Band 383, Heft 9

Kaposis Sarcoma (KS) is a highly angiogenic neoplasm associated with infection by the human γherpesvirus, HHV-8 or Kaposis sarcoma herpes virus (KSHV). When in 1872 the Hungarian scientist Moritz Kaposi described the sarcoma, which was later named after him, he was dealing with a rare dermatologic disease. Today, KS is a more common pathology due to its high incidence in AIDS, in immunosuppressed transplantation patients and, in its endemic form, in Africa. The introduction of highly active antiretroviral therapy (HAART) has led to a drastic reduction of KS incidence in HIVinfected patients, but in some cases KS resists the treatment. KS is more common in men than in women. The observation of spontaneous remissions during pregnancy stimulated investigations into the potential antiKS activity of the pregnancy hormone human chorionic gonadotropin (hCG). The variable effect in clinical trials using urinary preparations of the hormone (uhCG) has led to the hypothesis that contaminating moieties present in these preparations may account for the antiKS effect observed in vitro. While the discrepancy between laboratory tests and clinical trials remains a mystery, little is known about potential antiKS mechanisms of the hormone itself and/or other active moieties present in uhCG.

By targeting the highly conserved antiparallel βsheet formed by the interdigitation of the N and Cterminal strands of each monomer, dimerization inhibitors of HIV-1 protease may be useful to overcome the drug resistance observed with current activesite directed antiproteases. Sequestration of the monomer by the inhibitor (or disruption of the dimer interface) prevents the correct assembly of the inactive monomers to active enzyme. Strategies for the design of drugs targeting the dimer interface are described. Various dimerization inhibitors are reported including N and Cterminal mimetics, lipopeptides and crosslinked interface peptides.

A single amino acid substitution (Y78R) at the dimerdimer interface of homotetrameric single stranded DNA binding protein from E.coli (EcoSSB) renders the protein a stable dimer. This dimer can bind singlestranded DNA albeit with greatly reduced affinity. In vivo this dimeric SSB cannot replace homotetrameric EcoSSB. Amino acid changes at the rim of the dimerdimer interface nearby (Q76K, Q76E) show an electrostatic interaction between a charged amino acid at position 76 and bound nucleic acid. In conclusion, nucleic acid binding to homotetrameric SSB must take place across both dimers to achieve functionally correct binding.

Cdc37 associates with the heatshock protein 90 (Hsp90) molecular chaperone as one of several auxiliary proteins that are collectively referred to as Hsp90 cochaperones. Cdc37 has been proposed to be a specificity factor for Hsp90, directing it notably towards kinases. It is not known whether Cdc37 is essential for viability in the budding yeast Saccharomyces cerevisiae because of Hsp90-dependent or independent functions or both. Sti1 and Cpr7 are nonessential Hsp90 cochaperones that bind to a common surface on Hsp90 through tetratricopeptide repeats (TPR). We have found that Sti1 is specifically retained from yeast extracts by immobilized Cdc37. Similarly, the endogenous proteins are also found in a complex. Moreover, purified recombinant Sti1 and Cdc37 interact in the complete absence of Hsp90. Complexes between Cdc37 and Sti1 are not unique to this TPR protein since endogenous Cdc37 can be copurified with exogenously expressed Cpr7 fused to glutathioneStransferase. The heterogeneity of Cdc37 complexes, both with and without Hsp90, may expand the functional diversity of Cdc37. Here we show that the combination of cdc37 and sti1 mutations is synthetically lethal, suggesting that direct contacts between Cdc37 and Sti1 may at least contribute to vital functions in yeast.

Juvenile hormone binding proteins (JHBPs) serve as specific carriers of juvenile hormone (JH) in insect hemolymph. As shown in this report, Galleria mellonella JHBP is encoded by a cDNA of 1063 nucleotides. The preprotein consists of 245 amino acids with a 20 amino acid leader sequence. The concentration of the JHBP mRNA reaches a maximum on the third day of the last larval instar, and decreases fivefold towards pupation. Comparison of amino acid sequences of JHBPs from Bombyx mori, Heliothis virescens, Manduca sexta and G. mellonella shows that 57 positions out of 226 are occupied by identical amino acids. A phylogeny tree was constructed from 32 proteins, which function could be associated to JH. It has three major branches: (i) ligand binding domains of nuclear receptors, (ii) JHBPs and JH esterases (JHEs), and (iii) hypothetical proteins found in Drosophila melanogaster genome. Despite the close positioning of JHEs and JHBPs on the tree, which probably arises from the presence of a common JH binding motif, these proteins are unlikely to belong to the same family. Detailed analysis of the secondary structure modeling shows that JHBPs may contain a βbarrel motif flanked by αhelices and thus be evolutionary related to the same superfamily as calycins.

