Band 383, Heft 11

Pigment epitheliumderived factor is a member of the serpin superfamily of proteins, but one that lacks inhibitory properties against either serine or cysteine proteinases. Nevertheless it possesses a number of physiological properties that make it a potentially important protein in regulation of angiogenesis, in neuronal cell survival and in protection of neurons from neurotoxic agents. It is also a protein that is highly up regulated in the G0 phase of earlypassage cells compared with rapidly proliferating cells or senescent cells, and so is also linked to both the cell cycle and cell senescence. The determination of a high resolution Xray crystal structure of native PEDF provides insight into regions of the protein that may be involved in one or more of these functions.

DNAdependent protein kinase represses RNA polymerase I (Pol I) transcription in vitro. To investigate the mechanism underlying transcriptional repression, we compared Pol I transcription in extracts from cells that either contain or lack the catalytic subunit of DNAPK (DNAPKcs). ATPdependent repression of Pol I transcription was observed in extracts from DNAPKcscontaining but not deficient cells, required templates with free DNA ends, and was overcome by exogenous SL1, the factor that nucleates initiation complex formation. Orderofaddition experiments demonstrate that DNAPKcs does not inactivate component(s) of the PolI transcription machinery. Instead, phosphorylated Ku protein competes with SL1 for binding to the rDNA promoter and, as a consequence, prevents initiation complex formation. The results reveal a novel mechanism of transcriptional regulation by DNAPK. Once targeted to DNA, autophosphorylated Ku may displace positive or negativeacting factors from their target sites, thereby repressing or activating transcription in a genespecific manner.

Hepatocyte nuclear factor (HNF)1α is a homeodomaincontaining transcription factor participating in the regulation of gene expression in liver, kidney, gut and pancreas of vertebrates. In humans mutations in the HNF1α gene are responsible for one form of maturity onset diabetes of the young (MODY3). To define the molecular mechanism underlying MODY3 we investigated the functional properties of seven MODY3- associated mutations representing the spectrum of different kinds of mutations affecting all functional domains of the protein. The mutations introduced into an expression vector encoding human HNF1α include inframe deletion (ΔN127), nonsense (Q7X, R171X), frameshift (P291fsinsC) and missense (R229Q, P447L, T620I) mutations. Gel retardation and reporter gene assays showed that the functional properties of these mutants differ dramatically, but none of these mutants act in a dominant negative manner. Moreover, the mRNA stability of the mutants ΔN127, R171X, P291fsinsC and T547E548fsdelTG is impaired compared to the wildtype sequence in transfected cells. This decreased RNA stability is independent of the presence of an intron in the expression vector and thus differs from mechanisms known to be involved in nonsensemediated decay (NMD). Our results suggest that haploinsufficiency of HNF1α is responsible for the pathogenesis of MODY3.

Streptococcus pyogenes pSM19035-encoded (10.7 kDa) and ζ (32.4 kDa) proteins are necessary to secure stable plasmid inheritance in bacteria, with ζ acting as toxin that kills plasmiddeprived cells and as an antitoxin that neutralises the activity of ζ. The and ζ proteins copurify as a stable complex that, according to analytical ultracentrifugation and gel filtration, exists as 2ζ2 heterotetramer in solution. Cocrystals of the 2ζ2 complex contain and ζ in 1:1 molar ratio. Unfolding studies monitoring circular dichroic and fluorescence changes show that the ζ protein has a significantly lower thermodynamic stability than the protein both in free state and in the complex. Proteolytic studies indicate that ζ protein is more stable in the the 2ζ2 complex than in the free state. In vivo studies reveal a short halflife of the antitoxin (~18min) and a long lifetime of the ζ toxin (>60min). When transcriptiontranslation of a plasmid containing the and ζ genes was inhibited, cell death was observed after a short lag phase that correlates with the disappearance of the protein from the background.

