A negative regulator of telomere-length protein trf1 is associated with interstitial (TTAGGG)n blocks in immortal Chinese hamster ovary cells.

Telomeres of mammalian chromosomes are composed of long tandem repeats (TTAGGG)n which bind in a sequence-specific manner two proteins-TRF1 and TRF2. In human somatic cells both proteins are mostly associated with telomeres and TRF1 overexpression resulting in telomere shortening. However, chromosomes of some mammalian species, e.g., Chinese hamster, have large interstitial blocks of (TTAGGG)n sequence (IBTs) and the blocks are involved in radiation-induced chromosome instability. In normal somatic cells of these species chromosomes are stable, indicating that the IBTs are protected from unequal homologous recombination. In this study we expressed V5-epitope or green fluorescent protein (GFP)-tagged human TRF1 in different lines of mammalian cells and analyzed distribution of the fusion proteins in interphase nucleus. As expected, transient transfection of human (A549) or African green monkey cells with GFP-N-TRF1 or TRF1-C-V5 plasmids resulted in the appearance in interphase nuclei of multiple faint nuclear dots containing GFP or V5 epitope which we believe to represent telomeres. Transfection of immortalized Chinese hamster ovary (CHO) cell line K1 which have extremely short telomeres with GFP-N-TRF1 plasmid leads to the appearance in interphase nuclei of large GFP bodies corresponding in number to the number of IBTs in these cells. Simultaneous visualization of GFP and IBTs in interphase nuclei of transfected CHO-K1 cells showed colocalization of both signals indicating that expressed TRF1 actually associates with IBTs. These results suggest that TRF1 may serve as general sensor of (TTAGGG)n repeats controlling not only telomeres but also interstitial (TTAGGG)n sequences.

Association of some potential hormone response elements in human genes with the Alu family repeats.

Short interspersed repeats of the Alu family located in promoters of some human genes contain high-affinity binding sites for thyroid hormone receptor, retinoic acid receptor and estrogen receptor. The standard binding sites for the receptors represent variants of duplicated AGGTCA motif with different spacing and orientation (direct, DR, or inverted, IR), and Alu sequences were found to have functional DR-4, DR-2 or variant IR-3/IR-17 elements. In this study we analyzed distribution and abundance of the elements in a set of human genomic sequences from GenBank and their association with Alu repeats. Our results indicate that a major fraction of potentially active DR-4, DR-2 and variant IR-3/IR-17 elements in the genes is located within Alu repeats. Alu-associated DR-2 elements are conserved in primate evolution. However, very few Alu have potential DR-3 glucocorticoid-response elements. Gel-shift experiments with the probe (AUB) corresponding to the consensus Alu sequence just upstream of the RNA polymerase III promoter B-box and containing duplicated AGGTCA motif indicate that the probe interacts in a sequence-specific manner with human nuclear proteins which bind to standard IR-0, DR-1, DR-4 or DR-5 elements. The AUB sequence was also able to promote thyroid hormone-dependent trans-activation of a reporter gene. The results support the view that Alu retroposons played an important role in evolution of regulation of the primate gene expression by nuclear hormone receptors.

A novel human DNA-binding protein with sequence similarity to a subfamily of redox proteins which is able to repress RNA-polymerase-III-driven transcription of the Alu-family retroposons in vitro.

In this study we identified a novel protein which may contribute to the transcriptional inactivity of Alu retroposons in vivo. A human cDNA clone encoding this protein (ACR1) was isolated from a human expression library using South-western screening with an Alu subfragment, implicated in the regulation of Alu in vitro transcription and interacting with a HeLa nuclear protein down-regulated in adenovirus-infected cells. Bacterially expressed ACR1 is demonstrated to inhibit RNA polymerase III (Pol III)-dependent Alu transcription in vitro but showed no repression of transcription of a tRNA gene or of a reporter gene under control of a Pol II promoter. ACR1 mRNA is also found to be down-regulated in adenovirus-infected HeLa cells, consistent with a possible repressor function of the protein in vivo. ACR1 is mainly (but not exclusively) located in cytoplasm and appears to be a member of a weakly characterized redox protein family having a central, highly conserved sequence motif, PGAFTPXCXXXXLP. One member of the family identified earlier as peroxisomal membrane protein (PMP)20 is known to interact in a sequence-specific manner with a yeast homolog of mammalian cyclosporin-A-binding protein cyclophilin, and mammalian cyclophilin A (an abundant ubiquitously expressed protein) is known to interact with human transcriptional repressor YY1, which is a major sequence-specific Alu-binding protein in human cells. It appears, therefore, that transcriptional silencing of Alu in vivo is a result of complex interactions of many proteins which bind to its Pol III promoter.

