Band 382, Heft 6

Research on the regulation of transcription in mammals has focused in recent years mainly on the mechanism of transcriptional activation. However, transcriptional repression mediated by repressor proteins is a common regulatory mechanism in mammals and might play an important role in many biological processes. To understand the molecular mechanism of transcriptional repression, the activity of eight mammalian repressors or repressor domains was investigated using a set of model promoters in combination with two different transcriptional detection methods. The repressors studied were: REST, the thyroid hormone receptors α and β, the zinc finger protein NK10 containing a krüppelassociated box (KRAB), repressor domains derived from the proteins Egr-1, Oct2A and Dr1 and the repressor/activator protein YY1. Here we show that the repressor domains of REST, Egr-1, the thyroid hormone receptors α and β and NK10 were transferable to a heterologous DNAbinding domain and repressed transcription from proximal and distal positions. Moreover, these repressor domains also blocked the activity of a strong viral enhancer in a remote position. Thus, these domains are general transcriptional repressor domains. The krüppelassociated box was the most powerful repressor domain tested. In contrast, the repressor domains derived from Oct2A and Dr1 were inactive when fused to a heterologous DNAbinding domain. The repressor domain of YY1 exhibited transcriptional repression activity only in one of the transcriptional assay systems. The recruitment of histone deacetylases to the proximity of the basal transcriptional apparatus was recently discussed as a mechanism for some mammalian transcriptional repressor proteins. Here we show here that histone deacetylase 2, targeted to the reporter gene via DNAprotein interaction, functions as a transcriptional repressor protein regardless of the location of its binding site within the transcription unit.

The chlorophyll synthase gene from oat (Avena sativa) was cloned and expressed in Escherichia coli. The deduced amino acid sequence consists of 378 amino acids including a presequence of 46 amino acids. Deletion mutants show that a core protein comprising amino acid residues 88 to 377 is enzymatically active. The sequence of the mature protein shows 85% identity with the chlorophyll synthase of Arabidopsis thaliana and 62% identity with the chlorophyll synthase of Synechocystis PCC 6803. The gene is constitutively expressed as the same transcript level is found in darkgrown and in lightgrown seedlings. The enzyme requires magnesium ions for activity; manganese ions can reconstitute only part of the activity. Diacetyl and Nphenylmaleimide (NPM) inhibit the enzyme activity. Sitedirected mutagenesis reveals that, out of the 4 Arg residues present in the active core protein, Arg-91 and Arg-161 are essential for the activity. Five cysteine residues are present in the core protein, of which only Cys-109 is essential for the enzyme activity. Since the wildtype and all other Cysmutants with the exception of the mutant C304A are inhibited by Nphenylmaleimide, we conclude that the inhibitor binds to a nonessential Cys residue to abolish activity. The role of the various Arg and Cys residues is discussed.

5-Aminolevulinic acid (ALA) is a heme precursor that accumulates in lead poisoning and inborn porphyrias. It has been shown to produce reactive oxygen species upon metalcatalyzed aerobic oxidation and to cause oxidative damage to proteins, liposomes, DNA, and subcellular structures. Studies have also shown that ALA may condense to yield the cyclic product 3,6-dihydropyrazine 2,5-dipropanoic acid (DHPY). Here we propose that DHPY could be involved in DNA damage in the presence of high concentrations of ALA. Exposure of plasmid pUC19 DNA to low concentrations of DHPY (2 10 M) in the presence of 0.1 mM Cu[2+] ions causes DNA strand breaks, as demonstrated by agarose gel electrophoresis. It was also shown that in the presence of Cu[2+] ions DHPY is able to increase the oxidation of monomeric 2deoxyguanosine to form 8- oxo-7,8-dihydro-2deoxyguanosine as inferred from high performance liquid chromatography measurements using electrochemical detection. Addition of a metal chelator (bathocuproine, 0.5 mM), the DNA compacting polyamines spermidine (1 mM) and spermine (1 mM) or antioxidant enzymes such as superoxide dismutase (10 g/ml) and catalase (20 g/ml) protect the DNA against these damages. The data presented here are discussed with respect to the increased frequency of liver cancer in patients with acute intermittent porphyria.

