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

Toxoplasma gondii possesses sets of dense granule proteins (GRAs) that either assemble at, or cross the parasitophorous vacuole membrane (PVM) and exhibit motifs resembling the HT/PEXEL previously identified in a repertoire of exported Plasmodium proteins. Within Plasmodium spp., cleavage of the HT/PEXEL motif by the endoplasmic reticulum-resident protease Plasmepsin V precedes trafficking to and export across the PVM of proteins involved in pathogenicity and host cell remodelling. Here, we have functionally characterized the T. gondii aspartyl protease 5 (ASP5), a Golgi-resident protease that is phylogenetically related to Plasmepsin V. We show that deletion of ASP5 causes a significant loss in parasite fitness in vitro and an altered virulence in vivo. Furthermore, we reveal that ASP5 is necessary for the cleavage of GRA16, GRA19 and GRA20 at the PEXEL-like motif. In the absence of ASP5, the intravacuolar nanotubular network disappears and several GRAs fail to localize to the PVM, while GRA16 and GRA24, both known to be targeted to the host cell nucleus, are retained within the vacuolar space. Additionally, hypermigration of dendritic cells and bradyzoite cyst wall formation are impaired, critically impacting on parasite dissemination and persistence. Overall, the absence of ASP5 dramatically compromises the parasite's ability to modulate host signalling pathways and immune responses.

• MeSH
• aspartátové proteasy metabolismus   MeSH
• ELISA MeSH
• fluorescenční protilátková technika MeSH
• genový knockout MeSH
• Golgiho aparát enzymologie   MeSH
• interakce hostitele a parazita fyziologie   MeSH
• kultivované buňky MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• Toxoplasma enzymologie   patogenita   MeSH
• toxoplazmóza enzymologie   MeSH
• transfekce MeSH
• transmisní elektronová mikroskopie MeSH
• transport proteinů MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cathepsin research is one of the most progressive areas in proteolysis. Cathepsins are a diverse group of eukaryotic peptidases belonging to the aspartic, cysteine and serine classes. They act as both non-specific proteases and as specific processing enzymes in numerous physiological and pathological processes. In humans, their dysregulation is associated with severe pathologies, such as cancer, cardiovascular and neurodegenerative diseases, arthritis, and osteoporosis. Proteolytic systems controlled by cathepsins are a critical part of host-pathogen and host-parasite interactions as demonstrated for cathepsin-like peptidases from, e.g., hematophagous parasites, herbivorous insects and plants. Because of the broad involvement of cathepsins in pathological processes, they are among today’s top-priority drug targets. This review provides an update on the structure and function of pathology-associated cathepsins in humans and human parasites.

• MeSH
• aspartátové proteasy fyziologie   chemie   MeSH
• cysteinové proteasy fyziologie   chemie   MeSH
• endopeptidasy fyziologie   chemie   MeSH
• farmaceutická chemie MeSH
• inhibitory proteas * chemie   MeSH
• kathepsiny * biosyntéza   fyziologie   chemie   klasifikace   MeSH
• lidé MeSH
• parazitární nemoci * krev   MeSH
• patologické procesy * etiologie   MeSH
• proteolýza * MeSH
• racionální návrh léčiv MeSH
• serinové proteasy fyziologie   chemie   MeSH
• výzkum MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

Carnivorous plants of the genus Nepenthes produce their own aspartic proteases, nepenthesins, to digest prey trapped in their pitchers. Nepenthesins differ significantly in sequence from other aspartic proteases in the animal or even plant kingdoms. This difference, which also brings more cysteine residues into the structure of these proteases, can be a cause of uniquely high temperature and pH stabilities of nepenthesins. Their detailed structure characterization, however, has not previously been possible due to low amounts of protease present in the pitcher fluid and also due to limited accessibility of Nepenthes plants. In the present study we describe a convenient way for obtaining high amounts of nepenthesin-1 from Nepenthes gracilis using heterologous production in Escherichia coli. The protein can be easily refolded in vitro and its characteristics are very close to those described for a natural enzyme isolated from the pitcher fluid. Similarly to the natural enzyme, recombinant nepenthesin-1 is sensitive to denaturing and reducing agents. It also has maximal activity around pH 2.5, shows unusual stability at high pH and its activity is not irreversibly inhibited even after prolonged incubation in the basic pH range. On the other hand, temperature stability of the recombinant enzyme is lower in comparison with the natural enzyme, which can be attributed to missing N-glycosylation in the recombinant protein.