Protein-protein interaction of the human poly(ADP-ribosyl)transferase depends on the functional state of the enzyme.

Poly(ADP-ribosyl)transferase (pADPRT) is a nuclear protein which catalyzes the polymerization of ADP-ribose using NAD+ as substrate, as well as the transfer of ADP-ribose polymers to itself and other protein acceptors. The catalytic activity of pADPRT strictly depends on the presence of DNA single-strand breaks. In this report, protein-protein interaction of pADPRT was found to depend on both the extent of automodification with poly(ADP-ribose) and the presence of DNA. Specific binding of radiolabeled pADPRT to transblotted proteins was first tested in blot overlay experiments. For radiolabeling, use was made of the ability of the enzyme to incorporate [32P]ADP-ribose from [32P]NAD+. Varying the concentration of NAD+, two different forms of automodified pADPRT were obtained: oligo(ADP-ribosyl)ated pADPRT with less than 20 ADP-ribose units per chain, and poly(ADP-ribosyl)ated pADPRT with polymer lengths of up to 200 ADP-ribose residues. Interaction of these probes with transblotted HeLa nuclear extracts, purified histones, and distinct regions of recombinant pADPRT was investigated. While the oligo(ADP-ribosyl)ated enzyme associated preferentially with transblotted purified histones, or pADPRT present in HeLa nuclear extracts, poly(ADP-ribosyl)ated pADPRT bound to a variety of transblotted proteins in the nuclear extracts. In the presence of DNA, both the oligo- and the poly(ADP-ribosyl)ated enzymes bound to the transblotted recombinant zinc finger domain of pADPRT even at high salt concentrations. In the absence of DNA, the transblotted automodification domain of pADPRT appeared to be the region involved in self-association. In another set of experiments, unmodified or poly(ADP-ribosyl)ated pADPRT was immobilized on Sepharose. Affinity precipitation of recombinant pADPRT domains confirmed the specific interaction of pADPRT with its zinc finger region and the automodification domain, whereas no interaction was observed with the NAD+ binding domain. Affinity precipitation of HeLa nuclear extracts with poly(ADP-ribosyl)ated pADPRT-Sepharose led to the enrichment of a number of proteins, whereas nuclear proteins bound to the unmodified pADPRT-Sepharose in a smaller extent. The results suggest that protein-protein interaction of the human pADPRT is governed by its functional state.

NAD+ analogs substituted in the purine base as substrates for poly(ADP-ribosyl) transferase.

