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B Z Levi

Publications and source records attributed to B Z Levi.

At least 19 recordsLinked to original sources
PubMed2001

New isoforms of VEGF are translated from alternative initiation CUG codons located in its 5'UTR.

Vascular endothelial growth factor (VEGF) has a central role in normal as well as in tumor angiogenesis. As such, VEGF is subjected to multi-level regulation at the transcriptional, post-transcriptional, translational, and post-translational levels to ensure proper expression during embryogenesis and adulthood. Its mRNA contains an exceptionally long (1038 bp) 5' untranslated region (5'UTR), which has a role in transcriptional as well as translational regulation of VEGF expression. In this communication, we provide new evidence showing that an open reading frame (ORF) present in the 5'UTR encodes for new putative isoforms of VEGF due to alternative translational initiation from CUG codons. Like VEGF, the translation of the new isoforms is not sensitive to stress signals such as anoxia. Most likely, these isoforms either possess new capabilities, which are different from the activity of the classical VEGF isoforms, or affect the efficiency and capacity of translational initiation from the canonical AUG codon.

5' Untranslated Regions↗
PubMed2000

Interaction between interferon consensus sequence-binding protein and COP9/signalosome subunit CSN2 (Trip15). A possible link between interferon regulatory factor signaling and the COP9/signalosome.

Interferon consensus sequence-binding protein (ICSBP) is a member of the interferon regulatory factors (IRF) that has a pivotal role in mediating resistance to pathogenic infections in mice and in promoting the differentiation of myeloid cells. ICSBP exerts some of its transcriptional activities via association with other factors that enable its binding to a variety of promoters containing DNA composite elements. These interactions are mediated through a specific COOH-terminal domain termed IAD (IRF association domain). To gain a broader insight of the capacity of ICSBP to interact with other factors, yeast two-hybrid screens were performed using ICSBP-IAD as a bait against a B-cell cDNA library. Trip15 was identified as a specific interacting factor with ICSBP in yeast cells, which was also confirmed by in vitro glutathione S-transferase pull-down assays and by coimmunoprecipitation studies in COS7 cells. Trip15 was recently identified as a component of the COP9/signalosome (CSN) complex composed of eight evolutionary conserved subunits and thus termed CSN2. This complex has a role in cell-signaling processes, which is manifested by its associated novel kinase activity and by the involvement of its subunits in regulating multiple cell-signaling pathways and cell-cycle progression. We show that in vitro association of ICSBP with the CSN leads to phosphorylation of ICSBP at a unique serine residue within its IAD. The phosphorylated residue is essential for efficient association with IRF-1 and thus for the repressor activity of ICSBP exerted on IRF-1. This suggests that the CSN has a role in integrating incoming signals that affect the transcriptional activity of ICSBP.

3T3 Cells↗
PubMed2000

Cloning of an interferon regulatory factor 2 isoform with different regulatory ability.

Interferons (IFNs) are a family of multifunctional proteins involved in immune activation, regulation of cell growth and antiviral response. They exert their functions by induction of several IFN-stimulated genes, including IFN regulatory factors (IRFs), a family of transcriptional regulators. One of these factors, IRF-2, was initially cloned as an antagonistic counterpart to IRF-1 with oncogenic potential. Here we describe a second isoform of IRF-2, termed IRF-2s, cloned from human and murine cells. This isoform lacks two amino acids located C-terminal of the DNA-binding domain, which is conserved in all IRF family members, leading to a change in the predicted secondary structure. Both isoforms have similar binding affinities to known target sequences in electrophoretic mobility shift assays. Using reporter gene constructs with the type IV promoter region of the MHC class II transactivator (CIITA), which is the essential factor for IFN-gamma-induced MHC class II expression, we show that the short isoform IRF-2s exhibits a weaker activation ability compared to IRF-2. Thus, our data present the first evidence of two IRF-2 isoforms with different regulatory ability.

