Reconstruction of the evolutionary history of specific protein-coding genes is an essential component of the biological sciences toolkit and relies on identification of orthologs (a gene in different organisms related by vertical descent from a common ancestor and usually presumed to have the same or similar function) and paralogs (a gene related to another in the same organism by descent from a single ancestral gene which may, or may not, retain the same/similar function) across a range of taxa. While obviously essential for the reconstruction of evolutionary histories, ortholog identification is of importance for protein expression, modeling for drug discovery programs, identification of critical residues and other studies. Here we describe an automated system for searching for orthologs and paralogs in eukaryotic organisms. Unlike manual methods the system is fast, requiring minimal user input while still being highly configurable.

• MeSH
• fylogeneze * MeSH
• molekulární evoluce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The Leishmania major leucyl-aminopeptidase (LAPLm), a member of the M17 family of proteases, is a potential drug target for treatment of leishmaniasis. To better characterize enzyme properties, recombinant LAPLm (rLAPLm) was expressed in Escherichia coli. A LAPLm gene was designed, codon-optimized for expression in E. coli, synthesized and cloned into the pET-15b vector. Production of rLAPLm in E. coli Lemo21(DE3), induced for 4 h at 37 °C with 400 μM IPTG and 250 μM l-rhamnose, yielded insoluble enzyme with a low proportion of soluble and active protein, only detected by an anti-His antibody-based western-blot. rLAPLm was purified in a single step by immobilized metal ion affinity chromatography. rLAPLm was obtained with a purity of ~10% and a volumetric yield of 2.5 mg per liter, sufficient for further characterization. The aminopeptidase exhibits optimal activity at pH 7.0 and a substrate preference for Leu-p-nitroanilide (appKM = 30 μM, appkcat = 14.7 s-1). Optimal temperature is 50 °C, and the enzyme is insensitive to 4 mM Co2+, Mg2+, Ca2+ and Ba2+. However, rLAPLm was activated by Zn2+, Mn2+ and Cd2+ but is insensitive towards the protease inhibitors PMSF, TLCK, E-64 and pepstatin A, being inhibited by EDTA and bestatin. Bestatin is a potent, non-competitive inhibitor of the enzyme with a Ki value of 994 nM. We suggest that rLAPLm is a suitable target for inhibitor identification.

• MeSH
• aminopeptidasy * biosyntéza   chemie   genetika   izolace a purifikace   MeSH
• Escherichia coli * genetika   metabolismus   MeSH
• kinetika MeSH
• Leishmania major * enzymologie   genetika   MeSH
• protozoální proteiny * biosyntéza   chemie   genetika   izolace a purifikace   MeSH
• rekombinantní proteiny biosyntéza   chemie   genetika   izolace a purifikace   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Livestock diseases caused by Trypanosoma congolense, T. vivax and T. brucei, collectively known as nagana, are responsible for billions of dollars in lost food production annually. There is an urgent need for novel therapeutics. Encouragingly, promising antitrypanosomal benzoxaboroles are under veterinary development. Here, we show that the most efficacious subclass of these compounds are prodrugs activated by trypanosome serine carboxypeptidases (CBPs). Drug-resistance to a development candidate, AN11736, emerged readily in T. brucei, due to partial deletion within the locus containing three tandem copies of the CBP genes. T. congolense parasites, which possess a larger array of related CBPs, also developed resistance to AN11736 through deletion within the locus. A genome-scale screen in T. brucei confirmed CBP loss-of-function as the primary mechanism of resistance and CRISPR-Cas9 editing proved that partial deletion within the locus was sufficient to confer resistance. CBP re-expression in either T. brucei or T. congolense AN11736-resistant lines restored drug-susceptibility. CBPs act by cleaving the benzoxaborole AN11736 to a carboxylic acid derivative, revealing a prodrug activation mechanism. Loss of CBP activity results in massive reduction in net uptake of AN11736, indicating that entry is facilitated by the concentration gradient created by prodrug metabolism.

• MeSH
• dobytek MeSH
• karboxypeptidasy metabolismus   MeSH
• kyseliny karboxylové metabolismus   MeSH
• léková rezistence MeSH
• myši MeSH
• parazitemie veterinární   MeSH
• prekurzory léčiv metabolismus   MeSH
• protozoální proteiny metabolismus   MeSH
• sloučeniny boru metabolismus   MeSH
• trypanocidální látky metabolismus   MeSH
• Trypanosoma brucei brucei účinky léků   enzymologie   MeSH
• Trypanosoma congolense účinky léků   enzymologie   MeSH
• Trypanosoma vivax účinky léků   enzymologie   MeSH
• trypanozomóza africká farmakoterapie   parazitologie   veterinární   MeSH
• valin analogy a deriváty   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Leucyl aminopeptidases (LAPs) are involved in multiple cellular functions, which, in the case of infectious diseases, includes participation in the pathogen-host cell interface and pathogenesis. Thus, LAPs are considered good candidate drug targets, and the major M17-LAP from Trypanosoma cruzi (LAPTc) in particular is a promising target for Chagas disease. To exploit LAPTc as a potential target, it is essential to develop potent and selective inhibitors. To achieve this, we report a high-throughput screening method for LAPTc. Two methods were developed and optimized: a Leu-7-amido-4-methylcoumarin-based fluorogenic assay and a RapidFire mass spectrometry (RapidFire MS)-based assay using the LSTVIVR peptide as substrate. Compared with a fluorescence assay, the major advantages of the RapidFire MS assay are a greater signal-to-noise ratio as well as decreased consumption of enzyme. RapidFire MS was validated with the broad-spectrum LAP inhibitors bestatin (IC50 = 0.35 μM) and arphamenine A (IC50 = 15.75 μM). We suggest that RapidFire MS is highly suitable for screening for specific LAPTc inhibitors.

• MeSH
• Chagasova nemoc diagnóza   enzymologie   parazitologie   MeSH
• hmotnostní spektrometrie MeSH
• kinetika MeSH
• leucylaminopeptidasa genetika   izolace a purifikace   MeSH
• lidé MeSH
• rychlé screeningové testy * MeSH
• sekvence aminokyselin genetika   MeSH
• substrátová specifita MeSH
• Trypanosoma cruzi enzymologie   izolace a purifikace   patogenita   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH