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Fhit-nucleotide specificity probed with novel fluorescent and fluorogenic substrates
A Draganescu1, S C Hodawadekar, K R Gee
1Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Abstract:
Fhit, a member of the histidine triad superfamily of nucleotide-binding proteins, binds and cleaves diadenosine polyphosphates and functions as a tumor suppressor in human epithelial cancers. Function of Fhit in tumor suppression does not require diadenosine polyphosphate cleavage but correlates with the ability to form substrate complexes. As diadenosine polyphosphates are at lower cellular concentrations than mononucleotides, we sought to quantify interactions between Fhit and competitive inhibitors with the use of diadenosine polyphosphate analogs containing fluorophores in place of one nucleoside. Appp-S-(7-diethylamino-4-methyl-3-(4-succinimidylphenyl)) coumarin (ApppAMC), Appp-S-(4-4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacine-3-yl) methylaminoacetyl (ApppBODIPY), and GpppBODIPY, synthesized in high yield, are effective Fhit substrates, producing AMP or GMP plus fluorophore diphosphates. GpppBODIPY cleavage is accompanied by a 5.4-fold increase in fluorescence because BODIPY fluorescence is quenched by stacking with guanine. Titration of unlabeled diadenosine polyphosphates, inorganic pyrophosphate, mononucleotides, and inorganic phosphate into fluorescent assays provided values of K(m) and K(I) as competitive inhibitors. The data indicate that Fhit discriminates between good substrates via k(cat) and against cellular competitors in equilibrium binding terms. Surprisingly, pyrophosphate competes better than purine mononucleotides.
Insights
The Fhit protein, a tumor suppressor, binds diadenosine polyphosphates. Its tumor suppression role depends on substrate complex formation, not cleavage, with pyrophosphate surprisingly outcompeting mononucleotides.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- The histidine triad superfamily protein, Fhit, acts as a tumor suppressor in human epithelial cancers.
- Fhit's tumor suppressor function is linked to its ability to form substrate complexes, independent of its diadenosine polyphosphate cleavage activity.
Purpose of the Study:
- To quantify interactions between Fhit and competitive inhibitors.
- To investigate Fhit's substrate binding and inhibition using novel fluorophore-containing diadenosine polyphosphate analogs.
Main Methods:
- Synthesis of fluorophore-conjugated diadenosine polyphosphate analogs (ApppAMC, ApppBODIPY, GpppBODIPY).
- Utilizing fluorescence assays to measure Fhit substrate cleavage and competitive inhibition.
- Determining kinetic parameters (K(m), K(I)) for various inhibitors including diadenosine polyphosphates, pyrophosphate, and mononucleotides.
Main Results:
- Novel fluorophore analogs were synthesized and demonstrated to be effective Fhit substrates.
- GpppBODIPY cleavage resulted in a significant fluorescence increase due to BODIPY quenching by guanine.
- Pyrophosphate was found to be a more effective competitor than purine mononucleotides, indicating Fhit's discrimination against cellular competitors.
Conclusions:
- Fhit discriminates substrates based on catalytic efficiency (k(cat)) and cellular competitors based on binding affinity.
- The study provides insights into Fhit's molecular interactions relevant to its tumor suppressor role.
- Pyrophosphate's strong competitive binding highlights its potential significance in Fhit regulation.