Volume 383, Issue 12

Lysosomal proteinases are translated as preproenzymes, transferred through the Golgi apparatus as proenzymes, and localized in lysosomes as the mature enzymes. Pulsechase analyses and the immunoisolation of proenzymes or recombinant proenzymes are useful tools for analyzing this process, but the processing proteinases that participate in this pathway are largely unknown. Recently, we developed a new method for analyzing processing proteinases using Bafilomycin A1 and proteinase inhibitors. Here we summarize the recent progress including our results obtained using this method.

HB13 human myoblasts express physiological and biochemical markers associated with myoblast differentiation in nonhuman cell culture model systems. During differentiation, HB13 myoblasts also demonstrate fusionrelated increases in cathepsin B activity and protein levels. These increases are associated with an increase in levels of cathepsin B mRNA suggesting the involvement of transcriptional regulatory mechanisms. To examine these mechanisms human myoblasts were transfected with cathepsin B nested deletion promoter constructs within the 1.8 kb 5 promoter 1 region of the human catB gene. Transfected myoblasts that were maintained under differentiating conditions demonstrated higher promoter activity than those maintained in proliferating conditions. The highest activity was obtained with pSCB2-3 ( 1279/ +56 bp), a construct containing two putative upstream Ebox elements. Cotransfection experiments demonstrated that MyoD and myogenin transactivate cathepsin B promoter activity. Electrophoretic mobility shift assays of nuclear extracts incubated with an oligonucleotide containing two upstream Ebox elements found within the cathepsin B promoter demonstrated two band shifts. The band shifts were abolished using an oligonucleotide with mutations in both Ebox elements. Moreover, the shifted bands were supershifted and abolished when incubated with antimyogenin and antiMyoD, respectively. Collectively, these data support myogenic transcription factor mediated activation of cathepsin B expression during myogenesis.

Synovial fibroblasts from rheumatoid arthritis patients express elevated levels of the transcription factor Egr 1. The metabolic consequences of Egr-1 overexpression in fibroblasts are not known in detail. Therefore we searched for gene products that are differentially expressed in Egr-1high versus Egr-1low fibroblasts. Immortalized synovial fibroblasts were transfected with two different Egr-1 expression vectors. Expression of recombinant Egr-1 was confirmed by RTPCR and immunoblots. Random arbitrarily primed PCR revealed that Egr-1 induces enhanced transcription levels of the α1 chain of type I collagen. Increased expression of the α2 (I) chain could also be observed. We found enhanced levels of type I collagen propeptide in supernatants and stronger signals of α2 (I) protein in extracts of the Egr 1high expressing clone versus controls. Additionally, Egr-1 was transiently expressed in fibrosarcoma cells. These cells showed a pronounced elevation of type I collagen (α1) transcripts as well. Moreover, we could demonstrate that Egr-1 induces transcription of other genes including type II collagen (α1) and plateledderived growth factor β1. These data suggest that upregulation of Egr-1 might contribute to fibrosis observed in rheumatoid arthritis synovium by activation of genes encoding the α1 and α2 chains of type I collagen.

The soluble Nterminal ectodomain of amyloid precursor protein (sAPP), resulting from secretasemediated proteolytic processing, has been shown to function as a growth factor for epithelial cells, including keratinocytes and thyrocytes. Extracellularly applied sAPP binds to a cell surface receptor and exhibits a patchy binding pattern reminiscent of that observed for raft proteins. Here we show that (i) the receptorbound sAPP resides in a detergentinsoluble membrane microdomain which cofractionates in density gradients with cholesterolrich membrane rafts and caveolae; (ii) the sAPPbinding microdomains are different from caveolae; and (iii) sAPP is capable of binding to isolated rafts and inducing tyrosine phosphorylation of some raft proteins. These observations suggest that a novel type of membrane raft is involved in sAPP signaling.