Amino Acid Sequence↗
PubMed2000

Interplay between repressing and activating domains defines the transcriptional activity of IRF-1.

Interferon regulatory factor-1 (IRF-1) is a transcriptional activator with weak activation capacity. By defining the transcriptional activation domain of IRF-1 we identified two activator fragments located between amino acids 185 and 256 functioning in an additive manner. Another fragment of IRF-1, which has no activator function alone, acts as a strong enhancer element of these activator sequences. This enhancer element resides between the activator domains and the C-terminus. In addition, we identified a novel type of inhibitory domain in the N-terminal 60 amino acids of IRF-1 which strongly inhibits its transcriptional activity. Because this fragment is conserved in all interferon regulatory factors, we found similar repression effects in the corresponding fragments in IRF-2, IRF-3 and interferon consensus sequence binding protein (ICSBP/IRF-8). Interestingly, the corresponding sequence in p48/IRF-9 is divergent, so that it does not show this inhibitory activity. A five-amino-acid sequence distinguishes the p48/IRF-9 N-terminus from the homologous parts in other interferon regulatory factors containing the repressing function. Replacing the diverged amino acids in IRF-1 with the corresponding sequence of p48/IRF-9 resulted in a loss of inhibitory activity within IRF-1. The opposing activities within interferon regulatory factors may contribute to balanced or tuned regulation of gene activation, depending on the promoter context.

Amino Acid Sequence↗
PubMed1999

Protein-protein and DNA-protein interactions affect the activity of lymphoid-specific IFN regulatory factors.

IFN regulatory factors (IRFs) constitute a family of transcription factors that are involved in IFN signaling and the development and differentiation of the immune system. Targeted gene disruption studies in mice assigned their primary role to the immune system. Two lymphoid-specific IRF members, IFN consensus sequence binding protein (ICSBP) and IRF-4, bind target DNA with greater efficiency following interaction with two transcription factors, PU.1 and E47, leading to transcriptional synergy. PU.1 and E47 are essential for proper differentiation and maturation of lymphoid cells. In addition, ICSBP interacts with two IRF members, IRF-1 and IRF-2, which also have central roles in the regulation of cell-mediated immunity. Previously, we identified a region in ICSBP, termed the IRF association domain (IAD), that is conserved in all IRFs (excluding IRF-1 and IRF-2) and is essential for its interactions with other IRF proteins. Here we show that the IAD is an independent module used by ICSBP and IRF-4 for protein-protein interactions. In addition, an IAD of IRF-2 (IAD2), necessary for interaction with ICSBP, was identified and found to be conserved in IRF-1. The IAD2 shares similar characteristics with the PEST domain that is essential for the interaction of PU.1 with IRF-4. We also show that the ICSBP DNA binding domain is indispensable for the formation of DNA binding heterocomplexes and transcriptional activity. Therefore, our results shed light on the molecular mechanisms that affect IRF activities in the immune system via discrete functional domains.

3T3 Cells↗
PubMed1999

The use of bi-cistronic transfer vectors for the baculovirus expression system.

In this communication, we describe the construction of bi-cistronic transfer vectors for the baculovirus expression system (BVES), which are advantageous over the existing vectors. The new vectors provide a simple way to isolate recombinant viruses. More specifically, the gene of interest and the reporter gene luciferase (LUC), constitute the first and second cistrons, respectively, of the same transcript. Therefore, the LUC activity measured during infection of such a bi-cistronic virus, permits an on-line estimation of the recombinant protein level, a very useful feature for large-scale production of recombinant proteins. To achieve expression of the second cistron, the internal ribosome entry site (IRES) element of the encephalomyocarditis virus (EMCV) was employed. However, this element, which is highly efficient in mammalian systems, did not promote efficient internal translation of the second cistron in various insect cells lines originating from different insect species. The lack of efficient internal translation was not due to baculovirus propagation since the same phenomenon was also observed in a viral-free expression system. It seems that a component essential for efficient EMCV IRES activity is either missing or present in limiting amount in insect cells or not compatible. Nevertheless, LUC placed downstream to the IRES element, or immediately downstream to the first cistron, was expressed to a level that enabled the biotechnological application it was designed for.

