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Related Experiment Videos

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.

The Journal of Biological Chemistry
|February 15, 2000
PubMed
Summary

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.

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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.

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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.