* Corresponding author Two functionally redundant enzymes, trigger and the hsp70 chaperone DnaK, have been assist de novo protein folding in E. coli. Trigger is a peripheral peptidyl prolyl cis/trans isomerase (PPIase) of the large subunit of the ribosome. contrast, DnaK displays two catalytic features: secondary amide peptide bond cis/trans isomerase (APIase) function supplemented by the ATPase APIases accelerate the cis/trans isomerization nonprolyl peptide bonds. Both enzymes have for an unfolded polypeptide chain. The diminished temperature cell viability in the presence factor variants with impaired PPIase activity that the enhancement of folding rates plays role in protein folding in vivo. For the DnaK increase in the folding yield in vitro, the minimal model for APIase catalysis involves the catalyzed partitioning of a rapidly formed folding intermediate could be inferred from the DnaK/DnaJ/GrpE/ assisted refolding of GdmCldenatured luciferase. Using three different peptide bond cis/trans isomerization assays in vitro, we could show that no overlapping substrate specificity of trigger and DnaK. We propose that only if trigger recruits supplementing molecules is it capable exhibiting functional complementarity with DnaK protein folding.

Toc34 is a protein of the chloroplast outer envelope membrane that acts as receptor for preproteins containing a transit sequence. The recognition of preproteins by Toc34 is regulated by GTP binding and phosphorylation. The phosphorylation site of Toc34 is located at serine 113, close to the postulated triphosphate binding site. This can explain the downregulation of Toc34 by phosphorylation, resulting in the loss of GTP binding. Vice versa, GTP but not GDP binding of Toc34 influences the phosphorylation. The nucleotide specificity of Toc34 is not only determined by the classical nucleotide binding domains but by a nontypical region at the Nterminus of the protein. As a result, the GTP binding properties are unusual, since the triphosphate moiety of GTP is bound with higher affinity than the purine base. Purified Toc34 hydrolyses GTP at a low rate, which could regulate the receptor function. The rate of hydrolysis is greatly stimulated by a precursor protein.

Intracellular phosphorylation of cis-4-methylsphingosine was previously shown to result in a metabolically stable compound that accumulates in Swiss 3T3 fibroblasts and mimics the mitogenic effect induced by the shortlived sphingosine metabolite, sphingosine 1-phosphate. In the present study incubation of neuroblastoma B104 cells with cis-4-methylsphingosine (10 M) also resulted in an intracellular accumulation of its phosphorylated derivative that was, however, associated with the concentrationdependent induction of apoptosis, not observed after treatment with 10 M of sphingosine-1-phosphate or sphingosine, respectively. In B104 cells, cis-4-methylsphingosine stimulated p38 mitogenactivated protein kinase (p38 MAPK) and simultaneously inhibited extracellular signalregulated kinase (ERK), whereas sphingosine and sphingosine 1-phosphate only stimulated p38 MAPK without suppression of ERK. Inhibition of cis-4-methylsphingosine phosphorylation reduced both, apoptosis and concurrent regulation of mitogenactivated protein kinases (MAPKs), suggesting that the unusual accumulation of the phosphorylated sphingoid base was responsible for the biological effects. Furthermore, inhibition of p38 MAPK prevented cis-4-methylsphingosineinduced apoptosis, while suppression of the ERK pathway in the presence of sphingosine or sphingosine-1-phosphate resulted in apoptosis, indicating that the simultaneous opposite regulation of the two MAPKs was required for the induction of apoptosis.

Protein complexes formed by S100A8 and S100A9 represent the only AAbinding capacity in the human neutrophilic cytosol and are involved in the intracellular arachidonic acid metabolism. The formation of S100A8/A9 protein complexes and the binding of calcium to the complexes are prerequisites for the specific binding of polyunsaturated fatty acids. The present study was undertaken to characterize the fatty acid binding site within the protein complex. Deletions at both termini and point mutations of different basic amino acids especially within the extended Cterminal tail of human S100A9 were introduced. The S100A9 mutant proteins were then analyzed with respect to proteinprotein interaction (GST pull downassay and yeast twohybrid system) and functional properties (arachidonic acid and calcium binding). The data give strong evidence that the unique Ctail of S100A9 containing the three consecutive histidine residues (His103-His105) represents the region to which the fatty acid carboxygroup is bound to the protein complex. The localization of the AAbinding site within the unique Ctail of S100A9 correlates with the fact that fatty acid binding has not yet been reported for other S100 proteins.