Here we report on the study of the effects of different antineoplastic agents, including cytarabine, 4-hydroperoxyifosfamide, the activated form of ifosfamide, vincristine, and paclitaxel, with regard to their capacity to modulate the amount of cytoplasmic and membranebound heat shock protein 70 (Hsp70). Hsp70 levels were measured in the myelogenous leukemic cell line K562, in the human colon carcinoma cell line CX2, and in peripheral blood lymphocytes (PBL) under physiological conditions (37C), and following nonlethal heat shock at 41.8C. A concentration of 1 M and an incubation period of 2 h were determined as nonlethal, since none of the different antineoplastic agents induced necrosis or apoptosis in untreated or heatshocked cells under these conditions. Our results show that tubulininteracting agents, including vincristine and paclitaxel, but not DNAinteracting agents, including cytarabine and ifosfamide, selectively increase the amount of cytoplasmic Hsp70 in tumor and normal cells, as measured by semiquantitative Western blot analysis. Mechanistically, a vincristine and paclitaxelinduced tubulin assembly, as demonstrated by immunofluorescence microscopy, might be responsible for the elevated cytoplasmic Hsp70 levels. Interestingly, an increased membrane expression of Hsp70 following treatment with vincristine or paclitaxel was selectively observed on tumor cells, but not on normal cells.

The single transmembrane receptor sorLA/LR11 contains binding domains typical for the lowdensity lipoprotein receptors and a VPS10 domain which, in the related receptor sortilin, binds the neuropeptide neurotensin. SorLA is synthesized as a proreceptor which is processed to the mature form by a furinlike propeptidase. Endogenous sorLA and its hydra homologue HAB bind the neuropeptide head activator (HA). Transiently expressed partial sorLA constructs were investigated for ligand binding. We found that HA binds with nanomolar affinity to the VPS10 domain. The sorLA propeptide also bound to the VPS10 domain, whereas the receptorassociated protein RAP interacted both with the class A repeats and the VPS10 domain. The sorLA propeptide inhibited binding of HA to fulllength sorLA and to the VPS10 domain. It also interfered with binding of HA to hydra HAB, which is taken as evidence for a highly conserved tertiary structure of the VPS10 domains of this receptor in hydra and mammals. The propeptide inhibited HAstimulated mitosis and proliferation in the human neuroendocrine cell line BON and the neuronal precursor cell line NT2. We conclude that sorLA is necessary for HA signaling and function.

A gangliosidespecific sialidase that controls cellular functions such as growth, differentiation, and adhesion has been observed in a variety of cells, but its characterization proved difficult due to firm membrane attachment and lability of the purified enzyme. Here we report on the specificity toward gangliosides and susceptibility to certain inhibitors of a ganglioside sialidase solubilized and purified 5100-fold from human brain. The sialidase removed terminal sialic acids from gangliosides GM3, GM4, GD3, GD2, GD1a, GD1b, GT1b and GQ1b, but was inactive toward gangliosides with sialic acid in a branching position (as in GM1 and GM2). LysoGM3 and GD1a were good substrates, too, whereas Oacetylation of the sialic acid as in 9-OacetylGD3 caused strongly reduced cleavage. The new influenza virus drug 4-guanidino-2-deoxy-2,3- dehydroNacetylneuraminic acid (Zanamivir) exhibited an IC50 value of about 710 5 M that was in the range of the classical sialidase inhibitor 2-deoxy-2,3-dehydro Nacetylneuraminic acid; the bacterial sialidase inhibitor 4-nitrophenyloxamic acid, however, was ineffective. The glycosaminoglycans heparan sulfate, heparin, chondroitin sulfates A and B, as well as dextran sulfate and suramin, were all strongly inhibitory, suggesting that glycosaminoglycans present on the cell surface or in the extracellular matrix may influence the ability of the sialidase to alter the ganglioside composition of the membrane.

