Volume 383, Issue 5

Gap junctions are clustered channels between contacting cells through which direct intercellular communication via diffusion of ions and metabolites can occur. Two hemichannels, each built up of six connexin protein subunits in the plasma membrane of adjacent cells, can dock to each other to form conduits between cells. We have recently screened mouse and human genomic data bases and have found 19 connexin (Cx) genes in the mouse genome and 20 connexin genes in the human genome. One mouse connexin gene and two human connexin genes do not appear to have orthologs in the other genome. With three exceptions, the characterized connexin genes comprise two exons whereby the complete reading frame is located on the second exon. Targeted ablation of eleven mouse connexin genes revealed basic insights into the functional diversity of the connexin gene family. In addition, the phenotypes of human genetic disorders caused by mutated connexin genes further complement our understanding of connexin functions in the human organism. In this review we compare currently identified connexin genes in both the mouse and human genome and discuss the functions of gap junctions deduced from targeted mouse mutants and human genetic disorders.

Trypanosomes are the etiological agents of human sleeping sickness and livestock trypanosomosis (nagana), which are major diseases in Africa. Their cysteine proteases (CPs), which are members of the papain family, are expressed during the infective stages of the parasites life cycle. They are suspected to act as pathogenic factors in the mammalian host, where they also trigger prominent immune responses. Trypanosoma congolense, a major pathogenic species in livestock, possesses at least two families of closely related CPs, named CP1 and CP2. Congopain, a CP2-type of enzyme, shares structural and functional resemblances with cruzipain from T. cruzi and with mammalian cathepsin L. Like CPs from other Trypanosomatids, congopain might be an attractive target for trypanocidal drugs. Here we summarise the current knowledge in the two main areas of research on congopain: first, the biochemical properties of congopain were characterised and its substrate specificity was determined, as a first step towards drug design; second, the possibility was being explored that inhibition of congopain by hostspecific antibodies may mitigate the pathology associated with trypanosome infection.

Proteolytic maturation of lysosomal proteinases is initiated after receptormediated targeting to prelysosomal compartments, while terminal processing occurs upon delivery to lysosomes. These late processing events are impaired in patients suffering from inherited lysosomal disorders, such as sialic acid storage disease and mucolipidosis II (Icell disease). Lysosomes in the affected cells display marked changes in their physiological and morphological properties, with features reminiscent of prelysosomal compartments. This indicates that the absence of mature lysosomes interferes with the final processing steps during the biosynthesis of lysosomal proteinases. Thus, impaired proteinase maturation reflects an incompetent lysosomal apparatus and as such can be seen as a hallmark of lysosomal storage diseases.

Calpain is a calciumactivated non lysosomal neutral thiol protease (EC 3.4.22.17) present in a wide variety of eukaryotic cells. Calpain is usually present as an inactive form and is activated by calcium ions and phospholipids. The ability of calpain to alter, by limited proteolysis, the activity or function of numerous cytoskeletal proteins, enzymes, and receptors suggests its involvement in various Ca2+ regulated cellular functions. In this review we focus on the differentiation of mesenchymal stem cells, such as the myoblastic, osteoblastic, chondrocytic, and adipocytic lineages, and the biological significance of calpain in its regulation. Calpain has been implicated in the differentiation of myoblasts through the turnover of glycoproteins. In preosteoblastic cells, calpain is important in mediating the proliferative and prodifferentiating effects of parathyroid hormone and bone morphogenetic proteins. For the differentiation of chondrocytes, calpain is involved in cartilagematrix mineralization. Furthermore, calpain is required for the differentiation of 3T3-L1 preadipocytes into adipocytes, involving the transcriptional activation of the C/EBPα gene and the degradation of the cyclindependent kinase inhibitor p27 during the mitotic clonal expansion phase of adipocyte differentiation. We summarize these regulatory effects of calpain on the differentiation of mesenchymal stem cells and speculate on the function and location of calpain in the differentiation processes.

