Cholesterol and Its Derivatives Reversibly Inhibit Proteinase K

Singh, Namrata and Bhattacharyya, Debasish (2017) Cholesterol and Its Derivatives Reversibly Inhibit Proteinase K. Journal of Cellular Physiology, 232 (3). pp. 596-609. ISSN 0021-9541

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    Abstract

    Microorganisms express a variety of proteases that degrade many proteins of the host body and subvert host immune response. While elucidating the mechanism/s of an immune stimulatory drug that contains bile lipid, regulation of proteolytic activity was investigated. The drug and bile lipids both stabilize Proteinase K, an aggressive protease of fungal origin against auto-digestion. Among the components of bile lipids, only cholesterol and its derivatives stabilize the enzyme. Biophysical evidences such as scattering of light, intrinsic and extrinsic fluorescence emission spectra, circular dichroism spectra, atomic force microscopy, and transmission electron microscopy images indicated that cholesterol and its derivatives interact with Proteinase K. Inhibition kinetics using esterolysis of ATEE revealed noncompetitive inhibition by cholesterol. Surface Plasmon Resonance and mass spectrometric analysis indicated 1:1 stoichiometry of binding and with dissociation constant in the mM range. Further, the presence of four cholesterol recognition amino acid consensus motifs (CRAC motifs I–IV) was identified in Proteinase K. Bioinformatics analysis revealed that the whole stretch of cholesterol interacts very well with the hydrophobic groove of motif II only among the four CRAC motifs. Variation of cholesterol content of HepG2 human liver carcinoma cells showed positive correlation with binding of fluorescence tagged Proteinase K. Under these conditions, binding of Proteinase K to the cells did not affect their morphology, viability and growth kinetics. Cell bound Proteinase K could be released by an excess of its substrate, thereby restoring reversibly its proteolytic activity

    Item Type: Article
    URI: http://www.eprints.iicb.res.in/id/eprint/2613
    Subjects: Structural Biology & Bioinformatics
    Divisions: Indian Institute of Chemical Biology
    Depositing User: Ms Sutapa Ganguly
    Date Deposited: 20 Mar 2017 12:06
    Last Modified: 20 Mar 2017 16:30
    Official URL: http://dx.doi.org/10.1002/jcp.25457
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