• MeSH
• aspartátové proteasy chemie   genetika   metabolismus   MeSH
• disulfidy MeSH
• koncentrace vodíkových iontů MeSH
• Magnoliopsida enzymologie   genetika   MeSH
• masožravci MeSH
• redukční činidla MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• rostlinné proteiny chemie   genetika   metabolismus   MeSH
• stabilita enzymů MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Vacuoles play an important role in the physiology of pathogenic Candida spp. However, information on Candida albicans vacuolar enzymes, their properties, and regulation is scarce. Expression of the genes APR1 and CPY1 encoding vacuolar aspartic protease and serine carboxypeptidase, respectively, was analyzed using a clinical isolate of C. albicans. The transcription of both APR1 and CPY1 was upregulated in midexponential phase, together with increasing size of the vacuoles, when C. albicans was cultivated in yeast extract-peptone-dextrose agar at 30 °C. However, simultaneous upregulation of protein synthesis occurred only for Cpy1p. Analysis of APR1 and CPY1 expression under nitrogen-limited conditions revealed that the genes were regulated on both the transcriptional and translational levels and detectable amounts of Apr1p were synthesized only when C. albicans was grown in nitrogen-limited media.

• MeSH
• aspartátové proteasy genetika   metabolismus   MeSH
• Candida albicans enzymologie   genetika   růst a vývoj   MeSH
• dusík metabolismus   MeSH
• karboxypeptidasy genetika   metabolismus   MeSH
• kultivační média metabolismus   MeSH
• regulace genové exprese u hub MeSH
• upregulace MeSH
• vakuoly enzymologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The virulence of the Candida pathogens is enhanced by the production of secreted aspartic proteases, which therefore represent possible targets for drug design. Here, the crystal structure of the secreted aspartic protease Sapp2p from Candida parapsilosis was determined. Sapp2p was isolated from its natural source and crystallized in complex with pepstatin A, a classical aspartic protease inhibitor. The atomic resolution of 0.83 Å allowed the protonation states of the active-site residues to be inferred. A detailed comparison of the structure of Sapp2p with the structure of Sapp1p, the most abundant C. parapsilosis secreted aspartic protease, was performed. The analysis, which included advanced quantum-chemical interaction-energy calculations, uncovered molecular details that allowed the experimentally observed equipotent inhibition of both isoenzymes by pepstatin A to be rationalized.

• MeSH
• aspartátové proteasy chemie   genetika   izolace a purifikace   metabolismus   MeSH
• Candida chemie   enzymologie   genetika   MeSH
• exprese genu MeSH
• fungální proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• inhibitory proteas chemie   MeSH
• izoenzymy chemie   genetika   izolace a purifikace   metabolismus   MeSH
• katalytická doména MeSH
• kinetika MeSH
• krystalografie rentgenová MeSH
• kvantová teorie MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• pepstatiny chemie   MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• strukturní homologie proteinů MeSH
• substrátová specifita MeSH
• termodynamika MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