Lipoprotein(a) is composed of low density lipoprotein and apolipoprotein(a). Apolipoprotein(a) has evolved from plasminogen and contains 10 different plasminogen kringle 4 homologous domains [KIV(1 110)]. Previous studies indicated that lipoprotein(a) noncovalently binds the Nterminal region of lipoprotein B100 and the plasminogen kringle 4 binding plasma protein tetranectin. In this study recombinant KIV(2), KIV(7) and KIV(10) derived from apolipoprotein(a) were produced in E. coli and the binding to tetranectin and low density lipoprotein was examined. Only KIV(10) bound to tetranectin and binding was similar to that of plasminogen kringle 4 to tetranectin. Only KIV(7) bound to LDL. In order to identify the residues responsible for the difference in specificity between KIV(7) and KIV(10), a number of surfaceexposed residues located around the lysine binding clefts were exchanged. Ligand binding analysis of these derivatives showed that Y62, and to a minor extent W32 and E56, of KIV(7) are important for LDL binding to KIV(7), whereas R32 and D56 of KIV(10) are required for tetranectin binding of KIV(10).

Nanomolar concentrations of human amylin promote death of RINm5F cells in a time and concentrationdependent manner. Morphological changes of chromatin integrity suggest that cells are predominantly undergoing apoptosis. Human amylin induces significant activation of caspase-3 and strong and sustained phosphorylation of stressactivated protein kinases, cJun Nterminal kinase (JNK) and p38, that precedes cell death. Extracellular signalregulated kinase (ERK) activation was not concomitant with JNK and/or p38 activation. Activation of caspase-3 and mitogenactivated protein kinases (MAPKs) was detected by Western blot analysis. Addition of the MEK1 inhibitor PD 98059 had no effect on amylininduced apoptosis, suggesting that ERK activation does not play a role in this apoptotic scenario. A correlative inhibition of JNK activation by the immunosuppressive drug FK506, as well as a selective inhibition of p38 MAPK activation by SB 203580, significantly suppressed procaspase-3 processing and the extent of amylininduced cell death. Moreover, simultaneous pretreatment with both FK506 and SB 203580, or with the caspase-3 inhibitor AcDEVDCHO alone, almost completely abolished procaspase-3 processing and cell death. Thus, our results suggest that amylininduced apoptosis proceeds through sustained activation of JNK and p38 MAPK followed by caspase-3 activation.

A series of 2-alkyl-3-alkylamino-2Hbenzo and 2-alkyl-3-alkylamino-2Hpyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides, structurally related to BPDZ 44 and BPDZ 73, two potent pancreatic Bcells K+ ATP channel openers, were synthesized and tested on rat pancreatic islets (endocrine tissue) as well as on rat aorta rings (vascular smooth muscle tissue). Alkylation of the 2-position led to double bond tautomerization and formation of compounds with a 2Hconformation. In contrast to the previously described pyridothiadiazine dioxides, such as BPDZ 44, and 7-chlorobenzothiadiazine dioxides, such as BPDZ 73, the 2-alkylsubstituted analogs were found to be poorly active on the insulin releasing process although most drugs exhibited a vasorelaxant activity. As a result, the new 2-alkylsubstituted pyridinic compounds expressed a selectivity profile (vascular smooth muscle tissue vs pancreatic tissue) opposite to that of their nonalkylsubstituted counterparts, i.e. BPDZ 44. Additional investigations revealed that, in contrast to their non 2-alkylsubstituted analogs, the most interesting 2-methylsubstituted derivatives did not express the pharmacological profile of classical K+ATP channel openers. The pharmacological results rather suggest that alkylation of the 2-position of the thiadiazine ring led to drugs that could act as Ca2+ channel blockers rather than as potassium channel openers.

The reaction of recombinant chlorophyll synthase from Avena sativa, expressed in Escherichia coli, was investigated. To verify the identity of the recombinant and native enzymes, reaction rates were determined for both enzyme preparations with several chlorophyllide analogs. The rates of esterification of these modified substrates ranged from 0 to 100% of the rate with the natural substrate, and were nearly identical for both enzyme preparations. The Lineweaver Burk plot for variation of both chlorophyllide a and phytyl diphosphate concentration showed parallel lines, indicative of a 'pingpong' mechanism. Preincubation with phytyl diphosphate exhibited an initial rapid reaction phase, which did not occur after preincubation with chlorophyllide. We conclude that the tetraprenyl diphosphate must bind to the enzyme as the first substrate and esterification occurs when this preloaded enzyme meets the second substrate, chlorophyllide. Approximately 10 17% of the recombinant enzyme were preloaded with phytyl diphosphate under the experimental conditions. The rapid reaction phase is also observed for the chlorophyll synthase reaction in etiolated barley leaves in addition to the wellknown slow phase. This indicates that preloading of the enzyme with tetraprenyl diphosphate is also the basis for the reaction in vivo.

In late summer in Europe, pollen of mugwort is one of the major sources of atopic allergens. No information about the complete molecular structure of any mugwort allergen has been published so far. Here we report the isolation and characterization of mugwort pollen cDNA clones coding for two isoforms of the panallergen profilin. Thirtysix percent of the mugwort allergic patients tested displayed IgE antibodies against natural and recombinant profilin, and no significant differences were observed in the IgEbinding properties of the isoforms. One profilin isoform was purified to homogeneity and detailed structural analysis indicated that the protein exists in solution as dimers and tetramers stabilized by sulfydryl and/or ionic interactions. Profilin monomers were detectable only after exposure of multimers to harsh denaturing conditions. Dimers and tetramers did not significantly differ in their ability to bind serum IgE from mugwort pollenallergic patients. However, oligomeric forms might have a higher allergenic potential than monomers because larger molecules would have additional epitopes for IgEmediated histamine release. Profilin isolated from mugwort pollen also formed multimers. Thus, oligomerization is not an artifact resulting from the recombinant production of the allergen. Inhibition experiments showed extensive IgE crossreactivity of recombinant mugwort profilin and profilin from various pollen and food extracts.

The hyperthermoacidophilic archaeon Sulfolobus acidocaldarius has a unique respiratory system with at least two terminal oxidases. Genetic and preliminary biochemical studies suggested the existence of a unique respiratory supercomplex, SoxM. Here we show (i) that all respective genes are translated into polypeptides, and (ii) that the supercomplex can be separated from the alternative oxidase SoxABCD and in that way characterized in a catalytically competent form for the first time. It acts as a quinol oxidase and contains a total of seven metal redox centers. One of it the blue copper protein sulfocyanin functionally links two subcomplexes. One is a bb3-type terminal oxidase moiety containing CuA and CuB, whereas the other consists of a Rieske FeSprotein and a homolog to cytochrome b in this case hosting two hemes AS. Based on a 1:1 stoichiometry, 1 mol complex contains 6 mol Fe and 4 mol Cu. Its activity is completely inhibited by cyanide and strongly by aurachinC and D derivatives as inhibitors of the quinol binding site. These data suggest that the complex provides two proton pumping sites. Interestingly, subunitII reveals an unusual pH dependence and is proposed to act as a pH sensor as well as a regulator of catalytic activity via a reversible transition between two states of the CuA ligation. This is a novel hint at how S. acidocaldarius can adapt to and survive in its extreme natural environment.

We demonstrate the applicability of fluorescence correlation spectroscopy (FCS) for receptor binding studies using low molecular weight ligands on membranes of living nerve cells. The binding of benzodiazepine Ro 7 1986/602 (Ndesdiethylfluorazepam), labeled with the fluorophore Alexa 532, the benzodiazepine receptor was analyzed quantitatively at the membrane of single rat hippocampal neurons. The values obtained for the dissociation constant Kd = (9.9±1.9) nM and the rate constant for ligandreceptor dissociation kdiss = (1.28±0.08)10- 3 s 1 show that there is a specific and high affinity interaction between the dyelabeled ligand (RoAlexa) and the receptor site. The binding was saturated at approx. 100 and displacement of 10 nM RoAlexa, with a 1000- excess of midazolam, showed a nonspecific binding of 7 10%. Additionally, two populations of the benzodiazepine receptor that differed in their lateral mobility were detected in the membrane of rat neurons. The diffusion coefficients for these two populations [Dbound1 = (1.32±0.26) m2/s; Dbound2 = (2.63±0.63)10- 2 m2/s] related to binding sites, which shows a monoexponential decay in a timedependent dissociation of ligandreceptor complex.