Nitric oxide (NO) is synthesised by different nitric oxide synthases (NOS) from Larginine and acts as a signal transducer in a variety of cells. The neuronal isoenzyme of NOS (nNOS) was recently found in rodent βcells and βcell lines. We provide evidence that nNOS is also present in the human βcell line CM and that the specific inhibitor of nNOS PIN is expressed in CM and INS-1 cells. Furthermore, we investigated the influence of glucose on the activity of nNOS and the expression of PIN and are able to show that both are increased by glucose stimulation in the βcell lines but not in the mouse fibroblastic cell line LTK. This indicates that nNOS and PIN play a role in the specific function of βcells, not only in rodents but also in humans.

Sulfurtransferases/rhodaneses are a group of enzymes widely distributed in plants, animals, and bacteria that catalyze the transfer of sulfur from a donor molecule to a thiophilic acceptor substrate. Sulfurtransferases (STs) consist of two globular domains of nearly identical size and conformation connected by a short linker sequence. In plant STs this linker sequence is exceptionally longer than in sequences from other species. The Arabidopsis ST1 protein (AJ131404) contains five cysteine residues: one residue is universally conserved in all STs and considered to be catalytically essential; a second one, closely located in the primary sequence, is conserved only in sequences from eukaryotic species. Of the remaining three cysteine residues two are conserved in the so far known plant STs and one is unique to the Arabidopsis ST1. The aim of our study was to investigate the role of the twodomain structure, of the unique plant linker sequence and of each cysteine residue. The N and Cterminal domains of the Arabidopsis ST1, the fulllength protein with a shortened linker sequence and several pointmutated proteins were overexpressed in E. coli, purified and used for enzyme activity measurements. The Cterminal domain itself displayed ST activity which could be increased by adding the separately prepared Nterminal domain. The activity of an ST1 derivative with a shortened linker sequence was reduced by more than 60% of the wildtype activity, probably because of a drastically reduced protein stability. The replacement of each cysteine residue resulted in mutant forms which differed significantly in their stability, in the specific ST activities, and in their kinetic parameters which were determined for 3-mercaptopyruvate as well as thiosulfate as sulfur substrates: mutation of the putative active site cysteine (C332) essentially abolished activity; for C339 a crucial role at least for the turnover of thiosulfate could be identified.

The hair follicle consists of a complex system of multiple tissue compartments that are clearly distinguishable by their morphology and type of differentiation. We have synthesized hair folliclespecific keratins from the companion layer (K6hf, K17) and the hair cortex (Ha1, Hb3, Hb6) in Escherichia coli. The assembly of purified keratins in mixtures of K6hf/K17 and in mixtures of hair cortex keratins was compared in urea solutions, low ionic strength and physiological strength buffers, by urea melting gels, electron microscopy and analytical ultracentrifugation. Both types of keratin mixtures, keratins from the companion layer and keratins from the hair cortex, formed heterotypic complexes at 5 M urea. In low ionic strength buffers, the keratins from the companion layer were assembled to bona fide intermediate filaments. In contrast, mixtures of hair cortex keratins stayed in an oligomeric state with a mean s value of 9 as determined in sedimentation velocity experiments. Hair cortex keratins were, however, assembled into intermediate filaments at physiological salt conditions. A point mutated hair cortex keratin [Hb6(Glu402Lys)] formed no long filaments when mixed with Ha1; instead, the assembled structures showed a length distribution of 50.8±13.4 nm, comparable to the size distribution of assembly intermediates called unitlength filaments.