Animals↗
PubMed1999

Jerusalem of cytokines.

The Second Joint Meeting of the International Cytokine Society and the International Society for Interferon and Cytokine Research was held on October 25-30, 1998 in Jerusalem, Israel. The nature of this Joint Meeting dictated that it was intensive and covered topics that included receptor-ligand interactions, signal transduction, transcriptional regulation, antiviral action and apoptotic pathways induced by cytokines such as interferons, interleukines and chemokines. Their roles in infectious diseases and cancers were considered. This overview is by no mean comprehensive and covers only part of the many topics and subjects that were presented in the many plenary talks, symposia and poster sessions. The meeting was held in an excellent scientific atmosphere, that was probably affected by the "divine presence" in Jerusalem, and special thanks for the excellent organization are owed to Drs. Kaempfer, Revel, Wallach and Witz.

Animals↗
PubMed1998

Functional domains of interferon regulatory factor I (IRF-1).

Interferon (IFN) regulatory factors (IRFs) are a family of transcription factors among which are IRF-1, IRF-2, and IFN consensus sequence binding protein (ICSBP). These factors share sequence homology in the N-terminal DNA-binding domain. IRF-1 and IRF-2 are further related and have additional homologous sequences within their C-termini. Whereas IRF-2 and ICSBP are identified as transcriptional repressors, IRF-1 is an activator. In the present work, the identification of functional domains in murine IRF-1 with regard to DNA-binding, nuclear translocation, heterodimerization with ICSBP and transcriptional activation are demonstrated. The minimal DNA-binding domain requires the N-terminal 124 amino acids plus an arbitrary C-terminal extension. By using mutants of IRF-1 fusion proteins with green fluorescent protein and monitoring their distribution in living cells, a nuclear location signal (NLS) was identified and found to be sufficient for nuclear translocation. Heterodimerization was confirmed by a two-hybrid system adapted to mammalian cells. The heterodimerization domain in IRF-1 was defined by studies in vitro and was shown to be homologous with a sequence in IRF-2, suggesting that IRF-2 also heterodimerizes with ICSBP through this sequence. An acidic domain in IRF-1 was found to be required and to be sufficient for transactivation. Epitope mapping of IRF-1 showed that regions within the NLS, the heterodimerization domain and the transcriptional activation domain are exposed for possible contacts with interacting proteins.

Amino Acid Sequence↗
PubMed1998

Regulation of vascular endothelial growth factor (VEGF) expression is mediated by internal initiation of translation and alternative initiation of transcription.

Vascular Endothelial Growth Factor (VEGF) is a very potent angiogenic agent that has a central role in normal physiological angiogenesis as well as in tumor angiogenesis. VEGF expression is induced by hypoxia and hypoglycemia, and thus was suggested to promote neovascularization during tumor outgrowth. Yet, the molecular mechanism that governs VEGF expression is not fully characterized. VEGF induction is attributed in part to increased levels of transcription and RNA stability. Previously, we demonstrated that the 5' Untranslated Region (5' UTR) of VEGF has an important regulatory role in its expression. VEGF has an exceptionally long 5' UTR (1038 bp) which is highly rich in G+C nucleotides. This suggests that secondary structures in the 5' UTR might be essential for VEGF expression through transcriptional and post-transcriptional control mechanisms, as demonstrated for other growth factors. In this communication, we provide evidence that a computer predicted Internal Ribosome Entry Site (IRES) structure is biologically active and is located at the 3' end of the UTR. In addition, the results demonstrate that an alternative transcriptional initiation site for VEGF exists in the 5' UTR of VEGF. This alternative initiation site is 633 bp downstream of the main transcription start site and the resulting 5' UTR includes mainly the IRES structure. Therefore, our results suggest that VEGF is subjected to regulation at either translational level through a mechanism of ribosome internal initiation and/or transcriptional level through alternative initiation.