A replication fork barrier at the 3end of mouse ribosomal RNA genes blocks bidirectional fork progression and limits DNA replication to the same direction as transcription. This barrier is an inherent property of a defined DNAprotein complex including transcription termination factor I, and specific proteinprotein interactions occur between this factor and protein(s) of the replication machinery. Here we report that a second DNAbinding protein is essential for barrier activity. We have purified and functionally characterised the protein from HeLa cells. The final preparation contained two polypeptides with molecular masses of 70 and 86 kDa, respectively. Both polypeptides interact with a GCstretch adjacent to the binding site of transcription termination factor I. The specificity of binding to the barrier DNA was demonstrated in an electrophoretic mobility shift assay. The biochemical properties of this protein resemble that of Ku antigen, a human nuclear DNAbinding heterodimer that is the target of autoimmuneantibodies in several autoimmune diseases. Recombinant Ku protein, purified as heterodimer from coinfected insect cells, is able to partially rescue the barrier activity in Kudepleted HeLa cell extracts. These data demonstrate that transcription termination factor I and Ku act synergistically to prevent headon collision between the replication and the transcription machinery.

Constant levels of thyroid hormones in the blood are principal requirements for normal vertebrate development. Their release depends on the regulated proteolysis of thyroglobulin which is extracellularly stored in the follicle lumen under resting conditions. Thyroglobulin is proteolytically degraded to a major part in lysosomes, but in part also extracellularly leading to the release of thyroxine. Extracellularly occurring lysosomal enzymes are most probably involved in the proteolytic release of thyroxine. In this study we have analyzed the secretion of cathepsin B by thyroid follicle cells (primary cells as well as FRTL-5 cells) and its regulation by thyroid stimulating hormone, which stimulated the secretory release of the proenzyme as well as of mature cathepsin B. Within one to two hours of stimulation with thyroid stimulating hormone, the cathepsin B activity associated with the plasma membrane increased significantly. This increase correlated closely with the localization of lysosomes in close proximity to the plasma membrane of cultured thyrocytes as well as with the thyroxine liberating activity of thyrocyte secretion media. These observations indicate that thyroid stimulating hormone induces the secretion of cathepsin B, which contributes to the extracellular release of thyroxine by thyrocytes.

Activation of calpain results in the breakdown of α II spectrin (αfodrin), a neuronal cytoskeleton protein, which has previously been detected in various in vitro and in vivo neuronal injury models. In this study, a 150 kDa spectrin breakdown product (SBDP150) was found to be released into the cellconditioned media from SHSY5Y cells treated with the calcium channel opener maitotoxin (MTX). SBDP150 release can be readily quantified on immunoblot using an SBDP150- specific polyclonal antibody. Increase of SBDP150 also correlated with cell death in a timedependent manner. MDL28170, a selective calpain inhibitor, was the only protease inhibitor tested that significantly reduced MTXinduced SBDP150 release. The cellconditioned media of cerebellar granule neurons challenged with excitotoxins (NMDA and kainate) also exhibited a significant increase of SBDP150 that was attenuated by pretreatment with an NMDA receptor antagonist, R()-3-(2-carbopiperazine-4-yl)propyl-1- phosphonic acid (CPP), and MDL28170. In addition, hypoxic/hypoglycemic challenge of cerebrocortical cultures also resulted in SBDP150 liberation into the media. These results support the theory that an antibody based detection of SBDP150 in the cellconditioned media can be utilized to quantify injury to neural cells. Furthermore, SBDP150 may potentially be used as a surrogate biomarker for acute neuronal injury in clinical settings.

