RING (Really Interesting New Gene) Fingers (RNF) domain-containing proteins constitute the most widely represented type of E3 ubiquitin ligases in the human genome. They are involved both in the regulation of targeted-proteins’ function as well as in ubiquitin proteasome system (UPS)-mediated degradation (reviewed by 
). RING finger domains found in RNF proteins bind two zinc ions in a “cross-brace” fashion involving a distinctive cysteine and histidine residue-containing motif, a feature previously known to be shared only by zinc-finger DNA-binding proteins 
. Despite some DTX proteins having been found overexpressed in cancer, their mechanistic involvement in the underlying pathological process is still at an early stage of investigation 
. Most ubiquitin E3 ligases (UbE3L) studied until recently had been found to be involved in the N-degradation pathway responsible for recruiting specific UbE3L to the targeted proteins’ N-terminal region 
. Recently, a growing number of C-Degrons-bearing proteins have been identified, along with the underlying UbE3L-driven degradation complexes (reviewed by Varshawsky 
). The relatively recent field of C-Degron pathways had lead some authors to define this type of degradation mechanism with the acronym of “DesCEnd” (from “Destruction by the C-End”) 
. Although to date the RING motifs in DTX proteins have not been restricted to either of the N-Degron or C-Degron degradation pathways, the recently described involvement of DTX3c in the specific recruitment to the EphB4 C-pi
(phospho-inhibited) Degron 
assigns DTX3 to the C-Degron pathway.
2. DTX Proteins Structural and Functional Features: Phylogenetic and Intra-Species Considerations
DTX protein expression is almost ubiquitous in mammalians, although they display the highest expression in specific cell and tissue types like blood vessels, embryonic neural tissues, genital and reproductive apparatus, pancreas, lungs, liver, kidneys, adrenal gland, skin, intestinal epithelial components and muscle (see Uberon Anatomy Ontology at https://bioportal.bioontology.org/ontologies/UBERON
accessed on 17 May 2023 
). A first hint at the physiological role of DTX proteins has come from a DTX Drosophila
gene null mutant study which confirmed the tissue-specific requirement for DTX expression and its dispensable role in embryo development 
. This is consistent with the finding that T-cells, a DTX1 and 2 expressing mammalian cell type, can develop normally in absence of both DTX1 and DTX2 
. On the other hand, overexpression of DTX proteins in mammalian systems has been found to affect myogenesis, neurogenesis 
and the proper function of lymphoid organs 
. More recently, DTX protein expression in cancer cells has revealed a wide landscape of contextual functions linked, in part, to their regulation of NOTCH signaling. As graphically summarized in Figure 1
, mammalian DTX proteins share all or part of the Drosophila
DTX (dDTX) progenitor structure 
. As such, some phylogenetic and intra-species key comparative points can be made per each Drosophila
reference domain structure in order to better understand the emerging cellular and systemic functions of DTX proteins in mammalians. In particular, human DTX proteins are linked to the expression of five paralog genes along with their main isoform variants (Table 1
and Figure 1
). A number of human isoform variants have been reported for DTX proteins (Figure 1
). These are discussed below in relationship with the underlying protein domain features.
Figure 1. The Drosophila and human DTX protein domain structure. WWE, WWE tandem domains; DTX3-NTa, DTX3a N-Terminal variant (isoform 1); DTX3-NTb, DTX3b N-Terminal variant (isoform 2), D, D-Domain; H2-RING, canonical Ring finger motif (H2)-containing domain; HC-RING, C3HC4 finger motif-containing domain; DTC, Deltex C-Terminal Domain. DTX3c N-Terminal domain retains both 1a and 1b exons to generate a new isoform (isoform 3) with N-terminus 3D unique distribution differing from the canonical DTX3a and DTX3b isoforms.
Table 1. Human DTX genes and relative products features 1.
