Two di-leucine-based motifs account for the different subcellular localizations of the human endothelin-converting enzyme (ECE-1) isoforms.
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Endothelin-converting enzyme (ECE-1) is a type II integral membrane protein which plays a key role in the biosynthetic pathway of the vasoconstricting endothelins. Three ECE-1 isoforms, differing by their N-terminal cytoplasmic tails, are generated from a single gene. When expressed in CHO cells, they display comparable enzymatic activity but whereas ECE-1a is strongly expressed at the cell surface, ECE-1b is exclusively intracellular and ECE-1c presents an intermediate distribution. In the present study these different localizations were further described at the ultrastructural level, by electron microscope immunocytochemistry. To characterize the motifs responsible for the intracellular localization of ECE-1b we constructed chimeric proteins and point mutants. Two di-leucine-based motifs, contained in the N-terminal part of ECE-1b, were thus identified. One of these motifs (LV), displayed by both ECE-1b and ECE-1c, accounts for the reduced surface expression of ECE-1c as compared to ECE-1a. Mutation of both motifs (LL and LV) induces a very strong appearance of ECE-1b at the cell surface indicating that their presence in the N-terminal extremity of ECE-1b is critical for its exclusively intracellular localization. (+info)
The interaction of bioactive peptides with an immobilized phosphatidylcholine monolayer.
(50/2681)
The interaction of three bioactive peptides, bombesin, beta-endorphin, and glucagon with a phosphatidylcholine monolayer that was immobilized to porous silica particles and packed into a stainless steel column cartridge, has been studied using dynamic elution techniques. This immobilized lipid monolayer provides a biophysical model system with which to study the binding of peptides to a lipid membrane. In particular, the influence of temperature and methanol concentration on the affinity of each peptide for the immobilized lipid surface was assessed. For all test peptides, nonlinear retention plots were observed at all temperatures that contrasted sharply with the simple linear plots observed for the small unstructured control molecules N-acetyltryptophanamide and diphenylalanine. An analysis of the thermodynamics of the interaction of peptides with the immobilized monolayer was also carried out. The results revealed that while the peptides interacted with the monolayer predominantly through hydrophobic interactions, the relative contribution of DeltaH(assoc)(O) and DeltaS(assoc)(O) to the overall free energy of association was dependent on the temperature and methanol concentration. In particular, it was evident that under most conditions, the binding of the peptides to the immobilized lipid monolayer was enthalpy-driven, i.e., mediated by nonclassical hydrophobic interactions. Significant band-broadening and asymmetric and split peaks were also observed for bombesin, beta-endorphin, and glucagon at different temperatures and methanol concentrations. These changes in affinity and peak shape are consistent with the formation of multiple conformational species during the interaction of these peptides with the lipid monolayer. In addition, the binding behavior of the three test peptides on an n-octylsilica surface that lacked the phospho headgroups of the phospholipid was significantly different from that observed with the immobilized phosphatidylcholine surface, indicating a specificity of interaction between the peptides and the lipid surface. Overall, these experimental results demonstrate that the biomimetic phosphatidylcholine monolayer provides a stable and sensitive system with which to explore the molecular mechanism of peptide conformational changes during membrane interactions. (+info)
Functional overlap between two classes of matrix-degrading proteases in wound healing.
(51/2681)
Retarded wound healing was found in mice deficient in the serine protease precursor plasminogen, as well as in wild-type mice treated with the metalloprotease inhibitor galardin, but in both cases wound closure was ultimately completed in all mice within 60 days. The expression of several matrix metalloproteases in keratinocytes migrating to cover the wound was strongly enhanced by galardin treatment. However, when plasminogen-deficient mice were treated with galardin, healing was completely arrested and wound closure was not seen during an observation period of 100 days, demonstrating that protease activity is essential for skin wound healing. The requirement for both plasminogen deficiency and metalloprotease inhibition for complete inhibition of the healing process indicates that there is a functional overlap between the two classes of matrix-degrading proteases, probably in the dissection of the fibrin-rich provisional matrix by migrating keratinocytes. Each class alone is capable of maintaining sufficient keratinocyte migration to regenerate the epidermal surface, although this function would normally be performed by both classes acting in parallel. Since there are strong similarities between the proteolytic mechanisms in wound healing and cancer invasion, these results predict that complete arrest of this latter process in therapeutic settings will require the use of inhibitors of both classes of proteases. (+info)
Effect of metalloproteinase inhibitor on corneal cytokine expression after alkali injury.