Thyroglobulin contains 11 repeats of a motif called thyroglobulin type-1 domain that show sequence similarity to some proteins exhibiting inhibitory activity against cysteine proteinases. Here we report that thyroglobulin decreases the activity of cathepsins B, H, L, and papain. To examine the possible involvement of particular type-1 domains in that decrease of activity, some individual thyroglobulin type-1 domains were expressed in E. coli. These recombinant domains proved to be substrates for cathepsins B, H, L, and papain instead of inhibitors. The cleavage points with cathepsins B and L on the second and the fourth domains were determined. The possible reasons for degradation are discussed.

The mammalian legumain, also called asparaginyl endopeptidase (AEP), is critically involved in the processing of bacterial antigens for MHC class II presentation. In order to investigate the substrate specificity of AEP in the P1 position, we created a peptide library and digested it with purified pig kidney AEP. Digestion was less efficient only when proline was in the P1 position. Maximum AEP activity was found in lysosomal fractions of different types of antigen presenting cells (APC). When the multiple sclerosisassociated autoantigen myelin basic protein (MBP) was digested with AEP, the immunodominant epitope 8399 was destroyed. Myoglobin as an alternative substrate was AEP resistant. These results suggest an important, but not necessarily critical role for AEP in lysosomal antigen degradation.

The lysosomal cysteine protease cathepsin B is one of several proteases that have been linked to tumour progression. Its increased expression and secretion in tumour cells may facilitate the degradation of extra cellular matrix proteins, leading to tumour cell invasion and metastasis. Specific inhibitory monoclonal antibodies are a possible alternative to synthetic inhibitors as a therapeutic tool for cancer treatment. An inhibitory monoclonal antibody, which binds to an epitope near the active site of cathepsin B and inhibits its proteolytic activity, was prepared and its effect on invasion of rastransformed MCF-10A neoT cells was tested in vitro. Here we present the nucleotide sequences of the heavy and light chains of the inhibitory antibody and compare them to the murine immunoglobulin germline sequences for possible somatic hypermutations. Since no harmful mutations were found, a mouse/human chimeric antibody was constructed by fusing murine VH and VL variable regions of the inhibitory antibody with human γ 1 and κ constant regions, respectively. Chinese hamster ovary K1 cells were cotransfected with expression vectors pcDNA3L and pcDNA3H and the reactivity of the isolated chimeric antibody was tested by ELISA and Western blotting. We could demonstrate an inhibitory effect of the chimeric antibody.

Tumor necrosis factorα (TNFα) is presumably shed from cell membranes by TNFαcleaving enzyme (TACE). The peptides SPLAQAVRSSSR and DabcylLAQAVRSSSREdans, each encompassing the cleavage sequence of proTNFα recognized by TACE, were applied to intact umbilical vein endothelium (HUVEC), peripheral blood leukocytes (PBL) and the mast cell line HMC-1, which express TACE, to homogenates of rat heart tissue and to membrane and cytoplasmic extracts of PBL. Formation of SPLAQA (specific cleavage) was determined by HPLC, while cleavage (specific plus nonspecific) of DabcylTNFαEdans was followed over time by measuring fluorescence. Participation of TACE was assessed from inhibition due to the drug TAPI-2. Incubation with recombinant human TACE gave specific cleavage, fully inhibitable by TAPI-2 (IC50<0.1 M). HUVEC rapidly degraded TNFαpeptide, but in a nonspecific manner (no SPLAQA detectable) and 50 M TAPI-2 was without effect. Fluorescence was evoked when Dabcyl LAQAVRSSSREdans was incubated with HMC-1 or PBL and also with cytoplasmic and membrane fractions of lysed PBL, but in no case was there significant inhibition by TAPI-2. However, marginal (10%) inhibition of fluorescence by 50 M TAPI-2 was observed with homogenized heart tissue. This contained TACE, about 75% of which was without the inhibitory cysteine switch (Western blot). In conclusion, simple peptide analogs of proTNFα cannot be employed as substrates for measuring membrane TACE activity, largely due to extensive nonspecific proteolytic cleavage by whole cells and cell extracts.