Heparindeficient mice, generated by gene targeting of Ndeacetylase/Nsulfotransferase-2 (NDST-2), display severe mast cell defects, including an absence of stored mast cell proteases. However, the mechanism behind these observations is not clear. Here we show that NDST-2+/+ bone marrowderived mast cells cultured in the presence of IL-3 synthesise, in addition to highly sulphated chondroitin sulphate (CS), small amounts of equally highly sulphated heparinlike polysaccharide. The corresponding NDST-2/ cells produced highly sulphated CS only. Carboxypeptidase A (CPA) activity was detected in NDST+/+ cells but was almost absent in the NDST/ cells, whereas tryptase (mouse mast cell protease 6; mMCP-6) activity and antigen was detected in both cell types. Antigen for the chymase mMCP-5 was detected in NDST-2+/+ cells but not in the heparindeficient cells. Northern blot analysis revealed mRNA expression of CPA, mMCP-5 and mMCP-6 in both wildtype and NDST-2/ cells. A ~36 kDa CPA band, corresponding to proteolytically processed active CPA, as well as a ~50 kDa proCPA band was present in NDST-2+/+ cells. The NDST-2/ mast cells contained similar levels of proCPA as the wildtype mast cells, but the ~36 kDa band was totally absent. This indicates that the processing of proCPA to its active form may require the presence of heparin and provides the first insight into a mechanism by which the absence of heparin may cause disturbed secretory granule organisation in mast cells.

Carcinoembryonic antigenrelated cell adhesion molecule 1 (CEACAM1), an Iglike transmembrane protein, functions in cell adhesion, angiogenesis and epithelial cell morphogenesis, and has been identified as a tumor suppressor. For all of these functions, CEACAM1 requires signaling capabilities. However, the mechanisms of CEACAM1-mediated signaling are only poorly understood. Here we characterized for the first time CEACAM1 expression and signaling in the neuroendocrine rat pheochromocytoma PC12 cell line. Stimulation of CEACAM1 by ligation on the cell surface with antibodies induced formation of large CEACAM1 clusters and a rapid and transient CEACAM1 tyrosine dephosphorylation. Functionally, this dephosphorylation correlated with a reduced association between CEACAM1 and the tyrosine phosphatase SHP2. Clustering also stimulated binding of CEACAM1 to the actin cytoskeleton, measured by a partial translocation of CEACAM1 into the insoluble fraction after detergent extraction. Both tyrosine dephosphorylation and interaction with the cytoskeleton were sensitive to neuronal differentiation of PC12 cells. The first detected downstream activation of the mitogenactivated protein kinases ERK1 and ERK2, but not of JNK or p38, describes a novel target of CEACAM1-mediated signaling and contributes to the understanding of how CEACAM1 regulates cellular function.

The compound 5-(ethoxycarbonyl)-5-methyl-1-pyrroline Noxide (EMPO) is a hydrophilic cyclic nitrone spin trap, which, in contrast to DMPO, forms a relatively stable superoxide adduct (t1/2=8.6 min) with an EPR spectrum similar to the respective DMPO adduct. In order to find the optimal degree of lipophilicity of this novel type of spin trap with respect to the detection of radicals formed during lipid peroxidation, the ethoxy group of EMPO was replaced by alkoxy substituents of increasing chain length, leading to the methoxy (MeMPO), 1-propoxy (PrMPO), 1-butoxy (BuMPO), and 1-octyloxy (OcMPO) derivatives of EMPO. The stability of their superoxide adducts was found to be strongly dependent on the size of the alkoxycarbonyl group. Increasing chain length of the alkoxyl substituent decreased the stability of alkoxyl radical adducts of MeMPO, EMPO, and PrMPO, but increased the stability of OcMPO adducts. The stability of alkoxyl radical adducts of BuMPO, on the other hand, were practically independent of the size of the alkoxyl group. Detection of lipid alkoxyl radicals formed by peroxidizing linoleic acid in a stationary system was therefore only possible with the most lipophilic spin trap, OcMPO. However, with the more hydrophilic spin traps MeMPO, EMPO, PrMPO, and BuMPO optimal EPR signal intensity could be obtained when a slowflow system was used. Thus, within this series EMPO is the best spin trap for the detection of superoxide; OcMPO, on the other hand, is most suitable for the detection of lipid alkoxyl radicals.

Glutathione Stransferases (GSTs), which occur abundantly in most organisms, are essentially involved in the intracellular detoxification of numerous substances including chemotherapeutic agents, and thus play a major role in the development of drug resistance. A gene encoding a protein with sequence identity of up to 37% with known GSTs was identified on chromosome 14 of the malarial parasite Plasmodium falciparum. It was amplified using gametocyte cDNA and expressed in Escherichia coli as a hexahistidyltagged protein of 26 kDa subunit size. The homodimeric enzyme (PfGST) was found to catalyse the glutathione (GSH)dependent modification of 1-chloro-2,4-dinitrobenzene and other typical GST substrates such as onitrophenyl acetate, ethacrynic acid, and cumene hydroperoxide. The Km value for GSH was 164±20 M. PfGST was inhibited by cibacron blue (Ki=0.5 M), Shexylglutathione (Ki=35 M), and protoporphyrin IX (Ki=10 M). Hemin, a most toxic compound for parasitised erythrocytes, was found to be an uncompetitive ligand of PfGST with a Ki of 6.5 M. Based on the activity of PfGST in extracts of P. falciparum, the enzyme represents 1 to 10% of cellular protein and might therefore serve as an efficient in vivo buffer for parasitotoxic hemin. Destabilising ligands of GST are thus expected to be synergistic with the antimalarial drug chloroquine, which itself was found to be a very weak inhibitor of PfGST (IC50 >200 M). Xray quality crystals of PfGST (25020050 m) will serve as starting point for structurebased drug design.

The protein AUF1/hnRNP D was one of the first factors identified that binds to the AUrich region of certain mRNAs and mediates their fast degradation. Here we describe experiments to address the structural determinants for the binding of AUF1 to the RNA by combining comparative molecular modeling with gel shift assays. From our model of the RNA binding region of AUF1 we predicted that it interacts with RNA predominantly through stacking interactions that do not provide basespecific recognition. Only two RNA positions bound by AUF1 show base preferences: one for pyrimidine bases and the second for a conserved adenine residue. Gel shift assays with a panel of RNA oligonucleotides largely confirmed these modelbased binding determinants. An alignment with proteins of the hnRNP family demonstrated that the amino acids involved in the stacking interactions are conserved whereas those that confer a basespecific recognition in AUF1 are variable.

Various types of proteinases are implicated in the malignant progression of human and animal tumors. Proteinase inhibitors may therefore be useful as therapeutic agents in antiinvasive and antimetastatic treatment. The aims of this study were (1) to estimate the relative importance of proteinases in B16 cell invasion in vitro using synthetic, classspecific proteinase inhibitors and (2) to assess the inhibitory effect of some naturally occurring cysteine proteinase inhibitors. Serine proteinase inhibitor reduced invasiveness by up to 24%, whereas inhibition of aspartic proteinases reduced invasion by 11%. Synthetic inhibitors of cysteine proteinases markedly impaired invasion: cathepsin B inhibitors, particularly Ca 074Me, inhibited invasion from 20 40%, whereas cathepsin L inhibitor Clik 148 reduced invasion by 11%. The potato cysteine proteinase inhibitor PCPI 8.7 inhibited invasion by 21%, whereas another potato inhibitor, PCPI 6.6, and the mushroom cysteine proteinase inhibitor clitocypin had no effects. As the inhibitors that inhibited cathepsin B were in general more efficient at impairing the invasiveness, we conclude that of the two cysteine proteinases, cathepsin B plays a more important role than cathepsin L in murine melanoma cell invasion.

Cysteine proteases of the malaria parasite Plasmodium falciparum, known as falcipains, are promising targets for antimalarial chemotherapy. We evaluated cultured parasites for the stagespecific expression of cysteine proteases and sensitivity to cysteine protease inhibitors. Protease activity and inhibitor sensitivity varied markedly over time. Cysteine protease activity was greatest in early trophozoites, while sensitivity to cysteine protease inhibitors was greatest in mature trophozoites. Our results indicate the importance of considering the stagespecific effects of antimalarials and are consistent with the conclusion that the principal antimalarial activity of cysteine protease inhibitors is due to a block in hemoglobin hydrolysis.