Nucleotidase activities resembling subclass I and subclass II of human pyrimidine 5nucleotidases (P5N) were detected in chicken red blood cells (RBCs). In chicken RBCs from untreated controls, the activity of the subclass II enzyme was about one third of that of subclass I enzyme, whereas that ratio was approximately 5:1 in rat or human RBCs. The subclass I activity in chicken RBCs was increased 5- to 6- fold upon erythropoietic induction by phenylhydrazine administration, but the subclass II activity did not increase under these conditions. The subclass I enzyme was purified to near homogeneity. Its molecular mass was about 35 kDa as estimated by gel filtration and SDSpolyacrylamide gel electrophoresis. Its Nterminal 12 amino acids, PEFQKKTVHIKD, were also determined. The catalytic properties of the subclass I enzyme were very similar to those of the human enzyme with regard to substrate (preferential hydrolysis of CMP, dCMP, UMP), K values, optimum pH, and metal ion requirements. Antibodies against chicken P5N subclass I were raised in rats. The chicken P5NI as well as the rat P5NI proteins could be detected by antibodies in Western blot analyses, but not the P5NII proteins. These findings indicate that P5N subclass I may have an important function in chicken erythropoiesis.

Great attention has been devoted both to ageing phenomena at the mitochondrial level and to the antioxidant status of membrane structures. These kinds of investigations are difficult to perform in the brain because of its heterogeneity. It is known that synaptic heavy mitochondria (HM) may represent an aged mitochondrial population characterized by a partial impairment of their typical mitochondrial function. We arranged a novel system requiring no extraction procedure, very limited handling of the samples and their direct injection into the HPLC apparatus, to carry out, for the first time, a systematic and concomitant determination of vitamin E, Coenzyme Q[9] (CoQ[9]) and Coenzyme Q[10] (CoQ[10]) contents in rat brain mitochondria. The trends found for CoQ[9] and CoQ[10] levels in synaptic and nonsynaptic occipital cerebral cortex mitochondria during rat ageing are consistent with previous data. Hydroperoxides (HP) differed with age and it was confirmed that in the HM fraction the summation of contributions results in an oxidatively jeopardized subpopulation. We found that vitamin E seems to increase with age, at least in nonsynaptic free (FM) and synaptic light (LM) mitochondria, while it was inclined to remain substantially constant in HM.

One of the possible functions of lung surfactant protein B (SPB), an hydrophobic membraneassociated saposinlike protein, is to reduce the alveolar surface tension by promoting insertion of phospholipids into the air/liquid interface of the lung. SPB is a covalent homodimer; Cys48 of two polypeptides form an intermolecular disulphide bond. In order to test whether dimerisation of SPB is important for surfactant function, transgenic mice which express (Cys48Ser) human SPB in a mouse SPB null background were generated. In previous studies (Cys48Ser)SPB showed a concentrationdependent in vitro activity, suggesting that it may form noncovalent dimers. Here (Cys48Ser)SPB isolated from bronchoalveolar lavage of transgenic mice was studied at different concentrations by circular dichroism (CD) spectroscopy, pulsating bubble surfactometry, mass spectrometry and reversedphase HPLC. The results indicate that (Cys48Ser)SPB, both in a phospholipid environment and in organic solvents, is largely monomeric and exhibits low activity at concentrations lower than 1 2 M, while at higher concentrations it forms noncovalent dimers, which are nearly functionally equivalent to native SPB in vitro. Furthermore, electrospray mass spectrometry showed that more dimers were found relative to the monomer when the polarity of the solvent was decreased, and when the concentration of SPB increased. (Cys48Ser)SPB also eluted earlier than native SPB in reversedphase HPLC. Taken together, these results indicate that a polar surface is buried upon dimerisation, thereby promoting formation of interchain ion pairs between Glu51 Arg52 and Glu51Arg52.