The hemoglobin of the indigo snake (Drymarchon corais erebennus, Colubrinae) consists of two components, HbA and HbD, in the ratio of 1:1. They differ in both their α and β chains. The amino acid sequences of both α chains (αA and αD) and one β chain (βI) were determined. The presence of an αDchain in a snake hemoglobin is described for the first time. A comparison of all snake β chain sequences revealed the existence of two paralogous β chain types in snakes as well, which are designated as βI and βII type. For the discussion of the physiological properties of Drymarchon hemoglobin, the sequences were compared with those of the human α and β chains and those of the closely related water snake Liophis miliaris where functional data are available. Among the heme contacts, the substitution αD58(E7)His->Gln is unusual but most likely without any effect. The residues responsible for the main part of the Bohr effect are the same as in mammalian hemoglobins. In each of the three globin chains only two residues at positions involved in the α1/_2 interface contacts, most important for the stability and the properties of the hemoglobin molecule, are substituted with regard to human hemoglobin. On the contrary, nine, eleven, and six α1/β1 contact residues are replaced in the αA, αD, βI chains, respectively.

The serine protease, tissue kininogenase (kallikrein), belongs to a unique family of enzymes that cleaves the decapeptide, kallidin, from the endogenous substrate kininogen. By analysis of genealogy patterns rat KLK gene family members have been detected in ovarian luteinizing granulosa cells of both gonadotrophin treated and nontreated control rats. Recently, we demonstrated that tissue kininogenase showed intense immunolabeling in angiogenic endothelial cells isolated from bovine mature and regressing corpora lutea. Therefore, the question to answer was whether granulosa cells associated with ovarian vascularization possess the same capacity to express the kallikreinkinin cascade as do microvascular endothelial cells. As a first step, experiments were designed to determine the expression and visualization of tissue kininogenase (both active and pro forms) as well as kininogen and kinin receptors in granulosa cells of different developmental stages and segments of the ovarian follicle by immunoperoxidase assay, confocal fluorescent microscopy and in situ hybridization.

The acceleration effect of chondroitin-4-sulfate(CS) proteoglycan on the processing of procathepsin L in vitro was investigated using enzyme purified from the culture medium of MLC cells. Procathepsin L was slightly processed even when it was incubated without CSproteoglycan for 60 min in 50 mM acetate buffer, pH 5.5, and trace amounts of the 31 kDa mature form and 35 38 kDa intermediates of cathepsin L were formed. On the other hand, in the presence of CSproteoglycan, procathepsin L was completely converted to the mature form within the same 60 minute time period. Moreover, ZPheArgMCA hydrolyzing activity was increased significantly by the incubation with CSproteoglycan, while no considerable increase in the activity was observed during the incubation without CSproteoglycan. Since the specific cathepsin L inhibitor, CLIK-195, inhibited the processing of procathepsin L accelerated by CSproteoglycan, the trace amount of cathepsin L activity may participate in the processing. These results suggest that CSproteoglycan may play a role in accelerating the processing of procathepsin L as an endogenous enhancer in the extracellular environment in vivo.

Based on its monophenolic structure and given its pharmacological and toxicological importance, the ability of tyrosinase to oxidize acetaminophen was studied for the first time. Progress curves showed a transient phase characteristic of the monophenolase activity of tyrosinase prior to attaining the steadystate. The duration of this transient phase strongly increased with the drug concentration, which would partly explain why paracetamol oxidation by tyrosinase has not been studied hitherto. The pathway is enhanced by the presence of minute amounts of Ldopa, which shortens the length of the lag period. Acetaminophen oxidation was inhibited by tropolone, a selective inhibitor of tyrosinase. The presence of the corresponding odiphenol as intermediate was demonstrated with ascorbic acid by chemical oxidation using NaIO4 and by HPLC analysis, indicating that acetaminophen is oxidized by the monophenolase activity of tyrosinase to its corresponding oquinone. These results contribute to our knowledge of the oxidation mechanisms of acetaminophen.

Recombinant baculovirus particles displaying green fluorescent protein (GFP) fused to the major envelope glycoprotein gp64 of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) were characterized by fluorescence correlation spectroscopy (FCS). FCS detected Brownian motion of single, intact recombinant baculovirus display particles with a diffusion coefficient (D) of (2.89±0.74)10 8 cm2s 1 and an apparent hydrodynamic radius of 83.35±21.22 nm. In the presence of sodium dodecyl sulfate (SDS), Triton X-100, and octylglucoside, the diffusion time was reduced to the 0.2 ms range (D = 7.5710 7 cm2s 1), showing that the fusion proteins were anchored in the viral envelope. This allowed for a calculation of the number of single gp64 fusion proteins incorporated in the viral membrane. A mean value of 3.2 fluorescent proteins per virus particle was obtained. Our results show that FCS is the method of choice for studying enveloped viruses such as a display virus with one component being GFP.