Base Sequence↗
PubMed1997

Structural and functional features of a specific nucleosome containing a recognition element for the thyroid hormone receptor.

The Xenopus thyroid hormone receptor betaA (TRbetaA) gene contains an important thyroid hormone response element (TRE) that is assembled into a positioned nucleosome. We determine the translational position of the nucleosome containing the TRE and the rotational positioning of the double helix with respect to the histone surface. Histone H1 is incorporated into the nucleosome leading to an asymmetric protection to micrococcal nuclease cleavage of linker DNA relative to the nucleosome core. Histone H1 association is without significant consequence for the binding of the heterodimer of thyroid hormone receptor and 9-cis retinoic acid receptor (TR/RXR) to nucleosomal DNA in vitro, or for the regulation of TRbetaA gene transcription following microinjection into the oocyte nucleus. Small alterations of 3 and 6 bp in the translational positioning of the TRE in chromatin are also without effect on the transcriptional activity of the TRbetaA gene, whereas a small change in the rotational position of the TRE (3 bp) relative to the histone surface significantly reduces the binding of TR/RXR to the nucleosome and decreases transcriptional activation directed by TR/RXR. Our results indicate that the specific architecture of the nucleosome containing the TRE may have regulatory significance for expression of the TRbetaA gene.

Animals↗
PubMed1997

Phosphorylation events modulate the ability of interferon consensus sequence binding protein to interact with interferon regulatory factors and to bind DNA.

Two families of transcription factors mediate interferon (IFN) signaling. The first family, signal transducers and activators of transcription (STATs), is activated within minutes of IFN treatment. Specific phosphorylation events lead to their translocation to the nucleus, formation of transcriptional complexes, and the induction of the second family of transcription factors termed interferon regulatory factors (IRFs). Interferon consensus sequence binding protein (ICSBP) is a member of IRF family that is expressed only in cells of the immune system and acts as a transcriptional repressor. ICSBP binds DNA through the association with other transcription factors such as IRF-1 or IRF-2. In this communication, the domain that is involved in protein-protein interactions was mapped to the carboxyl terminus of ICSBP. This domain is also important for mediating ICSBP-repressing activity. In vitro studies demonstrated that direct binding of ICSBP to DNA is prevented by tyrosine (Tyr) phosphorylation. Yet, Tyr-phosphorylated ICSBP can bind target DNA only through the association with IRF-2 and IRF-1. This type of phosphorylation is essential for the formation of heterocomplexes. Tyr-phosphorylated ICSBP and IRF-2 are detected in expressing cells constitutively, and Tyr-phosphorylated IRF-1 is induced by IFN-gamma. These results strongly suggest that like the STATs, the IRFs are also modulated by Tyr phosphorylation that affects their biological activities.

Amino Acid Sequence↗
PubMed1996

A dominant negative mutant of an IFN regulatory factor family protein inhibits both type I and type II IFN-stimulated gene expression and antiproliferative activity of IFNs.

Type I (alpha,beta) and type II (gamma) IFNs elicit antiproliferative and antiviral activities through two distinct transcription pathways involving 1) IRF family proteins and ISGF3, and 2) STAT1. We have employed a dominant negative strategy to study the role of IRF family proteins in eliciting the biologic activities of IFN. A truncated IRF protein retaining the DNA-binding domain (DBD) of ICSBP (a member of the IRF family) was stably transfected into U937 monocytic cells. Clones expressing DBD had markedly reduced ISRE-binding activity and were defective in expressing several type I IFN-inducible genes. STAT1 was one such type I IFN-inducible gene whose expression was also inhibited in DBD clones. As a result, the expression of several IFN-gamma-inducible genes was also inhibited in these clones, indicating functional coupling of the type I and type II IFN transcription pathways. Furthermore, DBD clones grew more slowly than control clones and were refractory to antiproliferative effects of both types of IFNs. We found that IFN treatment of U937 cells leads to a G1 arrest and an increase in underphosphorylated retinoblastoma gene product. However, IFN treatment did not change the cell cycle profile, nor retinoblastoma gene product phosphorylation state in DBD clones. These data indicate that expression of DBD disrupts cell cycle regulatory mechanisms. Combined with the previously noted failure of DBD clones to elicit antiviral activity, the present work shows that IRF family proteins play an integral part in growth control activities of IFNs.

