Abstract
Ubiquitin is a post-translational modification that results in the covalent attachment of ubiquitin to cellular proteins. Modification of proteins with ubiquitin regulates various cellular processes. Ubiquitin is transferred through a three-step ubiquitin cascade, the final step is facilitated by the E3 ubiquitin ligase enzyme. One family of E3 ubiquitin ligases contain a Really Interesting New Gene (RING) domain that mediates the direct transfer of ubiquitin from an E2~ubiquitin conjugating partner. The RING domain primes ubiquitin, folding it back against the E2, exposing the thioester bond for nucleophilic attack by an incoming substrate. The RING Finger protein 167 (RNF167) is a type one transmembrane protein and a member of the protease-associated (PA)-transmembrane (TM)-RING subgroup. RNF167 sits in the endosomal and lysosomal membranes where it regulates organelle function. In particular, RNF167 regulates the neuronal lysosomal pathway by modifying proteins with ubiquitin and targeting them to the proteasome. Although important, the mechanism of ubiquitin transfer by RNF167 remains elusive. This study aimed to determine the activation and regulation of the RNF167 E3 ligase activity.
The study first aimed to characterise the domains of the RNF167, a RING-type E3 ligase with no reported structure. The boundaries of the RNF167 domains required for activity or regulation were identified. Activity assays showed that RNF167 RING domain promotes the transfer of ubiquitin with the UbcH5b, Ubc13, and Ube2eK E2 enzymes, a mechanism that is dependent on the conservation of the RING:E2 interface. Binding assays showed that RNF167 contacts ubiquitin as well as the E2 enzyme within the E2~ubiquitin conjugate. Furthermore, the full cytoplasmic domain of RNF167 contains a disordered C-terminal tail that when present results in the formation of higher-order poly-ubiquitin chains.
This study aimed to further understand the mechanism of substrate modification and recruitment by RNF167. The intrinsically disordered C-terminal tail of RNF167 was predicted to contain a substrate-binding domain. Recently reported substrates of RNF167 were assessed to see if they interact directly with purified RNF167 and to see if they were modified with ubiquitin. It was shown that Rab7 bound to purified RNF167, whereas DVL2 and SESN2 did not bind RNF167, suggesting that in cells they may interact indirectly. A mechanism of RNF167 substrate recruitment that depends on the disordered tail of RNF167 is proposed.