(52/2681)
PURPOSE: Interleukin (IL)-1alpha and IL-6 levels in the cornea are greatly elevated during the early stages after an alkali burn in mice. The authors investigated the effect of synthetic inhibitor of matrix metalloproteinase (SIMP) on the expression of inflammatory cytokines in alkali-burned murine corneas and evaluated the clinical appearance of the eyes. METHODS: After 0.5N NaOH-alkali burns to 400 corneas of ICR mice, 200 received 400 microg/ml of SIMP topically 4 times a day while 200 corneas were similarly treated with vehicle only. At days 4, 7 and 14 after injury, each cornea was assigned a clinical score for corneal opacity, corneal epithelial defect, hyphema and cataract. Extracts of injured corneas in each group were then assayed for cytokine production using ELISA systems for IL-1alpha, IL-1beta, IL-6 and tumor necrosis factor-alpha (TNF-alpha). RESULTS: The levels of IL-1alpha, IL-1beta and IL-6 were significantly lower in the SIMP-treated group than in the vehicle-treated group 7 days after the burn. However, levels of these cytokines were similar in the SIMP and non-SIMP groups at days 4 and 14. Levels of TNF-alpha did not differ between both groups at any postinjury time. In the SIMP-treated corneas, there was less opacification and hyphema formation and epithelial regeneration was faster. CONCLUSIONS: Topical application of SIMP in alkali-burned murine corneas reduced the expression of IL-1alpha, IL-1beta, and IL-6 and lessened the severity of the injury. (+info)
The proteasome inhibitor PS-341 in cancer therapy.
(53/2681)
The anticancer activity of the boronic acid dipeptide proteasome inhibitor PS-341 was examined in vitro and in vivo. PS-341 was a potent cytotoxic agent toward MCF-7 human breast carcinoma cells in culture, producing an IC90 of 0.05 microM on 24 h of exposure to the drug. In the EMT-6 tumor cell survival assay, PS-341 was equally cytotoxic administered p.o. or by i.p. injection up to a dose of 2 mg/kg. PS-341 was also toxic to the bone marrow colony-forming unit-granulocyte macrophage. PS-341 increased the tumor cell killing of radiation therapy, cyclophosphamide, and cisplatin in the EMT-6/Parent tumor, but was not able to overcome the in vivo resistance of the EMT-6/CTX and EMT-6/CDDP tumors. In the tumor growth delay assay, PS-341 administered p.o. had antitumor activity against the Lewis lung carcinoma, both primary and metastatic disease. In combination, regimens with 5-fluorouracil, cisplatin, Taxol and adriamycin, PS-341 seemed to produce primarily additive tumor growth delays against the s.c. tumor and was highly effective against disease metastatic to the lungs. The proteasome is an interesting new target for cancer therapy, and the proteasome inhibitor PS-341 warrants continued investigation in cancer therapy. (+info)
Novel TRH analog improves motor and cognitive recovery after traumatic brain injury in rodents.
