Dhouib R

References (8)

Title : Identification of a putative triacylglycerol lipase from papaya latex by functional proteomics - Dhouib_2011_FEBS.J_278_97
Author(s) : Dhouib R , Laroche-Traineau J , Shaha R , Lapaillerie D , Solier E , Ruales J , Pina M , Villeneuve P , Carriere F , Bonneu M , Arondel V
Ref : Febs J , 278 :97 , 2011
Abstract : Latex from Caricaceae has been known since 1925 to contain strong lipase activity. However, attempts to purify and identify the enzyme were not successful, mainly because of the lack of solubility of the enzyme. Here, we describe the characterization of lipase activity of the latex of Vasconcellea heilbornii and the identification of a putative homologous lipase from Carica papaya. Triacylglycerol lipase activity was enriched 74-fold from crude latex of Vasconcellea heilbornii to a specific activity (SA) of 57 mumol.min(-1).mg(-1) on long-chain triacylglycerol (olive oil). The extract was also active on trioctanoin (SA = 655 mumol.min(-1).mg(-1) ), tributyrin (SA = 1107 mumol.min(-1).mg(-1) ) and phosphatidylcholine (SA = 923 mumol.min(-1).mg(-1) ). The optimum pH ranged from 8.0 to 9.0. The protein content of the insoluble fraction of latex was analyzed by electrophoresis followed by mass spectrometry, and 28 different proteins were identified. The protein fraction was incubated with the lipase inhibitor [(14) C]tetrahydrolipstatin, and a 45 kDa protein radiolabeled by the inhibitor was identified as being a putative lipase. A C. papaya cDNA encoding a 55 kDa protein was further cloned, and its deduced sequence had 83.7% similarity with peptides from the 45 kDa protein, with a coverage of 25.6%. The protein encoded by this cDNA had 35% sequence identity and 51% similarity to castor bean acid lipase, suggesting that it is the lipase responsible for the important lipolytic activities detected in papaya latex.
ESTHER : Dhouib_2011_FEBS.J_278_97
PubMedSearch : Dhouib_2011_FEBS.J_278_97
PubMedID: 21114629

Title : Effects of surfactants on lipase structure, activity, and inhibition - Delorme_2011_Pharm.Res_28_1831
Author(s) : Delorme V , Dhouib R , Canaan S , Fotiadu F , Carriere F , Cavalier JF
Ref : Pharm Res , 28 :1831 , 2011
Abstract : Lipase inhibitors are the main anti-obesity drugs prescribed these days, but the complexity of their mechanism of action is making it difficult to develop new molecules for this purpose. The efficacy of these drugs is known to depend closely on the physico-chemistry of the lipid-water interfaces involved and on the unconventional behavior of the lipases which are their target enzymes. The lipolysis reaction which occurs at an oil-water interface involves complex equilibria between adsorption-desorption processes, conformational changes and catalytic mechanisms. In this context, surfactants can induce significant changes in the partitioning of the enzyme and the inhibitor between the water phase and lipid-water interfaces. Surfactants can be found at the oil-water interface where they compete with lipases for adsorption, but also in solution in the form of micellar aggregates and monomers that may interact with hydrophobic parts of lipases in solution. These various interactions, combined with the emulsification and dispersion of insoluble substrates and inhibitors, can either promote or decrease the activity and the inhibition of lipases. Here, we review some examples of the various effects of surfactants on lipase structure, activity and inhibition, which show how complex the various equilibria involved in the lipolysis reaction tend to be.
ESTHER : Delorme_2011_Pharm.Res_28_1831
PubMedSearch : Delorme_2011_Pharm.Res_28_1831
PubMedID: 21234659

Title : Watching intracellular lipolysis in mycobacteria using time lapse fluorescence microscopy - Dhouib_2011_Biochim.Biophys.Acta_1811_234
Author(s) : Dhouib R , Ducret A , Hubert P , Carriere F , Dukan S , Canaan S
Ref : Biochimica & Biophysica Acta , 1811 :234 , 2011
Abstract : The fact that Mycobacterium tuberculosis mobilizes lipid bodies (LB) located in the cytosol during infection process has been proposed for decades. However, the mechanisms and dynamics of mobilization of these lipid droplets within mycobacteria are still not completely characterized. Evidence in favour of this characterization was obtained here using a combined fluorescent microscopy and computational image processing approach. The decrease in lipid storage levels observed under nutrient depletion conditions was correlated with a significant increase in the size of the bacteria. LB fragmentation/condensation cycles were monitored in real time. The exact contribution of lipases in this process was confirmed using the lipase inhibitor tetrahydrolipstatin, which was found to prevent LB degradation and to limit the bacterial cell growth. The method presented here provides a powerful tool for monitoring in vivo lipolysis in mycobacteria and for obtaining new insights on the growth of cells and their entry into the dormant or reactivation phase. It should be particularly useful for studying the effects of chemical inhibitors and activators on cells as well as investigating other metabolic pathways.
ESTHER : Dhouib_2011_Biochim.Biophys.Acta_1811_234
PubMedSearch : Dhouib_2011_Biochim.Biophys.Acta_1811_234
PubMedID: 21238605

