Band 380, Heft 4

A detergent extract isolated from the enriched fraction of integral membrane proteins of Jurkat cells showed an enhanced tyrosine phosphate level when phosphorylated in the presence of phorbol 12-myristate 13-acetate (TPA) and phorbol 12,13-dibutyrate (PDBu). The enhanced tyrosine phosphorylation was observed when the reaction time exceeded 6 min; at shorter incubation times, however, TPA inhibited tyrosine phosphorylation. When the reaction proceeded for a constant time period longer than 6 min and phorbol esters were added at different times after the start of the reaction, two phases of an enhanced tyrosine phosphorylation of a 50 kDa protein were observed. An increased phosphorylation of the 50 kDa protein was correlated with an enhanced phosphorylation of poly-(Glu 4 ,Tyr 1 ). The two phases of enhanced phosphorylation differed in their TPA and PDBu requirement and in the proteins that were tyrosine phosphorylated. Studies with protein kinase C (PKC) inhibitors showed a negatively correlated effect on the enhanced tyrosine phosphorylation in phase I; tyrosine phosphorylation was further augmented. In phase II the regulation of tyrosine phosphorylation correlated with the efficiency of the PKC inhibitors on the α-isoform of PKC which was found in the cell extract. Separation of the proteins present in the investigated cell extract by gel filtration revealed a co-migration of the α-PKC and the 50 kDa protein. The metabolic labeling of intact Jurkat cells with 32 P i indicated that phorbol esters are also able to induce tyrosine phosphorylation of the 50 kDa protein under in vivo conditions. These data suggest an activation of two different tyrosine phosphorylation pathways by phorbol esters involving tyrosine phosphorylation/autophosphorylation of a 50 kDa kinase, as confirmed by 5′-p-fluorosulfonylbenzoyladenosine (FSBA) labeling, that are accurately regulated by α-PKC.

Expression of gangliosides in the liver was examined in primary cultures of hepatocytes from adult rats and liver tissues from rats of different ages. Hepatocytes were isolated from 7-week-old rat liver and cultured in L-15 medium containing insulin, dexamethasone and 10% fetal bovine serum. Hepatocytes proliferated only on the first day, and then ceased proliferation. The content of GD3 and GD1a increased during the period of active proliferation and reached a nearly constant level, whereas GM1, GD1b, GT1b, and GQ1b gradually increased throughout culture. Addition of EGF to the culture medium caused significant increases in the content of GD3, and to a lesser degree of GM3, but exhibited little effect on the expression of other ganglioside species. The specific induction of GD3 and GM3 expression by EGF was reproduced under serum-free conditions, despite the lack of hepatocyte proliferation. Expression of gangliosides in cultured hepatocytes was also modulated by cell density; higher cell density brought about increased content of GM1, GD1a, GD1b, GT1b, and GQ1b with concomitant reduction of GM3 in cells. The composition of gangliosides in liver tissues demonstrated a unique developmental pattern. GD3 and GD1a were strongly expressed in E-16 embryonic tissue and rapidly decreased with increasing age. GD1b, GT1b, and GQ1b were found only in postnatal liver tissues. These findings suggest that the expression of gangliosides in rat hepatocytes and liver tissues are regulated by growth- and development-dependent factors.

The yeast Gal80 protein inhibits the transcription activation function of Gal4p by physically interacting with the activation domain (Gal4-AD). Gal80p interaction with Gal1p or Gal3p is required to relieve Gal4p inhibition in response to galactose. Gal80p orthologs of Saccharomyces cerevisiae and Kluyveromyces lactis , ScGal80p and KlGal80p, can also inhibit the heterologous Gal4p variants; however, heterologous Gal3p/Gal1p only regulate ScGal80p but not KlGal80p. To compare KlGal80p and ScGal80p, point mutations known to affect ScGal80p function were introduced at corresponding positions in KlGal80p, and Gal4p regulation in vivo and KlGal80p-binding to Gst-Gal1p and Gst-Gal4-AD in vitro were analysed. The in vitro binding properties of the KlGal80p mutants were similar to those of ScGal80p, but two out of four mutants differed in Gal4p regulation. E. g. KlGAL80 s-0 (G302R) but not ScGAL80 s-0 (G301R) alleviates Gal4p inhibition. Possibly, this difference is related to a role of phosphorylation in the regulation of Gal80p function in K. lactis . Wild-type and mutant forms of KlGal80p are shown to be subject to carbon source regulated phosphorylation whereas no evidence for ScGal80p phosphorylation exists. (Hyper-)phosphorylation of KlGal80p is strongly reduced in galactose-containing medium. This reduction requires KlGal1p but no interaction with KlGal4p. The inhibition deficient KlGal80s-0p (G302R) variant is under-phosphorylated. We thus propose that phosphorylation of Gal80p in Kluyveromyces lactis contributes to the regulation of Gal4p mediated transcription.

