Spanish flu, polio epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases caused by RNA viruses. The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands affordable and reliable assays for testing antivirals. To test inhibitors of viral proteases, we have developed an inexpensive high-throughput assay based on fluorescent energy transfer (FRET). We assayed an array of inhibitors for papain-like protease from SARS-CoV-2 and validated it on protease from the tick-borne encephalitis virus to emphasize its versatility. The reaction progress is monitored as loss of FRET signal of the substrate. This robust and reproducible assay can be used for testing the inhibitors in 96- or 384-well plates.
- MeSH
- antivirové látky farmakologie MeSH
- COVID-19 farmakoterapie MeSH
- fluorescenční barviva chemie MeSH
- inhibitory proteas farmakologie MeSH
- koronavirové proteasy podobné papainu antagonisté a inhibitory chemie genetika metabolismus MeSH
- lidé MeSH
- preklinické hodnocení léčiv MeSH
- rezonanční přenos fluorescenční energie metody MeSH
- RNA-helikasy antagonisté a inhibitory chemie genetika metabolismus MeSH
- RNA-viry enzymologie MeSH
- rychlé screeningové testy metody MeSH
- SARS-CoV-2 enzymologie MeSH
- serinové endopeptidasy chemie genetika metabolismus MeSH
- virové nestrukturální proteiny antagonisté a inhibitory chemie genetika metabolismus MeSH
- viry klíšťové encefalitidy enzymologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The protein sequences found in nature represent a tiny fraction of the potential sequences that could be constructed from the 20-amino-acid alphabet. To help define the properties that shaped proteins to stand out from the space of possible alternatives, we conducted a systematic computational and experimental exploration of random (unevolved) sequences in comparison with biological proteins. In our study, combinations of secondary structure, disorder, and aggregation predictions are accompanied by experimental characterization of selected proteins. We found that the overall secondary structure and physicochemical properties of random and biological sequences are very similar. Moreover, random sequences can be well-tolerated by living cells. Contrary to early hypotheses about the toxicity of random and disordered proteins, we found that random sequences with high disorder have low aggregation propensity (unlike random sequences with high structural content) and were particularly well-tolerated. This direct structure content/aggregation propensity dependence differentiates random and biological proteins. Our study indicates that while random sequences can be both structured and disordered, the properties of the latter make them better suited as progenitors (in both in vivo and in vitro settings) for further evolution of complex, soluble, three-dimensional scaffolds that can perform specific biochemical tasks.
- MeSH
- cirkulární dichroismus MeSH
- databáze proteinů MeSH
- datové soubory jako téma MeSH
- molekulární modely * MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- peptidová knihovna * MeSH
- proteinové agregáty MeSH
- rekombinantní proteiny chemie izolace a purifikace toxicita MeSH
- rozpustnost MeSH
- sbalování proteinů MeSH
- sekundární struktura proteinů * MeSH
- sekvence aminokyselin MeSH
- výpočetní biologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Reactive α,β-unsaturated aldehydes, including 4-oxoalk-2-enals, are known to be present in volatile secretions of numerous heteropteran insect species. Because the aldehydes are likely to originate from metabolism of fatty acids (FAs), the present study aimed to examine and compare the aldehyde and FA profiles of four model heteropteran species. The model species consisted of adult family group representatives within the infraorder Pentatomomorpha (Hemiptera: Heteroptera): seed bug (Lygaeus equestris (Lygaeoidea)), dock leaf bug (Coreus marginatus (Coreoidea)), red firebug (Pyrrhocoris apterus (Pyrrhocoroidea)), and European stink bug (Graphosoma lineatum (Pentatomoidea)). Solid-phase microextraction combined with two-dimensional gas-chromatography/time-of-flight mass spectrometry was used to establish the profiles of volatile secretions in stressed living insects. The FA profiles of acylglyceride and phospholipid fractions deposited in fat body and/or hemolymph were obtained by liquid chromatography/mass spectrometry and gas chromatography with flame ionization detection techniques. Our results based on multivariate statistical analyses of the data imply that volatile secretion blends as well as fat body and/or hemolymph lipid profiles are species specific but the differences in volatile blends between different species do not mirror the changes in corresponding fat body and/or hemolymph lipid profiles of stressed and non-stressed individuals.
- MeSH
- aldehydy analýza MeSH
- druhová specificita MeSH
- fyziologický stres MeSH
- hemolymfa MeSH
- Heteroptera chemie MeSH
- mastné kyseliny analýza MeSH
- mikroextrakce na pevné fázi MeSH
- plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
- tukové těleso MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