Almost 500 million people worldwide suffer from Type I allergy, a genetically determined immunodisorder which is based on the production of IgE antibodies against per se harmless antigens (allergens). Due to their worldwide distribution and heavy pollen production, grasses represent a major allergen source for approximately 40% of allergic patients. We purified Phl p 4, a major timothy grass (Phleum pratense) pollen allergen with a molecular mass of 61.3 kDa and a pI of 9.6 to homogeneity. Circular dichroism spectroscopical analysis indicates that Phl p 4 contains a mixed αhelical/βpleated secondary structure and, unlike many other allergens, showed no reversible unfolding after thermal denaturation. We show that Phl p 4 is a major allergen which reacts with IgE antibodies of 75% of grass pollen allergic patients (n=150) and induces basophil histamine release as well as immediate type skin reactions in sensitized individuals. Phl p 4-specific IgE from three patients as well as two rabbitanti Phl p 4 antisera crossreacted with allergens present in pollen of trees, grasses, weeds as well as plantderived food. Rabbit antibodies raised against Phl p 4 also inhibited the binding of allergic patients IgE to Phl p 4. Phl p 4 may thus be used for diagnosis and treatment of sensitized allergic patients.

Two novel proteins, BM8 and BM14, were isolated from Bungarus multicinctus (Taiwan banded krait) venom using the combination of chromatography on a SPSephadex C-25 column and a reversephase HPLC column. Both proteins contained 82 amino acid residues including 10 cysteine residues, but there were two amino acid substitutions at positions 37 and 38 (Glu37Ala38 in BM8; Lys37Lys38 in BM14). CD spectra and acrylamide quenching studies revealed that the gross conformation of BM8 and BM14 differed. In contrast to BM8, BM14 inhibited the binding of [3H]quinuclidinyl benzilate to the M2 muscarinic acetylcholine (mAchR) receptor subtype. Trinitrophenylation of Lys residues abolished the mAchRbinding activity of BM14, indicating that the Lys substitutions at positions 37 and 38 played a crucial role in the activity of BM14. The genomic DNA encoding the precursor of BM14 was amplified by PCR. The gene shared virtually identical structural organization with αneurotoxin and cardiotoxin genes. The intron sequences of these genes shared a sequence identity up to 84%, but the proteincoding regions were highly variable. These results suggest that BM8, BM14, neurotoxins and cardiotoxins may have originated from a common ancestor, and the evolution of snake venom proteins shows a tendency to diversify their functions.

Degradation of extracellular matrix proteins by proteases such as the cysteine protease cathepsin B is critical to malignant progression. We have established that procathepsin B presents on the surface of tumor cells through its interaction with the annexin II tetramer [Mai et al., J. Biol. Chem. 275 (2000), 12806 12812]. Cathepsin B activity can also be detected on the tumor cell surface and in their culture medium. Interestingly, the annexin II tetramer also interacts with extracellular matrix proteins, such as collagen I, fibrin and tenascinC. Both cathepsin B and tenascinC are expressed at high levels in malignant tumors, especially at the invasive edges of tumors, and are implicated in tumor angiogenesis. In this study, we report that tenascinC can be degraded by cathepsin B in vitro. We demonstrate by immunohistochemistry that both cathepsin B and tenascinC are expressed highly in malignant anaplastic astrocytomas and glioblastomas as compared to normal brain tissues. Interestingly, cathepsin B and tenascinC were also detected in association with tumor neovessels. We suggest that interactions between cathepsin B and tenascinC are involved in the progression of gliomas including the angiogenesis that is a hallmark of anaplastic astrocytomas.

The calpains form a growing family of structurally related intracellular multidomain cysteine proteinases containing a papainrelated catalytic domain, whose activity depends on calcium. The calpains are believed to play important roles in cytoskelatel remodeling processes, cell differentiation, apoptosis and signal transduction, but are also implicated in a number of diseases. Recent crystal structures of truncated rat and fulllength human apomcalpain revealed the domain arrangement and explained the inactivity of mcalpain in the absence of calcium by a disrupted catalytic domain. Proteolysis studies have indicated several susceptible sites, in particular in the catalytic subdomain IIb and in the following domain III, which are more accessible to attacking proteinases in the presence than in the absence of calcium. The current view is that mcalpain exhibits a number of calcium binding sites, which upon calcium binding cooperatively interact, triggering the reformation of a papainlike catalytic domain, accompanied by enhanced mobilisation of the whole structure. To further analyse the flexibility of mcalpain, we have determined and refined the human fulllength apomcalpain structure of a second crystal form to 3.15 å resolution. Here we present this new structure, compare it with our first structure now rerefined with tighter constrain parameters, discuss the flexibility in context with the proteolysis and calcium binding data available, and suggest implications for the calciuminduced activation process.