HIV protease (PR) represents a prime target for rational drug design, and protease inhibitors (PI) are powerful antiviral drugs. Most of the current PIs are pseudopeptide compounds with limited bioavailability and stability, and their use is compromised by high costs, side effects, and development of resistant strains. In our search for novel PI structures, we have identified a group of inorganic compounds, icosahedral metallacarboranes, as candidates for a novel class of nonpeptidic PIs. Here, we report the potent, specific, and selective competitive inhibition of HIV PR by substituted metallacarboranes. The most active compound, sodium hydrogen butylimino bis-8,8-[5-(3-oxa-pentoxy)-3-cobalt bis(1,2-dicarbollide)]di-ate, exhibited a K(i) value of 2.2 nM and a submicromolar EC(50) in antiviral tests, showed no toxicity in tissue culture, weakly inhibited human cathepsin D and pepsin, and was inactive against trypsin, papain, and amylase. The structure of the parent cobalt bis(1,2-dicarbollide) in complex with HIV PR was determined at 2.15 A resolution by protein crystallography and represents the first carborane-protein complex structure determined. It shows the following mode of PR inhibition: two molecules of the parent compound bind to the hydrophobic pockets in the flap-proximal region of the S3 and S3' subsites of PR. We suggest, therefore, that these compounds block flap closure in addition to filling the corresponding binding pockets as conventional PIs. This type of binding and inhibition, chemical and biological stability, low toxicity, and the possibility to introduce various modifications make boron clusters attractive pharmacophores for potent and specific enzyme inhibition.

• MeSH
• aspartátové endopeptidasy chemie   MeSH
• borany farmakologie   chemická syntéza   chemie   MeSH
• financování organizované MeSH
• HIV-proteasa chemie   MeSH
• inhibitory HIV-proteasy farmakologie   chemická syntéza   chemie   MeSH
• krystalografie rentgenová MeSH
• kvantitativní vztahy mezi strukturou a aktivitou MeSH
• racionální návrh léčiv MeSH

Candida parapsilosis is an emerging non-albicans Candida species that largely affects low-birth-weight infants and immunocompromised patients. Fungal pathogenesis is promoted by the dynamic expression of diverse virulence factors, with secreted proteolytic enzymes being linked to the establishment and progression of disease. Although secreted aspartyl proteases (Sap) are critical for Candida albicans pathogenicity, their role in C. parapsilosis is poorly elucidated. In the present study, we aimed to examine the contribution of C. parapsilosisSAPP genes SAPP1, SAPP2, and SAPP3 to the virulence of the species. Our results indicate that SAPP1 and SAPP2, but not SAPP3, influence adhesion, host cell damage, phagosome-lysosome maturation, phagocytosis, killing capacity, and cytokine secretion by human peripheral blood-derived macrophages. Purified Sapp1p and Sapp2p were also shown to efficiently cleave host complement component 3b (C3b) and C4b proteins and complement regulator factor H. Additionally, Sapp2p was able to cleave factor H-related protein 5 (FHR-5). Altogether, these data demonstrate the diverse, significant contributions that SAPP1 and SAPP2 make to the establishment and progression of disease by C. parapsilosis through enabling the attachment of the yeast cells to mammalian cells and modulating macrophage biology and disruption of the complement cascade.IMPORTANCE Aspartyl proteases are present in various organisms and, among virulent species, are considered major virulence factors. Host tissue and cell damage, hijacking of immune responses, and hiding from innate immune cells are the most common behaviors of fungal secreted proteases enabling pathogen survival and invasion. C. parapsilosis, an opportunistic human-pathogenic fungus mainly threatening low-birth weight neonates and children, possesses three SAPP protein-encoding genes that could contribute to the invasiveness of the species. Our results suggest that SAPP1 and SAPP2, but not SAPP3, influence host evasion by regulating cell damage, phagocytosis, phagosome-lysosome maturation, killing, and cytokine secretion. Furthermore, SAPP1 and SAPP2 also effectively contribute to complement evasion.

