Specific Synthetic Inhibitors Research Articles

Skin Malignant Melanoma and Matrix Metalloproteinases: Promising Links to Efficient Therapies

Skin malignant melanoma (MM) is one of the most frequent and aggressive neoplasia worldwide. Its associated high mortality rates are mostly due to its metastases, while diagnosis and treatment of MM in its early stages is of favorable prognostic. Even skin superficial MMs at incipient local stages can already present with lymph node invasion and distant metastases. Therefore, knowledge of the controllable risk factors and pathogenic mechanisms of MM development, spreading, and metastatic pattern, as well as early diagnosis, are essential to decrease the high mortality rates associated with cutaneous malignant melanoma. Genetic factors are incriminated, although lifetime-acquired genetic mutations appear to be even more frequently involved in the development of MM. Skin melanocytes divide only twice per year and have time to accumulate genetic mutations as a consequence of environmental aggressive factors, such as UV exposure. In the search for more promising therapies, matrix metalloproteinases have become of significant interest, such as MMP-1, MMP-2, MMP-9, and MMP-13, which have been linked to more aggressive forms of cancer and earlier metastases. Therefore, the development of specific synthetic inhibitors of MMP secretion or activity could represent a more promising and effective approach to the personalized treatment of MM patients.

Differentiated modulation of signaling molecules AMPK and SIRT1 in experimentally drug-induced hepatocyte injury.

Currently available medicines have little to offer in terms of supporting the regeneration of injured hepatic cells. Previous experimental studies have shown that resveratrol and metformin, less specific activators of AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1), can effectively attenuate acute liver injury. The aim of this experimental study was to elucidate whether modulation of AMPK and SIRT1 activity can modify drug/paracetamol (APAP)-induced hepatocyte damage in vitro. Primary rat hepatocytes were pretreated with mutual combinations of specific synthetic activators and inhibitors of SIRT1 and AMPK and followed by a toxic dose of APAP. At the end of cultivation, medium samples were collected for biochemical analysis of alanine-aminotransferase and nitrite levels. Hepatocyte viability, thiobarbituric reactive substances, SIRT1 and AMPK activity and protein expression were also assessed. The harmful effect of APAP was associated with decreased AMPK and SIRT1 activity and protein expression alongside enhanced oxidative stress in hepatocytes. The addition of AMPK activator (AICAR) or SIRT1 activator (CAY10591) significantly attenuated the deleterious effects of AMPK inhibitor (Compound C) on the hepatotoxicity of APAP. Furthermore, CAY10591 but not AICAR markedly decreased the deleterious effect of APAP in combination with SIRT1 inhibitor (EX-527). Our findings demonstrate that decreased AMPK activity is associated with the hepatotoxic effect of APAP which can be significantly attenuated by the administration of a SIRT1 activator. These findings suggest that differentiated modulation of AMPK and SIRT1 activity could therefore provide an interesting and novel therapeutic opportunity in the future to combat hepatocyte injury.

HIV-1 Tat and Heparan Sulfate Proteoglycans Orchestrate the Setup of in Cis and in Trans Cell-Surface Interactions Functional to Lymphocyte Trans-Endothelial Migration.

