNA sequences containing the PEA3/Ets motif ((C/A)GGA(A/T)(G/A)). We demonstrate here that GABP DNA binding activity and GABP-dependent gene expression in 3T3 cells are inhibited by pro-oxidant conditions. DNA binding of recombinant GABP-alpha was activated by chemical reduction (dithiothreitol) and by thioredoxin; however, GSSG inhibited GABP DNA binding activity. Treatment of GABP-alpha, but not GABP-beta1, with sulfhydryl-alkylating agents also inhibited GABP DNA binding activity. Our results suggest that GABP DNA binding activity is redox-regulated in vivo, possibly by thioredoxin-mediated reduction and by GSSG-mediated oxidation of the GABP-alpha subunit. The regulation of GABP (nuclear respiratory factor 2) DNA binding activity by cellular redox changes provides an important link between mitochondrial and nuclear gene expression and the redox state of the cell.

Identification of functional domains and evolution of Tc1-like transposable elements.

Tc1-like transposable elements from teleost fish have been phylogenetically examined to determine the mechanisms involved in their evolution and conserved domains of function. We identified two new functional domains in these elements. The first is a bipartite nuclear localization signal, indicating that transposons can take advantage of the transport machinery of host cells for nuclear uptake of their transposases. The second is a novel combination of a paired domain-related protein motif juxtaposed to a leucine zipper-like domain located in the putative DNA-binding regions of the transposases. This domain coexists with a special inverted repeat structure in certain transposons in such phylogenetically distant hosts as fish and insects. Our data indicate that reassortment of functional domains and horizontal transmission between species are involved in the formation and spread of new types of transposable elements.

Biophysical characterization of recombinant proteins expressing the leucine zipper-like domain of the human immunodeficiency virus type 1 transmembrane protein gp41.

Envelope oligomerization is thought to serve several crucial functions during the life cycle of human immunodeficiency virus type 1 (HIV-1). We recently reported that virus entry requires coiled-coil formation of the leucine zipper-like domain of the HIV-1 transmembrane envelope glycoprotein gp41 (C. Wild, T. Oas, C. McDanal, D. Bolognesi, and T. Matthews, Proc. Natl. Acad. Sci. USA 89:10537-10541, 1992; C. Wild, J. W. Dubay, T. Greenwell, T. Baird, Jr., T. G. Oas, C. McDanal, E. Hunter, and T. Matthews, Proc. Natl. Acad. Sci. USA 91:12676-12680, 1994). To determine the oligomeric state mediated by this region of the envelope, we have expressed the zipper motif as a fusion partner with the monomeric maltose-binding protein of Escherichia coli. The biophysical properties of this protein were characterized by velocity and equilibrium sedimentation, size exclusion chromatography, light scattering, and chemical cross-linking analyses. Results indicate that the leucine zipper sequence from HIV-1 is capable of multimerizing much larger and otherwise monomeric proteins into extremely stable tetramers. Recombinant proteins containing an alanine or a serine substitution at a critical isoleucine residue within the zipper region were also generated and similarly analyzed. The alanine- and serine-substituted proteins behaved as tetrameric and monomeric species, respectively, consistent with the influence of these same substitutions on the helical coiled-coil structure of synthetic peptide models. On the basis of these findings, we propose that the fusogenic gp4l structure involves tetramerization of the leucine zipper domain which is situated approximately 30 residues from the N-terminal fusion peptide sequence.

Propensity for a leucine zipper-like domain of human immunodeficiency virus type 1 gp41 to form oligomers correlates with a role in virus-induced fusion rather than assembly of the glycoprotein complex.

For a number of viruses, oligomerization is a critical component of envelope processing and surface expression. Previously, we reported that a synthetic peptide (DP-107) corresponding to the putative leucine zipper region (aa 553-590) of the transmembrane protein (gp41) of human immunodeficiency virus type 1 (HIV-1) exhibited alpha-helical secondary structure and self-associated as a coiled coil. In view of the tendency of this type of structure to mediate protein association, we speculated that this region of gp41 might play a role in HIV-1 envelope oligomerization. However, later it was shown that mutations which should disrupt the structural elements of this region of gp41 did not affect envelope processing, transport, or surface expression (assembly oligomerization). In this report we compare the effects of amino acid substitutions within this coiled-coil region on structure and function of both viral envelope proteins and the corresponding synthetic peptides. Our results establish a correlation between the destabilizing effects of amino acid substitutions on coiled-coil structure in the peptide model and phenotype of virus entry. These biological and physical biochemical studies do not support a role for the coiled-coil structure in mediating the assembly oligomerization of HIV-1 envelope but do imply that this region of gp41 plays a key role in the sequence of events associated with viral entry. We propose a functional role for the coiled-coil domain of HIV-1 gp41.

Molecular cloning and expression of cDNA encoding a galactose/N-acetylgalactosamine-specific lectin on mouse tumoricidal macrophages.

We previously reported that the mouse macrophage galacose and N-acetylgalactosamine-specific lectin (MMGL) may participate in the binding of the macrophages to tumor cells [Oda, S., Sato, M., Toyoshima, S., & Osawa, T. (1989) J. Biochem. 105, 1040-1043]. We now report the cloning and characterization of a cDNA encoding MMGL. The MMGL gene encoded a protein consisting of 304 amino acid residues with a molecular weight of 34,595. The deduced amino acid sequence indicated that MMGL had a single membrane-spanning region, three leucine zipper-like domains, and a carbohydrate recognition domain. Two N-glycosylation sites were found in the extracellular region of MMGL, corresponding to the heavy N-glycosylation in the native MMGL. Comparison of the amino acid sequence of MMGL with those of rat hepatic lectins revealed a high overall sequence homology. The sequence homology was especially high in the putative membrane-spanning region and carbohydrate recognition domain. There was, however, a region of 25 amino acids which did not exist on hepatic lectins. The MMGL cDNA without the region encoding the putative membrane-spanning region and intracellular region was expressed in Escherichia coli. The expressed protein had galactose-binding activity and its sugar-binding specificity was same as that of the native lectin.