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C Skerka

Publications and source records attributed to C Skerka.

32 records · Page 2Linked to original sources
PubMed1997

Coordinate expression and distinct DNA-binding characteristics of the four EGR-zinc finger proteins in Jurkat T lymphocytes.

The Early Growth Response Genes (EGR-1 to AT133/EGR-4) encode a family of proteins that are composed of three homologous consecutive zinc fingers of the Cys2-His2 type and different flanking sequence. Upon growth stimulation of resting cells the four EGR-genes are simultaneously transcribed. We have analyzed the expression of the four EGR-proteins in Jurkat T cells and show by Western blot analysis that the four EGR-proteins are coordinately induced upon treatment with a combination of PHA and PMA. As the individual proteins are reported to bind to identical target sequences, we have analyzed the DNA-binding of the native proteins. Using nuclear extract in which we have demonstrated expression of all four EGR-proteins, only EGR-1, but no other member of this protein family is found to bind to the EGR-consensus site (GCG GGG GCG). In addition, DNA-binding of both native EGR-1 and of recombinant EGR-1 and AT133/EGR-4 proteins expressed in insect cells was analyzed. This comparison revealed distinct binding properties of recombinant EGR-1 and AT133/EGR-4 to oligonucleotides that include the EGR-consensus sites. The distinct binding affinities suggest that in vivo EGR-proteins bind to different target sequences and that each EGR-protein regulates distinct target genes. This is underlined by demonstrating that EGR-1 but not AT133/EGR-4 binds to a related G-rich promoter element with the sequence GGG GTG GGG. This G-rich sequence serves as an overlapping binding site for the two zinc finger proteins EGR-1 and Sp1. As similar overlapping binding sites for EGR-1 and Sp1 have been identified in several human and mouse gene promoters, we raise the question whether the Sp1 binding sites described in a large number of eukaryotic gene promoters also represent binding sites for EGR-1.

Animals↗
PubMed1995

Mapping of the complement regulatory domains in the human factor H-like protein 1 and in factor H1.

The human factor H-like protein 1 (FHL-1) is composed of seven repetitive domains (short consensus repeats; SCRs) that are identical in sequence to the seven NH2-terminal SCRs of the complement regulatory protein factor H. We have identified the native FHL-1 protein as a 42-kDa human plasma protein by immunoblotting and by comparing the mobility to that of a recombinant FHL-1 protein. Here, we demonstrate the existence of two distinct co-migrating human plasma proteins that represent the 42-kDa FHL-1 protein and the previously identified 43-kDa factor H-related 1 beta protein. Similar to factor H, the recombinant FHL-1 protein displays cofactor activity in factor I-mediated cleavage of C3b. To identify relevant SCRs of factor H and FHL-1, we recombinantly expressed the domains shared between the two proteins in the baculovirus expression system. Recombinant FHL-1 and all truncated forms that include SCRs 1 to 4 displayed cofactor activity. All four NH2-terminal SCRs are essential, as deletion mutants composed of SCR 1 and 4 only; of SCRs 1, 2, and 4 only, or of SCRs 1, 3, and 4 only were functionally inactive. Similarly, the distance between these individually folding domains is critical for function, as a recombinant protein that had two and four amino acids inserted between SCRs 1 and 2 or between SCRs 3 and 4, respectively, had no activity. These results demonstrate that all four NH2-terminal SCRs of FHL-1 (and of factor H) are required for cofactor activity in factor I-mediated cleavage of C3b, and that the distance between these SCRs is essential.

Base Sequence↗
PubMed1995

A regulatory element in the human interleukin 2 gene promoter is a binding site for the zinc finger proteins Sp1 and EGR-1.

