TGF-β Prodomain Alignments Reveal Unexpected Cysteine Conservation Consistent with Phylogenetic Predictions of Cross-Subfamily Heterodimerization

Abstract

Evolutionary relationships between prodomains in the TGF-β family have gone unanalyzed due to a perceived lack of conservation. We developed a novel approach, identified these relationships, and suggest hypotheses for new regulatory mechanisms in TGF-β signaling. First, a quantitative analysis placed each family member from flies, mice, and nematodes into the Activin, BMP, or TGF-β subfamily. Second, we defined the prodomain and ligand via the consensus cleavage site. Third, we generated alignments and trees from the prodomain, ligand, and full-length sequences independently for each subfamily. Prodomain alignments revealed that six structural features of 17 are well conserved: three in the straitjacket and three in the arm. Alignments also revealed unexpected cysteine conservation in the “LTBP-Association region” upstream of the straitjacket and in β8 of the bowtie in 14 proteins from all three subfamilies. In prodomain trees, eight clusters across all three subfamilies were present that were not seen in the ligand or full-length trees, suggesting prodomain-mediated cross-subfamily heterodimerization. Consistency between cysteine conservation and prodomain clustering provides support for heterodimerization predictions. Overall, our analysis suggests that cross-subfamily interactions are more common than currently appreciated and our predictions generate numerous testable hypotheses about TGF-β function and evolution.

Fourth Chromosome Resource Project: a comprehensive resource for genetic analysis in Drosophila that includes humanized stocks

“…Rescue crosses were planned with UAS. Actβ and its 4 closest human relatives INHBA, INHBB, INHBC, and INHBE ( Wisotzkey and Newfeld 2020 ). Among the 4 human Inhibin-β proteins, the one with the most direct connection to Actβ is not discernable based on amino acid similarity.…”

Molecular Evolution of Transforming Growth Factor-β (TGF-β) Gene Family and the Functional Characterization of Lamprey TGF-β2

“…To investigate in detail the evolutionary position of the TGFbs in lampreys, we constructed a phylogenetic tree including the full-length sequences of TGF-bs identified in lampreys and the TGF-b sequences of representative species in the deuterostomes using the Maximum Likelihood method (Figure 1A). Consistent with previous studies (40), we used mouse GDNF (glial-derived neurotrophic factor) as an outgroup to root the phylogenetic tree. GDNF shares key structural similarities with mature TGF-b family ligands, such as having the same cysteine pattern as TGFb family ligands and belonging to dimeric secretory proteins, but signal through a Ret tyrosine kinase receptor that is distinct from the TGF-b family receptors.…”

Bone morphogenetic protein signaling: the pathway and its regulation

“…BMP family proteins are initially synthesized as inactive proproteins, which consist of a prodomain and a highly conserved ligand domain at the C-terminus. While the prodomains of different family members are less conserved, sequence comparisons show regions of conservation highlighting the evolutionary relatedness of specific ligand genes ( Wisotzkey and Newfeld 2020 ). After translation, the proproteins dimerize via a disulfide bond between the ligand domains and undergo proteolytic processing to generate 110–140 amino acid bioactive BMP ligands comprised of the highly conserved C-terminal domain ( Fig.…”