Putative Leucine Zipper Domain Research Articles

Molecular structure, expression, and functional role of Clec11a in skeletal biology and cancers.

C-type lectin domain family 11 member A (Clec11a), also known as stem cell growth factor (SCGF), C-type lectin superfamily member 3 (CLECSF3), or osteolectin was initially identified as a growth factor for hematopoietic progenitor cells. The human Clec11a gene encodes a polypeptide of 323 amino acids with characteristics of a secreted glycoprotein encompassing two integrin-binding motifs, RGD (Arg-Gly-Asp) and LDT (Leu-Asp-Thr), a putative leucine zipperdomain, and a functional C-type lectin domain. It regulates hematopoietic differentiation and homeostasisand exhibits a protective effect against severe malarial anemia and lipotoxicity. Furthermore, Clec11a promotes the differentiation of mesenchymal progenitors into mature osteoblasts in vitro and plays an important role in the maintenance of adult skeleton age-related bone loss and fracture repair. Receptor ligand binding results in activation of downstream signaling cascades including glycogen synthase kinase 3 (GSK3), β-catenin, and Wnt, resulting in the expression of osteoblast-related gene transcripts including Alp, Runx2, Lef1, and Axin2. In addition, Clec11a is also associated with the development of several cancers, including leukemia, multiple myeloma, and gastrointestinal tract tumors. To date, however, the mechanisms governing transcription regulation of the Clec11a gene arenot known and remainto be uncovered. Understanding the function and mechanism of action of Clec11a will pave the way for the development of Clec11a as a novel therapeutic target for conditions such as cancer, anemia, and skeletal diseases.

Cloning, molecular characterization and expression analysis of a SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE gene (CitSERK1-like) in Valencia sweet orange

Somatic embryogenesis receptor-like kinase (SERK) belonging to the receptor-like kinases (RLKs) has been shown to be implicated in somatic embryogenesis (SE). In this study, a somatic embryogenesis receptor-like gene CitSERK1-like was cloned and characterized from Citrus sinensis cv. ‘Valencia’, a genotype with high somatic embryogenesis capacity for over 26 years. Fifteen consecutive amino acids in putative leucine zipper domain of CitSERK1-like gene were different from the reported CitSERK1 gene. Homology search and sequence analysis demonstrated that the deduced CitSERK1-like protein shared a high degree of identity with SERKs from other species in sequence and structure. Real-time PCR analysis revealed that the transcript of CitSERK1-like was enhanced during the induction of SE. At subsequent embryo-transition phase, a moderate level of expression was detected in heart-torpedo and cotyledon embryos, while low expression was detected in globular embryo. Among the different tissues, the expression of CitSERK1-like was highest in young leaves. Further analysis of its spatial expression by in situ hybridization revealed that CitSERK1-like was mainly located in the embryogenic callus and vascular cells of different embryos or tissues. The results of temporal and spatial expression of CitSERK1-like showed that it played critical roles throughout the process of SE and had a broader role in plant development. In addition, CitSERK1-like expression was up-regulated by 2, 4-D and NAA at the early stage, but down-regulated afterward. Taken together, it suggested that CitSERK1-like activated complex developmental pathways associated with somatic embryogenesis and plant growth.

