Most of the data indicating that the six ankyrin

Most of the data indicating that the six-ankyrin repeat domain-containing Asb genes drive compartment expansion come from zebrafish and concern Asb11. However, there is evidence to suggest that these data can be expanded to the entire vertebrate phylum and to all six-ankyrin repeat domain-containing Asb genes. As discussed above, in human neuronal systems, heterologous expression of ASB11 can also drive compartment expansion, whereas in murine myoblasts, all six-ankyrin repeat domain-containing Asb genes drive such expansion. In addition, Asb5, Asb9, Asb11, and Asb13 can all drive compartment expansion in the untransformed mammalian gastrointestinal system and show possible deregulation in gastrointestinal cancer [20]. The apparent importance of these six-ankyrin repeat domain-containing ASB proteins in compartment expansion prompts us to review their function here.
Asb genes were originally identified in mice, but have since been found in all members of the Chordata investigated; however, no obvious relatives of Asb genes have been identified in nonchordates. Nevertheless, a bioinformatical search (M. Peppelenbosch, 2018) revealed more than 4000 different Asb orthologs and paralogs that have been found in humans, mice, rabbits, and even the African toad. Asb genes are classified as belonging to the SOCS-box superfamily, which is a group of genes acting as ubiquitin E3 ligases and mediating ubiquitination (Box 2). Within the Asb family, the six-ankyrin repeat domain-containing proteins are evolutionarily the most ancient because they are the only ones found in nonvertebrates. The remarkable differences in primary sequence and gene structure suggest that the non-six-ankyrin repeat domain-containing Asb proteins arose as a result of parallel AGN 194310 rather than being descended from the six-ankyrin repeat domain-containing Asb genes. Functionally, however, there are important similarities [21]. Asb genes are characterized by the presence of a conserved SOCS box motif and a variable number of ankyrin repeats. The SOCS box is a structural C-terminal domain that interacts with Elongin C via its B/C box conserved motif. In turn, Elongin C binds Elongin B to form a dimer that bridges the substrate bound by the SOCS box protein to a Cullin protein. This step is thought to be supported by a conserved Cullin box motif, located downstream of the B/C box in the SOCS box. Cullin then recruits a RING finger-containing protein, Rbxl/2, completing the assembly of the ElonginC–Cul2–SOCS box (ECS) E3-type ubiquitin ligase complex. Therefore, SOCS box proteins are the substrate recognition units of ECS complexes, regulating the turnover of a range of proteins by targeting them for polyubiquitination and subsequent proteasomal degradation [22]. Deletion of the cullin domain abrogates the biochemical and biological functions of Asb9 (the only SOCS box-containing protein for which this has explicitly been shown) [19]. An important focus of research is now the identification of the specific substrates of Asb gene products in vivo. In vitro, Asb proteins appear to be promiscuous and capable of ubiquitinating a variety of substrates (Box 2). In vivo, however, their enzymatic activities appear to be specific to a small number of substrates [23]. In human heterologous expression studies, both zebrafish d-asb11 and human ASB11 ubiquitinate DeltaA but not the highly homologous DeltaD [7]. A mass spectrometry analysis of human ASB11-binding proteins also suggested a limited set of substrates [24], with Tankyrase being a particularly interesting find because this protein is an important regulator of Wnt/β-catenin signaling; thus, this could provide a rational explanation of how ASB11 can specifically support progenitor expansion without affecting the size of the stem cell compartment per se25, 26.
Detailed Action of Asb Paralogs in the Regulation of Compartment Size The mechanism whereby Asb genes may affect compartment size can be explained by examining the relationship between zebrafish d-asb11 and DeltaA (the ligands of Notch signaling). In canonical Delta/Notch signaling, Delta ligands binding with Notch receptors promotes Notch signaling activities in surrounding cells, which induce the downregulation of Delta in this group of cells 27, 28. This process of lateral inhibition leads to distinct cell populations (Delta-signaling cells or Notch-signaling cells), in turn affecting cell fate and organ development. In zebrafish embryos, morpholino-mediated knockdown of d-asb11 resulted in repression of different Delta/Notch elements and their transcriptional targets, while these were induced upon d-asb11 misexpression. d-asb11 expression activated Notch reporters cell nonautonomously in vitro and in vivo when co-expressed with a Notch reporter, but repressed Notch reporters when expressed in DeltaA-expressing cells. In agreement, d-asb11 was able to specifically ubiquitinate and degrade DeltaA both in vitro and in vivo[9]. A more recent study showed that a zebrafish mutant line lacking the Cul5 box of d-asb9/11 induced the impaired expression of target genes of Notch signaling, suggesting that the Cul5 box domain is critical for substrate ubiquitination and neurogenesis via its effects on Notch signaling [19].