Activation of the interleukin 2 (IL-2) gene after antigen recognition is a critical event for T cell proliferation and effector function. Prior studies have identified several transcription factors that contribute to the activity of the IL-2 promoter in stimulated T lymphocytes. Here we describe a novel regulatory element within the IL-2 promoter located immediately upstream of the nuclear factor of activated T cell (NFAT) domain. This region (termed the zinc finger protein binding region (ZIP)) serves as binding site for two differently regulated zinc finger proteins: the constitutively expressed transcription factor Sp1 and the inducible early growth response protein EGR-1. In unstimulated cells which do not secrete IL-2, only Sp1 binds to this region, while in stimulated IL-2 secreting cells the inducible EGR-1 protein recognizes this element. In Jurkat T cells, the ZIP site serves as an activator for IL-2 gene expression, and a combination of ZIP and NFAT binding sites is required for maximal IL-2 promoter activity. These results suggest a critical role of the ZIP site for IL-2 promoter activity.

Animals↗
PubMed1995

The human factor H-related gene 2 (FHR2): structure and linkage to the coagulation factor XIIIb gene.

The human factor H-related gene 2 (FHR2) encodes a serum protein structurally and immunologically related to complement factor H. We describe the isolation and genomic organization of the human FHR2 gene from a yeast artificial chromosome library. The FHR2 gene is organized in five exons and spans about 7 kilobases (kb) of human genomic DNA. A comparison with the corresponding cDNA sequence (clone DDESK59) shows that the analyzed FHR2 gene has a deleted region within exon 4. A new splice acceptor site created in the truncated exon indicates that the analyzed gene could be translated to a truncated protein. Further, we demonstrate that the genes for FHR2 and beta subunit of coagulation factor XIII are located in the same 165 kb YAC DNA. Thus, the three structurally related genes FXIIIb, FHR2, and factor H are linked on human chromosome 1 in the regulators of complement activation (RCA) gene cluster. The physical linkage of the FHR2 and the factor H genes provides additional evidence for a close relatedness of complement factor H and the factor H-related proteins. The linkage and the almost exclusive organization in short consensus repeat-containing domains indicates a close evolutionary relationship of the FXIIIb, FHR2, and factor H genes.

Amino Acid Sequence↗
PubMed1994

Complement factor H and related proteins: an expanding family of complement-regulatory proteins?

Recently, several factor-H-related molecules have been identified in humans and mice, initiating new interest in this group of serum proteins. Although no complement-regulatory activity has been demonstrated yet for the human factor-H-related proteins, the immunological and structural similarities to factor H suggest overlapping functions. Here, Peter Zipfel and Christine Skerka review these similarities and suggest a new nomenclature to identify members of the factor-H-related family.

Amino Acid Sequence↗
PubMed1993

A novel short consensus repeat-containing molecule is related to human complement factor H.

We have identified a novel factor H-related cDNA, which was isolated from a human liver cDNA library. The DOWN16 clone is 1269 base pairs in size and hybridized to a mRNA of 1.4 kilobases. Similar to the previously described factor H-related proteins, the predicted translation product of 331 amino acids contains a hydrophobic signal sequence followed by a stretch of five short consensus repeats (SCRs). These five SCRs display homology to SCRs of factor H: SCRs1-3 (DOWN16) are homologous to SCRs6-8 of factor H, while SCRs4 and -5 are related to SCRs19 and -20. In vitro translation demonstrated that the DOWN16 cDNA encodes a primary translation product of an apparent molecular mass of 37,500 Da which is directed to the secretory pathway and is glycosylated. Thus, we propose that the protein will be present in human serum. The relatedness of structural elements between this novel gene and factor H may suggest common functions of these proteins not yet determined.

Amino Acid Sequence↗
PubMed1993

Cell type specific expression of members of the IL-8/NAP-1 gene family.