HIV-1 transactivating factor Tat is released by infected cells. Extracellular Tat homodimerizes and engages several receptors, including integrins, vascular endothelial growth factor receptor 2 (VEGFR2) and heparan sulfate proteoglycan (HSPG) syndecan-1 expressed on various cells. By means of experimental cell models recapitulating the processes of lymphocyte trans-endothelial migration, here, we demonstrate that upon association with syndecan-1 expressed on lymphocytes, Tat triggers simultaneously the in cis activation of lymphocytes themselves and the in trans activation of endothelial cells (ECs). This “two-way” activation eventually induces lymphocyte adhesion and spreading onto the substrate and vascular endothelial (VE)-cadherin reorganization at the EC junctions, with consequent endothelial permeabilization, leading to an increased extravasation of Tat-presenting lymphocytes. By means of a panel of biochemical activation assays and specific synthetic inhibitors, we demonstrate that during the above-mentioned processes, syndecan-1, integrins, FAK, src and ERK1/2 engagement and activation are needed in the lymphocytes, while VEGFR2, integrin, src and ERK1/2 are needed in the endothelium. In conclusion, the Tat/syndecan-1 complex plays a central role in orchestrating the setup of the various in cis and in trans multimeric complexes at the EC/lymphocyte interface. Thus, by means of computational molecular modelling, docking and dynamics, we also provide a characterization at an atomic level of the binding modes of the Tat/heparin interaction, with heparin herein used as a structural analogue of the heparan sulfate chains of syndecan-1.

Histone deacetylases up-regulate C/EBPα expression through reduction of miR-124-3p and miR-25 in hepatocellular carcinoma

BackgroundCCAAT enhancer binding protein α (C/EBPα), as an important transcription factor involved in cell proliferation, differentiation and metabolism, was up-regulated in primary hepatocellular carcinoma (HCC) and predicted poorer prognosis. In this study, we explored how histone deacetylases (HDACs) up-regulated C/EBPα in HCC. MethodsThe protein expressions of HDAC1, HDAC2 were associated with C/EBPα by immunohistochemistry staining in a HCC tissue microarray. HCC cells were then treated with HDAC inhibitors or siRNAs to determine the roles of miR-124-3p and miR-25 in the regulation of C/EBPα mRNA expression. ResultsBoth HDAC1 and HDAC2 proteins were significantly associated with C/EBPα. Inhibition of HDAC by either pharmacological inhibitors or siRNAs decreased C/EBPα mRNA expression in dose-dependent manners in HCC cells. HDAC inhibitors reduced C/EBPα mRNA stability as shown by pmiRGLO luciferase reporter assays. HDAC inhibition consistently induced miR-124-3p and miR-25 expression. Conversely, blockage of miR-124-3p and/or miR-25 by treatment with specific synthetic inhibitors abolished C/EBPα reduction. More importantly, C/EBPα mRNA stability could be rescued by site-directed mutations of miR-124-3p or miR-25 recognition sites in the C/EBPα 3′UTR sequence. In summary, HDAC may up-regulate C/EBPα expression through miR-124-3p and miR-25 in HCC.

Targeting host calpain proteases decreases influenza A virus infection.

Influenza A viruses (IAV) trigger contagious acute respiratory diseases. A better understanding of the molecular mechanisms of IAV pathogenesis and host immune responses is required for the development of more efficient treatments of severe influenza. Calpains are intracellular proteases that participate in diverse cellular responses, including inflammation. Here, we used in vitro and in vivo approaches to investigate the role of calpain signaling in IAV pathogenesis. Calpain expression and activity were found altered in IAV-infected bronchial epithelial cells. With the use of small-interfering RNA (siRNA) gene silencing, specific synthetic inhibitors of calpains, and mice overexpressing calpastatin, we found that calpain inhibition dampens IAV replication and IAV-triggered secretion of proinflammatory mediators and leukocyte infiltration. Remarkably, calpain inhibition has a protective impact in IAV infection, since it significantly reduced mortality of mice challenged not only by seasonal H3N2- but also by hypervirulent H5N1 IAV strains. Hence, our study suggests that calpains are promising therapeutic targets for treating IAV acute pneumonia.

Cloning and functional expression of a chitinase cDNA from the apple leaf miner moth Lithocolletis ringoniella.