Animals↗
PubMed1996

Selective binding of VEGF121 to one of the three vascular endothelial growth factor receptors of vascular endothelial cells.

VEGF121 and VEGF165 are vascular endothelial growth factor splice variants that promote the proliferation of endothelial cells and angiogenesis. VEGF165 contains the 44 additional amino acids encoded by exon 7 of the VEGF gene. These amino acids confer upon VEGF165 a heparin binding capability which VEGF121 lacks. 125I-VEGF165 bound to three vascular endothelial growth factor (VEGF) receptors on endothelial cells, while 125I-VEGF121 bound selectively only to the flk-1 VEGF receptor which corresponds to the larger of the three VEGF receptors. The binding of 125I-VEGF121 to flk-1 was not affected by the removal of cell surface heparan sulfates or by heparin. Both VEGF165 and VEGF121 inhibited the binding of 125I-VEGF121 to a soluble extracellular domain of the flk-1 VEGF receptor in the absence of heparin. However, heparin potentiated the inhibitory effect of VEGF165 by 2-3-fold. These results contrast with previous observations which have indicated that the binding of 125I-VEGF165 to the flk-1 receptor is strongly dependent on heparin-like molecules. Further experiments showed that the receptor binding ability of VEGF165 is susceptible to oxidative damage caused by oxidants such as H2O2 or chloramine-T. VEGF121 was also damaged by oxidants but to a lesser extent. Heparin or cell surface heparan sulfates restored the flk-1 binding ability of damaged VEGF165 but not the receptor binding ability of damaged VEGF121. These observations suggest that alternative splicing can generate a diversity in growth factor signaling by determining receptor recognition patterns. They also indicate that the heparin binding ability of VEGF165 may enable the restoration of damaged VEGF165 function in processes such as inflammation or wound healing.

Animals↗
PubMed1996

Interleukin 6 induces the expression of vascular endothelial growth factor.

Angiogenesis, the formation of new blood vessels, is induced by various growth factors and cytokines that act either directly or indirectly. Vascular endothelial growth factor (VEGF) is a specific mitogen for vascular endothelial cells and therefore has a central role in physiological events of angiogenesis. Interleukin-6 (IL-6) expression on the other hand is elevated in tissues that undergo active angiogenesis but does not induce proliferation of endothelial cells. We demonstrate using Northern analysis that treatment of various cell lines with IL-6 for 6-48 h results in a significant induction of VEGF mRNA. The level of induction is comparable to the documented induction of VEGF mRNA by hypoxia or cobalt chloride, an activator of hypoxia-induced genes. In addition, it is demonstrated by transient transfection assays that the effect of IL-6 is mediated not only by DNA elements at the promoter region but also through specific motif(s) located in the 5'-untranslated region (5'-UTR) of VEGF mRNA. Our results imply that IL-6 may induce angiogenesis indirectly by inducing VEGF expression. It is also shown that the 5'-UTR is important for the expression of VEGF. The 5'-UTR of VEGF is exceptionally long (1038 base pairs) and very rich in G + C. This suggests that secondary structures in the 5'-UTR might be essential for VEGF expression through transcriptional and post-transcriptional control mechanisms.

Animals↗
PubMed1995

Platelet factor-4 inhibits the mitogenic activity of VEGF121 and VEGF165 using several concurrent mechanisms.

