- Publikační typ
- abstrakt z konference MeSH
- MeSH
- antifungální látky farmakologie škodlivé účinky terapeutické užití MeSH
- Candida albicans metabolismus patogenita růst a vývoj MeSH
- Candida metabolismus patogenita virologie MeSH
- echinokandiny terapeutické užití MeSH
- flukonazol terapeutické užití MeSH
- ketokonazol terapeutické užití MeSH
- kvasinky * patogenita MeSH
- mykózy MeSH
- nemoci imunitního systému komplikace MeSH
Secreted aspartic proteases (Saps) are extracellular proteolytic enzymes that enhance the virulence of Candida pathogens. These enzymes therefore represent possible targets for therapeutic drug design. Saps are inhibited by nanomolar concentrations of the classical inhibitor of aspartic proteases pepstatin A and also by the inhibitors of the HIV protease, but with the K(i) of micromolar values or higher. To contribute to the discussion regarding whether HIV protease inhibitors can act against opportunistic mycoses by the inhibition of Saps, we determined the structure of Sapp1p from Candida parapsilosis in complex with ritonavir (RTV), a clinically used inhibitor of the HIV protease. The crystal structure refined at resolution 2.4 Å proved binding of RTV into the active site of Sapp1p and provided the structural information necessary to evaluate the stability and specificity of the protein-inhibitor interaction.
- MeSH
- aspartátové endopeptidasy antagonisté a inhibitory chemie MeSH
- Candida enzymologie MeSH
- fungální proteiny antagonisté a inhibitory chemie MeSH
- inhibitory HIV-proteasy chemie farmakologie MeSH
- krystalografie rentgenová MeSH
- molekulární modely MeSH
- ritonavir chemie farmakologie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Opportunistic pathogens of the genus Candida cause infections representing a major threat to long-term survival of immunocompromised patients. Virulence of the Candida pathogens is enhanced by production of extracellular proteolytic enzymes and secreted aspartic proteases (Saps) are therefore studied as potential virulence factors and possible targets for therapeutic drug design. Candida parapsilosis is less invasive than C. albicans, however, it is one of the leading causative agents of yeast infections. We report three-dimensional crystal structure of Sapp1p from C. parapsilosis in complex with pepstatin A, the classical inhibitor of aspartic proteases. The structure of Sapp1p was determined from protein isolated from its natural source and represents the first structure of Sap from C. parapsilosis. Overall fold and topology of Sapp1p is very similar to the archetypic fold of monomeric aspartic protease family and known structures of Sap isoenzymes from C. albicans and Sapt1p from C. tropicalis. Structural comparison revealed noticeable differences in the structure of loops surrounding the active site. This resulted in differential character, shape, and size of the substrate binding site explaining divergent substrate specificities and inhibitor affinities. Determination of structures of Sap isoenzymes from various species might contribute to the development of new Sap-specific inhibitors.
- MeSH
- aspartátové endopeptidasy chemie MeSH
- Candida enzymologie patogenita MeSH
- fungální proteiny chemie MeSH
- katalytická doména MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- pepstatiny chemie MeSH
- sbalování proteinů MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Publikační typ
- práce podpořená grantem MeSH
The predicted secretome of the opportunistic fungal pathogen Candida albicans contains more than 200 ORFs of diverse and often unknown function. Majority of the secreted proteins that have been experimentally evaluated to date are hydrolytic enzymes (proteinases, phospholipases and lipases). Acting on the interface between the pathogen and the host, the secreted hydrolases may enable invasion of host tissues, help the pathogen to avoid host defense mechanisms, or allow the microorganism to utilize host cell macromolecules as a source of nutrients. Aspartic proteinases constitute the best-characterized family of the C. albicans secreted proteins. Number of studies addressed also secreted phospholipases. Lipases and N-acetylhexosaminidase have received less attention thus far. Many ORFs that are predicted to encode secreted proteins await characterization.
Secreted aspartic proteases (Saps) of pathogenic Candida spp. represent a specific target for antifungal drug development. We synthesized a series of peptidomimetic inhibitors with different isosteric groups and modifications at individual positions and tested them with purified Saps from C. albicans (Sap2p), C. tropicalis (Sapt1p), and C. parapsilosis (Sapp1p). The kinetic parameters indicated that all three proteases prefer binding of inhibitors containing bulky hydrophobic residues between positions P3 and P3'. The most divergent specificity was found for Sapp1p. The sequence alignment of Sap2p, Sapt1p, and Sapp1p, and homology modeling of Sapp1p with the crystal structure of Sapt1p and the complex of Sap2p with a peptidomimetic inhibitor showed that the overall folds of Sap2p, Sapt1p, and Sapp1p are similar. However, the N- and C-terminal loops formed by disulfide bonds between residues 47-53 and 258-292 are significantly shorter in Sapp1p, and a unique insertion following Tyr 129 in Sapp1p results in the formation of a loop that can interact with inhibitor residues. These Sapp1p structural differences might lead to its altered susceptibility to inhibition.
- MeSH
- aspartátové endopeptidasy chemie účinky léků MeSH
- Candida enzymologie MeSH
- financování organizované MeSH
- inhibitory proteas farmakologie chemická syntéza chemie MeSH
- izoenzymy chemie účinky léků MeSH
- molekulární konformace MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- stereoizomerie MeSH
- substrátová specifita MeSH
- terciární struktura proteinů MeSH
- vztahy mezi strukturou a aktivitou MeSH