The expression and regulation of seven human members of a family of related cytokines, which play a role as effectors of inflammation, were analysed in hemopoietic cells and in fibroblasts. In T lymphocytes all genes: platelet basic protein (PBP); platelet factor 4 (PF-4); IL-8/NAP-1; IP-10; GRO; pAT464 and pAT744 were induced by stimulation with phytohemagglutinin and phorbol 12-myristate 13-acetate (PHA/PMA). In contrast to T cells, only some of the genes were induced upon terminal differentiation of pro-monocytic cells and upon serum stimulation of resting fibroblasts. This distinct expression indicates functional differences of the individual proteins. The expression of inflammatory mediators in fibroblasts suggests the involvement of these cells in inflammatory reactions.

Cell Differentiation↗
PubMed1993

Genomic organization, chromosomal localization and promoter function of the human zinc-finger gene pAT133.

We report the genomic structure and chromosomal localization of the human zinc-finger gene pAT133. This gene belongs to a family of four human immediate-early genes (pAT133, pAT225/EGR1, pAT591/EGR2 and EGR3), which have almost identical zinc-finger domains, but distinct flanking regions. The human pAT133 gene is organized in two exons, and has been mapped to human chromosome 2p13. We determined the transcription start site by primer extension analysis and identified several regulatory elements in the upstream regulatory sequence. The pAT133-promoter functions as an inducible promoter in human T cells and in fibroblasts, as constructs containing the bacterial chloramphenicol acetyl transferase (CAT) gene driven by a 943 bp pAT133-promoter fragment were induced in these cells by stimulation with PHA/PMA or by serum, respectively. The serum inducible pAT133-promoter lacks consensus serum response elements. However, we could demonstrate that two CArG-like motifs with a single base exchange confer serum inducibility to a reporter construct. Due to their serum responsiveness, these elements may serve as a high affinity binding sites for the recently described human serum response factor-related proteins.

Amino Acid Sequence↗
PubMed1993

Two regulatory domains are required for downregulation of c-myc transcription in differentiating U937 cells.

In many differentiating cells, a reduction of c-myc proto-oncogene expression is a prerequisite for terminal differentiation. The downmodulation of c-myc in differentiating cells is due to at least two different mechanisms: (i) an elongation block to c-myc transcription activated during an early phase of differentiation and (ii) an inhibition of transcription initiation activated during a later phase. In order to determine cis-acting target structures of the c-myc gene required for the late-phase downregulation of transcriptional initiation, we permanently transfected U937 cells with constructs containing the bacterial chloramphenicol acetyl transferase (CAT) gene driven by a 2.8 kb c-myc promoter region or deletions thereof. We determined two distinct domains in the c-myc promoter region both of which are essential for efficient terminal downregulation: a proximal domain and a distal domain which are located between base pairs -606 to -101, and between -2392 to -1396, respectively, relative to P1. The identification of two distinct regulatory elements suggests the requirement and cooperation of two regulatory factors as an essential event for mediating differentiation-induced downregulation of c-myc in monocytic cells. The implications of these results for deregulation of the translocated c-myc allele in Burkitt's lymphoma are discussed.

Cell Differentiation↗
PubMed1992

Two additional human serum proteins structurally related to complement factor H. Evidence for a family of factor H-related genes.

We identify and characterize two human serum proteins with an apparent molecular mass of 24 and 29 kDa, which are antigenically related to complement factor H. These proteins represent differently glycosylated forms and are encoded by the same mRNA. The corresponding cDNA clone is 1051 bp in size and hybridized to a 1.4-kb mRNA derived from human liver. The predicted translation product represents a protein of 270 amino acids, which displays a hydrophobic leader sequence, indicative of a secreted protein. The secreted part is organized in four short consensus repeats (SCR) and has a single putative N-linked glycosylation site. This predicted sequence is closely related to that of the previously described factor H-related proteins h37 and h42, which are also derived from a 1.4-kb mRNA. Amino acid comparison of these factor H-related proteins showed identical leader sequences, an exchange of three amino acids in SCR1, identical sequences of SCR2, and a lower degree of homology between SCR3-4 (h24 and h29) and SCR4-5 (h37 and h42). In addition, SCR3-4 of h24 and h29 display homology to SCR19-20 of human complement factor H. The relatedness of structural elements of the factor H-related proteins h24, h29, h37, and h42 and of factor H, suggests a function common to these proteins and indicates the existence of a gene family consisting of factor H and at least two factor H-related genes.