The DNAbased quantitative analysis of genetic chimerism is becoming increasingly more important for molecular biology in general and molecular medicine in particular. Useful genomic targets for these analyses are polymorphic sequences, but here the problem of a reliable quantification with high dynamic range is not yet satisfactorily solved. To this end we have combined the allelespecific amplification with a realtime PCRbased quantification for rapid allelotyping and chimerism analysis. The sequence variations are discriminated by the 3end of the allelespecific primer. Amplification is monitored by SYBRGreen I fluorescence. We demonstrate the efficiency of this method for two clinically relevant targets: (i) the 10 bp insertion/deletion polymorphism in the promoter of the factor VIIc (FVIIc) gene and (ii) the 4G/5G single nucleotide polymorphism in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene. Both polymorphisms are associated with clinical risk factors. Allelotyping results were in complete agreement with those obtained by reference methods. Mixed chimeric DNA samples could be quantified reliably with a dynamic range of 1:3000 for an easy target (FVIIc) and of 1:64 for a difficult target (PAI-1). Our protocol is particularly useful for rapid, reliable and inexpensive genotyping and quantitative chimerism analysis without requiring expensive fluorophor dye labelled probes.

Phospholipid flipping in biogenic membranes is a key feature of membrane bilayer assembly. Flipping is facilitated by proteinaceous transporters (flippases) that do not need metabolic energy to function. No flippase has yet been identified. The architecture of the E. coli protein translocon suggests that it could account for the flippase activity in the bacterial inner membrane. To test this possibility, we used E. coli cells depleted of SecYE or YidC to assay flipping in proteoliposomes reconstituted from detergent extracts of their inner membranes. We conclude that the protein translocon contributes minimally, if at all, to phospholipid flippase activity in the inner membrane.

Group 4 grass pollen allergens represent 60 kDa glycoproteins recognized by 70% of patients sensitive to these pollens. An antiserum against purified Phl p 4 from timothy grass pollen was used to investigate various pollens, fruits, and vegetables for Phl p 4-related allergens by immunogold electron microscopy. In timothy grass, mugwort, and birch pollens, allergens were located in the wall, and in timothy grass and birch pollens additionally in the cytoplasm. In peanut, apple, celery root, and carrot root, only cytoplasmic areas were labeled. Group 4-related allergens thus occur in pollens of unrelated plants and in plant food and may therefore contribute to crossreactivities in patients allergic to various pollens and plant food.

The large extracellular loop of human CD81, a tetraspanin mediating hepatitis C virus envelope protein E2 binding to human cells, has been crystallized in a hexagonal form. The threedimensional structure, solved and refined at 2.6 å resolution (Rfactor = 22.8%), shows that the protein adopts a dimeric assembly, based on an association interface built up by tetraspaninconserved residues. Structural comparisons with the tertiary structure of human CD81 large extracellular loop, previously determined in a different crystal form, show marked conformational fluctuations in the molecular regions thought to be involved in binding to the viral protein, suggesting rules for recognition and assembly within the tetraspan web.

Folding of cathepsin S, like other cathepsin Llike proteases, depends on its proregion. The major part of the proregion forms a small domain distal from the catalytic centre, suggesting function(s) beyond activesite shielding. Using an optimised in vitro transrefolding assay, we compared reactivation of denatured cathepsin S by the genuine propeptide, wildtype and ten selected mutants. Including structural data and binding constants, we identified the prodomain core and the hairpin region to be important for the foldase function.

We present a method that allows preparing long DNA containing defined mismatches without the use of gel electrophoretic or chromatographic purification steps. The preparation starts with the synthesis of two PCR products, which are identical except for those positions that will later form the mismatches. One of the PCR primers must be 5phosphorylated, such that in two separate reactions two PCR products are obtained, which are 5phosphorylated in one strand. After removal of the phosphorylated strands by λexonuclease, the resulting single strands are hybridized to form the mismatchcontaining heteroduplex. The application of this procedure is demonstrated for the analysis of the Escherichia coli MutHLS system.

In recent years there has been great interest in quantitative polymerase chain reaction. Consequently, a large number of assays have been developed, of which the one using nonhomologous competitors is arguably the most precise. Despite widespread applications, currently there is no simple method to synthesize such competitors. Here a facile and costeffective, singlestep method for synthesis of recombinant, nonhomologous, competitor complementary DNA is described. The method can be adapted to generate competitors of any size and sequence. The entire procedure is quick, straightforward and does not require any specialized equipment except a standard thermocycler.