Abstract
LNX (Ligand of Numb Protein-X) proteins, LNX1 and LNX2, are RING- and PDZ-based E3-ubiquitin ligases known to interact with Numb. Silencing of LNX2 has been reported to down-regulate WNT and NOTCH, two key signaling pathways in tumorigenesis. Here we report the identification of the domain boundary of LNX2 to confer its ubiquitination activity, its crystal structure along with functional studies. We show that the RING domain in LNX2 is flanked by two Zinc-binding motifs (Zn-RING-Zn), in which the N-terminal Zinc-binding motif adopts novel conformation. Although this motif follows the typical Cys2His2-type zinc finger configuration, it is devoid of any secondary structure and forms an open circle conformation, which has not been reported yet. This unique N-terminal Zn-finger motif is indispensable for the activity and stability of LNX2, as verified using mutational studies. The Zn-RING-Zn domain of LNX2 is a dimer and assumes a rigid elongated structure that undergoes autoubiquitination and undergoes N-terminal polyubiquitination. The ubiquitin chains consist of all seven possible isopeptide linkages. These results were validated using full-length LNX2. Moreover we have demonstrated the ubiquitination of cell fate determinant protein, Numb by LNX2. Our study provides a structural basis for the functional machinery of LNX2 and thus provides the opportunity to investigate suitable drug targets against LNX2.
Highlights
Among the numerous posttranslational modifications that exist for proteins, ubiquitination has been revealed to be an important mechanism for the spatiotemporal regulation of many cellular processes
To identify the nature of the isopeptide linkages formed by fulllength LNX2 (FL-LNX2), we performed a ubiquitination assay using single lysine ubiquitin mutants, each of which containing only one lysine residue with six other lysine residues replaced by arginine
This demonstrated that FL-LNX2 formed polyubiquitin chains containing all seven possible isopeptide linkages, which is consistent with our previous results (Figure 1C)
Summary
Among the numerous posttranslational modifications that exist for proteins, ubiquitination has been revealed to be an important mechanism for the spatiotemporal regulation of many cellular processes. The E2-Ub conjugate cooperates with an ubiquitin ligase (E3) to transfer Ub to the substrate [1, 2] or to the E3 itself, in the case of autoubiquitination. The RING-type E3 ligases are known to regulate key cellular processes, such as apoptosis [7], p53 signaling [8], and DNA repair pathways [9], among others. Abnormal regulation in these processes lead to dysregulated cell growth signals or genomic instability, which are associated with oncogenic progression [10]. It is important to know how these RING-type E3 ligases regulate so many processes as well as themselves
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