878. Toward ‘Pseudotyping’ Adenovirus Vectors: Efficient Generation of pIX-Expressing Helper Cell Lines

Specificity of gene delivery is a key prerequisite for gene therapy. We have previously demonstrated that modification of the minor capsid protein IX (pIX) can be used to modify cell type specificity of human adenovirus type-5 (HAdV-5) particles. The 14.3 kDa hexon-associated pIX is strongly conserved between the different members of the mastadenoviruses. Protein IX is believed to function as a capsid cement by linking the hexons in groups-of-nine (GONs). Although viruses lacking pIX do not form GONs, and are less heat-stable than wild-type viruses, they can be propagated with the same kinetics and yields as the wild-type viruses. Deletion mutants lacking (parts of) the conserved N-terminal domain are not incorporated into the virion, implicating this domain in capsid formation. In contrast, pIX molecules with mutations in other conserved domains (i.e. the central alanine stretch, and the putative leucine-zipper domain) are efficiently incorporated into virions. The carboxyl terminal domain contains the putative leucine-zipper domain that may be responsible for trimerization via the formation of a coiled-coil structure. Mutants in this region are efficiently incorporated into the capsid, suggesting that trimerization is not essential for capsid formation. Further analysis of these mutants revealed that deletion of the putative leucine-zipper domain does not affect capsid stabilization by pIX, demonstrating that trimerization is not necessary for the heat-stability phenotype1. To facilitate pseudotyping adenoviral vectors with pIX variants we have set up an efficient system for the generation of pIX-expressing helper cell lines. With an optimized pIX-expression cassette, we generated monoclonal and polyclonal helper cell lines that express wt as well as modified pIX genes to levels equivalent as in a wt HAdV-5 infected cells. Immune-affinity electron microscopy on viruses lacking the pIX gene demonstrated that more than 96% of the particles contain pIX protein incorporated in their capsids after propagation on the pIX-expressing helper cell lines. In addition, the pIX amounts in the helper cells are sufficient to generate heat-stable particles. Finally, the ratio between the pIX protein and other capsid proteins is identical to those found in wt particles. The cell lines are very stable: cell lines grown for two months in the absence of selection contain pIX in amounts identical to the parental line. This demonstrates that pIX is not toxic to cells. These data, together with our previous demonstration that alpha-helical spacers can be fused to pIX to expose new ligands on the outer surface of particles2, establishes that modification of pIX is feasible for pseudotyping adenovirus vectors.

134. Structural Requirements of Adenovirus Targeting Via by Modification of Minor Capsid Protein IX

In mastadenoviruses, the hexon-associated protein IX is a 14.3kDa protein that is strongly conserved between the different members of the genus. It associates with nine hexon capsomers to form group-of-nine hexons (GON). These make up the central region of each of the facets of the icosahedron. Protein IX is believed to function as a capsid 'cement'. So far, its structure in not known. Although viruses lacking pIX do not form GONs and are less heat-stable than wild-type viruses, they can be propagated with the same kinetics and yields as the wild-type viruses. Deletion mutants lacking (parts of) the conserved N-terminal domain are not incorporated into the virion, implicating this domain in capsid formation. In contrast, pIX molecules with mutations in other conserved domains (i.e. the central alanine stretch, and the putative leucine zipper domain) are efficiently incorporated into virions. The carboxyl terminal domain contains a putative leucine-zipper domain that may be responsible for trimerization via the formation of a coiled-coil structure. Mutants in this region are efficiently incorporated into the capsid, suggesting that trimerization is not essential for capsid formation. To get more insight into the structure/function relationship we studied the incorporation of heterologous pIX molecules into the HAdV-5 capsid. Our data show that the pIX proteins of HAdV-12 (species A) and HAdV-7 (species B) can be efficiently incorporated into the capsid of pIX-deleted HAdV-5 (species C). In addition, further deletion mutagenesis confirmed that the N-terminus of the HAdV-5 pIX is required and sufficient for incorporation into the capsid. These data further our knowledge on pIX and facilitated the development of pIX as an anchor for the incorporation of heterologous peptides into the capsid. In this way receptor-binding ligands were incorporated in the capsid. Inclusion of alphahelical spacers of up to 7.5 nm between the pIX anchor and the ligand resulted in more efficient binding of the virions to antibodies and cells. These data conform that modification of pIX can be used to incorporate heterologous peptides into the adenoviral capsid, and support the feasibility of this approach to amend the tissue tropism of the adenovirus vectors.

Identification and characterization of NIF3L1 BP1, a novel cytoplasmic interaction partner of the NIF3L1 protein

The NIF3L1 protein is strongly conserved during evolution from bacteria to mammals and recently its function in neuronal differentiation has been demonstrated. In the present study we identified novel binding partners of human NIF3L1 by screening a HeLa cDNA-library using the yeast two-hybrid system. We could show that the NIF3L1 protein is interacting with itself and with the NIF3L1 binding protein 1 (NIF3L1 BP1), a novel protein of 23.67 kDa bearing a putative leucine zipper domain. Furthermore, both interactions were confirmed using the mammalian two-hybrid system. Deletion analyses clearly demonstrated that a C-terminal region of 100 amino acids of the NIF3L1 BP1 is sufficient for the interaction with NIF3L1. The NIF3L1 BP1 is ubiquitously expressed and cotransfection experiments revealed that NIF3L1 and NIF3L1 BP1 interact in the cytoplasm of human LNCaP cells. This study provides novel insights into the cellular function of the NIF3L1 protein.