The protective antigen (PA) component of anthrax toxin translocates the catalytic moieties lethal factor (LF) and edema factor (EF) into the cytosol. The proteolytically activated 63 kDa form of PA (PA63) has the ability to oligomerize and bind LF/EF. PA has four distinct domains performing specialized functions; whereas the function of domains I, II and IV has been well characterized, domain III has no known role in the biological activity of PA. Here we report the role of amino acid residues lining an exposed hydrophobic patch of domain III in the biological activity of PA. The residues Phe[552], Phe[554], Ile[562], Leu[566] and Ile[574] were individually substituted with alanine and the effect was studied. All mutant PA proteins except Phe552Ala were equally active as wildtype PA in exhibiting a toxic phenotype to J774A.1 cells in the presence of LF. Substitution of Ala for Phe[552] reduced the ability of PA to intoxicate cells by more than 250-fold. However, Phe552Ala was equally active in receptor binding and susceptibility to trypsin and chymotrypsin as wildtype PA, the activities that have been shown to be essential for the biological activity of PA. This mutated PA protein had a decreased ability to bind LF, oligomerize on cells and to induce release of [86]Rb+ from Chinese hamster ovary cells. These results suggest that the residue Phe[552] in PA plays an important role in LF binding and oligomerization. Our study provides a basis for further exploration of the biological significance of domain III of PA.

A novel antifungal protein, designated chrysancorin, was isolated from seeds of Chrysanthemum coronarium var. spatiosum with a procedure involving ion exchange chromatography on DEAEcellulose, affinity chromatography on Affigel blue resin, ion exchange chromatography on SPSepharose and FPLCgel filtration on Superdex 75. The Nterminus of chrysancorin displays sequence similarity to the genomic sequence of chromosome 1 from Arabidopsis thaliana BAC T19E23. Chrysancorin exhibits a molecular mass of 13.4 kDa in gel filtration and SDSpolyacrylamide gel electrophoresis. It stimulates the proliferation of mouse splenocytes and inhibits the activity of human immunodeficiency virus-1 reverse transcriptase. The protein possesses antifungal activity against Botrytis cinerea, Mycosphaerella arachidicola and Physalospora piricola, but not against Rhizoctonia solani, Fusarium oxysporum and Coprinus comatus. However, we could not detect antibacterial activity against a variety of bacteria.

We purified forms of legumain from a plant source (seeds of kidney bean, Phaseolus vulgaris) and a mammal (kidney of pig, Sus scropha) for comparison of their properties. Both forms were found to be stable only under moderately acidic pH conditions, and were maximally active at about pH 6; the plant enzyme was somewhat less stable and had a slightly higher pH optimum. With benzyloxycarbonylXaa AlaAsnaminomethylcoumarylamide substrates, the two forms of legumain showed distinctly different specificities for the P3 residue, the plant legumain preferring amino acids with bulky hydrophobic side chains because of lower K values. Both forms of legumain were highly specific for hydrolysis of asparaginyl bonds in the arylamide substrates and in neurotensin. Aspartyl bonds were hydrolysed about 100-fold more slowly with lower pH optima. Potential substrates containing other amino acids structurally similar to asparagine were not hydrolysed. There were clear differences in specificity of hydrolysis of protein substrates. The plant legumain differed from pig legumain in its action on tetanus toxoid Cfragment, cleaving at Asn[97] but not at Asn[337], and produced more extensive digestion of phaseolin. The plant form of legumain was much more weakly inhibited by eggwhite cystatin than was the mammalian form.

