Biomedical subjects

N Rousselet

Publications and source records attributed to N Rousselet.

9 recordsLinked to original sources

PubMed2006

Expression of cathepsin L in human tumor cells is under the control of distinct regulatory mechanisms.

Cathepsin L, a cysteine protease, is overexpressed in human tumor cells and plays a major role in melanoma progression. Our aim was herein to identify molecular mechanisms, which contribute to its overexpression. We found that cathepsin L protein expression correlated with mRNA level in tumor cells. Therefore, we focused on mechanisms involved in cathepsin L mRNA regulation. CpG island was localized in the 5' region of cathepsin L gene that encompassed regulatory regions identified as essential for promoter activity. CpG dinucleotides, not methylated in any melanoma cells analysed, were methylated in a B lymphoma cell line, which poorly express cathepsin L. Our data demonstrate that in lymphoma cells, cathepsin L silencing was methylation-dependent. Furthermore, gene amplification was involved in cathepsin L overexpression in one melanoma cell line, while transcriptional mechanisms but not mRNA stability are responsible of cathepsin L overexpression in others melanoma cells. In addition, NF-Y, Sp1, Sp2 and Sp3 transcription factors, essential to basal cathepsin L transcription, are not directly involved in overexpression. Thus, our data provides the first demonstration that cathepsin L expression in tumor cells is under the control of distinct molecular mechanisms.

Animals↗

PubMed1995

The role of histidine 231 in thermolysin-like enzymes. A site-directed mutagenesis study.

In the zinc metallopeptidases produced by the genus Bacillus, an active site histidine has been proposed to either stabilize the transition state in catalysis by donating a hydrogen bond to the hydrated peptide (Matthews, B. W. (1988) Acc. Chem. Res. 21, 333-340) or to polarize a water molecule, which subsequently attacks the peptidyl bond (Mock, W. L., and Aksamawati, M. (1994) Biochem. J. 302, 57-68). Site-directed mutagenesis techniques have been used to change this residue in the zinc endopeptidase from Bacillus stearothermophillus to either phenylalanine or alanine. At pH 7.0, the kcat/Km values of the substrate leucine enkephalin for the phenylalanine and alanine mutants were reduced by factors of 430- and 500-fold, respectively, as compared with the wild-type enzyme, mostly due to changes in kcat. In addition, the enzymatic activities of the mutant enzymes showed little pH dependence in the alkaline range, unlike the wild-type enzyme. The mutations did not greatly alter the binding affinities of inhibitors containing sulfydryl groups to chelate the active site zinc, while those of inhibitors containing hydroxamate or carboxylate zinc-chelating groups were increased between 80- and 250-fold. The largest change in the binding affinity of an inhibitor (> 5 orders of magnitude) was found with the proposed transition state mimic, phosphoramidon. The results are generally in agreement with x-ray crystallography studies and favor the involvement of the active site histidine in transition state binding.

Amino Acid Sequence↗

PubMed1994

New dual inhibitors of neutral endopeptidase and angiotensin-converting enzyme: rational design, bioavailability, and pharmacological responses in experimental hypertension.

In the treatment of cardiovascular diseases, it could be of therapeutic interest to associate the hypotensive effects resulting from the inhibition of angiotensin II formation, ensured by endothelial angiotensin-converting enzyme (ACE), with the diuretic and natriuretic responses due to the protection of the endogenous atrial natriuretic peptide (ANP) from inactivation by epithelial neutral endopeptidase (NEP). However, an investigation of this hypothesis requires an orally active compound able to jointly inhibit ACE and NEP. Dual inhibitors have therefore been designed by a rational approach, based on the characteristics of the active sites of both enzymes, which belong to the same family of zinc metallopeptidases, and on the structures of their most potent and selective inhibitors. As both NEP and ACE contain a large S'1-S'2 domain able to accommodate aromatic residues, the cyclic ACE inhibitor 3-(mercaptomethyl)-3,4,5,6-tetrahydro-2-oxo-1H-1-benzazocine-1-ace tic acid was selected as a template. Various aliphatic constraints were introduced on the benzyl moiety of the potent NEP inhibitor N-[2-(mercaptomethyl)-3-phenylpropanoyl]-L-tyrosine (IC50 NEP = 2 nM, IC50 ACE = 25 nM) to improve the fit between the computed most stable conformers of these molecules and the ACE template. New dual inhibitors, of general formula, N-[2(R,S)-(mercaptomethyl)-3(R,S)-phenylbutanoyl]-L-amino acid with IC50 values in the nanomolar range for both enzymes were generated by this approach. The separation of the four stereoisomers using chiral amines and the stereoselective synthesis of the 2-(mercaptomethyl)-3-phenylbutanoyl moiety showed that inhibitors with the 2S,3R configuration are the most potent on both NEP and ACE. The "in vivo" potency of various prodrugs of these inhibitors to inhibit ACE activity in lung and NEP activity in kidney was measured after oral administration in mice. From this pharmacokinetical study the most potent dual inhibitor RB 105 (N-[(2S,3R)-2-(mercaptomethyl)-3-phenylbutanoyl-L-alanine (compound 44c) (KI NEP 1.7 nM, KI ACE 4.5 nM) and its most efficient in vivo prodrug mixanpril, [N-[(2S,3R)-2-[(benzoylthio)methyl]-3-phenylbutanoyl]-L-alan ine (compound 18) (ED50 NEP approximately 1 mg/kg, ED50 ACE approximately 7 mg/kg) were selected. Competition experiments with a tritiated inhibitor of ACE or NEP bound to mouse lung and kidney membranes respectively showed that mixanpril has a long duration of action (> 8 h). As expected, after i.v. administration in the spontaneously hypertensive rat (SHR), RB 105 decreased blood pressure and increased diuresis and natriuresis.(ABSTRACT TRUNCATED AT 400 WORDS)

Alanine↗

PubMed1983