MacroH2A1.2 binds the nuclear protein Spop

X-chromosome inactivation is a phenomenon by which one of the two X chromosomes in somatic cells of female mammals is inactivated for life. The inactivated X chromosomes are covered with Xist (X-inactive specific transcript) RNA, and also enriched with the histone H2A variant, macroH2A1.2. The N-terminal one-third of macroH2A1.2 is homologous to core histone H2A, but the function of the C-terminal two-thirds, which contains a basic, putative leucine zipper domain, remains unknown. In this study, we tried analyzing protein–protein interaction with a yeast two-hybrid system to interact with the nonhistone region of mouse macroH2A1.2. The results showed that macroH2A1.2 interacts with mouse nuclear speckled type protein Spop. The Spop protein has a unique composition: an N-terminal MATH, and a C-terminal BTB/POZ domain. Further binding domain mapping in a glutathione- S-transferase (GST) pull-down experiment revealed that macroH2A1.2 binds the MATH domain of Spop, which in turn binds to the putative leucine zipper domain of macroH2A1.2.

Leucine Zipper-mediated Homodimerization of the p21-activated Kinase-interacting Factor, βPix: IMPLICATION FOR A ROLE IN CYTOSKELETAL REORGANIZATION

Pix, a p21-activated kinase-interacting exchange factor, is known to be involved in the regulation of Cdc42/Rac GTPases. The 85-kDa betaPix-a protein contains an Src homology 3 domain, the tandem Dbl homology and Pleckstrin homology domains, a proline-rich region, and a GIT1-binding domain. In addition to those domains, betaPix-a also contains a putative leucine zipper domain at the C-terminal end. In this study, we demonstrate that the previously identified putative leucine zipper domain mediates the formation of betaPix-a homodimers. Using in vitro and in vivo methodologies, we show that deletion of the leucine zipper domain is sufficient to abolish betaPix-a homodimerization. In NIH3T3 fibroblast cells, expression of wild type betaPix-a induces the formation of membrane ruffles. However, cells expressing the leucine zipper domain deletion mutant could not form membrane ruffle structures. Moreover, platelet-derived growth factor-mediated cytoskeletal changes were completely blocked by the leucine zipper domain deletion mutant. The results suggest that the leucine zipper domain enables betaPix-a to homodimerize, and homodimerization is essential for betaPix-a signaling functions leading to the cytoskeletal reorganization.

Polymorphisms of the Ro52 gene associated with anti-Ro 52-kd autoantibodies in patients with primary Sjögren's syndrome.

To screen for the Ro52 gene encoding the 52-kd Ro autoantigen for possible mutations and polymorphisms associated with primary Sjogren's syndrome (SS). The restriction enzyme fragment-single-strand conformation polymorphism method was used to search for mutations and polymorphisms in the Ro52 gene in 97 patients with primary SS and 72 healthy control subjects. The results were verified by automated DNA sequencing and natural or amplification-created restriction site tests. A single-nucleotide polymorphism (SNP) was discovered in intron 3 (137 bp upstream of exon 4). The C/T genotype was significantly more prevalent among patients who were positive for anti-Ro 52-kd (20 of 38) than among healthy controls (9 of 72) (P = 0.00003); significant differences were not seen in patients who were negative for anti-Ro 52-kd. Furthermore, the frequency of the T allele in this position among groups of anti-Ro 52-kd-positive patients, anti-Ro 52-kd-negative patients, and control subjects was significantly increased in the patients who were positive for anti-Ro 52-kd compared with the controls. We present the results of a complete screening for the Ro52 gene in patients with primary SS and the results of an association study. An SNP in intron 3 was found to be strongly associated with the presence of anti-Ro 52-kd autoantibodies in primary SS. This finding is interesting in light of the fact that an alternative messenger RNA is made by deleting exon 4, which encodes a putative leucine zipper domain, to generate a shorter version of the Ro 52-kd protein.

Latency-associated nuclear antigen of Kaposi’s sarcoma-associated herpesvirus (human herpesvirus-8) binds ATF4/CREB2 and inhibits its transcriptional activation activity

Latency-associated nuclear antigen (LANA), encoded by ORF 73 of Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus-8), may play an important role in the persistence of the viral episome by tethering it to host chromosomes during mitosis. It also has been suggested from its amino acid sequence features that LANA may have transcription-regulatory activity. Here, it is reported that LANA interacts with activating transcription factor (ATF) 4/cAMP response element-binding protein (CREB) 2, a member of the ATF/CREB family of transcription factors, and represses the transcriptional activation activity of ATF4/CREB2. Repression by LANA is independent of the DNA-binding ability of ATF4/CREB2, since LANA also represses transactivation of ATF4/CREB2 fused to the GAL4 DNA-binding domain and does not affect the DNA-binding ability of ATF4/CREB2 in an electrophoretic mobility shift assay. The putative leucine zipper domain of LANA is required for binding to the relatively conserved basic region/leucine zipper domain (bZIP) of ATF4/CREB2, suggesting that the interaction may involve leucine zipper dimerization.