Amino Acid Sequence↗
PubMed1991

Induction of members of the IL-8/NAP-1 gene family in human T lymphocytes is suppressed by cyclosporin A.

Human members of a family of structurally related cytokines, which play a role as effectors of inflammation, were analyzed for their expression and regulation in T lymphocytes. Members of this gene family include Platelet Basic Protein (PBP); Platelet Factor 4 (PF-4); IL-8/NAP-1; IP-10, a gamma interferon induced protein; GRO; pAT 464 and pAT 744. In resting T lymphocytes the RNAs of the individual genes could not be detected, but all genes were induced upon stimulation with PHA or with PHA/PMA. The induction of five genes was blocked by the immunosuppresive drug cyclosporin A (CSA), which appears to affect initial events in T cell activation. This expression in T lymphocytes, especially the sensitivity to CSA, indicates a common immunmodulatory role of these structural related proteins.

Blotting, Northern↗
PubMed1991

Clone pAT 133 identifies a gene that encodes another human member of a class of growth factor-induced genes with almost identical zinc-finger domains.

We report the structure and regulation of a gene represented by clone pAT 133, which is induced upon transition from a resting state (G0) through the early phase of the cell cycle (G1). The pAT 133 gene is immediately induced, with FOS-like kinetics, in human T cells and in fibroblasts. Primary structure analysis showed that the encoded protein contains three tandem zinc-finger sequences of the type Cys2-Xaa12-His2. This zinc-finger region, which is thought to bind DNA in a sequence-specific manner, is similar (greater than 80% on the amino acid level) to two previously described transcription factors pAT 225/EGR1 and pAT 591/EGR2. Except for the conserved zinc-finger domains, the amino acid sequences of the three proteins are distinct. This structural similarity suggests that the pAT 133 gene encodes a transcription factor with a specific biological function. Comparing the regulation of these related zinc-finger-encoding genes showed coordinate induction upon mitogenic stimulation of resting T lymphocytes and of resting fibroblasts. However, upon transition from a proliferating (G1) to a resting state of the cell cycle the three genes were differently regulated. In human histiocytic U937 cells mRNA of clone pAT 133 was constitutively expressed, whereas mRNA of pAT 225/EGR1 was induced upon induction of terminal differentiation. In contrast mRNA representing pAT 591/EGR2 was not expressed in these cells. This difference in gene regulation suggests distinct biological roles in the control of cell proliferation for the respective proteins.

Amino Acid Sequence↗
PubMed1991

Molecular cloning of a human serum protein structurally related to complement factor H.

Two cDNA clones termed H36-1 and H36-2 were isolated from a human liver cDNA library. Clone H36-1 appears to represent the recently isolated human serum proteins h37 and h42, which are two differently glycosylated forms of a protein antigenically related to human complement factor H. The H36-1 deduced protein sequence is 327 amino acid long and possesses a leader sequence. The secreted part of the protein is comprised of five tandem repeating units, termed short consensus repeats (SCRs). SCR 1 and 2 display high homology to the corresponding region of the recently isolated murine factor H-related cDNA clone 13G1. In contrast, the 3'-end of the H36-1 clone shows sequence homology to the 3'-end of human complement factor H. The second clone, H36-2, is nearly identical to H36-1. Within 1148 base pairs, where the two clones overlap, their nucleotide sequences differed at nine positions. One nucleotide exchange in the sequence of H36-2 which was located within SCR 1 creats a stop codon (TAA). Consequently, the corresponding mRNA cannot code for a functional protein, suggesting that this clone is a transcribed pseudogene. These two clones represent new human members of the family of proteins structurally related to complement factor H.

Amino Acid Sequence↗