- Diffusion based kinetic selectivity modulation of enzymatic proteolysis in a microfluidic reactor: Experimental analysis and stochastic modeling
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Microreactors enable new experimental ways for enzyme engineering. In this context, we show that the liquid-liquid parallel laminar flows in microchannels cause a kinetic selectivity modification of proteolytic enzymatic reaction involving hemoglobin and pepsin, reaction that generates numerous bioactive peptides at different advancement state. Here we show that this diffusion based kinetic modulation induces an altered peptides appearance kinetics for a part of the initial substrate population. Indeed, microfluidic and conventional batch experiments performed in the same reaction conditions lead to strong differences in the resulting peptidic profiles obtained by reversed-phase high-performance liquid chromatography. Such differences are explained by the laminar flow diffusive conditions inside microchannels and are supported by a stochastic algorithm based on the Michaelis-Menten equation. Several bioactive peptides are identified by mass spectrometry and show the potential of such methodology in peptides screening from a complex proteolysis but also for selective peptides preparation by microfluidics.
- Elagli, Adil,Laurette, Simon,Treizebre, Anthony,Bocquet, Bertrand,Froidevaux, Renato
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p. 3873 - 3882
(2014/01/06)
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- Cathepsin D activity and selectivity in the acidic conditions of a tumor microenvironment: Utilization in the development of a novel Cathepsin D substrate for simultaneous cancer diagnosis and therapy
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Pro-Cathepsin D (pCD) is an aspartyl endopeptidase which is over expressed in many cancers. This over expression generally led to its secretion into the extracellular culture medium of cancer cells. Moreover, pCD can auto activate and cleave its substrates at an acidic pH compatible with that found in tumor microenvironments (TME). Thus, exploiting these two pathological characteristics of TME offers the opportunity to develop new protease-activated vector on the basis of their specific substrate structures. The aim of this study was to validate new pCD substrates in the extracellular pH conditions of TME. As a first step, we investigated the effect of pH on the catalytic activity and selectivity of mature Cathepsin D (CD). It was found that the increase in the pH of the media led to a decrease in the reaction rate. However, the specificity of mature CD was not affected by a variation in pH. In the second step, the effect of the substrate structure was studied. We demonstrated that the substrate structure had a significant effect on the catalytic activity of CD. In fact, some modifications in peptide structure induced a change in the catalytic behavior that involved a substrate activation phenomenon. We suggest that this activation may be related to the amphiphilic nature of the modified peptide that may induce an interfacial activation mechanism. Finally, pCD, which is the major form found in the extracellular culture medium of cancer cells, was used. We demonstrated that the proform of CD cleave the modified peptide 5 at pH 6.5 with the same cleavage selectivity obtained with the mature form of the protease. These data provide a better understanding of CD behavior in tumor microenvironment conditions and this knowledge can be used to develop more specific tools for diagnosis and drug delivery.
- Achour, Oussama,Bridiau, Nicolas,Kacem, Meriem,Delatouche, Régis,Bordenave-Juchereau, Stéphanie,Sannier, Frédéric,Thiéry, Valérie,Piot, Jean-Marie,Maugard, Thierry,Arnaudin, Ingrid
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p. 2010 - 2017
(2013/10/22)
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