Skeletal muscle dystrophin is a 427 kDa protein thought to act as a link between the actin cytoskeleton and the extracellular matrix. Perturbations of the dystrophin-associated complex, for example, between dystrophin and the transmembrane glycoprotein β-dystroglycan, may lead to muscular dystrophy. Previously, the cysteine-rich region and first half of the carboxy-terminal domain of dystrophin were shown to interact with β-dystroglycan through a stretch of fifteen amino acids at the carboxy-terminus of β-dystroglycan. This region of dystrophin implicated in binding β-dystroglycan contains four modular protein domains: a WW domain, two putative Ca 2+ -binding EF-hand motifs, and a putative zinc finger ZZ domain. The WW domain is a globular domain of 38–40 amino acids with two highly conserved tryptophan residues spaced 20–22 amino acids apart. A subset of WW domains was shown to bind ligands that contain a Pro-Pro-x-Tyr core motif (where x is any amino acid). Here we elucidate the role of the WW domain of dystrophin and surrounding sequence in binding β-dystroglycan. We show that the WW domain of dystrophin along with the EF-hand motifs binds to the carboxy-terminus of β-dystroglycan. Through site-specific mutagenesis and in vitro binding assays, we demonstrate that binding of dystrophin to the carboxyterminus of β-dystroglycan occurs via a β-dystroglycan Pro-Pro-x-Tyr core motif. Targeted mutagenesis of conserved WW domain residues reveals that the dystrophin/β-dystroglycan interaction occurs primarily through the WW domain of dystrophin. Precise mapping of this interaction could aid in therapeutic design.

Glycoconjugates have a whole spectrum of biological roles, from those that appear trivial to those that are crucial. Results accumulated in the past years indicate they might also play an important role in the response to stress, a complex physiological response of the human organism to various threats. We have recently identified stressin, a human serum glycoprotein, which was found to be increased under stress conditions. Here we report the purification of stressin from sera of professional soldiers and partial characterization of its protein and carbohydrate parts using lectin blotting and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Stressin was purified using a combination of ammonium sulfate precipitation, ion exchange chromatography, preparative gel electrophoresis and reverse-phase HPLC. It was found to be a highly glycosylated protein. Only 21.9 kDa (out of 36.7 kDa) was the protein part, whereas the remaining 40% of the mass originated from N-linked oligosaccharides. The carbohydrate part contained 12 sialic acids moieties, nearly 90% of which were lost due to post-source decay in the field-free tube. Tryptic fragments were produced from glycosylated and deglycosylated stressin, separated by reverse-phase HPLC and their exact molecular masses were determined using MALDI-MS. Comparison with tryptic maps of other proteins in computer databases indicated that stressin does not correspond to any already described protein.

Understanding receptor-ligand interactions, and the signal transduction pathways they activate, is of great interest for the discovery of novel antagonists and agonists. In this report we describe a rapid and efficient procedure to evaluate the importance of several different epidermal growth factor (EGF) residues for the binding and activation of its receptor (EGFR). We constructed an EGF mutant library randomized at positions 13, 15 and 16 and expressed them on filamentous phages. Phage display is a powerful system, allowing rapid isolation of binding mutants. Since many of the most pharmacologically interesting receptors cannot be produced in a soluble form, we developed a technique to rapidly select receptor-binding molecules directly on cells. A luciferase assay, simple to perform, was then used to test their biological transduction activity and to rapidly detect mutants of interest. Analysis of the resulting sequences revealed that the wild-type amino acids at positions 13, 15 and 16 are optimized for binding and activity. EGF mutants with agonist properties were also isolated and tolerated substitutions were identified.

Previous DSC and X-ray studies on RM6, a loop deletion mutant of wtROP protein, have shown that removal of five amino acids from the loop causes a dramatic reorganization of the wild-type structure. The new tetrameric molecule exhibits a significantly higher stability (Lassalle, M.W. et al ., J. Mol. Biol., 1998, 279 , 987–1000) and unfolds in a second order reaction (Lassalle, M.W. and Hinz, H.-J., Biochemistry, 1998, 37 , 8465–8472). In the present investigation we report extensive refolding studies of RM6 at different temperatures and GdnHCl concentrations monitored by CD and fluorescence to probe for changes in secondary and tertiary structure, respectively. The measurements permitted us to determine activation parameters as a function of denaturant concentration. The results demonstrate convincingly that the variation with GdnHCl concentration of the activation parameters ∆H # , ∆S # and ∆G # is very similar for unfolding and refolding. For both processes the activation properties approach a maximum in the vicinity of the denaturant concentration, c (K=1) , where the equilibrium constant equals 1, i.e. ∆G 0 equals zero. CD and fluorescence refolding kinetics are described by identical constants suggesting that the formation of secondary and tertiary structure occurs simultaneously. Refolding is, however, characterized by a more complex mechanism than unfolding. Although the general pattern is dominated by the sequence monomers to dimers to tetramers, parallel side reactions involving dimers and monomers have to be envisaged in the initial folding phase, supporting the view that the native state of RM6 can be reached by several rather than a single pathway.