Insect chitinase plays essential roles in chitin catabolism involved in digestion and molting during insect development. In the current work, we cloned a chitinase cDNA, LrCht5, from the apple leaf miner moth Lithocolletis ringoniella and characterized its amino acid sequence and protein properties. The L. ringoniella chitinase cDNA was 2136 bp in length with an open reading frame of 1737 bp that encodes a polypeptide of 579 amino acid residues with a predicted molecular mass of 64.4 kDa and pI of 5.49. The catalytic domain has several phosphorylation and glycosylation sites. The recombinant LrCht5 was expressed in Escherichia coli and the Spodoptera frugiperda cell line Sf9, and the LrCht5 expressed in insect cells exhibited chitinolytic activity. LrCht5 was most stable at pH 6.0 and 45°C. This work has potential application in the development of novel and more specific synthetic chitinase inhibitors for use as bioinsecticides.

In Vivo Imaging of Matrix Metalloproteinase 12 and Matrix Metalloproteinase 13 Activities in the Mouse Model of Collagen‐Induced Arthritis

To develop enzyme-activatable Förster resonance energy transfer (FRET) substrate probes to detect matrix metalloproteinase 12 (MMP-12) and MMP-13 activities in vivo in mouse models of inflammatory arthritis. Peptidic FRET probes activated by MMP-12 and MMP-13 were reverse designed from inhibitors selected from a phosphinic peptide inhibitor library. Selectivity of the probes was demonstrated in vitro using MMP-1, MMP-2, MMP-3, MMP-12, and MMP-13. In vivo activation of the probes was tested in the zymosan-induced mouse model of inflammation, and probe specificity was evaluated by the MMP inhibitor GM6001 and specific synthetic inhibitors of MMP-12 and MMP-13. The probes were used to monitor these enzyme activities in the collagen-induced arthritis (CIA) model in vivo. The MMP-12 and MMP-13 activity probes (MMP12ap and MMP13ap, respectively) discriminated between the activities of the 2 enzymes. The in vivo activation of these probes was inhibited by GM6001 and by their respective specific inhibitors. In the CIA model, MMP12ap activation peaked 5 days after disease onset and showed strong correlation with disease severity during this time (r = 0.85, P < 0.0001). MMP13ap activation increased gradually after disease onset and correlated with disease severity over a longer period of 15 days (r = 0.58, P < 0.0001). We generated two selective FRET probes that can be used to monitor MMP-12 and MMP-13 activities in live animals. MMP12ap follows the initial stage of inflammation in CIA, while MMP13ap follows the progression of the disease. The specificity of these probes is useful in monitoring the efficacy of MMP inhibitors.

Targeting Inflammation in Cardiovascular Diseases. Still a Neglected field?

Prevention and treatment of atherosclerosis is still a clinical challenge in the cardiovascular medicine. The classical belief that atherosclerotic lesion development solely depends on lipid deposition has been replaced by the current concept that activation of immune and inflammatory responses plays a central role in plaque initiation and progression. In this review we summarize studies on human and genetically modified animals describing a finite number of cellular and molecular mechanisms that underlie immunoinflammation in atherosclerotic plaques. We focus on the pro- and antiinflammatory mediators activated during atherogenesis and the intracellular signaling pathways regulating these events. Besides the advances on established pharmacological agents, we propose potential strategies for reduction/stabilization of atherosclerotic plaques based on the clinical data in inflammatory-associated pathologies and on the encouraging studies in experimental models of atherosclerosis. We emphasize the potential of such novel inhibitors comprising receptor antagonists, neutralizing antibodies, kinase inhibitors, peptide-based technologies, and chemicals as emerging antiinflammatory strategies for the treatment of atherosclerotic disease complications.

