title: Interactions of cellular histidine triad nucleotide binding protein 1 with nucleosides 5'-O-monophosphorothioate and their derivatives - Implication for desulfuration process in the cell.
abstract: BACKGROUND
  One of the activities of histidine triad nucleotide-binding protein 1 (Hint1) under in vitro conditions is the conversion of nucleoside 5'-O-phosphorothioate (NMPS) to its 5'-O-phosphate (NMP), which is accompanied by the release of hydrogen sulfide.
  
  
  METHODS
  Non-hydrolyzable derivatives of AMPS and dCMPS, each containing the residue able to form a covalent bond in nucleic acid-protein complexes via photocrosslinking (at 308nm), were applied at the complexing experiments with recombinant and cellular Hint1. The cellular lysates prepared after RNAi-mediated knockdown of Hint1 were incubated with AMPS and the level of desulfuration was measured.
  
  
  RESULTS
  Recombinant Hint1 and Hint1 present in the cellular lysate of A549 cells, formed complexes with the used substrate analogs. Computer modeling experiments, in which the ligand was docked at the binding pocket, confirmed that direct interactions between Hint1 and the screened analogs are possible. Using RNAi technology, we demonstrated lowered levels of AMPS substrate desulfuration in reactions that employed the cell lysates with a reduced Hint1 level.
  
  
  CONCLUSIONS
  The enzymatic conversion of AMPS to AMP occurred with the participation of cellular Hint1, the protein, which is present in all organisms.
  
  
  CONCLUSIONS
  The intracellular Hint1 could be responsible for the in vivo desulfuration of nucleosides-5'-monophosphorothioate, thus it can contribute to the phosphorothioate oligonucleotides metabolism. H2S released during this process may participate in several physiological processes, thus NMPSs can be precursors/donors of H2S in vivo and can be used to study the effects of this gas in biological systems. Moreover, the controlled delivery of (d)NMPSs into cells may be of medicinal utility.
authors.affiliation: Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Department of Bioorganic Chemistry, Lodz, Poland. Electronic address: akrakow@cbmm.lodz.pl.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Krakowiak
authors.affiliation: Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Department of Bioorganic Chemistry, Lodz, Poland.
authors.forename: Róża
authors.initials: R
authors.lastname: Pawłowska
authors.affiliation: Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Department of Bioorganic Chemistry, Lodz, Poland.
authors.forename: Beata
authors.initials: B
authors.lastname: Kocoń-Rębowska
authors.affiliation: Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Department of Bioorganic Chemistry, Lodz, Poland.
authors.forename: Rafał
authors.initials: R
authors.lastname: Dolot
authors.affiliation: Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Department of Bioorganic Chemistry, Lodz, Poland.
authors.forename: Wojciech J
authors.initials: WJ
authors.lastname: Stec
keywords: (d)NMPS; Hint1; Hydrogen sulfide; PS-oligo metabolism; Photocrosslink; siRNA