• MeSH
• aspartátové endopeptidasy genetika   metabolismus   MeSH
• buněčné linie MeSH
• Candida parapsilosis enzymologie   patogenita   MeSH
• faktory virulence genetika   metabolismus   MeSH
• fungální proteiny genetika   metabolismus   MeSH
• imunitní únik MeSH
• komplement imunologie   MeSH
• lidé MeSH
• makrofágy mikrobiologie   MeSH
• virulence MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The opportunistic fungal pathogen Cryptococcus neoformans is a major cause of mortality in immunocompromised individuals, resulting in more than 600,000 deaths per year. Many human fungal pathogens secrete peptidases that influence virulence, but in most cases the substrate specificity and regulation of these enzymes remains poorly understood. The paucity of such information is a roadblock to our understanding of the biological functions of peptidases and whether or not these enzymes are viable therapeutic targets. We report here an unbiased analysis of secreted peptidase activity and specificity in C. neoformans using a mass spectrometry-based substrate profiling strategy and subsequent functional investigations. Our initial studies revealed that global peptidase activity and specificity are dramatically altered by environmental conditions. To uncover the substrate preferences of individual enzymes and interrogate their biological functions, we constructed and profiled a ten-member gene deletion collection of candidate secreted peptidases. Through this deletion approach, we characterized the substrate specificity of three peptidases within the context of the C. neoformans secretome, including an enzyme known to be important for fungal entry into the brain. We selected a previously uncharacterized peptidase, which we term Major aspartyl peptidase 1 (May1), for detailed study due to its substantial contribution to extracellular proteolytic activity. Based on the preference of May1 for proteolysis between hydrophobic amino acids, we screened a focused library of aspartyl peptidase inhibitors and identified four high-affinity antagonists. Finally, we tested may1Δ strains in a mouse model of C. neoformans infection and found that strains lacking this enzyme are significantly attenuated for virulence. Our study reveals the secreted peptidase activity and specificity of an important human fungal pathogen, identifies responsible enzymes through genetic tests of their function, and demonstrates how this information can guide the development of high affinity small molecule inhibitors.

• MeSH
• aspartátové proteasy metabolismus   MeSH
• Cryptococcus neoformans enzymologie   patogenita   MeSH
• faktory virulence metabolismus   MeSH
• fungální proteiny metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• imunoblotting MeSH
• koncentrace vodíkových iontů MeSH
• kryptokokóza enzymologie   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• modely nemocí na zvířatech MeSH
• myši MeSH
• proteasy metabolismus   MeSH
• proteomika MeSH
• stanovení celkové genové exprese MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Parazitární onemocnění postihují stovky milionů lidí po celém světě, a představují tak závažný zdravotní a socioekonomický problém. Proteolytické enzymy (proteasy) parazitů se účastní řady klíčových procesů spojených s patogenezí, což z nich činí perspektivní cílové molekuly pro vývoj antiparazitárních léčiv. Aspartátové proteasy tvoří relativně malou, ale funkčně významnou součást proteolytické výbavy parazitů. Mají nezastupitelnou roli v trávicím systému při degradaci krevních proteinů hostitele, které slouží parazitům jako výhradní zdroj potravy. Kromě toho plní i další zásadní úlohy v interakci parazita s hostitelem nebo při vývoji vajíček parazitů. V tomto referátu jsou shrnuty dostupné poznatky o aspartátových proteasach parazitů, jejich regulaci a využití v biomedicíně.

Parasitic diseases affect hundreds of millions people around the world and represent a serious health and socioeconomic problem. Proteolytic enzymes (proteases) of parasites are involved in many processes associated with pathogenesis, which makes them promising molecular targets for the development of antiparasitic drugs. Aspartic proteases comprise a relatively small, but functionally significant part of the proteolytic system of parasites. They play a critical role in the digestion of host blood proteins that serve as an ultimate source of nutrients for blood -feeding parasites. Furthermore, they have essential functions in host -parasite interactions or parasite egg development. This review article summarizes current knowledge on aspartic proteases of parasites, their regulation and use in biomedicine.

• MeSH
• aspartátové proteasy * MeSH
• interakce hostitele a patogenu MeSH
• lidé MeSH
• paraziti * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH