Regenerative protein thymosin beta-4 is a novel regulator of purinergic signaling

FASEB J. 2011 Mar;25(3):907-15. doi: 10.1096/fj.10-169417. Epub 2010 Nov 24.


By an unknown mechanism, β-thymosins are extracellular modulators of angiogenesis, inflammation, wound healing, and development. We were interested in identifying β-thymosin interactors and determining their importance in β-thymosins signaling in human vein endothelial cells (HUVECs). We performed pulldown experiments with biotinylated thymosin β-4 (Tβ4) in comparison to neutravidin beads alone and used mass spectrometric analysis to identify differentially interacting proteins. By this method, we identified F1-F0 ATP synthase, a known target of antiangiogenic angiostatin. By surface plasmon resonance, we determined for Tβ4 binding to the β subunit of ATP synthase a K(D) of 12 nM. Blocking antibodies and antagonists (oligomycin, IC(50) ∼1.8 μM; piceatannol, IC(50) ∼1.05 μM; and angiostatin, IC(50) ∼2.9 μg/ml) of ATP synthase inhibited the Tβ4-induced increase in cell surface ATP levels, as measured by luciferase assay, and the Tβ4-induced increase in HUVEC migration, as measured by transwell migration assay. Silencing of the ATP-responsive purinergic receptor P2X4 with siRNA also blocked Tβ4-induced HUVEC migration in a transwell assay. Furthermore, in silico we identified common amphiphilic α-helical structural similarities between β-thymosins and the inhibitory factor 1 (IF1), an inhibitor of ATP synthase hydrolysis. In summary, we have identified an extracellular signaling pathway where Tβ4 increases cell surface ATP levels via ATP synthase and have shown further that ATP-responsive P2X4 receptor is required for Tβ4-induced HUVEC migration.

MeSH terms

  • Amino Acid Sequence
  • Cell Movement / physiology
  • Cells, Cultured
  • Endothelial Cells / cytology
  • Endothelial Cells / metabolism*
  • Humans
  • Molecular Sequence Data
  • Neovascularization, Physiologic / physiology*
  • Protein Structure, Tertiary
  • Proteins / metabolism
  • Proton-Translocating ATPases / metabolism
  • Receptors, Purinergic P2X4 / metabolism*
  • Signal Transduction / physiology*
  • Thymosin / chemistry
  • Thymosin / genetics
  • Thymosin / metabolism*
  • Umbilical Veins / cytology
  • Wound Healing / physiology


  • ATPase inhibitory protein
  • Proteins
  • Receptors, Purinergic P2X4
  • thymosin beta(4)
  • Thymosin
  • Proton-Translocating ATPases