Pathogenic Candida albicans yeasts frequently cause infections in hospitals. Antifungal drugs lose effectiveness due to other Candida species and resistance. New medications are thus required. Secreted aspartic protease of C. parapsilosis (Sapp1p) is a promising target. We have thus solved the crystal structures of Sapp1p complexed to four peptidomimetic inhibitors. Three potent inhibitors (Ki: 0.1, 0.4, 6.6 nM) resembled pepstatin A (Ki: 0.3 nM), a general aspartic protease inhibitor, in terms of their interactions with Sapp1p. However, the weaker inhibitor (Ki: 14.6 nM) formed fewer nonpolar contacts with Sapp1p, similarly to the smaller HIV protease inhibitor ritonavir (Ki: 1.9 µM), which, moreover, formed fewer H-bonds. The analyses have revealed the structural determinants of the subnanomolar inhibition of C. parapsilosis aspartic protease. Because of the high similarity between Saps from different Candida species, these results can further be used for the design of potent and specific Sap inhibitor-based antimycotic drugs.

• Klíčová slova
• Inhibitor, crystal structure, hydrogen bonds, noncovalent interactions, peptidomimetics,
• MeSH
• aspartátové endopeptidasy antagonisté a inhibitory   metabolismus   MeSH
• Candida parapsilosis enzymologie   MeSH
• fungální proteiny antagonisté a inhibitory   metabolismus   MeSH
• inhibitory proteas chemická syntéza   chemie   farmakologie   MeSH
• molekulární modely MeSH
• molekulární struktura MeSH
• peptidomimetika chemická syntéza   chemie   farmakologie   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aspartátové endopeptidasy MeSH
• fungální proteiny MeSH
• inhibitory proteas MeSH
• peptidomimetika MeSH
• SAPP1 protein, Candida parapsilosis MeSH Prohlížeč

Cryptococcosis is an invasive infection that accounts for 15% of AIDS-related fatalities. Still, treating cryptococcosis remains a significant challenge due to the poor availability of effective antifungal therapies and emergence of drug resistance. Interestingly, protease inhibitor components of antiretroviral therapy regimens have shown some clinical benefits in these opportunistic infections. We investigated Major aspartyl peptidase 1 (May1), a secreted Cryptococcus neoformans protease, as a possible target for the development of drugs that act against both fungal and retroviral aspartyl proteases. Here, we describe the biochemical characterization of May1, present its high-resolution X-ray structure, and provide its substrate specificity analysis. Through combinatorial screening of 11,520 compounds, we identified a potent inhibitor of May1 and HIV protease. This dual-specificity inhibitor exhibits antifungal activity in yeast culture, low cytotoxicity, and low off-target activity against host proteases and could thus serve as a lead compound for further development of May1 and HIV protease inhibitors.

• MeSH
• antifungální látky chemie   metabolismus   farmakologie   MeSH
• aspartátové proteasy antagonisté a inhibitory   genetika   metabolismus   MeSH
• Cryptococcus neoformans enzymologie   MeSH
• fungální proteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• HIV-proteasa chemie   metabolismus   MeSH
• HIV enzymologie   MeSH
• houby účinky léků   MeSH
• katalytická doména MeSH
• krystalografie rentgenová MeSH
• preklinické hodnocení léčiv MeSH
• rekombinantní proteiny biosyntéza   chemie   izolace a purifikace   MeSH
• simulace molekulární dynamiky MeSH
• substrátová specifita MeSH
• vazebná místa MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• antifungální látky MeSH
• aspartátové proteasy MeSH
• fungální proteiny MeSH
• HIV-proteasa MeSH
• rekombinantní proteiny MeSH

Nepenthesins are aspartic proteases secreted by carnivorous pitcher plants of the genus Nepenthes. They significantly differ in sequence from other plant aspartic proteases. This difference, which provides more cysteine residues in the structure of nepenthesins, may contribute to their unique stability profile. Recombinantly produced nepenthesin 1 (rNep1) from N. gracilis in complex with pepstatin A was crystallized under two different crystallization conditions using a newly formulated low-pH crystallization screen. The diffraction data were processed to 2.9 and 2.8 Å resolution, respectively. The crystals belonged to space group P212121, with unit-cell parameters a = 86.63, b = 95.90, c = 105.40 Å, α = β = γ = 90° and a = 86.28, b = 97.22, c = 103.78 Å, α = β = γ = 90°, respectively. Matthews coefficient and solvent-content calculations suggest the presence of two molecules of rNep1 in the asymmetric unit. Here, the details of the crystallization experiment and analysis of the X-ray data are reported.

