ABSTRACT
We discovered that the abundant human nuclear protein poly(ADP-ribose) polymerase-1 (hPARP-1) binds to intramolecular DNA quadruplexes in vitro with high affinity and with a stoichiometry of two proteins for one quadruplex. Using an enzymatic assay, we have shown that hPARP-1 gets catalytically activated upon binding to G-quadruplexes localized at the c-kit promoter and human telomere regions. This is the first example of a truly functional quadruplex-protein interaction, which has possible implications in understanding hPARP-1 mediated mechanisms of transcription regulation and telomere end protection.
Subject(s)
G-Quadruplexes , Poly(ADP-ribose) Polymerases/metabolism , Humans , Microscopy, Atomic Force , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/ultrastructure , Protein Binding , Proto-Oncogene Proteins c-kit/genetics , Proto-Oncogene Proteins c-kit/metabolismABSTRACT
Ionisation of trimethylphosphate (TMP), dimethylphosphate (DMP) and diethylphosphate (DEP) is investigated by acidic titration in water by Raman (R), Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopies. The vibrational frequencies of the PO(2)(-) ionic form and the neutral form were found in accord with the literature. While increasing further H(+) concentration, the PO band disappears in the benefit of new ones. These results, together with deuteration experiments indicate the presence of a new ionic form positively charged with general formula R(1)R(2)R(3)P(OH)(+) or R(1)R(2)P(OH)(+)(2). The pK of this phosphonium entities is lying in the range -2, -4. These results were confirmed by (31)P NMR titration. The occurrence of such a phosphonium ion in aqueous solutions might be of crucial importance for biochemical reactions and interactions, owing to the large spread of phosphoryl group in biomolecules and keeping in mind that intracellular compartments are more likely concentrated media with little free water than real aqueous solutions. Furthermore, pK's can be shifted by physical-chemical parameters like dielectric constant and electric field. This may involve at least fractional positive charge apparition that might be important in biochemical regulation by charge-charge and charge-dipole interactions. This finding will gain to be further explored on more complex molecules like phospholipids, nucleic acids and proteins.
Subject(s)
Organophosphates/chemistry , Organophosphonates/chemistry , Phosphites/chemistry , Magnetic Resonance Spectroscopy/methods , Molecular Conformation , Organophosphates/chemical synthesis , Solutions , Spectroscopy, Fourier Transform Infrared/methods , WaterABSTRACT
Various phosphono-phosphates and diphosphonates were synthesized as 1,3-diphosphoglycerate (1,3-diPG) analogues by using a beta-ketophosphonate, an alpha-fluoro,beta-ketophosphonate or a beta-ketophosphoramidate to mimic the unstable carboxyphosphate part of the natural substrate. The inhibitory effect of these analogues on glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from Trypanosoma brucei (Tb) and rabbit muscle were measured with respect to both substrates, glyceraldehyde-3-phosphate (GAP) and 1,3-diPG. Interestingly, all 1,5-diphosphono,2-oxopentanes without substitution at the C-3 position selectively inhibit the Tb GAPDH with respect to 1,3-diPG and are without effect on Rm GAPDH. All 1-phospho,3-oxo,4-phosphonobutanes show themselves to be non-selective inhibitors either with regard to substrates or organisms, but they will be of a great interest as 1,3-diPG stable models for structural studies of co-crystals with GAPDHs.