Poly(ADP-ribosyl) transferase (pADPRT) catalyzes the transfer of the ADP-ribose moiety from NAD+ onto proteins as well as onto protein-bound ADP-ribose. As a result, protein-bound polymers of ADP-ribose are formed. pADPRT itself contains several acceptor sites for ADP-ribose polymers and may attach polymers to itself (automodification). In this study the influence of substitutions in the purine base of NAD+ on the polymerization reaction was investigated. The adenine moiety of NAD+ was replaced by either guanine, hypoxanthine or 1,N6-ethenoadenine. These analogs served as substrates for polymer synthesis as judged from the extent of automodification of the enzyme and the sizes of the polymers formed. Time course experiments revealed that 1,N6-etheno NAD+ (epsilon-NAD+) and nicotinamide hypoxanthine dinucleotide (NHD+) were rather poor substrates as compared to NAD+. Synthesis of GDP-ribose polymers from nicotinamide guanine dinucleotide (NGD+) was more efficient, but still significantly slower than poly(ADP-ribosyl)ation of the enzyme using NAD+. The size of the different polymers appeared to correlate with these observations. After 30 min of incubation in the presence of 1 mM substrate, polymers formed from epsilon-NAD+ or NHD+ contained up to 30 epsilon-ADP-ribose or IDP-ribose units, respectively. Using NGD+ as substrate polymers consisted of more than 60 GDP-ribose units, an amount similar to that achieved by poly(ADP-ribosyl)ation in the presence of only 0.1 mM NAD+ as substrate. These results suggest that the presence of an amino group in the purine base of NAD+ may facilitate catalysis. Substitution of the nicotinamide moiety of NAD+ with 3-acetylpyridine had no detectable effect on polymer formation. Oligomers of GDP-ribose and epsilon-ADP-ribose exhibited a slower mobility in polyacrylamide gels as compared to ADP-ribose or IDP-ribose oligomers. This feature of the two former analogs as well as their markedly attenuated polymerization by pADPRT provide valuable tools for the investigation of the enzymatic mechanism of this protein. Moreover, polymers of epsilon-ADP-ribose may be useful for studying enzymes degrading poly(ADP-ribose) owing to the fluorescence of this analog. Digestion of epsilon-ADPR polymers with snake venom phosphodiesterase was accompanied by a significant fluorescence enhancement.

A trypsin sensitive stromelysin isolated from rheumatoid synovial fluid is an activator for matrix metalloproteinases.

The processing of synovial fluids of patients suffering from rheumatoid arthritis led to the characterization of a neutral metalloproteinase with polymorphonuclear leukocyte progelatinase and polymorphonuclear leukocyte procollagenase activating properties. The activator exhibits a relative molecular mass of M(r) 27,000 and is an active form of stromelysin. Thus, it reacts specifically with antibodies raised against human stromelysin, splits polymorphonuclear leukocyte progelatinase in a manner characteristic of stromelysin, and is inhibited by EDTA as well as by a tissue inhibitor of metalloproteinases (TIMP-2). The activator shows a high specificity for the matrix metalloproteinases, polymorphonuclear leukocyte progelatinase and polymorphonuclear leukocyte procollagenase. It shows only weak hydrolysis of casein and gelatin, and it does not activate fibroblast M(r) 72,000 progelatinase. Brief treatment with trypsin does not lead to a significant change in the activator's relative molecular mass, but induces a rapid loss of its activating activity for polymorphonuclear leukocyte progelatinase, while its proteolytic activity against the synthetic substrate, N-(2,4)-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg, is increased about 3-fold. The same tryptic treatment does not affect the activator's proteolytic activity towards casein and gelatin.

Characterization of a gelatinase from human rheumatoid synovial fluid cells.

A metalloproteinase with a specificity for gelatin was isolated from serum-free medium of cultures of rheumatoid synovial fluid. The enzyme showed all the properties of a leukocyte gelatinase. In addition to gelatin this proteinase cleaved the synthetic substrate dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg (Dnp-peptide) rapidly, while casein was a much poorer substrate. This proteinase showed no enzymatic activity against collagen type I, was secreted in a latent form and could be activated by trypsin or organomercurial compounds, such as mersalylic acid or 4-aminophenyl-mercury acetate. The latent enzyme had an apparent molecular mass of 130,000-150,000 estimated by gel filtration or 97,000 by electrophoresis on polyacrylamide gel containing sodium dodecyl sulphate. When analysed by immunoblotting the enzyme was recognized by antibodies raised against human polymorphonuclear leukocyte gelatinase. Although we found synovial fibroblasts to be largely present in the cell cultures we could not detect any fibroblast gelatinase activity.

Purification of histone F3 by covalent chromatography.

The arginine-rich histone F3 has been purified by covalent affinity chromatography. By the use of activated Thiol-Sepharose 4B for the purification of cysteine containing histone F3 a highly purified protein was obtained. The simple purification procedure offers the opportunity to get larger amounts of pure histone F3 within short time.