Title : Two cutinase-like proteins secreted by Mycobacterium tuberculosis show very different lipolytic activities reflecting their physiological function - Schue_2010_FASEB.J_24_1893
Author(s) : Schue M , Maurin D , Dhouib R , Bakala N'Goma JC , Delorme V , Lambeau G , Carriere F , Canaan S
Ref : FASEB Journal , 24 :1893 , 2010
Abstract : Cutinases are extracellular enzymes that are able to degrade cutin, a polyester protecting plant leaves and many kinds of lipids. Although cutinases are mainly found in phytopathogenic fungi or bacteria, 7 genes related to the cutinase family have been predicted in the genome of Mycobacterium tuberculosis. These genes may encode proteins that are involved in the complex lipid metabolism of the bacterium. Here, we report on the biochemical characterization of two secreted proteins of M. tuberculosis, Rv1984c and Rv3452, belonging to the cutinase family. Although their amino acid sequence shows 50% identity with that of the well-characterized cutinase from Fusarium solani pisi, and a high level of homology has been found to exist between these two enzymes, they show distinct substrate specificities. Rv1984c preferentially hydrolyzes medium-chain carboxylic esters and monoacylglycerols, whereas Rv3452 behaves like a phospholipase A(2), and it is able to induce macrophage lysis. The tetrahydrolipstatin inhibitor, a specific lipase inhibitor, abolishes the activity of both enzymes. Site-directed mutagenesis was performed to identify the catalytic triad of Rv1984c. Structural models for Rv1984c and Rv3452 were built, based on the crystal structure of F. solani cutinase, with a view to investigating the contribution of specific residues to the substrate specificity. Our findings open new prospects for investigating the physiological roles of cutinase-like proteins in the lipid metabolism and virulence of M. tuberculosis.
ESTHER : Schue_2010_FASEB.J_24_1893
PubMedSearch : Schue_2010_FASEB.J_24_1893
PubMedID: 20103719
Gene_locus related to this paper: myctu-cutas1 , myctu-RV3452

Title : A monoacylglycerol lipase from Mycobacterium smegmatis Involved in bacterial cell interaction - Dhouib_2010_J.Bacteriol_192_4776
Author(s) : Dhouib R , Laval F , Carriere F , Daffe M , Canaan S
Ref : Journal of Bacteriology , 192 :4776 , 2010
Abstract : MSMEG_0220 from Mycobacterium smegmatis, the ortholog of the Rv0183 gene from M. tuberculosis, recently identified and characterized as encoding a monoacylglycerol lipase, was cloned and expressed in Escherichia coli. The recombinant protein (rMSMEG_0220), which exhibits 68% amino acid sequence identity with Rv0183, showed the same substrate specificity and similar patterns of pH-dependent activity and stability as the M. tuberculosis enzyme. rMSMEG_0220 was found to hydrolyze long-chain monoacylglycerol with a specific activity of 143 +/- 6 U mg(-1). Like Rv0183 in M. tuberculosis, MSMEG_0220 was found to be located in the cell wall. To assess the in vivo role of the homologous proteins, an MSMEG_0220 disrupted mutant of M. smegmatis (MsDelta0220) was produced. An intriguing change in the colony morphology and in the cell interaction, which were partly restored in the complemented mutant containing either an active (ComMsDelta0220) or an inactive (ComMsDelta0220S111A) enzyme, was observed. Growth studies performed in media supplemented with monoolein showed that the ability of both MsDelta0220 and ComMsDelta0220S111A to grow in the presence of this lipid was impaired. Moreover, studies of the antimicrobial susceptibility of the MsDelta0220 strain showed that this mutant is more sensitive to rifampin and more resistant to isoniazid than the wild-type strain, pointing to a critical structural role of this enzyme in mycobacterial physiology, in addition to its function in the hydrolysis of exogenous lipids.
ESTHER : Dhouib_2010_J.Bacteriol_192_4776
PubMedSearch : Dhouib_2010_J.Bacteriol_192_4776
PubMedID: 20601476
Gene_locus related to this paper: mycs2-a0qnz7

