Effective DNA Inhibitors of Cathepsin G by In Vitro Selection
| Autor: | Moltrasio Danilo, Rodolfo Pescador, Elena Vianini, Claudia Sissi, Barbara Gatto, Roberto Porta, Manlio Palumbo, Lorena Lucatello |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2008 |
| Předmět: |
Cathepsin G
Sequence analysis medicine.medical_treatment Biology defibrotide alternating polynucleotides Catalysis Article Inorganic Chemistry lcsh:Chemistry chemistry.chemical_compound medicine Physical and Theoretical Chemistry Molecular Biology lcsh:QH301-705.5 PCR: polymerase chain reaction Spectroscopy chemistry.chemical_classification Protease CatG: Cathepsin G Organic Chemistry General Medicine Selex SPR: Surface Plasmon Resonance In vitro Computer Science Applications genomic DNA ssDNA: single strand DNA PAGE: Polyacrilamide gel electrophoresis Enzyme Biochemistry chemistry lcsh:Biology (General) lcsh:QD1-999 Selex: Systematic evolution of ligands by exponential enrichment TG repeats DNA Systematic evolution of ligands by exponential enrichment |
| Zdroj: | International Journal of Molecular Sciences Volume 9 Issue 6 Pages 1008-1023 International Journal of Molecular Sciences, Vol 9, Iss 6, Pp 1008-1023 (2008) |
| ISSN: | 1422-0067 |
| DOI: | 10.3390/ijms9061008 |
| Popis: | Cathepsin G (CatG) is a chymotrypsin-like protease released upon degranulation of neutrophils. In several inflammatory and ischaemic diseases the impaired balance between CatG and its physiological inhibitors leads to tissue destruction and platelet aggregation. Inhibitors of CatG are suitable for the treatment of inflammatory diseases and procoagulant conditions. DNA released upon the death of neutrophils at injury sites binds CatG. Moreover, short DNA fragments are more inhibitory than genomic DNA. Defibrotide, a single stranded polydeoxyribonucleotide with antithrombotic effect is also a potent CatG inhibitor. Given the above experimental evidences we employed a selection protocol to assess whether DNA inhibition of CatG may be ascribed to specific sequences present in defibrotide DNA. A Selex protocol was applied to identify the single-stranded DNA sequences exhibiting the highest affinity for CatG, the diversity of a combinatorial pool of oligodeoxyribonucleotides being a good representation of the complexity found in defibrotide. Biophysical and biochemical studies confirmed that the selected sequences bind tightly to the target enzyme and also efficiently inhibit its catalytic activity. Sequence analysis carried out to unveil a motif responsible for CatG recognition showed a recurrence of alternating TG repeats in the selected CatG binders, adopting an extended conformation that grants maximal interaction with the highly charged protein surface. This unprecedented finding is validated by our results showing high affinity and inhibition of CatG by specific DNA sequences of variable length designed to maximally reduce pairing/folding interactions. |
| Databáze: | OpenAIRE |
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