Besides its physiological role in lysosomal protein breakdown, extralysosomal cathepsin B has recently been implicated in apoptotic cell death. Highly specific irreversible cathepsin B inhibitors that are readily cellpermeant should be useful tools to elucidate the effects of cathepsin B in the cytosol. We have covalently functionalised the poorly cellpermeant epoxysuccinyl based cathepsin B inhibitor [RGlyGlyLeu(2S, 3S)tEpsLeuProOH; R=OMe] with the Cterminal heptapeptide segment of penetratin (R=AhxArg ArgNleLysTrpLysLysNH2). The high inhibitory potency and selectivity for cathepsin B versus cathepsin L of the parent compound was not affected by the conjugation with the penetratin heptapeptide. The conjugate was shown to efficiently penetrate into MCF-7 cells as an active inhibitor, thereby circumventing an intracellular activation step that is required by other inhibitors, such as the prodruglike epoxysuccinyl peptides E64d and CA074Me.

We explored the unique substrate specificity of the primary S1 subsite of human urinary kallikrein (hK1), which accepts both Phe or Arg synthesizing and assaying peptides derived from PhenylacetylPheSer ArgEDDnp, a previously described inhibitor with analgesic and antiinflammatory activities [Emim et al., Br. J. Pharmacol. 130 (2000), 1099 1107]. Phe was substituted by amino acids containing larger aliphatic or aromatic side chains as well as by nonnatural basic amino acids, which were designed to combine a large hydrophobic and/or aromatic group with a positivelycharged group at their side chains. In general, all peptides with basic amino acids represented better inhibitors than those with hydrophobic amino acids. Furthermore, the S1 subsite specificity proved to be much more selective than the mere distinction between Phe and Arg, for minor differences in the side chains of the nonnatural amino acids resulted in major differences in the Ki values. Finally, we present a series of peptides that were assayed as competitive inhibitors for human tissue kallikrein that may lead to the development of novel peptides, which are both more potent and selective.

The mechanism by which proteins form amyloid fibrils is of high interest to the scientific community as its understanding could resolve questions relevant to conformational diseases. The structural and energetic basis of the process is still largely unknown. The main controversial issue is the coexistence of several protein conformations. Three models for the mechanism of protein fibrillogenesis have been proposed which need to be tested by experiments. In this report, amyloid fibrils grown from human stefin B (type I cystatin) are described. This physiologically relevant protein readily forms fibrils in vitro, in contrast to the homologue human stefin A which forms fibrils under extreme conditions only. In order to specifically label stefin B fibrils in vitro, rabbit polyclonal antibody and mouse monoclonal antibody A6/2 against human stefin B were used for immunogold labelling. Samples were examined by transmission electron microscopy. Fibrils of stefin B were strongly labelled using polyclonal antibody and Protein A gold, whereas no positive reaction was observed with monoclonal antibody A6/2.

Lysosomal serine and cysteine proteases are reported to play a role in collagen degradation. In this study, the activities of the lysosomal cysteine proteases cathepsin B and H, dipeptidyl peptidase I, and the serine protease tripeptidyl peptidase I and dipeptidyl peptidase II, all ascribed a role in collagen digestion, were compared with those of the aspartate protease cathepsin D, and lysosomal glycosidases in leukocytes from rheumatoid arthritis patients at different stages of the disease. In all patients the activities of cysteine protease cathepsin B, dipeptidyl peptidase I, aspartate protease cathepsin D, and two glycosidases were elevated, but the activities of the serine proteases tripeptidyl peptidase I, dipeptidyl peptidase II, and the cysteine protease cathepsin H was unchanged. The magnitude of the increased activity was correlated with the duration of the disease. Patients with longstanding RA (10 years or more) had higher cysteine protease activity in their leukocytes than did those with disease of shorter duration. This tendency suggests that elevated lysosomal cysteine protease activities, together with aspartate protease cathepsin D and lysosomal glycosidases (but not serine proteases), are associated with progression of rheumatoid arthritis.