While the biochemical mechanisms mediating repression of NFκB activity by glucocorticoids (GCs) are relatively well studied, the role of promoter architecture for the effects of GCs on NFκB remains poorly characterized. Therefore we constructed a set of synthetic promoter reporter constructs containing various numbers of GCresponsive elements (GREs) in distinct distances to NFκB binding sites. TNFαinduced activity of a synthetic promoter controlled by three NFκB binding sites was repressed by dexamethasone. The presence of only one GRE in the vicinity of the κB sites abolished this repression and allowed synergistic transcriptional activation by NFκB and the glucocorticoid receptor (GR). The synergism identified here was not affected by the number of GREs, but strictly depends on the spacing between GREs and κB sites. These experiments reveal that the functional interplay between NFκB and the GR also involves dependent on the promoter context synergistic stimulation of transcription.

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) and type 2 (HAI-2) are recently identified integral membrane Kunitztype proteinase inhibitors. They have important regulatory roles in pericellular activation of hepatocyte growth factor/scatter factor (HGF/SF) which is critically involved in the development and regeneration of various tissues. Recent reports suggest that HGF/SF is also involved in testicular development and spermatogenesis. In this study, we analyzed the expression of HAIs in the testis. In human testis, HAI-2 was strongly expressed whereas HAI-1 mRNA was hardly detectable. Of interest was the observation that the mRNA size of HAI-2 was shorter in the testis (1.2 kb) than those in the other tissues such as placenta (1.5 kb). Subsequent experiments revealed that there are two major transcription start sites of the HAI-2 gene, which are ~30 bp and ~360 bp upstream from the translation initiation ATG codon. Although the latter site appeared to be mainly used in the placenta and other nontesticular organs, only the former site is used in testis, resulting in the ~300 bp shorter mRNA. An immunohistochemical study using a specific monoclonal antibody raised against human HAI-2 protein indicated that HAI-2 is expressed exclusively in primary spermatocytes. These results suggest a distinct regulation of HAI 2 gene expression in testis and that HAI-2 may play a role in the process of spermatogenesis.

A merocrine released protein (named 115k protein) was highly enriched from the secretion of the rat coagulating gland. The protein has a molecular mass of 115 kDa as calculated by SDSPAGE under reducing conditions. Furthermore, the 115 kDa protein is glycosylated, and carries Man, GlcNAc, Gal, Fuc and sialic acid residues. For identification, Nterminal amino acid and nucleotide sequence analyses were performed. The sequences obtained showed 86 to 100% identity with human and mouse IgGFc binding proteins. The functional capacity of IgG binding of the 115 kDa protein was shown by overlay experiments, indicating its membership in the IgG binding protein family.

Incubation of mitochondrial outer membrane porin with citraconic anhydride prior to treatment with fluorescein isothiocyanate (FITC) resulted in the labeling of a set of lysines located at a boundary between the water phase and lipid phase. The elution pattern of porin from the cation exchanger has been considered as indicative for the location of lysines. Electrical measurements after reconstitution of the modified protein in lipid bilayer membranes revealed that certain specific lysine residues are more susceptible to alterations. The innermost positive residues were only slightly influenced, while the outermost lysines exhibited a substantial change in channel properties. These results suggest the presence of critical charged residues in mitochondrial outer membrane porin that may be responsible for both the channels selectivity and its voltage dependence.

A cysteine endopeptidase from rat spleen was purified, characterized and its gene cloned. This enzyme was originally recognized by its action of producing kininpotentiating peptide from a plasma protein. We named it cathepsin Y due to its localization, acidic pH optimum and the presence of the same set of active site amino acids as in other thiol cathepsins. Here we show the total sequence of the mRNA obtained by means of TaKaRa 5 Full RACE Core Set and complete the previously reported sequence. This completion of the mRNA sequence resulted in the omission of the strangely attached Cterminal peptide from cathepsin Y.