Trypsin inhibitors secreted by human colorectal adenocarcinoma cell lines were analyzed by reverse zymography. Among eleven cell lines analyzed, the major inhibitor secreted was protease nexin-II (PN-II), a secreted form of amyloid Β protein precursor (APP) containing a Kunitz-type serine proteinase inhibitor domain. Expression of the APP gene was also confirmed in the cell lines and the main APP mRNA species were PN-II types. The APP gene expression was constant during cell growth in vitro . On the other hand, the rate of extracellular PN-II accumulation markedly increased after long-term serum-free maintenance of the confluent culture. The extracellular accumulation of PN-II was also strongly stimulated either by interleukin-1β (IL-1β) treatment or to a lesser extent by basic fibroblast growth factor, tumor necrosis factor-α, hepatocyte growth factor or epidermal growth factor. Neither serum depletion-nor IL-1β-induced stimulation of extracellular PN-II accumulation were accompanied by obvious alteration of the levels of APP mRNA and cellular APP holoprotein, suggesting that the enhanced extracellular accumulation of PN-II might result from up-regulation of the secretory pathway of APP. The IL-1β-induced PN-II secretion was significantly inhibited by relatively high concentrations (50–200μg/ml) of aprotinin, a serine proteinase inhibitor, in a dose-dependent manner without obvious cell-toxic effects.

The dried latex of the mountain papaya, Carica candamarcensis , was chromatographed on CM-Sephadex C50, giving rise to three peaks (CCI, CCII and CCIII) with amidase activity on N-α-benzoyl-DL-arginine-4-nitroanilide. The less basic, most active, peak, CCI, was separated into two components, CCIa and CCIb, by reverse-phase HPLC under denaturing conditions. The primary structures of CCla and CCIb are presented. They were deduced from sequence analysis of the whole proteins and peptides resulting from enzymatic digestions. Both proteinases are made of 213 amino acid residues, CCIb sharing 88–89% similarity with the three subvariants (G 90 /R 212 , E 90 /R 212 , E 90 /K 212 ) of CCIa. 139–140 amino acid residues (65.8%) of CCIa and 141 residues (66.5%) of CCIb are common to papain. The seven cysteine residues are aligned with those of papain and the catalytic triad (Cys 25 , His 159 , Asn 175 ) of all cysteine peptidases of the papain family is conserved. The similarity with the other cysteine proteases from Carica papaya is discussed.

Recently interest has been focused on secretory leucocyte protease inhibitor (SLPI) and its role in immediate hypersensitive reactions, possibly by inhibiting mast cell chymase. The purpose of this investigation was to show whether or not SLPI is produced in mast cells. Double-immunolabelling revealed that SLPI coexists with mast cell tryptase (60%) and chymase (37%). On the other hand, in situ hybridisation studies demonstrated the expression of SLPI mRNA in all mast cells. The differences in results can be attributed to the fact that in situ hybridisation is a more sensitive method than immunohistochemistry. Hence, we conclude that SLPI is produced in human tonsillar mast cells.

The stereochemical features of the interaction between the sulfur atom of methionine residues and surrounding atoms are examined on a large set of known protein crystal structures. It appears that the minimum energy conformations observed in small molecule crystals are not observed within the protein core. This suggests that these interactions are either of little intensity, though they might contribute to regulate the protein physiological behavior, or physicochemically different from their counterpart in small molecule crystals.

Trypsin was purified from crayfish, Pacifastacus leniusculus , hepatopancreas, and the gene that encoded this enzyme was cloned from a hepatopancreas cDNA library. Crayfish trypsin is synthesized as a zymogen according to the sequence of the putative precursor peptide. The authenticity of the trypsinogen is supported by the deduced amino acid sequence and confirmed by the N-terminal amino acid sequence of the mature protein. The enzyme has features characteristic of a trypsin, such as a specific binding pocket. Sequence comparison shows that crayfish trypsin is similar to those of other species, with the exception that six cysteine residues present in vertebrates are missing. Some structural characteristics, such as the length of the signal peptide and a calcium binding site, are similar to bacterial trypsin.