Aa-Pri2, a single-copy gene from Agrocybe aegerita, specifically expressed during fruiting initiation, encodes a hydrophobin with a leucine-zipper domain.

The Aa-Pri2 gene, specifically expressed during basidiocarp differentiation of the mushroom Agrocybe aegerita, was cloned. Sequence analysis showed a 525-nt ORF interrupted by three introns (51, 48, 54 bp). One TATA box (-94), two CAAT boxes (-382, -398) and one CT-rich motif (-149) were present. The transcription start point was located by primer extension at position -60. The hydrophobic Aa-PRI2 protein (123 aa) possessed a putative signal peptide (aa1-aa18), eight cysteine residues characteristic of the hydrophobin proteins, and one putative leucine-zipper domain (aa50-aa71). Hybridization of the genomic DNA of several edible mushrooms with the Aa-Pri2 cDNA, showed a 2.7-kb DNA fragment highly similar to the Aa-Pri2 gene in the species Agrocybe chaxingu; sequences showing slight hybridization signals were detected in Pleurotus eryngii, Lentinula edodes and Agaricus bisporus.

Disruption of PML-associated nuclear bodies mediated by the human cytomegalovirus major immediate early gene product.

The PML gene product is associated with a defined nuclear structure (10-20 per cell) known variously as PML-bodies, ND10, PODs or Kr bodies. Certain conditions are known to compromise the integrity of PML-bodies; these include environmental stress (e.g. heat shock), a chromosomal translocation-associated acute promyelocytic leukaemia, and infection with certain viruses [including human cytomegalovirus (HCMV), herpes simplex virus type 1 and adenovirus]. Expression of the HCMV major immediate early (IE) protein (IE1(491aa)) is by itself sufficient to cause disruption of PML-bodies, resulting in the dispersal of the PML antigen uniformly throughout the nucleus. In uninfected cells undergoing mitosis PML is excluded from chromatin. However, both IE1(491aa) and PML were observed to associate with mitotic chromosomes in cells infected with HCMV or transfected with the IE1 gene. A series of in-frame IE1 deletion mutants was used in DNA transfection experiments to identify two large sequence elements (aa 132-274 and the C-terminal aa 347-491) not required for dispersal of the PML antigen. However, a putative leucine-zipper domain (aa 105-139), a putative zinc-finger domain (aa 267-286) and exon 2 and 3 coding sequences (aa 6-85) were required. The association of the IE1 gene product with chromatin required an acidic domain near the C terminus (aa 421-486). The interaction of IE1(491aa) with chromatin was therefore not required for the disruption of PML-bodies. Exon 2 (aa 1-24) was shown to encode a nuclear localization signal.

Characterization and chromosomal localization of rat scavenger receptor class B type I, a high density lipoprotein receptor with a putative leucine zipper domain and peroxisomal targeting sequence.

High density lipoprotein (HDL) participates in reverse cholesterol transport and in the delivery of cholesterol to steroid-producing tissues. Scavenger receptor class B type I (SR-BI) was recently shown to bind HDL and mediate internalization of its cholesterol content. We have cloned the rat homolog of this receptor, determined its chromosomal location, and examined its expression in rat tissues and in a model of follicular development, ovulation, and luteinization. The predicted protein contained two transmembrane domains, a leucine zipper motif, and a peroxisomal targeting sequence. The rat and human SR-BI genes were mapped to a region previously linked between rat and human chromosomes 12. SR-BI gene expression was detected in several rat tissues, with high levels in ovarian tissue, liver, and adrenal cortex, as determined by ribonuclease protection assay and in situ hybridization. A significant increase in SR-BI gene expression was detected in the late phase of corpus luteum formation, and transcripts were abundant in corpus luteum and in thecal cells at all stages of follicular development. In conclusion, the rat SR-BI complementary DNA predicted a protein with several conserved motifs, including a putative leucine zipper and a peroxisomal targeting sequence. The chromosomal locations of the rat and human SR-BI homologs suggest that this gene is a new member of a previously reported, conserved synteny group. SR-BI gene expression was high in steroid-producing tissues and in the liver, consistent with a role of this receptor in the uptake of HDL cholesterol.

