Open Access Thesis
Date of Award
William W. Branford
In vertebrates, the TGF #914; superfamily of secreted peptides are stringently regulated since they are responsible for multiple cell processes and behaviors that give rise to the proper patterning of embryonic tissues. Examples of members of the TGF #914; superfamily are Nodal and Lefty. Dysregulation of these proteins can lead to many diseases and developmental syndromes in humans.
Lefty functions by antagonizing Nodal an essential organizer signal that patterns dorsal mesoderm and the embryonic axes. Loss of Lefty expression results in excess Nodal signaling which has been shown to cause several perturbations including metastatic cancer. Although studies have clearly shown that Lefty antagonizes Nodal signaling, there is a lack of understanding of the regulatory mechanism of Lefty itself. Previous studies have shown that proteolytic cleavage of the prodomain (PD) from mature Lefty (Mat-Lefty) is necessary for Lefty activity.
Here we present studies carried out using the Xenopus laevis embryo that demonstrate that PD over-expression causes exogastrulation, a phenotype also resulting from a loss of Xenopus Lefty (Xlefty) function. Furthermore, when the PD and Xlefty are co-expressed, the effects of Xlefty over-expression are rescued. Our biochemical studies also showed that the PD interacts with Mat-Xlefty but a PD mutated (PDmut) molecule does not. The sites mutated in the PDmut are evolutionarily-conserved residues that mediate the interaction between the prodomain and the mature ligand in other TGF #914; proteins making our results consistent with previously observed behavior of TGF #914; prodomains but new to Lefty. Our study also shows that the PDmut is unable to rescue the effects of a Xlefty over-expression phenotype unlike the un-mutated PD.
In addition we show here that mutations of the PD affect the secretion of these Xlefty-mutated derivatives. Taken together, these results suggest that the PD negatively regulates Xlefty activity by interacting with Mat-Xlefty and cleavage of the PD releases regulation allowing proper secretion and function. This new insight into the regulatory role of the Xlefty PD provides potential therapeutic value to address dysfunctional Nodal signaling. Furthermore, our secretion studies of the PD and Xlefty revealed that Xlefty is secreted but the PD is not. The Xlefty-mutated derivatives, including a PD Less, Xlefty, Xlefty cleavage mutants and the PDmut, resulted in no secreted products. These secretion results open a Pandora's box and further studies are warranted to elucidate the mechanism of PD regulation of Xlefty and whether it occurs in an intracellular or extracellular context.
Vasquez, Adrian Amelio, "The Xenopus Lefty (xlefty) Prodomain Negatively Regulates Xlefty Activity And Is Necessary For Proper Xlefty Secretion" (2013). Wayne State University Theses. 318.