Protease Inhibitors with Antileishmanial Activity

Proteases make up the largest class of enzymes, with more than 1600 proteases identified from over 1700 organisms. These proteases participate in numerous physiological and pathological processes. In Leishmania, these enzymes play important roles during their life cycle, and in different steps of the microorganism-host interplay. They have become important targets for the rational development of drugs. Proteolytic enzyme activity is closely regulated in many ways, including substrate selectivity, specific cellular location, and it can also be regulated by specific synthetic inhibitors. Protease inhibitor interactions are involved in the regulation of processes such as protein digestion, various physiological and pathological processes and microbial diseases. There are pathological conditions in which uncontrolled proteolytic activity of exogenous proteases derived from infectious agents such as protozoa play a role in the onset and perpetuation of infection, making protease inhibitors potentially applicable as therapeutic agents in the battle against these diseases. Current chemotherapy of leishmaniasis still relies on drugs, which have limited efficiency, toxic side effects and now the emergence of drug resistance. Here in this work some aspects of the relation between antileishmanial and protease inhibitor activities are discussed. Keywords: Leishmania, protease, inhibitors, leishmaniasis, microorganism-host interplay, Rational development, chemotherapu, drug resistance, proteolytic enyme, synthetic inhibtors

Substrate specificity and inhibition of brassinin hydrolases, detoxifying enzymes from the plant pathogens Leptosphaeria maculans and Alternaria brassicicola

Blackleg (Leptosphaeria maculans and Leptosphaeria biglobosa) and black spot (Alternaria brassicicola) fungi are devastating plant pathogens known to detoxify the plant defence metabolite, brassinin. The significant roles of brassinin as a crucifer phytoalexin and as a biosynthetic precursor of several other plant defences make it important in plant fitness. Brassinin detoxifying enzymes produced by L. maculans and A. brassicicola catalyse the detoxification of brassinin by hydrolysis of its dithiocarbamate group to indolyl-3-methanamine. The purification and characterization of brassinin hydrolases produced by L. maculans (BHLmL2) and A. brassicicola (BHAb) were accomplished: native BHLmL2 was found to be a tetrameric protein with a molecular mass of 220 kDa, whereas native BHAb was found to be a dimeric protein of 120 kDa. Protein characterization using LC-MS/MS and sequence alignment analyses suggested that both enzymes belong to the family of amidases with the catalytic Ser/Ser/Lys triad. Furthermore, chemical modification of BHLmL2 and BHAb with selective reagents suggested that the amino acid serine was involved in the catalytic activity of both enzymes. The overall results indicated that BHs have new substrate specificities with a new catalytic activity that can be designated as dithiocarbamate hydrolase. Investigation of the effect of various phytoalexins on the activities of BHLmL2 and BHAb indicated that cyclobrassinin was a competitive inhibitor of both enzymes. On the basis of pH dependence, sequence analyses, chemical modifications of amino acid residues and identification of headspace volatiles, a chemical mechanism for hydrolysis of the dithiocarbamate group of brassinin catalysed by BHLmL2 and BHAb is proposed. The current information should facilitate the design of specific synthetic inhibitors of these enzymes for plant treatments against blackleg and black spot fungal infections.

Post-Translational Regulation of Cathepsin B, but not of Other Cysteine Cathepsins, Contributes to Increased Glioblastoma Cell Invasiveness In Vitro

Cells that migrate away from a central tumour into brain tissue are responsible for inefficient glioblastoma treatment. This migratory behaviour depends partially on lysosomal cysteine cathepsins. Reportedly, the expression of cathepsins B, L and S gradually increases in the progression from benign astrocytoma to the malignant glioblastoma, although their specific roles in glioma progression have not been revealed. The aim of this study was to clarify their specific contribution to glioblastoma cell invasion. The differences between the matrix invading cells and non-invading core cells from spheroids derived from glioblastoma cell culture and from glioblastoma patients' biopsies, and embedded in type I collagen, have been studied at the mRNA, protein and cathepsin activity levels. Analyses of the two types of cells showed that the three cathepsins were up-regulated post-translationally, their specific activities increasing in the invading cells. The cystatin levels were also differentially altered, resulting in higher ratio of cathepsins B and L to stefin B in the invading cells. However, using specific synthetic inhibitors and silencing strategies revealed that only cathepsin B activity was involved in the invasion of glioblastoma cells, confirming previous notion of cathepsin B as tumour invasiveness biomarker. Our data support the concept of specific roles of cysteine cathepsins in cancer progression. Finally the study points out on the complexity of protease regulation and the need to include functional proteomics in the systems biology approaches to understand the processes associated with glioma invasion and progression.

IS THE UPREGULATION OF VEGF BY hCG IN HUMAN LUTEAL CELLS DEPENDENT ON HIF-1ALPHA EXPRESSION?

Angiogenesis is essential for normal growth and function of the corpus luteum. Several lines of research have demonstrated the importance of Vascular Endothelial Growth Factor (VEGF) in corpus luteum angiogenesis. Generally it is believed that hypoxia is the primary inducer of VEGF production in most tissues. Indeed, experiments using non-human primate cells suggested that hypoxia was a major regulator of luteal VEGF expression. However we have previously shown that VEGF is upregulated by human chorionic gonadotropin (hCG) in the human corpus luteum. As Hypoxia Inducible Factor-1alpha (HIF-1alpha) has been shown to regulate the expression of VEGF under non-hypoxic (i.e. ligand stimulated) conditions, as well as under hypoxic conditions, in cell lines, we hypothesised that the effect of hCG on luteal VEGF expression was mediated through HIF-1alpha. We therefore studied the effect of hCG on VEGF and HIF-1á expression in cultured luteinized granulosa cells (LGCs) in vitro and in human corpora lutea in vivo. LGCs were obtained from women undergoing oocyte recovering as part of an assisted conception program. Isolated LGCs were cultured under serum-free conditions, on Matrigel-coated plastic tissue culture plates, for one week and the culture medium was refreshed every two days. Cells were then treated with hCG alone (100 ng/ml) or hCG in the presence of synthetic inhibitory molecules before mRNA was collected. The expression of VEGF and HIF-1alpha mRNA was analyzed by Taqman quantitative real-time PCR. In vitro, hCG up-regulated both VEGF (P<0.0001) and HIF-1alpha (P<0.0001) mRNA transcripts in LGCs. In vivo, expression was investigated in corpora lutea from the late-luteal phase in the presence (n=5) or absence (n=5) of exogenous hCG concentrations similar to those seen in early pregnancy. Both VEGF (P=0.02) and HIF-1alpha (P=0.034) mRNA expression was upregulated by hCG during luteal rescue when compared to that in the late-luteal phase. To investigate whether the effect of hCG on HIF-1alpha/VEGF expression was under influence of progesterone, LGCs were stimulated with hCG in the presence or absence of the progesterone synthesis inhibitor aminoglutethamide (100 μM). The up-regulation of both HIF-1alpha and VEGF by hCG was independent of progesterone in vitro. As both the Raf/MEK/MAPK and the PI3 kinase/Akt signalling pathways have been shown to stimulate VEGF mRNA expression through the regulation of HIF-1alpha activity in a range of cell lines, LGCs were stimulated with hCG in the presence or absence of specific synthetic inhibitors of the Raf/MEK/MAPK (PD98059 (50 μM)) and PI3 kinase/Akt (LY294002 (50 μM)) pathways. PD98059 and LY294002 inhibited both VEGF and HIF-1alpha mRNA expression, whereas the mRNA expression of steroidogenic acute regulatory protein (StAR), which is regulated through a cAMP-dependent PKA pathway, was not affected. In summary, our results suggest that the expression of HIF- 1alpha is hormonally regulated in human luteal cells under normoxic conditions in vitro and in vivo and this may in turn regulate VEGF expression. (poster)

Specificity of an extracellular proteinase from Conidiobolus coronatus and its inhibition by an inhibitor from insect hemolymph

The relatively little-investigated entomopathogen Conidiobolus coronatus secretes several proteinases into culture broth. Using a combination of ion-exchange and size-exclusion chromatography, we purified to homogeneity a serine proteinase of Mr 30,000-32,000, as ascertained by SDS-PAGE. The purified enzyme showed subtilisin-like activity. It very effectively hydrolyzed N-Suc-Ala(2)-Pro-Phe-pNa with a Km-1.36 x 10(-4) M and Kcat-24 s(-1), and N-Suc-Ala(2)-Pro-Leu-pNa with Km-6.65 x 10(-4) M and Kcat-11 s(-1). The specificity index k(cat)/K(m) for the tested substrates was calculated to be 176,340 s(-1) M(-1) and 17,030 s(-1) M(-1), respectively. Using oxidized insulin B chain as a substrate, the purified proteinase exhibited specificity to aromatic and hydrophobic amino-acid residues, such as Phe, Leu, and Gly at the P1 position, splitting primarily the peptide bonds: Phe(1)-Val(2), Leu(15)-Tyr(16), and Gly(23)-Phe(24). The proteinase appeared to be sensitive to the specific synthetic inhibitors of the serine proteinases DFP (diisopropyl flourophosphate) and PMSF (phenyl-methylsulfonyl fluoride) as well as to some naturally occurring protein inhibitors of chymotrypsin. It is worth noting that the enzyme exhibited the highest sensitivity to inhibition by AMCI-1 (with an association constant of 3 x 10(10) M(-1)), an inhibitor of cathepsin G/chymotrypsin from the larval hemolymph of Apis mellifera, reinforcing the possibility of involvement of inhibitors from hemolymph in insect innate immunity. The substrate specificity and proteinase inhibitor effects indicate that the purified proteinase from the fermentation broth of Conidiobolus coronatus is a subtilisin-like serine proteinase.

The role of dipeptidyl peptidase IV (DP IV, CD26) in T cell activation and multiple sclerosis

AbstractThe ectoenzyme dipeptidyl peptidase IV (DP IV; E.C. 3.4.14.5; CD26) plays a crucial role in T cell activation, representing a new type of costimulatory T cell structure. Effectors of DP IV‐like activity, including naturally occurring and specific synthetic inhibitors, suppress DNA synthesis as well as cytokine production (IL‐2, IL‐10, IL‐12, IFN‐γ) of stimulated T cells. These effects are in part caused by the immunosuppressive cytokine TGF‐β1, leading to blockade of the cell cycle at the restriction point G1/S via p27kip. At the same time, DP IV inhibitors provoke tyrosine phosphorylation and p38 MAP kinase activation. Elevated numbers of CD26+ T cells were observed in patients with autoimmune diseases such as rheumatoid arthritis or multiple sclerosis (MS). The expression of DP IV/CD26 in resting T cell clones derived from patients with MS was found to be 3‐ to 4‐fold higher than on resting peripheral blood T cells from healthy persons. In myelin‐specific T cells from MS patients, DP IV inhibitors suppress DNA synthesis, as well as IFN‐γ, IL‐4 and TNF‐α production. Moreover, in experimental autoimmune encephalomyelitis (EAE), a well characterized CD4+ T cell‐mediated autoimmune disease leading to CNS inflammation and demyelinization, administration of a DP IV/CD26 inhibitor prevents clinical and neuropathological signs of EAE and suppresses ongoing disease. This review summarizes evidence for the role of DP IV enzyme activity in regulation of T cell activation, outlines signal transduction mechanisms used or affected by this enzyme and provides support for the concept of a novel peptidase‐based immunosuppressive approach to treat autoimmune diseases like MS.

Three small integrin binding ligand N-linked glycoproteins (SIBLINGs) bind and activate specific matrix metalloproteinases.

Matrix metalloproteinases (MMPs) are critical for development, wound healing, and for the progression of cancer. It is generally accepted that MMPs are secreted in a latent form (proMMP) and are activated only upon removal of their inhibitory propeptides. This report shows that three members of the SIBLING (Small, Integrin-Binding LIgand, N-linked Glycoprotein) family can specifically bind (Kd approximately equal nM) and activate three different MMPs. Binding of SIBLING to their corresponding proMMPs is associated with structural changes as indicated by quenching of intrinsic tryptophan fluorescence, increased susceptibility to plasmin cleavage, and decreased inhibition by specific natural and synthetic inhibitors. Activation includes both making the proMMPs enzymatically active and the reactivation of the TIMP (tissue inhibitors of MMP) inhibited MMPs. Bone sialoprotein specifically binds proMMP-2 and active MMP-2, while osteopontin binds proMMP-3 and active MMP-3, and dentin matrix protein-1 binds proMMP-9 and active MMP-9. Both pro and active MMP-SIBLING complexes are disrupted by the abundant serum protein, complement Factor H, thereby probably limiting SIBLING-mediated activation to regions immediately adjacent to sites of secretion in vivo. These data suggest that the SIBLING family offers an alternative method of controlling the activity of at least three MMPs.

Porphyromonas gingivalis gingipain-R enhances interleukin-8 but decreases gamma interferon-inducible protein 10 production by human gingival fibroblasts in response to T-cell contact.

Proteases produced by Porphyromonas gingivalis, an oral pathogen, are considered important virulence factors and may affect the responses of cells equipped with proteinase-activated receptors. The aim of this study was to investigate the effect of the arginine-specific cysteine protease gingipain-R produced by P. gingivalis on chemokine production by human gingival fibroblasts (HGF) and the effect of gingipain-R treatment on the subsequent contact-dependent activation of HGF by T cells. HGF incubated in the presence of purified 47-kDa gingipain-R showed increased levels of interleukin-8 (IL-8) mRNA. Cyclooxygenase-2 (COX-2) mRNA was also induced. Further exposure of HGF to activated T cells resulted in the dose- and time-dependent enhancement of IL-8 transcription and release. T-cell membrane-bound tumor necrosis factor (TNF) was the ligand inducing IL-8 production by HGF, since TNF neutralization abrogated HGF responses to T-cell contact. The enhanced IL-8 release was due, at least in part, to prostaglandin-E(2) production, which was mostly blocked by indomethacin. Gingipain-R proteolytic activity was required since heat inactivation, specific synthetic protease inhibitors, and the natural substrate competitor histatin 5 abrogated its effects. The enhanced production of IL-8 in response to T-cell contact was specific since monocyte chemotactic protein-1 (MCP-1) production was unaffected while interferon-gamma-inducible protein-10 (IP-10) was inhibited. The sum of these activities may result in the recruitment of differential cell types to sites of inflammation since IL-8 preferentially recruits neutrophils and IP-10 attracts activated T cells and may be relevant to the pathogenesis of periodontitis.

Circulating sex hormones and endometrial stromelysin-1 (matrix metalloproteinase-3) at the start of bleeding episodes in levonorgestrel-implant users.

Unpredictable endometrial bleeding is the major side-effect of levonorgestrel-releasing s.c. implants (Norplant), otherwise a method of choice for long-term contraception. The mechanisms responsible for bleeding are still unknown and no reliable treatment is available. Several matrix metalloproteinases (MMP) are expressed and activated in human endometrium only at menstruation and specific synthetic inhibitors of MMP fully prevent the tissue breakdown that occurs in menstrual-like endometrial explants. To investigate whether MMP are inappropriately expressed and activated in Norplant-treated endometria during bleeding episodes, volunteers were recruited to provide blood and endometrial biopsies at the start of bleeding episodes and during non-bleeding intervals. Whereas serum concentrations of levonorgestrel and sex hormones showed no change at bleeding, except for a slight decrease of oestradiol concentration, the expression and activation of stromelysin-1 released by explants cultured for 1 day were consistently increased at the start of bleeding episodes. Furthermore, stromelysin-1 was immunolocalized in stromal cells within breakdown areas of several bleeding endometria, but not in non-bleeding endometria. These observations suggest that the expression and activation of stromelysin-1 participate in the initiation of bleeding episodes upon Norplant contraception. New strategies in the prevention and treatment of abnormal bleeding based on MMP control should be envisaged.

