Protease characterization
Dotaz
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- MeSH
- elektroforéza metody MeSH
- krysa rodu rattus MeSH
- proteasy MeSH
- střeva enzymologie MeSH
- Check Tag
- krysa rodu rattus 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
Byla purifikována a charakterizována protease stabilni v organických rozpoštědlech. Její molekulová hmotnost byla stanovena pomocí SDS-PAGE a SEC na 26 kDa. Purifikovaná proteasa měla nejvyšší aktivitu při 70°C a její pH optimum bylo velmi široké (5,0 – 12.0). Po ošetření 5 mM EDTA a β-mercaptoethanolem zůstával enzym aktivní. Proteasa měla po hodinové inkubaci při 30°C v přítomnosti 20, 40 a 60 % (v/v) organických rozpouštědel, jako jsou například DMSO, DMF, aceton, ethanol, iso-propanol a toluen vyšší aktivitu a stabilitu. Proteolytická aktivita byla v přítomnosti Mn2+ iontů prokazatelně vyšší a v přítomnosti 5, 10 a 15 mM Pb2+, Zn2+, K+, Fe2+, Co2+, Cd2+, Mg2+, Ca2+, Fe3+ iontů po dobu 1h při 30°C si zachovávala více než 90 % aktivity.
In the present research, stable in organic solvent protease from a new thermophilic actinomycete isolate, was purified and characterized. The approximate molecular mass of 26 kDa was determined by SDS-PAGE and SEC. The purified protease showed maximum activity at 70°C and exhibited broad pH optimum (5.0 – 12.0). After treatment with 5 mM EDTA and β-mercaptoethanol the enzyme remained fully active. The protease showed an increased activity and stability in the presence of 20, 40 and 60 % (v/v) organic solvents such as DMSO, DMF, acetone, ethanol, iso-propanol and toluene when incubated for 1h at 30°C. The proteolytic activity was significantly enhanced in presence of Mn2+ and remained more than 90 % active in the presence of 5, 10 and 15 mM Pb2+, Zn2+, K+, Fe2+, Co2+, Cd2+, Mg2+, Ca2+, Fe3+ ions for 1h at 30°C. The kinetic constants were also determined.
Darunavir is the most recently approved human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) and is active against many HIV type 1 PR variants resistant to earlier-generation PIs. Darunavir shows a high genetic barrier to resistance development, and virus strains with lower sensitivity to darunavir have a higher number of PI resistance-associated mutations than viruses resistant to other PIs. In this work, we have enzymologically and structurally characterized a number of highly mutated clinically derived PRs with high levels of phenotypic resistance to darunavir. With 18 to 21 amino acid residue changes, the PR variants studied in this work are the most highly mutated HIV PR species ever studied by means of enzyme kinetics and X-ray crystallography. The recombinant proteins showed major defects in substrate binding, while the substrate turnover was less affected. Remarkably, the overall catalytic efficiency of the recombinant PRs (5% that of the wild-type enzyme) is still sufficient to support polyprotein processing and particle maturation in the corresponding viruses. The X-ray structures of drug-resistant PRs complexed with darunavir suggest that the impaired inhibitor binding could be explained by change in the PR-inhibitor hydrogen bond pattern in the P2' binding pocket due to a substantial shift of the aminophenyl moiety of the inhibitor. Recombinant virus phenotypic characterization, enzyme kinetics, and X-ray structural analysis thus help to explain darunavir resistance development in HIV-positive patients.
- MeSH
- genové produkty env - virus lidské imunodeficience metabolismus MeSH
- genové produkty gag - virus lidské imunodeficience metabolismus MeSH
- HIV infekce virologie MeSH
- HIV-1 izolace a purifikace účinky léků MeSH
- HIV-proteasa genetika chemie metabolismus MeSH
- inhibitory HIV-proteasy farmakologie MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- missense mutace MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- mutační analýza DNA MeSH
- polyproteiny metabolismus MeSH
- sekvence aminokyselin MeSH
- substituce aminokyselin MeSH
- sulfonamidy farmakologie MeSH
- terciární struktura proteinů MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- virová léková rezistence MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Protease inhibitors (PIs) are ubiquitous regulatory proteins present in all kingdoms. They play crucial tasks in controlling biological processes directed by proteases which, if not tightly regulated, can damage the host organism. PIs can be classified according to their targeted proteases or their mechanism of action. The functions of many PIs have now been characterized and are showing clinical relevance for the treatment of human diseases such as arthritis, hepatitis, cancer, AIDS, and cardiovascular diseases, amongst others. Other PIs have potential use in agriculture as insecticides, anti-fungal, and antibacterial agents. PIs from tick salivary glands are special due to their pharmacological properties and their high specificity, selectivity, and affinity to their target proteases at the tick-host interface. In this review, we discuss the structure and function of PIs in general and those PI superfamilies abundant in tick salivary glands to illustrate their possible practical applications. In doing so, we describe tick salivary PIs that are showing promise as drug candidates, highlighting the most promising ones tested in vivo and which are now progressing to preclinical and clinical trials.
- MeSH
- inhibitory proteas izolace a purifikace terapeutické užití MeSH
- interakce hostitele a parazita genetika imunologie MeSH
- klíšťata metabolismus MeSH
- lidé MeSH
- slinné žlázy metabolismus MeSH
- sliny chemie metabolismus MeSH
- transkriptom genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
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
Proteases are typical key enzymes that hydrolyze proteins into amino acids and peptides. Numerous proteases have been studied, but the discovery of metagenome-derived proteases is still significant for both commercial applications and basic research. An unexplored protease gene sep1A was identified by function-based screening from a plasmid metagenomic library derived from uncultured contaminated agricultural soil microorganisms. The putative protease gene was subcloned into pET-32a (+) vector and overexpressed in E. coli BL21(DE3) pLysS, then the recombinant protein was purified to homogeneity. The detailed biochemical characterization of the Sep1A protein was performed, including its molecular characterization, specific activity, pH-activity profile, metal ion-activity profile, and enzyme kinetic assays. Furthermore, the protein engineering approach of random mutagenesis via error-prone PCR was applied on the original Sep1A protein. Biochemical characterization demonstrated that the purified recombinant Ep48 protein could hydrolyze casein. Compared with the original Sep1A protein, the best variant of Ep48 in the random mutagenesis library, with the Gln307Leu and Asp391Gly changes, exhibited 2.62-fold activity at the optimal reaction conditions of 50 °C and pH 9.0. These results are the first step toward a better understanding of the properties of Sep1A protein. Protein engineering with error-prone PCR paves the way toward the metagenome-derived genes for biotechnological applications.
- MeSH
- Bacteria chemie enzymologie genetika izolace a purifikace MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- genová knihovna MeSH
- kinetika MeSH
- klonování DNA MeSH
- metagenom MeSH
- molekulární sekvence - údaje MeSH
- mutageneze MeSH
- proteasy chemie genetika metabolismus MeSH
- půdní mikrobiologie * MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- stabilita enzymů MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
While the selection of amino acid insertions in human immunodeficiency virus (HIV) reverse transcriptase (RT) is a known mechanism of resistance against RT inhibitors, very few reports on the selection of insertions in the protease (PR) coding region have been published. It is still unclear whether these insertions impact protease inhibitor (PI) resistance and/or viral replication capacity. We show that the prevalence of insertions, especially between amino acids 30 to 41 of HIV type 1 (HIV-1) PR, has increased in recent years. We identified amino acid insertions at positions 33 and 35 of the PR of HIV-1-infected patients who had undergone prolonged treatment with PIs, and we characterized the contribution of these insertions to viral resistance. We prepared the corresponding mutated, recombinant PR variants with or without insertions at positions 33 and 35 and characterized them in terms of enzyme kinetics and crystal structures. We also engineered the corresponding recombinant viruses and analyzed the PR susceptibility and replication capacity by recombinant virus assay. Both in vitro methods confirmed that the amino acid insertions at positions 33 and 35 contribute to the viral resistance to most of the tested PIs. The structural analysis revealed local structural rearrangements in the flap region and in the substrate binding pockets. The enlargement of the PR substrate binding site together with impaired flap dynamics could account for the weaker inhibitor binding by the insertion mutants. Amino acid insertions in the vicinity of the binding cleft therefore represent a novel mechanism of HIV resistance development.
- MeSH
- buněčné linie MeSH
- chemické modely MeSH
- difrakce rentgenového záření MeSH
- financování organizované MeSH
- HIV-1 enzymologie fyziologie genetika MeSH
- HIV-proteasa genetika chemie izolace a purifikace metabolismus MeSH
- inhibitory reverzní transkriptasy chemie MeSH
- inzerční mutageneze MeSH
- katalýza MeSH
- kinetika MeSH
- konsenzuální sekvence MeSH
- látky proti HIV terapeutické užití MeSH
- ledviny cytologie MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- rekombinantní proteiny chemie izolace a purifikace metabolismus MeSH
- replikace viru MeSH
- RNA virová analýza MeSH
- sekvence aminokyselin MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- virová léková rezistence MeSH
- Check Tag
- lidé MeSH