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Thyrotropin-releasing hormone (TRH) and certain TRH analogs show substantial neuroprotective effects in experimental brain or spinal cord trauma but also have other physiological actions (autonomic, analeptic, and endocrine) that may be undesirable for the treatment of neurotrauma in humans. We developed a novel TRH analog (2-ARA-53a), with substitutions at the NH(2)-terminus and imidazole ring, that preserves the neuroprotective action of TRH-like compounds while decreasing or eliminating their autonomic, analeptic, and endocrine effects. Rats administered 2-ARA-53a (1.0 mg/kg, n = 17) intravenously 30 min after lateral fluid percussion brain injury showed marked improvement in motor recovery compared with vehicle-treated controls (n = 14). Treatment of mice subjected to moderate controlled cortical impact brain injury, at the same dose and time after trauma (n = 8), improved both motor recovery and cognitive performance in a water maze place learning task compared with vehicle-treated controls (n = 8). In injured rats, no autonomic or analeptic effects were observed with this compound, and endocrine effects were significantly reduced with 2-ARA-53a, in contrast to those found with a typical NH(2)-terminal-substituted TRH analog (YM-14673). These findings demonstrate that the neuroprotective effects of TRH-related compounds can be dissociated from their other major physiological actions and suggest a potential role for dual-substituted TRH analogs in the treatment of clinical neurotrauma. (+info)
Recognition of L-amino acid ester compounds by rat peptide transporters PEPT1 and PEPT2.
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Peptide transporters (PEPT1 and PEPT2) in epithelia play an important role in the absorption of small peptides and peptide-like drugs. Recently, it was demonstrated that various nonpeptidic compounds can be transported by these transporters. In the present study, we focused on the L-amino acid ester compounds and examined the mechanisms of their interaction with rat PEPTs (rPEPTs) using stable transfectants. Valacyclovir, the L-valyl ester prodrug of the antiherpetic agent acyclovir, competitively inhibited [(14)C]glycylsarcosine uptake in the rPEPT1- or rPEPT2-expressing cells. Dixon plot analyses showed that the inhibition constant (K(i)) values of valacyclovir were 2.7 and 0.22 mM for rPEPT1 and rPEPT2, respectively, suggesting that rPEPT2 had higher affinity for this agent. Various L-valine alkyl esters significantly inhibited [(14)C]glycylsarcosine uptake. L-Valine methyl ester (Val-OMe) competitively inhibited [(14)C]glycylsarcosine uptake with K(i) values of 3.6 and 0.83 mM for rPEPT1 and rPEPT2, respectively, indicating that Val-OMe is also a high-affinity substrate for rPEPT2. Val-OMe had a trans-stimulation effect on [(14)C]glycylsarcosine efflux from both transfectants, suggesting the translocation of L-valine methyl ester via rPEPTs. Val-OMe showed the most potent inhibitory effect among the several L-amino acid methyl esters examined. We conclude that Val-OMe, as well as valacyclovir, could be recognized and transported by rPEPT1 and rPEPT2 and that these L-valyl esters showed higher affinity for rPEPT2 as do most substrates of these transporters. Our results suggest that L-valine is a desirable L-amino acid for the esterification of poorly permeable drugs to enhance their oral bioavailability targeting intestinal PEPT1. (+info)
Substrate specificity of the periplasmic dipeptide-binding protein from Escherichia coli: experimental basis for the design of peptide prodrugs.
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Pure dipeptide-binding protein (DppA) from Escherichia coli was studied in a filter binding assay to determine its binding specificity. A substrate:DppA stoichiometry of 1:1 was found with both [14C]AlaAla and Ala[14C]Phe. Surprisingly, substrate binding did not vary over the pH range pH 3-9.5. Different dipeptides yielded liganded protein with various pI values, implying that DppA can undergo subtly different conformational changes to accommodate different substrates. Using [125I]Tyr-peptides as substrates in competition assays, the relative binding affinities for a range of dipeptides were found to parallel their overall transport rates into E. coli through the dipeptide permease (Dpp), showing that DppA alone controls the specificity of Dpp. With a series of substituted glycyl peptides, binding affinity was progressively enhanced by alkylation (with methyl to butyl) of the N-terminal alpha-amino group. Thus, results from this approach provide an essential experimental basis, which complements the information from the crystal structure of DppA, for the design of peptidomimetic antibacterials targeted for transport through Dpp. (+info)