Characterization and Chromosomal Localization of Rat Scavenger Receptor Class B Type I, a High Density Lipoprotein Receptor with a Putative Leucine Zipper Domain and Peroxisomal Targeting Sequence

Characterization and Chromosomal Localization of Rat Scavenger Receptor Class B Type I, a High Density Lipoprotein Receptor with a Putative Leucine Zipper Domain and Peroxisomal Targeting Sequence

A novel repressor, par-4, modulates transcription and growth suppression functions of the Wilms' tumor suppressor WT1.

The tumor suppressor WT1 represses and activates transcription. The loss and/or imbalance of the dual transcriptional activity of WT1 may contribute to Wilms' tumor. In this study, we identified par-4 (for prostate apoptosis response) as a WT1-interacting protein that itself functions as a transcriptional repressor. par-4 contains a putative leucine zipper domain and is specifically upregulated during apoptosis of prostate cells (S. F. Sells, D. P. Wood, Jr., S. S. Joshi-Barve, S. Muthukkumar, R. J. Jacob, S. A. Crist, S. Humphreys, and V. M. Rangnekar, Cell Growth Differ. 5:457-466, 1994). The leucine repeat domain of par-4 was shown to interact with the zinc finger DNA binding domain of WT1. Immunoprecipitation-Western blot (immunoblot) analyses demonstrated in vivo WT1-par-4 interactions. par-4 was ubiquitously expressed, and the protein was found in both the nucleus and the cytoplasm. Functionally, par-4 inhibited transcription activated by WT1, but not by the related protein EGR1. Inhibition of WT1-mediated transcription was dependent on the domain of par-4 that mediates its physical association with WT1. In addition, par-4 augmented WT1-mediated repression, possibly by contributing an additional repression domain. Consistent with these results, par-4 functioned as a transcriptional repressor when brought to a promoter via a heterologous DNA binding domain. Significantly, par-4, but not a mutant unable to interact with WT1, rescued growth suppression caused by WT1. Thus, we identified a novel repressor that modulates transcription as well as growth suppression functions of WT1.

Complementation of a yeast top2ts mutation by a cDNA encoding rat DNA topoisomerase II alpha.

A series of yeast expression plasmids which comprise segments of the cDNA sequences encoding rat topo II alpha have been constructed. The transcription of these constructs is under the control of the yeast GAL1 promoter. Galactose-dependent expression of the cloned rat topo II alpha cDNA complemented a yeast top2ts mutation, as well as a deletion mutation at the yeast TOP2 locus. Truncation of 12 N-terminal amino acids and/or 158 C-terminal amino acids of rat topo II alpha had no effect on its ability functionally to substitute for top2ts. Moreover, a cDNA construct with mutated putative leucine zipper domain (amino acids 993-1013) retained the complementation activity. These observations suggest that transformants capable of conditional topo II alpha expression can be exploited as a useful model system for studies on the structure-function relationships of wild-type and mutated topo II alpha, as well as the interplay of potential antitumor drugs with the enzyme.

A role for the KP leucine zipper in regulating P element transposition in Drosophila melanogaster.

The KP element can repress P element mobility in Drosophila melanogaster. Three mutant KP elements were made that had either two amino acid substitutions or a single amino acid deletion in the putative leucine zipper domain found in the KP polypeptide. Each KP element was expressed from the actin 5C proximal promoter. The wild-type control construct strongly repressed P element mobility, measured by the GD sterility and sn(w) mutability assays, in a position-independent manner. The single amino acid deletion mutant failed to repress P mobility by the double amino acid substitution mutants was position dependent. The results show that the leucine zipper of the KP polypeptide is important for P element regulation. This supports the multimer-poisoning model of P element repression, because leucine zipper motifs are involved in protein-protein interactions.

Does a synthetic peptide containing the leucine-zipper domain of c-myb form an α-helical structure in solution?

We have examined a synthetic peptide containing the putative leucine zipper domain of the chicken c-myb proto-oncogene using circular dichroism (CD) spectroscopy. The peptide adopts an α-helical structure only at low temperatures and in the presence 2,2,2-trifluoroethanol.