Digestive proteolytic activity in larvae of tomato moth, Lacanobia oleracea; effects of plant protease inhibitors in vitro and in vivo

Three distinct digestive protease activities, with strongly alkaline pH optima, were identified in the gut of tomato moth ( Lacanobia oleracea) larvae, and characterised using specific synthetic substrates and inhibitors. These were; a trypsin-like activity, a chymotrypsin-like activity specific for substrates and inhibitors containing more than one amino acid residue, and an elastase-like activity, accounting for 40%, 30% and 20% of overall proteolysis respectively. The protease activities differed in their sensitivities to inhibition by different plant protein protease inhibitors (PIs), as estimated by I 50 values. Soya bean Kunitz trypsin inhibitor (SKTI) was the only plant PI tested to inhibit all three digestive protease activities at concentrations <40 μg/ml (approx. 5×10 −6M). Incorporation of SKTI into a potato leaf-based artificial diet at 2% of total protein, decreased larval survival and growth (by approx. 33% and 40% respectively after 21 days) and retarded development (by approx. 2 days). However, when SKTI was expressed in transgenic potato plants at approx. 0.5% of total protein, only marginal effects on L. oleracea larvae were observed, which decreased with time. Whilst the presence of SKTI in artificial diet increased endogenous larval trypsin-like activity by up to four-fold, no effects on this activity were observed in larvae feeding on transgenic plants.

TNF-α converting enzyme (TACE) is inhibited by TIMP-3

TNF-α converting enzyme (TACE; ADAM-17) is a membrane-bound disintegrin metalloproteinase that processes the membrane-associated cytokine proTNF-α to a soluble form. Because of its putative involvement in inflammatory diseases, TACE represents a significant target for the design of specific synthetic inhibitors as therapeutic agents. In order to study its inhibition by tissue inhibitors of metalloproteinases (TIMPs) and synthetic inhibitors of metalloproteinases, the catalytic domain of mouse TACE (rTACE) was overexpressed as a soluble Ig fusion protein from NS0 cells. rTACE was found to be well inhibited by peptide hydroxamate inhibitors as well as by TIMP-3 but not by TIMP-1, -2 and -4. These results suggest that TIMP-3, unlike the other TIMPs, may be important in the modulation of pathological events in which TNF-α secretion is involved.

Crystal structure of the catalytic domain of human tumor necrosis factor-alpha-converting enzyme.

Tumor necrosis factor-alpha (TNFalpha) is a cytokine that induces protective inflammatory reactions and kills tumor cells but also causes severe damage when produced in excess, as in rheumatoid arthritis and septic shock. Soluble TNFalpha is released from its membrane-bound precursor by a membrane-anchored proteinase, recently identified as a multidomain metalloproteinase called TNFalpha-converting enzyme or TACE. We have cocrystallized the catalytic domain of TACE with a hydroxamic acid inhibitor and have solved its 2.0 A crystal structure. This structure reveals a polypeptide fold and a catalytic zinc environment resembling that of the snake venom metalloproteinases, identifying TACE as a member of the adamalysin/ADAM family. However, a number of large insertion loops generate unique surface features. The pro-TNFalpha cleavage site fits to the active site of TACE but seems also to be determined by its position relative to the base of the compact trimeric TNFalpha cone. The active-site cleft of TACE shares properties with the matrix metalloproteinases but exhibits unique features such as a deep S3' pocket merging with the S1' specificity pocket below the surface. The structure thus opens a different approach toward the design of specific synthetic TACE inhibitors, which could act as effective therapeutic agents in vivo to modulate TNFalpha-induced pathophysiological effects, and might also help to control related shedding processes.