Title : Lipolytic enzymes in Mycobacterium tuberculosis - Cotes_2008_Appl.Microbiol.Biotechnol_78_741
Author(s) : Cotes K , Bakala N'goma J C , Dhouib R , Douchet I , Maurin D , Carriere F , Canaan S
Ref : Applied Microbiology & Biotechnology , 78 :741 , 2008
Abstract : Mycobacterium tuberculosis is a bacterial pathogen that can persist for decades in an infected patient without causing a disease. In vivo, the tubercle bacillus present in the lungs store triacylglycerols in inclusion bodies. The same process can be observed in vitro when the bacteria infect adipose tissues. Indeed, before entering in the dormant state, bacteria accumulate lipids originating from the host cell membrane degradation and from de novo synthesis. During the reactivation phase, these lipids are hydrolysed and the infection process occurs. The degradation of both extra and intracellular lipids can be directly related to the presence of lipolytic enzymes in mycobacteria, which have been ignored during a long period particularly due to the difficulties to obtain a high expression level of these enzymes in M. tuberculosis. The completion of the M. tuberculosis genome offered new opportunity to this kind of study. The aim of this review is to focus on the recent results obtained in the field of mycobacterium lipolytic enzymes and although no experimental proof has been shown in vivo, it is tempting to speculate that these enzymes could be involved in the virulence and pathogenicity processes.
ESTHER : Cotes_2008_Appl.Microbiol.Biotechnol_78_741
PubMedSearch : Cotes_2008_Appl.Microbiol.Biotechnol_78_741
PubMedID: 18309478

Title : Characterization of an exported monoglyceride lipase from Mycobacterium tuberculosis possibly involved in the metabolism of host cell membrane lipids - Cotes_2007_Biochem.J_408_417
Author(s) : Cotes K , Dhouib R , Douchet I , Chahinian H , de Caro A , Carriere F , Canaan S
Ref : Biochemical Journal , 408 :417 , 2007
Abstract : The Rv0183 gene of the Mycobacterium tuberculosis H37Rv strain, which has been implicated as a lysophospholipase, was cloned and expressed in Escherichia coli. The purified Rv0183 protein did not show any activity when lysophospholipid substrates were used, but preferentially hydrolysed monoacylglycerol substrates with a specific activity of 290 units x mg(-1) at 37 degrees C. Rv0183 hydrolyses both long chain di- and triacylglycerols, as determined using the monomolecular film technique, although the turnover was lower than with MAG (monoacyl-glycerol). The enzyme shows an optimum activity at pH values ranging from 7.5 to 9.0 using mono-olein as substrate and is inactivated by serine esterase inhibitors such as E600, PMSF and tetrahydrolipstatin. The catalytic triad is composed of Ser110, Asp226 and His256 residues, as confirmed by the results of site-directed mutagenesis. Rv0183 shows 35% sequence identity with the human and mouse monoglyceride lipases and well below 15% with the other bacterial lipases characterized so far. Homologues of Rv0183 can be identified in other mycobacterial genomes such as Mycobacterium bovis, Mycobacterium smegmatis, and even Mycobacterium leprae, which is known to contain a low number of genes involved in the replication process within the host cells. The results of immunolocalization studies performed with polyclonal antibodies raised against the purified recombinant Rv0183 suggested that the enzyme was present only in the cell wall and culture medium of M. tuberculosis. Our results identify Rv0183 as the first exported lipolytic enzyme to be characterized in M. tuberculosis and suggest that Rv0183 may be involved in the degradation of the host cell lipids.
ESTHER : Cotes_2007_Biochem.J_408_417
PubMedSearch : Cotes_2007_Biochem.J_408_417
PubMedID: 17784850
Gene_locus related to this paper: myctu-rv0183

Title : Assaying lipase activity from oil palm fruit (Elaeis guineensis Jacq.) mesocarp - Ngando_2006_Plant.Physiol.Biochem_44_611
Author(s) : Ngando Ebongue GF , Dhouib R , Carriere F , Amvam Zollo PH , Arondel V
Ref : Plant Physiol Biochem , 44 :611 , 2006
Abstract : The mesocarp of mature oil palm fruit undergoes intensive triglycerides hydrolysis upon abscission and bruising. This generates such a high amount of free fatty acids that the oil might become unfit for human consumption without appropriate refining. The lipase (EC 3.1.1.3) involved in the breakdown of the oil is not stable after homogenization of the tissue in aqueous buffers. In this study, we have devised a solvent-based procedure that allowed us to obtain fractions with stable lipase activity. Using these fractions, we have determined the optimal conditions for assaying mesocarp lipase activity. The activity was highest at a temperature of 35 degrees C and a pH of 9. The lipase was found to be strictly calcium dependent. The specific activity of the lipase measured in optimal conditions was found to be 33 mumol fatty acids released min(-1) mg(-1) protein using olive oil as substrate. The mesocarp contains about 190 U of lipase g(-1) fresh weight. This activity was found to be inhibited by the lipase inhibitor tetrahydrolipstatin (THL), suggesting that the lipase is a serine hydrolase.
ESTHER : Ngando_2006_Plant.Physiol.Biochem_44_611
PubMedSearch : Ngando_2006_Plant.Physiol.Biochem_44_611
PubMedID: 17064925