• Klíčová slova
• Nepenthes gracilis, aspartic proteases, low-pH crystallization screen, nepenthesins,
• MeSH
• aspartátové endopeptidasy chemie   MeSH
• koncentrace vodíkových iontů MeSH
• krystalizace MeSH
• krystalografie rentgenová MeSH
• Magnoliopsida enzymologie   MeSH
• pepstatiny chemie   MeSH
• rostlinné proteiny chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aspartátové endopeptidasy MeSH
• pepstatin MeSH Prohlížeč
• pepstatiny MeSH
• rostlinné proteiny MeSH

To identify the gut-associated tick aspartic hemoglobinase, this work focuses on the functional diversity of multiple Ixodes ricinus cathepsin D forms (IrCDs). Out of three encoding genes representing Ixodes scapularis genome paralogs, IrCD1 is the most distinct enzyme with a shortened propeptide region and a unique pattern of predicted post-translational modifications. IrCD1 gene transcription is induced by tick feeding and is restricted to the gut tissue. The hemoglobinolytic role of IrCD1 was further supported by immunolocalization of IrCD1 in the vesicles of tick gut cells. Properties of recombinantly expressed rIrCD1 are consistent with the endo-lysosomal environment because the zymogen is autoactivated and remains optimally active in acidic conditions. Hemoglobin cleavage pattern of rIrCD1 is identical to that produced by the native enzyme. The preference for hydrophobic residues at the P1 and P1' position was confirmed by screening a novel synthetic tetradecapeptidyl substrate library. Outside the S1-S1' regions, rIrCD1 tolerates most amino acids but displays a preference for tyrosine at P3 and alanine at P2'. Further analysis of the cleavage site location within the peptide substrate indicated that IrCD1 is a true endopeptidase. The role in hemoglobinolysis was verified with RNAi knockdown of IrCD1 that decreased gut extract cathepsin D activity by >90%. IrCD1 was newly characterized as a unique hemoglobinolytic cathepsin D contributing to the complex intestinal proteolytic network of mainly cysteine peptidases in ticks.

• MeSH
• genetická transkripce fyziologie   MeSH
• genom fyziologie   MeSH
• hemoglobiny genetika   metabolismus   MeSH
• kathepsin D genetika   metabolismus   MeSH
• klíště enzymologie   genetika   MeSH
• posttranslační úpravy proteinů fyziologie   MeSH
• proteiny členovců genetika   metabolismus   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• střeva enzymologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hemoglobiny MeSH
• kathepsin D MeSH
• proteiny členovců MeSH
• rekombinantní proteiny MeSH

Trypsin proteinase inhibitors (TPIs) of Nicotiana attenuata are major antiherbivore defenses that increase dramatically in leaves after attack or methyl jasmonate (MeJA) elicitation. To understand the elicitation process, we characterized the proteolytic fragmentation and release of TPIs from a multidomain precursor by proteases in MeJA-elicited and unelicited plants. A set of approximately 6-kD TPI peptides was purified from leaves, and their posttranslational modifications were characterized. In MeJA-elicited plants, the diversity of TPI structures was greater than the precursor gene predicted. This elicited structural heterogeneity resulted from differential fragmentation of the linker peptide (LP) that separates the seven-domain TPI functional domains. Using an in vitro fluorescence resonance energy transfer assay and synthetic substrates derived from the LP sequence, we characterized proteases involved in both the processing of the TPI precursor and its vacuolar targeting sequence. Although both a vacuolar processing enzyme and a subtilisin-like protease were found to participate in a two-step processing of LP, only the activity of the subtilisin-like protease was significantly increased by MeJA elicitation. We propose that MeJA elicitation increases TPI precursor production and saturates the proteolytic machinery, changing the processing pattern of TPIs. To test this hypothesis, we elicited a TPI-deficient N. attenuata genotype that had been transformed with a functional NaTPI gene under control of a constitutive promoter and characterized the resulting TPIs. We found no alterations in the processing pattern predicted from the sequence: a result consistent with the saturation hypothesis.

• MeSH
• acetáty farmakologie   MeSH
• cyklopentany farmakologie   MeSH
• geneticky modifikované rostliny MeSH
• inhibitory proteas metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• listy rostlin metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• oxylipiny MeSH
• posttranslační úpravy proteinů MeSH
• proteasy metabolismus   MeSH
• proteinové prekurzory metabolismus   MeSH
• regulace genové exprese enzymů MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné proteiny metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• stabilita enzymů MeSH
• tabák enzymologie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acetáty MeSH
• cyklopentany MeSH
• inhibitory proteas MeSH
• methyl jasmonate MeSH Prohlížeč
• oxylipiny MeSH
• proteasy MeSH
• proteinové prekurzory MeSH
• rostlinné proteiny MeSH