The chemical assessment of the complete disulphide bridge pattern in the βchain of human recombinant follicotropin (βFSH) was accomplished by integrating classical biochemical methodologies with mass spectrometric procedures. A proteolytic strategy consisting of a double digestion of native βFSH using the broadspecificity protease subtilisin first, followed by trypsin, was employed. The resulting peptide mixture was directly analysed by FABMS, leading to the assignment of the first three disulphide bridges. The remaining SS bridges were determined by HPLC fractionation of the proteolytic digest followed by ESMS analysis of the individual fractions. The pattern of cysteine couplings in βFSH was determined as: Cys3-Cys51, Cys17-Cys66, Cys20-Cys104, Cys28-Cys82, Cys32-Cys84 and Cys87-Cys94, confirming the arrangement inferred from the crystal structure of the homologous βCG. A subset of the SS bridge pattern comprising Cys3-Cys51, Cys28- Cys82 and Cys32-Cys84 constitutes a cysteine knot motif similar to that found in the growth factor superfamily.

PC12 cells interact with several growth factors (e. g. EGF, FGF, and NGF) via specific tyrosine receptor kinases, resulting in cell proliferation or neuronal differentiation. The small GTPase Ras is known to be involved in downstream signaling of these growth factor receptors. Furthermore, cellmatrix interactions mediated by integrins, as well as integrininduced signaling, are also involved in growth factorstimulated signal transduction in PC12 cells. In this study we determined the expression of the α1 integrin subunit in response to EGF and NGF in PC12 wildtype (wt) cells, and in PC12 cells overexpressing an inactive HRas protein (RasN17). In PC12 wt cells, α1 integrin expression is upregulated by EGF and NGF. Cell surface expression of α1β1 integrin is also enhanced in growth factortreated cells. This upregulation leads to increased α1β1- specific adhesion to collagen. In cells expressing the dominantnegative RasN17 variant, α1 integrin expression and α1β1-specific adhesion remain unchanged in response to both growth factors.

A novel mouse gap junction gene, coding for a presumptive protein of 258 amino acids (molecular mass: 28981 Da), has been designated connexin29. This single copy gene was mapped to distal mouse chromosome 5 and shows 75% sequence identity to a human connexin30.2 sequence in the database. Connexin29 mRNA (4.4 kb) is highly expressed in mouse sciatic nerve and less abundant in spinal cord as well as in adult brain, where it increased 12-fold between day 7 and 14 post partum. Our expression data suggest that the new connexin gene is active in myelinforming glial cells.

A strongly [75]Selabeled 22 kDa protein detected previously showed in its Nterminal sequence the highest similarity to the family of thioldependent peroxidases, now called peroxiredoxins. The respective gene prxU was cloned and analyzed. prxU encodes a protein of 203 amino acids (22470 Da) and contains an inframe UGA codon (selenocysteine) at the position of the so far strictly conserved and catalytically active Cys47. The second conserved cysteine present in 2-Cys peroxiredoxins was replaced by alanine. Heterologous expression of the Eubacterium acidaminophilum PrxU as a recombinant selenoprotein in Escherichia coli was not possible. A cysteineencoding mutant gene, prxU47C, containing UGC instead of UGA was strongly expressed. This recombinant PrxU47C mutant protein was purified to homogeneity by its affinity tag, but was not active as a thioldependent peroxidase. The identification of prxU reveals that the limited class of natural selenoproteins may in certain organisms also include isoenzymes of peroxiredoxins, previously only known as nonselenoproteins containing catalytic cysteine residues.

The tight regulation of extracellular matrix remodeling and degradation is of great importance in physiological processes like development and morphogenesis, as well as in pathological situations like tumor invasion and metastasis. Tissue inhibitors of metalloproteinases (TIMPs) are the naturally occuring inhibitors of matrix metalloproteinases, which are involved in matrix turnover. In this report we describe the cloning of human TIMP-4 from a human adenocarcinoma and an osteosarcoma cell line and the expression of the inhibitory domain in the methylotrophic yeast Pichia pastoris. The inhibition of MMP-8, -9, -12, -13 and -14 by the Nterminal domain of TIMP-4 was analysed. Using a fluorescent MCApeptide, K values for each subclass of MMPs were determined. With dissociation constants in the nanomolar range, TIMP-4 seems to be a good inhibitor for all classes of MMPs without remarkable preference for special MMPs.