The 121-amino acid form of vascular endothelial growth factor (VEGF121) and the 165-amino acid form (VEGF165) are mitogenic for vascular endothelial cells and induce angiogenesis in vivo. VEGF165 possesses a heparin binding ability and in the absence of heparin-like molecules does not bind efficiently to the VEGF receptors of vascular endothelial cells. The binding of 125I-VEGF165 to the VEGF receptors of endothelial cells, and the heparin-dependent binding of 125I-VEGF165 to a soluble extracellular domain of the VEGF receptor KDR/flk-1, were inhibited by the angiogenesis inhibitor platelet factor-4 (PF4). In contrast, PF4 was not able to inhibit the binding of VEGF121, a VEGF isoform which lacks a heparin binding capacity, to the VEGF receptors of the cells or to KDR/flk-1. These results indicate that PF4 may inhibit VEGF165 binding to VEGF receptors by disrupting the interaction of VEGF165 with cell surface heparan sulfates. Since PF4 mutants lacking a heparin binding ability retain their anti-angiogenic activity, alternative inhibitory mechanisms were also examined. 125I-PF4 bound with high affinity (Kd 5 x 10(-9) M) to VEGF165-coated wells. The binding of 125I-PF4 to the VEGF165-coated wells was inhibited by several types of heparin binding proteins, including unlabeled PF4 and unlabeled VEGF165. The binding was not inhibited by proteins which lack a heparin binding capacity, nor was it inhibited by VEGF121. Heparinase did not inhibit the binding of 125I-PF4 to VEGF165, indicating that heparin-like molecules are not required. These experiments suggest that PF4 can bind to heparin binding proteins such as VEGF165 leading to an inhibition of their receptor binding ability. In agreement with these results, we have observed that PF4 inhibits efficiently the VEGF165 induced proliferation of vascular endothelial cells. Unexpectedly, PF4 also inhibited efficiently the VEGF121-induced proliferation of the cells, indicating that PF4 can disrupt VEGF receptor mediated signal transduction using an unknown mechanism which does not interfere with VEGF121 binding.

Animals↗
PubMed1995

Functional domain analysis of interferon consensus sequence binding protein (ICSBP) and its association with interferon regulatory factors.

Interferon consensus sequence binding protein (ICSBP) is a member of the interferon regulatory factor (IRF) family of proteins that include IRF-1, IRF-2, and ISGF3gamma which share sequence similarity at the putative DNA binding domain (DBD). ICSBP is expressed exclusively in cells of the immune system and acts as a repressor of interferon consensus sequence (ICS) containing promoters that can be alleviated by interferons. In this communication, we have searched for functional domains of ICSBP by dissecting the DBD from the repression activity. The putative DBD of ICSBP (amino acids 1-121) when fused in frame to the transcriptional activation domain of the herpes simplex VP16 (ICSBP-VP16) is a very strong activator of ICS-containing promoters. In addition, ICSBP-VP16 fusion construct transfected into adenovirus (Ad) 12 transformed cells enabled cell surface expression of major histocompatibility complex class I antigens as did treatment with interferon. On the other hand, the DBD of the yeast transcriptional activator GAL4 was fused in frame to a truncated ICSBP in which the DBD was impaired resulting in a chimeric construct GAL4-ICSBP. This construct is capable of repressing promoters containing GAL4 binding sites. Thus, ICSBP contains at least two independent domains: a DBD and a transcriptional repressor domain. Furthermore, we have tested possible interactions between ICSBP and IRFs. The chimeric construct GAL4-ICSBP inhibited the stimulated effect of IRF-1 on a reporter gene, implying for a possible interaction between IRF-1 and ICSBP. Electromobility shift assays, demonstrated that ICSBP can associate with IRF-2 or IRF-1 in vitro as well as in vivo. Thus, ICSBP contains a third functional domain that enables the association with IRFs. These associations are probably important for the fine balance between positive and negative regulators involved in the interferon-mediated signal transduction pathways in cells of the immune system.

Base Sequence↗