Abstract
Thymosin proteins are well known for their actin-binding activity. Thymosin beta 4 (Tβ4) has been associated with biological activities in tissue repair and cell migration via interaction with ATP-synthase in vertebrates, while the information of similar thymosin functions in invertebrates is limited. We have shown previously that ATP-synthase is present on the surface of crayfish hematopoietic tissue (HPT) cells, and that astakine 1 (Ast1, an invertebrate cytokine) was found to interact with this β-subunit of ATP synthase. Here, we identified five different β-thymosins from Pacifastacus leniusculus, designated Pl-β-thymosin1-5. The two dominant isoforms in brain, HPT and hemocytes, Pl-β-thymosin1 and 2, were chosen for functional studies. Both isoforms could bind to the β-subunit of ATP-synthase, and Pl-β-thymosin1, but not Pl-β-thymosin2, significantly increased extracellular ATP formation. Moreover, Pl-β-thymosin1 stimulated HPT cell migration in vitro and Ast1 blocked this effect. Pl-β-thymosin2 increased the circulating hemocyte number at an early stage after injection. Additionally, in vivo injection of Pl-β-thymosin1 resulted in significant reduction of reactive oxygen species (ROS) production in crayfish HPT whereas Pl-β-thymosin2 had a similar but transient effect. Both Pl-β-thymosins induced the expression of Ast1 and superoxide dismutase (SOD) transcripts, while silencing of endogenous Pl-β-thymosin 1 and 2 by RNAi resulted in significant reduction of the Ast1 and SOD transcripts. The diverse effects exhibited by Pl-β-thymosin1 and Pl-β-thymosin2 indicates that these proteins are involved in a complex interaction that regulates the hematopoietic stem cell proliferation and differentiation.
Highlights
Thymosins are ubiquitous intracellular proteins and are considered as major actin sequestering proteins, which binds monomeric G-actin in a 1:1 complex
It was reported that Thymosin b4 (Tb4) interacts with F1-F0ATP synthase on the plasma membrane of human umbilical vein endothelial cells (HUVECs), and that interaction resulted in an increase of the extracellular ATP concentration, which led to an increase in cell migration [16]
We have demonstrated that a b-subunit of F1ATP synthase may function as a receptor for astakine 1 (Ast1), a hematopoietic cytokine, and that this receptor is present on the surface of a small subpopulation of the crayfish hematopoietic tissue (HPT) cells
Summary
Thymosins are ubiquitous intracellular proteins and are considered as major actin sequestering proteins, which binds monomeric G-actin in a 1:1 complex. Thymosin b4 (Tb4) is a highly conserved member of the bthymosin family, and it is a small peptide of about 5 kDa molecular mass This abundant peptide influences numerous cellular functions, including migration, attachment and spreading of endothelial and cancer cells [4,5,6,7]. Most studies deal with Drosophila Ciboulot, a 14.4 kDa protein that contains three thymosin domains, and plays a major role in axonal growth during brain metamorphosis [17]. Another interesting invertebrate member of the b-thymosin family is thypedin a protein that is involved in foot regeneration in Hydra (Cnidaria) and which contains 27 copies of a b-thymosin-like domain [18]
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