2.1. DTX Domain I: WWE Domain-Bearing and WWE-Domain-Less DTX Proteins
The domain structure of human DTX proteins reflects that observed in Drosophila
DTX which share a phylogenetically conserved three-domains structural organization. Starting from the N-terminal region, Drosophila
DTX bears a tandem WWE domain which is conserved in DTX1, −2, and −4 human paralogs (Figure 1
). WWE tandem domain in dDTX was shown to bind NOTCH ankyrin repeats 
, and its presence in human DTX1, DTX2 and DTX4 N-terminal domain supports their ability to physically interact with NOTCH ankyrin motifs 
. This finding further strengthens the functional link between DTX proteins and Notch signaling regulation. Interestingly, WWE tandem domains are frequently found in ubiquitin UbE3Ls and ADP-ribose polymerases 
although the direct implication of such occurrence has not yet found a mechanistic explanation. In light of this finding, it is not surprising that some WWE-domain bearing DTX members such as DTX2 have been found associated to the recruitment and regulation of ADP-ribosylation targets 
2.2. Domain II: The Proline-Rich Region in DTX Proteins
The N-terminal WWE tandem domain in dDTX, DTX1, DTX2 and DTX4 (part of the interspecies domain-I), is followed by a proline-rich region which stands as the constitutive component of DTX domain-II. This proline-rich region is present also in DTX3, following a short unique N-terminus domain. The only DTX member missing this proline-rich region is DTX3L, which displays a two-domain structural organization (Figure 1
) with a long and unique N-terminus domain. DTX3L N-terminus domain mediates homo- and hetero-dimeric binding, and it enhances its heterodimeric self-ubiquitination when bound to DTX1 in vitro 
. A dDTX study found DTX proline-rich region (domain-II) to contribute to NOTCH regulation since a deletion mutant spanning the full dDTX proline-rich domain (aa 476–484) exhibited a dominant negative effect on Notch activation 
. Another hint on the function of DTX domain-II has come from the identification of an SH3 motif in dDTX (aa 480–484) along with its ability to bind the signaling adaptor GRB2 
. Interestingly, the SH3 motif is conserved in DTX1, DTX3 and DTX4 but is not present in human DTX2 proline-rich domain (see Figure 1
), offering the first structural hint to DTX2’s physical interaction differences underlying its diversified cellular functions. Indeed, a hint of this SH3 motif potential functions in DTX2 proline-rich domain may come from the study of its isoform (DTX2B), which lacks this SH3 motif. Specifically, DTX2B displays the exclusion of DTX2A fourth exon (aa 337–383), which contains a serine residue (S360) which has been found phosphorylated by cell-cycle-related kinases (Aurora and polo-like kinases) during mitosis by a large-scale phospho-proteomic study 
. Therefore, this DTX2 residue embedded in its proline-rich domain could, per se
, offer a first hint at DTX2’s potential isoform-specific functional consequences with regard to proliferative disfunctions such as in cancer.
2.3. Domain III: The Deltex C-Terminal Domain (DTC) and the H2- and HC-RING Domains in DTX Family Members
A common feature shared by all DTX proteins relates to the presence of a RING domain in their DTC region 
. Although the UbE3L enzymatic activity has not been yet demonstrated for all RING-motifs containing proteins, the putative UbE3L enzymatic nature of DTX proteins has received sufficient experimental support in that all of them have been shown to (a) bear self-ubiquitination capability as homodimer or heterodimers 
; (b) bind and increase the ubiquitination of at least one cellular target either in cell-free or in vivo cellular assays; and (c) interact with an E2-ubiquitin conjugase (URL source: UNIPROTKB -> BIOGRID [https://www.uniprot.org/database/DB-0184
; Release 2023_02 accessed 7 May 2023 
). Since two types of RING motifs are found in DTX proteins, the RING-type motif present in DTX ligases can be used to further sub-categorize them. Specifically, the H2-RING motif is found in DTX1, DTX2 and DTX4, while an HC-RING (synonym of C3HC4-RING) domain is present in DTX3 and DTX3L (Figure 1
and Figure 2
). The name of the H2-RING refers to the histidine residues present in the “HxxH” core motif spanning the central part of the RING domain as shown in Figure 2 
. Similarly, the C3HC4-RING motif featured in DTX3 and DTX3L at the same conserved core location, bears an “HxxC” motif from which originates its acronym of HC-RING. The biochemical and biological significance associated to these two types of RING motifs in the context of DTX proteins biological function is still unclear. However, the finding that the HC-RING of DTX3L is able to enhance self-ubiquitination of the DTX3L when dimerizing with DTX1 containing an H2-RING in vitro 
, suggests that HC-RING and H2-RING DTX heterodimers may also play a synergistic role in vivo. Additionally, these two RING motifs functions are likely to be affected by intra-molecular interaction with other DTX domains ultimately conferring them specificity for their cellular binding partners and substrates. Interestingly, the DTX RING bears also non enzymatic functions as suggested by the mutagenesis of aa 571 located within the Drosophila
DTX RING-H2 (spanning aa 548–603) which impairs dDTX multimerization when associated to residue 574 (->A) replacement 
. This finding suggests a cooperative function of specific residues within the RING structure towards modulating DTX homologous and heterologous configuration.
Figure 2. Sequence comparison and consensus (bold) of the RING motifs in DTX proteins. Bolded sequences, consensus sequence within each RING category, red letters under the asterisk indicate consensus residues shared among all DTX proteins RING motifs; black asterisks are the conserved C residues concurring to the Ring domain numbering; red asterisks correspond to positions in the RING domain alignment supporting the H2 versus HC RING acronym.